TAURINE STIMULATION OF CALCIUM UPTAKE

IN THE RETINA: MECHANISM OF ACTION

by

JULIUS D. MILITANTS, B.S., M.S.

A DISSERTATION

IN

PHARMACOLOGY

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center

in Partial Fulfillment of the Requfrements for the Degree of

DOCTOR OF PHILOSOPHY

Advisory Committee

John B. Lombardini (Chairperson) Michael P. Blanton

Howard K. Strahlendorf Jean C. Strahlendorf Thomas E. Tenner

Accepted

Associate D § ^ of the Gradu§ft>echool of Biomedical Sciences Texas Tech University Health Sciences Center

May, 2003

ACKNOWLEDGEMENTS

I would like to thank God, our father in heaven, who has taken care of

me all these years and who has helped me be hopeful in spite of adversity

and happy in spite of difficulty. Through my long journey. He has always

truly made His loving presence known to me, and for this I feel so

privileged. I can only wish that everyone allows Him into their heart as He

has come into mine. All that we see will pass, and God's love will live

forever.

Next, I would like to thank Dr. John B. Lombardini, who gave me a

job when I had nothing else to do and nowhere else to go. Working for him

as his technician and his PhD student was reward enough in it by itself It is

only right that he be the one to mentor me to the most important

achievement I have in my life. I truly hope I have made him proud.

As with Dr. Lombardini, Dr. Tina K. Machu gave me a job when I

needed it, and yet again, my work had become replete with fulfillment and

satisfaction. It is akin to having lightning strike twice. I thank her for all

her help and friendship, and for the wonderful shine she has added to my

life and my future.

The members of my committee have been most encouraging,

especially in light of the unusual circumstances of my education. It was

very easy for me to give up on nodon that I should get the PhD, but my

committee made cle tr their support and urged me on. At rimes, I feel it is

more of their will that propelled me forward than it was mine. Without

them, I truly would have given up in despair and friastration. Thank you so

11

much, Dr. Michael P. Blanton, Dr. Howard K. Strahlendorf, Dr. Jean C.

Strahlendorf and Dr. Thomas E. Tenner. I pray that in the fiiture, I can live

up to the faith you had in me.

I must thank all my fi-iends in Lubbock, in the department, among the

students, in the whole school and outside. There are too many to mention.

Your love and fi"iendship give me life and fill my life. I cannot imagine not

having y'all there... imagining all of you there makes all hardship seem so

small. Just the thought of my friends truly makes me happy. Thank you so

much for being there.

Lastly, to my family, my mom, sister and brothers especially, your

love is unfailing, and I know it isn't because you are family. It is truly

wonderful to have you always with me, in thought and spirit. I hope my

work brings you joy and pride, for you deserve all the good that comes your

way. My future I offer to you and I hope that there will be greater things for

us all. To my departed father and brother, I offer this little achievement,

too, and hope you can be proud of me there in heaven, if it be allowed. I do

wish that you both were here.

Ill

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii

ABSTRACT vii

LIST OF TABLES x

LIST OF FIGURES xi

CHAPTER

L INTRODUCTION 1

Background 1

Physiologic significance of taurine 1

Taurine depletion experiments 3

Immunolocalization of taurine 6

Taurine uptake in retinal tissue 8

Taurine binding to retinal dssue 11

Taurine and calcium uptake in the retina 12

Calcium uptake and calcium binding

in the retina 14

Calcium flux and phototransduction 15

Taurine and protein phosphorylation 16 Hypotheses 17

iv

Main Interest 17

ATPase activity in the retina 18

Modulation of taurine uptake 19

Possible modulation of calcium channels 19

Calcium uptake and calcium binding 20

IL METHODS 22

Preparation of dssue samples 22

Protein assay 23

Calcium uptake assay 23

ATPase assay 24

Taurine uptake assay 25

Calcium binding assay 25

Stadstical analysis 26

m. RESULTS 29

Preliminary data 29

Hypothesis 1 37

Hypothesis 2 43

Hypothesis 3 59

Hypothesis 4 68

IV. DISCUSSION 72

Taurine effects of calcium uptake 72

ATPase activity in the retina 76

Taurine uptake versus taurine binding 79

The experimental use of chelerythrine (CHT) 83

Modulation of calcium channels 84

Calcium uptake versus calcium binding 90

Conclusions 93

REFERENCES 95

VI

ABSTRACT

Taurine is a fi-ee amino acid found in millimolar concentrations inside

most animal cells, and the retina appears to possess the greatest amount of

taurine compared to the other cell types. Taurine modulates calcium uptake

in retinal dssue, suggesting that the physiologic function of taurine may be

related to calcium. Taurine is known to stimulate calcium uptake in the

presence of low calcium levels (-10-500 |iM) in the presence of ATP, and

the mechanism behind this effect of taurine was studied. Much is unknown

about this specific effect of taurine. What is the site of acdon of taurine?

What is the nature of the calcium uptake? Most importantly, is this

pardcular type of calcium uptake, and in turn the effects of taurine,

physiologically relevant? Truly, the main interest of this thesis is the

possible physiologic relevance of the sdmulation produced by taurine. The

specific questions that were addressed relate to the nature of the ATP-

dependence of stimulation by taurine, the site of action of taurine, and the

nature of the calcium uptake that taurine increases.

Chelerythrine (CHT) is a potent protein kinase C (PKC) and ATPase

inhibitor that has been previously shown to inhibit taurine-related effects,

specifically in vitro CHT treatment produced an increased in the

phosphorylation of proteins that taurine specifically inhibited. The

discovery of the possible interaction between taurine and CHT suggested

the use of CHT as a possible pharmacological tool in the study of the effects

of taurine. Experiments using CHT were conducted that tested the

hypothesis that taurine modulates ATPase activity in the retina. CHT

VII

inhibited the stimulatory effects of taurine on retinal calcium uptake, and it

was used to help define the mechanism of acdon of taurine.

Among other effects, CHT inhibits ATPase activity in the retina, and

because the stimulatory effects of taurine are ATP-dependent, the data

suggested that ATPase activity may be involved in taurine stimulation of

calcium uptake. Thus, the role of ATPase activity in the mechanism of

action of taurine was studied. Taurine had no direct effect on ATPase

acdvity and so the involvement of ATPase activity was discounted.

CHT also inhibited taurine uptake, suggesting another mechanism by

which CHT may antagonize the stimulatory effects of taurine on calcium

uptake. Taurine uptake was studied relative to its stimulatory effects. The

kiendcs of taurine uptake were determined in both whole retinal

homogenate and in isolated rod outer segments (ROS). In the whole retina,

two uptake components were defined, one of low-affinity and the other of

high-affinity. In contrast, only one uptake system of high-affinity was

observed in ROS. Another series of experiments were conducted to address

the second hypothesis that the inhibidon of taurine uptake abolishes or

attenuates the stimulatory effects of taurine on calcium uptake. An

analogue of taurine, guanidinoethane sulfonic acid (GES) was found to

effecdvely inhibit taurine uptake. Inhibidon of taurine uptake with GES

surprisingly did not produce any effects, eliminating taurine uptake as a

necessary event behind taurine-dependent sdmulation of calcium uptake.

The data suggested that taurine binds to the membrane to produce its

effects.

Vll l

The nature of the calcium uptake was a logical succeeding question to

the stimulatory effects of taurine. Reference literature described the

modulatory effects of taurine on ion channels in the heart and in the skeletal

muscle, which suggested the possible involvement of calcium channel

activation in the mechanism of action of taurine. Experiments were

conducted to test the third hypothesis that specific inhibition of calcium

channels abolishes or attenuates the stimulatory effects of taurine. The

effects of taurine were antagonized by cation channel blockers, specifically

by pharmacologic blockers of cGMP-gated channels. The data strongly

suggested that taurine exerts a stimulatory effect on this channel to increase

calcium uptake. The mechanism behind this effect on ion channels is

unknown.

Lasdy, experiments were conducted to test the fourth hypothesis that

taurine does not affect calcium binding to rednal membranes. Taurine is

known to modulate calcium binding to sarcolemmal membranes and so the

stimulatory effects of taurine may include the stimuladon of calcium

binding. The involvement of calcium binding was ruled out with the use of

binding experiments and calcium ionophore treatments, suggesdng that the

increase in calcium uptake induced by taurine is solely due to increased flux

through calcium channels. The mechanism of taurine, thus, can be

summarized: Taurine binds to membranes to modulate the activadon of

calcium channels and increase calcium uptake, a process which does not

involve ATPase acdvity, taurine uptake or calcium binding.

IX

LIST OF TABLES

1. Inhibitory potency (IC50) of CHT against calcium uptake and ATPase activity in rat retinal homogenate 33

2. Kinetic constants for taurine uptake calculated from data shown in Figures 12 and 14 46

3. Inhibitory potency (IC50) for CHT inhibition of high- and low-affinity uptake in whole retinal homogenate, and of high-affinity taurine uptake in isolated ROS 51

4. Half-maximal inhibition constant values (IC50) for GES inhibition of high- and low-affinity uptake in whole rednal homogenate, and of high-affinity taurine uptake in isolated ROS 55

LIST OF FIGURES

1. Calcium uptake in whole retinal homogenate in the presence of taurine alone 29

2. Calcium uptake in whole retinal homogenate in the presence of 1.2 mM ATP ± 32 mM 30

3. The inhibitory effects of CHT on calcium uptake in whole rednal homogenate in the absence and presence of 1.2 mM ATP ± 32 mM taurine.

(A) Raw uptake data (mean ± SEM, N = 5-7) 31

(B) Calcium uptake in the whole retina homogenate in the presence of 1.2 mM ATP presented in Figure 3(A) converted to % control, and best-fit non-linear regression curve (N = 6-7) 32

(C) Calcium uptake in the whole retina homogenate in the presence of 1.2 mM ATP and 32 mM taurine presented in Figure 3(A) converted to % control, and best-fit non-linear regression curve (N = 6-7) 32

4. Calcium uptake in whole retinal homogenate in the presence of increasing concentrations of ATP ± 100 |iM CHT 34

5. Calcium uptake in whole rednal homogenate in the presence of increasing concentradons oftaurineandl.2mMATP ilOOflMCHT 35

6. The effect of the PKC inhibitor K252b on calcium uptake in whole retinal homogenate in the presence of 1.2 mM ATP ± 32 mM taurine 36

XI

7. ATPase activity in whole retinal homogenate in the presence of increasing concentradons of ATP 37

8. ATPase activity in whole retinal homogenate in the presence of 1.2 mM ATP ± 32 mM taurine and of increasing concentradons of CHT.

(A) Raw ATPase data (mean ± SEM, N = 3) 39

(B) Data presented in Figure 8(A) expressed in terms of % control, and best-fit non-linear regression curve 39

9. ATPase acdvity in whole retinal homogenate in the presence of increasing concentradons of ouabain 41

10. ATPase acdvity in the presence of 100 |iM CHT, 1 mM ouabain (OUA), 1.2 mM ATP ± 32 mM taurine 41

11. ATPase activity in whole retinal homogenate in the presence of increasing concentradons of thapsigargin 42

12. Taurine uptake in whole retinal homogenate in the

presence of increasing concentradons of total taurine 43

13. Eadie Hofstee plot of data fi"om Figure 12 44

14. Taurine uptake in isolated ROS in the presence of increasing concentrations of total taurine 45

15. Eadie Hofstee plot ofdata from Figure 14 46

16. High-affinity taurine uptake in whole retinal homogenate in the presence of increasing concentradons of CHT.

(A) Raw uptake data (mean ± SEM, N = 3, *p < 0.05 vs. 0 liM CHT, Dunnett's posttest) 48

Xll

(B) Data presented in Figure 16(A) expressed in terms of % cond-ol, and best-fit non-linear regression curve (N = 3) 48

17. Low-affinity taurine uptake in whole retinal homogenate in the presence of increasing concentradons of CHT.

(A) Raw uptake data (mean ± SEM, N = 3, *p < 0.05 vs. 0 |iM CHT, Dunnett's posttest) 49

(B) Data presented in Figure 17(A) expressed in terms of % control, and best-fit non-linear regression curve (N = 3) 49

18. High-affinity taurine uptake in isolated ROS in the presence of increasing concentradons of CHT.

(A) Raw uptake data (mean ± SEM, N = 3, p < 0.05 vs. 0 |iM CHT, Dunnett's posttest) 50 *

(B) Data presented in Figure 18(A) expressed in terms of % control, and best-fit non-linear regression curve (N = 3) 50

19. Eadie-Hofstee transformation of taurine uptake data as measured in the presence of 25-750 |J,M total taurine ± 5 |iM CHT 52

20. High-affinity taurine uptake in whole retinal homogenate in the presence of phorbol myristate acetate (PMA) and staurosporine (STAU) 53

21. High-affinity taurine uptake in whole retinal homogenates inhibited by GES 54

22. Low-affinity taurine uptake in whole retinal homogenates inhibited by GES 55

23. High-affinity taurine uptake in isolated ROS inhibited by GES 56

Xll l

24, Calcium uptake in whole retinal homogenate in the presence of GES, a taurine uptake inhibitor 57

25, Calcium uptake in isolated ROS in the presence of GES, a taurine uptake inhibitor 58

26, Calcium uptake in whole retinal homogenate in the presence of 1.2 mM ATP ±32 mM taurine, and cadmium, a non-specific cation channel inhibitor 59

27, Calcium uptake in whole retinal homogenate in the presence of 1,2 mM ATP ± 32 mM taurine, and of increasing concentrations of nifedipine, a blocker of voltage-gated L-type calcium channels 60

28, Calcium uptake in whole retinal homogenate in the presence of 1.2 mM ATP ± 32 mM taurine, and of LY83583, a blocker of cGMP-gated channels 61

29, Calcium uptake in isolated ROS in the presence of 1.2 mM ATP ± 32 mM taurine, and of LY83586, a blocker of cGMP-gated channels 62

30, Calcium uptake in isolated ROS in the presence of 1,2 mM ATP ± 32 mM taurine, and Rp-8-Br-PET-cGMPS, a specific blocker of cGMP-gated channels 63

31, Calcium uptake in isolated ROS in the presence of 1,2 mM ATP ±32 mM taurine, and of increasing concentrations of dibutyryl cGMP, a membrane-permeant analogue of cGMP 65

32, Calcium uptake in the isolated ROS in the presence of 1,2 mM ATP and increasing concend-adons of Rp-cGMP, an analogue of cGMP 65

XIV

33. Calcium uptake in the isolated ROS in the presence of 1,2 mM ATP ± 32 mM taurine, and of increasing concentrations of zaprinast, an inhbitor of cGMP-specific phosphodiesterase 67

34, Calcium binding in whole retinal homogenate in the presence of 1,2 mM ATP and/or 32 mM 69

35, Calcium binding in whole retinal homogenate in the presence of increasing concentrations of ATP 69

36, Calcium binding in whole retinal lysate in the presence of ATP and increasing concentrations of taurine 70

37, Calcium uptake in whole retinal lysate in the presence of A23187, a calcium ionophore 71

38, Calcium uptake in lysed ROS in the presenceofA23187, a calcium ionophore 71

XV

CHAPTER I

INTRODUCTION

Background

Physiologic significance of taurine

The bulk of the first chapter was adapted fi-om a review published in

Nutritional Neuroscience (Militante and Lombardini, 2002). Taurine (2-

aminoethanesulfonic acid) is a fi-ee amino acid found in high millimolar

concentradons in animal dssue to which an ever growing number of cellular

and physiologic functions have been atd-ibuted (Wright et al., 1986;

Huxtable, 1992; Sturman, 1993). Taurine has been sUidied as a

neurodansmitter, as an osmoregulator and as an antioxidant, among others.

In addition, the effects of taurine to protect against hepatotoxicity and to

modulate ion channels have been reviewed (Timbrell et al,, 1995; Satoh and

Sperelakis, 1998). However, reviews on the significance of taurine seemed

to have decreased considerably among peer-reviewed journals this past

decade.

Special attendon has been paid to the mechanism of action of taurine

in the retina, especially in the decades of the 70s and 80s, mainly because of

experiments which suggest that taurine is most abundant in the retina

compared to other dssue types and because of studies which linked taurine

deficiency with visual dysflincdon (reviewed in Lombardini, 1991), More

importandy, the simple supplementation of the diet with taurine proved to

be sufficient in alleviating the vision problem, theoretically through the

replenishment of the depleted taurine levels. Taurine is a naUiral product

that is readily available in food and nutrition stores, and is relatively

inexpensive. Thus, the understanding of its actions may provide an

inexpensive health option for many people.

There are other arguments for the physiologic importance of taurine

in the retina. Numerous reports validate the effect of taurine to modulate

cellular processes in vitro. The synthesis, uptake and release of taurine are

tightly regulated, suggesting that the maintenance of taurine levels is crucial

in the normal funcdon of the redna (reviewed in Lombardini, 1991; in

Militante and Lombardini, 2002). Also, taurine exhibits d-ophic effects in

animal models of rednal regeneration (reviewed in Lima, 1999). Moreover,

the decrease in retinal taurine levels appears to be one of the earliest lesions

observed in the degeneration of the retina in rat models of rednitis

pigmentosa (Okada et al,, 2000), However, scientific interest and research

on the physiologic function of taurine have been, for all intents and

purposes, insufficient to the extent that there is still more speculation than

certainty as to the exact mechanism of action of taurine, in the redna or in

other tissues. More studies have to be done, especially in light of the

potential taurine has to affect cell function.

Calcium ions provide a crucial signal in the photodansducdon

process in the rod outer segments (ROS) of the retinal photoreceptor cells

(Baylor, 1996), The acdvadon and deacdvadon of the cGMP-gated cation

channels found in the plasma membrane of the ROS are involved in this

calcium flux. Taurine concend-adons in retinal tissues have been estimated

to be as high as 79 mM (Voaden et al,, 1977), and so it is of great interest

that taurine is known to exert some condol over calcium flux in the redna

(reviewed in Lombardini, 1991). This may be an important physiologic

funcdon of taurine. The research presented in this thesis concerns the

mechanism of acdon behind the specific sdmulatory effect of taurine under

condidons of low calcium concentradon.

Taurine depletion experiments

An important model through which the native function of taurine in

the retina may be established is through the use of animals which have been

nutritionally deprived or pharmacologically depleted of taurine. In fact, this

is the most common methodology used to study the importance of taurine in

the retina. One of the earliest and more well-known models is that of the cat

which is fed a taurine-free casein diet (Schmidt et al., 1976). In this model,

the animal exhibited taurine levels of only 6% and 60% of normal in the

plasma and in the retina, respectively. A similar nutritional protocol has

been used in Rhesus monkeys which utilized taurine-fi-ee human infant

formula (Imaki et al,, 1987; Neuringer and Sturman, 1987), In human

padents, taurine deficiencies were found to occur with total parenteral

nudition that did not include taurine in the food formula (Geggel et al,

1985), and this particular model has also been used to sdidy the physiologic

function of taurine in the retina.

The rat model presents a different metabolic type as the use of a

taurine-fi-ee diet is not effecdve in decreasing the level of taurine in the

retina (Lake, 1982), Antagonists of taurine transport have been used in the

rat to deplete taurine in the redna, specifically, guanidinoethanesulfonic

acid (GES) and p-alanine (Lake, 1981, 1982; Lake and Cocker, 1983;

Quesada et al,, 1984; Lake and de Marte, 1988). The inclusion of GES in

the drinking water (1%) of rats causes a significant decrease in the amino

acid level (>50%) and in the uptake of radiolabeled taurine in the retina

(-60%). It is interesting to note that the retina appears to be particulariy

sensitive to taurine depletion as compared to other neural-type tissues as

GES d-eatment had no effect on the taurine levels in the cerebellum and in

the cerebral cortex (Quesada et al, 1984). Also, other amino acids (GABA,

glycine, glutamate, aspartate, glutamine) do not appear to be affected by

GES d-eatment (Lake, 1981, 1982). Taurine uptake in the retina is also

inhibited by P-alanine but tissue taurine levels are only slighdy decreased

(Lake and de Marte, 1988).

The depledon of taurine has been invariably linked to retinal cell

damage and elecfroretinogram (ERG) abnormalities. Depletion of taurine

results in cellular degeneration and death in the rat (Pasantes-Morales et al,,

1983; Lake, 1986; Lake and Malik, 1987; Cocker and Lake, 1987; Quesada

et al,, 1988; Shimada et al,, 1992; Lombardini et al,, 1996), the cat (Berson

et al,, 1976; Jacobson et al,, 1987; Leon et al, 1995) and in the Rhesus

monkey (Neuringer and Sturman, 1987; Imaki et al , 1993), ERG patterns

were studied in the rat and in the cat, and expectedly, abnormalities were

observed. Behavioral studies revealed deficits in visual acuity in rhesus

monkeys nudidonally depleted of taurine (Neuringer and Sturman, 1987),

In all cases, the reind-oduction of taurine or the withdrawal of the taurine

uptake antagonist reverses or prevents the negadve effects of taurine

depletion. It is in these experiments that the nud-idonal value of taurine has

been most cleariy defined, as the use of taurine supplementadon to reverse

lesions associated with taurine deficiency was proven to be easy, safe and

effecdve.

Of pardcular note is the work by Geggel et al. (1985) which described

ERG abnormalities in human patients suffering taurine deficiency. Patients

on total parenteral nutrition which did not include taurine were found to

have decreased levels of taurine in the blood. Moreover, ERG deficits were

observed in some of the subjects. The authors found that only young

children and not adult patients were affected. While the reason behind this

difference is unclear, it can be surmised that taurine metabolism changes

with age, Indoduction of taurine to the intravenous solutions proved

effective in normalizing ERG activity in three out of four children tested.

In various rat studies, it has been demonsdated that the ERG deficits

occur before retinal cell loss. Also, the deleterious effects of taurine

deficiency on cell survival and ERG acdvity have been demonsdated to be

either dependent on or synergistic with light exposure. Rapp et al. (1988)

described the interaction between light exposure and GES treatment in rats

exposed to either low- or high-intensity cyclic lighting for 10 weeks. No

outer nuclear layer (ONL) abnormalities were observed with GES treatment

and simultaneous low-intensity light exposure. However, ERG

abnormalities were observed, indicating the precedence of ERG dysfuncdon

over frank cell loss. Significant cell loss in the ONL of the retina and

greater reducdon in the a- and b-wave were observed with GES freatment

and simultaneous exposure to high-intensity light. Measurements of

taurine content demonstrated lower taurine levels with exposure to high-

intensity light, with and without GES treatment, suggesting light-induced

loss of taurine.

Other studies which used a light-cycle protocol against a dark-

maintenance protocol support the putative interaction between light

exposure and GES treatment in the deterioration of ERG activity (Quesada

et al , 1988; Cocker and Lake, 1989). GES-related ERG deficits were

significantly less in dark-maintained rats than in rats exposed to light.

Taurine levels were also higher with dark maintenance. Similar to the low-

and high-intensity light experiments previously discussed (Rapp et al,

1988), DNA measurements indicate no cell loss with GES deatment with

both light-cycle and dark maintenance protocols. In general, ERG deficits

preceded cell loss, were associated with decreased taurine levels and with

exposure to light.

Immunolocalization of taurine

The localizadon of a substance to specific cell types in histological

preparations is key to idendfying its physiological significance. Usually,

distribution to specific cell-types suggests a requirement for the substance in

the unique fijnction or funcdons of that cell type. The scientific proof of the

abundance of taurine in the redna nadirally led to great interest as to the

idendty of the specific rednal cells which contained this amino acid. Early

studies made use of radiolabeled taurine and autoradiography of retinal

dssue secdons. Through this method, the high-affinity uptake of taurine in

photoreceptor cells and pigment epithelium was demonsfrated in the rat,

mouse, guinea pig, baboon, pigeon, cat and frog (Lake et al , 1978), To a

lesser degree, taurine was also taken up in bipolar and amacrine neurons,

and in glial cells.

Taurine uptake studies readily identify cells that actively transport

taurine, but cells may slowly fransport taurine yet retain high amounts

infracellulariy. The labeling of taurine in fixed dssue through

immunochemical procedures provides a more accurate visualization of

steady-state levels of taurine. The use of andsera in specifically detecting

taurine was first indoduced with brain secdons in 1985 (Madsen et al,

1985; Ottersen et al , 1985), Taurine-like immunoreacdvity (TLI) was first

demonsfrated in insect photoreceptors in 1988 (Schafer et al, 1988) and in

the mammalian retina in 1989 (Lake and Verdone-Smith, 1989) with the cat,

rat and guinea pig. The procedure has since been used as the method of

choice in studying taurine distribution in the retina.

Various animal species have been studied, e.g., honeybee

(Apis)(SchsifeT et al , 1988), marine snail (Bulla gouldiana) (Michel et al,

2000), Australian lungfish (Neoceratodus forsteri){?ow, 1994), goldfish

(Marc, et al, 1995; Omura and Inagaki, 2000), eel (Anguilla

japonica){Omum, Inagaki, 2000), rat (Fletcher and Kalloniatis, 1996), cat

(Marc et al , 1998), cynomologous monkey (Kalloniads et al, 1996), and

human (Nag et al , 1998), Photoreceptor cells or analogous cell types

consistendy exhibit TLI, but other cell types like Muller glial cells also

exhibited reacdvity. Photoreceptor cells, specifically the rod outer segments

(ROS) of the rod cell, are responsible for visual sensation, fransforming

light signals into neuronal signals. It is particulariy noteworthy that very

few studies with human retina have been performed, and that the available

data indicate that photoreceptor ROS do not contain significant levels of

taurine (Nag et al , 1998), in condast to data from lower animal forms like

the monkey (Kalloniatis et al , 1996), Data from the eel, however,

demonstrated that ROS were not immunostained in light-adapted

conditions, but were intensely stained in dark-adapted conditions, implying

that taurine flux in the ROS is dynamic in nadire. Cleariy, the experimental

conditions under which tissue sampling is done would influence the results

of taurine analysis, at least in the ROS.

Taurine uptake in retinal tissue

In the rat and cat, the uptake of taurine has been associated with

Muller, photoreceptor and amacrine cells (reviewed in Huxtable, 1989;

Lombardini, 1991), The affinity of the uptake system for taurine is

measured by the concenfration of taurine subsdate (Km) needed to produce

half-maximal activity. The kinedcs of taurine uptake in the retina have been

studied to a fairly significant degree. It is fairly well-accepted that there are

at least two sites for taurine uptake characterized by two discernible uptake

components with different affinities for taurine, a system that seems to also

exist in brain slices and synaptosomal preparations (reviewed in Huxtable,

1989; Lombardini, 1991). Usually, there is at least a 10-fold difference in

the affinity and the maximum velocity (Vmax) exhibited by the two uptake

systems. There is evidence that high-affinity taurine uptake is specifically

associated with photoreceptor cells (Schmidt and Berson, 1978), High-

affinity uptake was observed to disappear with the loss of photoreceptor

cells in rats with hereditary age-related rednal degeneradon called RCS

(Royal College of Surgeons) rats. However, the exact nadire of uptake in

the ROS vis-a-vis the whole retina was unknown.

Salceda and colleagues have done considerable work with taurine

uptake in the retinal pigment epithelium (RPE) cells (Salceda and Saldana,

1993). Experiments with RPE cells isolated from rat eyes revealed uptake

systems functioning with Km values of less than 100 |iM, Later sdidies

revealed the existence of another uptake system with lower affinity (Km =

-400 |iM) (Salceda, 1999). Thus, it appears that, in addition to the

photoreceptor cells, RPE cells may also account for high-affinity uptake in

the retina. RPE cells are intimately associated with the ROS and form an

important component of the rednal-blood barrier which separates the blood

from the outer retinal layer. It is possible that, in the RCS rats, RPE cells

degenerate along with photoreceptor cells as they age, hence, the

disappearance of high-affinity taurine uptake with age (Schmidt and Berson,

1978). Regardless, the data suggest that taurine uptake is tighdy regulated

as it is fransported from the intravascular space to the photoreceptor cells.

The taurine transporter has been cloned using DNA from the Mabin-

Darby canine kidney (MDCK) cell line (Uchida et al , 1992, 1993), mouse

brain (Liu et al , 1992), rat brain (Smith et al , 1992), human thyroid (Jhiang

et al , 1993), human placenta (Ramamoorthy et al , 1994), and bovine

endothelial cells (Qian et al , 2000). Using the DNA of the taurine

fransporter from the human thyroid, the taurine fransporter in human RPE

cell line was also cloned through reverse franscripdon-polymerase chain

reacdon (RT-PCR) (Miyamoto et al , 1996). The DNA contained 1863 base

pairs, predicdng a protein 620 amino acids in length. The DNA was also

found to be almost identical in sequence to the taurine transporter cloned in

human thyroid and placenta. The DNA sequence has been deposited as

human RPE taurine fransporter in GenBank under accession No, U09220.

The fransporter was expressed in frog oocytes, and the fiincdon of this

fransporter was found to be sodium- and chloride-dependent, and

susceptible to inhibition by P-alanine and GABA. In similar fashion, the

taurine fransporter in mouse retina (mTAUT) was cloned (accession No.

AF020194) using the DNA of the MDCK taurine fransporter (Vinnakota et

al , 1997). The DNA was 2163 base pairs long and coded for a 621-amino

acid protein. It exhibited >93% homology with the mouse brain fransporter.

The protein was expressed in frog oocytes and its funcdon was found to be

sodium- and chloride-dependent, and sfrongly inhibited by P-alanine,

hypotaurine and guanidinoethanesulfonic acid (GES), a taurine analogue.

Surprisingly, when in situ hybridization studies were done, the highest

expression of the mTAUT mRNA was not found in the retina but in the

ciliary body, specifically in the outer layer (Vinnakota et al , 1997). The

iris, conjunctiva and the cornea showed little signal. The retina per se

showed only a modest level of silver grains. Cells in the ganglion cell layer,

the inner nuclear layer, inner plexiform layer, inner segment layer and the

RPE cells were labeled. In the rat retina, the mRNA for the taurine

fransporter was also found in the same areas (Morimura et al , 1997). The

transporter mRNA in the rat retina was also found to increase in chronic, but

not in acute, hypemafremia (Morimura et al , 1997). In condast, antibodies

directed against two cloned high-affinity fransporters (TAUTl and TAUT2)

revealed that these fransporters were expressed in photoreceptor and bipolar

10

cells in the retina (Pow et al , 2002), Regardless of the localizadon and

regulation of taurine fransporters, deletion of the mTAUT fransporter gene

results in vision loss due to severe retinal degeneration, suggesting that the

function of taurine fransporters to take up taurine into the cell is crucial in

the retina (Heller-Sdlb et al , 2002),

Taurine binding to rednal issue

Similar to taurine uptake, binding studies with retina membrane

reveal at least two sites of varying affinities for taurine (Huxtable, 1989), It

was assumed that taurine binding receptors corresponded, at least in part, to

fransporter proteins responsible for taurine uptake, Ligand affinity is

characterized by the ligand concenfration at which half-maximal binding is

observed, a value defined as the dissociadon constant (Kd), Sodium-

dependent high affinity sites have been studied in the retina of rat and

chicken, and have been found to have a Kd value of > 10 |iM (Lopez-

Colome and Pasantes-Morales, 1980; Lombardini and Prien, 1983),

Binding sites that are sodium-independent and of lower affinity have also

been demonsfrated (Salceda and Pasantes-Morales, 1982), A low-affinity

sodium-dependent binding site has also been idendfied which co-exists with

the high-affinity site (Lombardini and Prien, 1983).

Lopez-Colome et al, (1991) used osmodcally shocked membrane

preparadons from chick RPE cells in culdire for taurine binding

experiments and found a single saturable binding site (Kd = -237 nM

taurine). In fresh membranes, taurine binding increased with sodium and

with higher incubation temperature. Freezing and thawing of the membrane

11

appeared to eliminate sodium- and temperature-dependent increases in

binding. Taurine uptake was also sdidied in fresh and frozen samples, and

uptake was not compromised by the freeze-thaw procedure. Thus, there

appears to be distinction between uptake and binding sites, at least in RPE

cells. The distinction was verified by studies with GES, which is a taurine

uptake inhibitor, GES was to found to have very weak effects on taurine

binding. The data suggested that in some cases, taurine binding sites do not

correlate with taurine uptake sites.

Taurine and calcium uptake in the retina

It is well-established that taurine is a biphasic modulator of rednal

calcium uptake in in vitro experiments (reviewed in Lombardini, 1991),

These in vitro experiments, in brief, entail the incubation of tissue with

radiolabeled calcium in the presence of varying concenfrations of unlabelled

calcium. Calcium uptake increases as calcium levels are increased in the

buffer (Liebowitz et al, 1989). The exact nature of this uptake is unknown,

although it is reasonable to assume that it involves more than one

biochemical mechanism. As with any experimental model, there are

limitations to its use as a physiological system and conclusions drawn from

its use must be taken in the proper context.

At low concenfradons of calcium in the reacdon buffer (10-500 |iM),

both ATP and taurine act as stimulatory agents. However, taurine is

effecdve only in the presence of ATP. It is unclear if taurine potentiates the

effect of ATP to stimulate calcium uptake, or if taurine and ATP together

sdmulate uptake through an altogether different mechanism. The effect of

12

taurine is also dependent on the presence of bicarbonate (Pasantes-Morales

and Ordonez, 1982), hence the use of at least 25 mM bicarbonate in most

calcium uptake experiments. Half-maximal stimulation by taurine has been

estimated at anywhere from -8 to -30 mM concentradon (Liebowitz et al,

1987; Liebowitz et a l , 1988; Lombardini et al, 1989).

ATP and taurine singly are inhibitory at higher calcium

concenfradons (> 1,2 mM) (Liebowitz et al, 1989). At these

concenfrations, basal calcium uptake increases in propordon to the calcium

included in the buffer. The data suggest that in the presence of higher

calcium concenfrations, uptake is increased through additional mechanisms

that are modulated by taurine in a manner disdnct from its effects on

calcium uptake at lower calcium concenfrations.

Within the intermediate calcium concenfration range, the ATP-

dependent stimulatory effect of taurine on calcium uptake diminishes. It is

probably more accurate to say that ATP and taurine stabilize calcium uptake

in the face of changing calcium levels, sdmulatory when there is less

calcium and inhibitory when there is more (Militante and Lombardini,

2000), The phenomenon may be physiologically relevant as calcium levels

change within the cell as a mechanism for cellular signaling.

Taurine characterisdcally acts at the millimolar level which is

indiitive if not predictable given that, in nature, animal cells act to maintain

millimolar quandties of taurine infracellulariy. Taurine, thus, is considered

to be an osmotic agent, first and foremost. However, the effects of taurine

on calcium uptake in the retina are mimicked by some taurine analogues and

not by others, and the opposite effect is seen with yet others (reviewed in

13

Militante and Lombardini, 2002), The data strongly suggest that the effects

of taurine are not mediated by osmotic mechanisms. As of yet, the

mechanism behind these effects of taurine is not yet fully understood.

Taurine depletion in the rat with both GES and P-alanine did not

result in any change in the effect of taurine to stimulate calcium uptake at

low calcium concenfration (Militante and Lombardini, 2002), The data are

interesting in that one might expect some form of cellular adaptation to

occur in response to taurine depledon which would render the retina more

sensidve to the effects of taurine. However, this is not the case, at least with

this pardcular effect of taurine on the redna.

Calcium uptake and calcium binding in the retina

Care must be taken in the analysis of the effects of taurine as the

modulation of both calcium fransport and binding to membranes may

explain the specific effects of taurine on calcium uptake. In fact, eariy data

from rat retina revealed the reladve lack of effect of A23187, a divalent

ionophore, in increasing calcium uptake in the retina at high buffer

concenfradons of calcium, and thus, calcium uptake as such was atfributed

to membrane surface components rather than acdial influx of calcium

(Lombardini and Liebowitz, 1990). However, these sdidies were preceded

by sdidies that demonstrated increases in calcium uptake in PI and P2

fracdons from chick retina with A23187 freatment (Pasantes-Morales and

Quesada, 1980), All the preceding uptake assays were done at 36-37°C for

5 minutes or less to select for uptake acdvity over binding acdvity.

However, the "uptake" measured may still have included some level of

14

calcium to membranes. In whole rat retinal membranes, two putative

"uptake" sites have been described, exhibidng Km values of 35 and 2076

\XM (Lombardini, 1983), The high affinity system has been logically

identified with specific calcium transporter proteins. The lower affinity

"uptake" system may actually be membrane binding, as fransporter systems

usually work at subsfrate levels in the low micromolar range. Suffice to say,

the calcium activity measured should be attributed to more than one specific

mechanism and may not involve the acdial flux of calcium through the

plasma membrane. Thus, the term calcium accumulation is probably more

appropriate and precise than is calcium uptake, the more commonly used

term in the scientific literature.

Calcium flux and phototransduction

The biphasic effects of taurine on calcium uptake are observed both

in whole retinal samples and in isolated ROS (Liebowitz et al , 1989;

Militante and Lombardini, 1998a, b, 2000), It is important to note that the

ROS is a very specialized segment of the photoreceptor cell which is

responsible for the conversion of photonic signals to neural signals which

the brain can understand (Baylor, 1996), The whole retinal sample, on the

other hand, would contain the whole photoreceptor cell plus all the other

types of neuronal and glial cells which are found in the retina. Clearly, the

nature and the physiologic significance of the calcium uptake assayed in the

ROS and the whole rednal samples would be radically different.

The precise regulation of the inward flow of calcium through cGMP-

gated channels is crucial in the light-sensing function of the ROS (Baylor,

15

1996), In fact, the photofransducdon process and the reguladon of the

cGMP-gated calcium channel may provide a physiologic significance for

the biphasic effects of taurine on calcium uptake, in the ROS at least. In the

absence of light stimulus, the cGMP-gated channels are acdvated in the

ROS, allowing for the flow of calcium and sodium into the ROS. At this

point, taurine would be inhibitory to the uptake of calcium and presumably

is not acting on channel function. With light stimulus, cGMP levels drop

drastically, inactivating the channel. Calcium levels then drop as active

extrusion proceeds with the NaVCa^-K^ exchanger. The lower calcium

levels signal an increase in both the synthesis of cGMP and the affinity of

the channel for cGMP, resulting in the reopening of the channel, an event

necessary for continued photofransduction in the ROS, At this stage when

calcium levels have dropped, taurine may then assist in the process of

recovery by increasing calcium uptake through the cGMP-gated channels.

Taurine and protein phosphorylation

The most important funcdonal effect of taurine is most probably

associated with the reversible elecfroretinogram deficits observed after

taurine depledon in the cat, monkey, and rat (Lombardini, 1991), This

deficit is coincident with an increase in the phosphoryladon of a specific

-20 K protein in the mitochondrial fracdon of the rat retina. Conversely,

the phosphoryladon of this protein has been demonsfrated to be inhibited by

taurine in vitro (Lombardini, 1992). However, the idendty of the protein and

its link to the fiancdonal deficit observed are unknown.

16

Chelerythrine (CHT) is a benzophenanthridine alkaloid that exhibits

several biological effects including the inhibidon of protein kinase C (PKC)

(Herbert et al , 1990) and adenosinefriphosphatase (ATPase) (Cohen et al,

1978). Previously, it was demonsfrated that CHT causes sdmulation, both

in the presence and absence of taurine, of the in vitro phosphorylation of the

same -20 K mitochondrial protein in the rat retina that taurine depledon

stimulates (Lombardini, 1995; Lombardini, 1996; Lombardini et al, 1996),

Half-maximal stimuladon was estimated at approximately 37 |iM. PKC

acdvation was shown to have no effect on the phosphorylation of the -20 K

protein (Lombardini, 1993), and so it was assumed that CHT was acdng

through some mechanism other than PKC inhibition to cause the increase in

phosphorylation. Because of these data, CHT was then considered as a

possible pharmacologic tool in studying the effects of taurine on calcium

uptake.

Hypotheses

Main Interest

The mechanism of action of taurine is the primary interest of these

experiments. CHT was used as a pharmacologic tool as CHT exhibited

modulatory effects of the phosphorylation of the -20K protein in the retina,

the same protein which taurine appears to also modulate. In preliminary

experiments, CHT was found to inhibit the sdmulatory effects of taurine on

calcium uptake in the retina, and was thus used to study the mechanism of

acdon of taurine. The data presented in this thesis all revolve around a

blanket hypothesis: The stimulatory effect of taurine on ATP-dependent

17

calcium uptake in the retina is dependent on the moduladon of ATPase

acdvity, on the uptake of taurine into retina tissue, and on the activation of

calcium channels, and not on the modulation of calcium binding to

membranes. This hypothesis can be divided into several sub-hypotheses

under which all the experiments are organized.

ATPase activity in the retina

The first sub-hypothesis is: Taurine modulates ATPase activity in the

retina. The data pertinent to this sub-hypothesis were previously published

in the Biochemical Pharmacology joumal (Militante and Lombardini,

1998a), The sdmulatory effects of taurine are profoundly dependent on the

presence of ATP, One of the earliest studies which described this specific

dependence suggested that the hydrolysis of ATP was necessary in the

potentiating effects of taurine (Pasantes-Morales, 1982), implying the need

for ATPase activity. In addidon, the effects of CHT, an inhibitor of PKC

and ATPase activity, were tested, and CHT was found to antagonize the

effects of taurine. Thus, it was surmised that the ATPase acdvity of the

retinal dssue is involved in the effects of taurine. As the activity of the

different forms of ATPase is dependent on the specific ions present in the

buffer, ATPase acdvity was measured in the rednal dssue under buffer

conditions that were identical to that under which calcium uptake

experiments were conducted, ATPase activity was then studied reladve to

the effects of taurine in the retina.

18

Moduladon of taurine uptake

The second sub-hypothesis is: The specific inhibidon of taurine

uptake abolishes or attenuates the sdmulatory effects of taurine on calcium

uptake. Most of the data presented in this secdon were previously

published in the Brain Research iowmdX (Militante and Lombardini, 1999a)

and in the Journal of Pharmacology and Experimental Therapeutics

(1999b). In all of the calcium uptake experiments, the retinal dssue was

exposed to taurine in solution and, accordingly, two possibilities exist: the

dssue may actively take up taurine and taurine may bind to dssue

membrane. Similarly, calcium uptake was studied in bovine ROS, and

specifically the sdmulatory effect of ATP was characterized (Hemminki,

1975), The data suggested that the uptake of ATP into the dssue was

necessary in ATP-dependent stimulation. The same question could be asked

of taurine: Is taurine uptake necessary in taurine-stimulated calcium uptake?

Taurine is thought to bind to membranes and to alter calcium binding by

altering the membrane environment (Huxtable and Sebring, 1986), but

taurine uptake into the dssue most certainly occurs, too. Taurine uptake

kinetics were characterized in the whole retina and in isolated ROS, and the

inhibidon of uptake was studied. The inhibition of uptake was then

associated with the effects of taurine on calcium uptake.

Possible moduladon of calcium channels

The third sub-hypothesis is: The specific inhibition of calcium

channels abolishes or adenuates the stimulatory effects of taurine. Most of

the data presented in this section come from a research ardcle published in

19

the Amino Acids journal (Militante and Lombardini, 1998b), One

possibility is that the stimulatory effects of taurine on calcium uptake may

be due to the opening of calcium channels. It is strange that the effects of

taurine on calcium channels in the retina have not been studied extensively

given that the modulatory effects of taurine on ion channels in the heart are

well-documented (Satoh and Sperelakis, 1998). In the retina, the most

important calcium channel is the cGMP-gated calcium channel.

Experiments were performed to sdidy the involvement of cGMP-gated

channel activation in the effects of taurine. Modulators of the channel were

used in conjuncdon with taurine freatment in calcium uptake experiments.

Calcium uptake and calcium binding

The last subhypothesis is: Taurine does not affect calcium binding to

retinal membranes. Experiments were done to study the nadire of the

calcium uptake that taurine modulates, specifically to differendate between

calcium uptake through retinal membrane and calcium binding to retinal

membrane. Calcium uptake was measured in retinal tissue by incubating

retinal samples with radiolabeled calcium and measuring the amount that

remained after filtering the sample through a glass fiber filter. In theory, the

acdvity measured would be comprised of calcium taken up into the dssue

and calcium that bound to the membrane, and is more correcdy described as

calcium accumulation. It is possible that taurine binds to the membrane and

increases the binding of calcium to the membrane in addidon to modulating

calcium uptake. In these experiments, retinal dssue was osmodcally lysed

to denadire acdve calcium fransport. Calcium uptake was also measured

20

with and without treatment with A23187, a calcium ionophore that makes

membranes freely permeable to calcium. A23187 would allow the calcium

gradient produced by uptake systems to dissipate when the tissue samples

are washed and filtered, leaving only calcium bound to membranes. The

effects of taurine on calcium binding can thus be studied. Calcium binding

was also measured at 0°C in the presence of ATP and taurine. The use of

lower incubadon temperatures has been fraditionally used to study binding

as acdve uptake systems usually require a physiologic 37°C temperature to

funcdon adequately.

21

CHAPTER II

METHODS

Preparation of dssue samples

Adult Sprague-Dawley or Wistar rats were anesthetized with CO2 and

killed by cervical dislocation or decapitation, after which the eyes were

removed and either used immediately or frozen at -80°C to be thawed later

for experimental use (Militante and Lombardini, 1998ab), Whole retinal

dssue samples were isolated by cutting the comea open and by gently

teasing the tissue out of the eye cup into a 0.32 mM sucrose soludon while

on ice. All subsequent procedures were done on ice. The dssue was then

cenfrifliged for 15 minutes at 16,000 x g (4°C), washed in 20 mM sodium-

bicarbonate, recentrifuged as before and then washed in sodium-bicarbonate

buffer [50 mM NaHCOj, 50 mM NaCl, 50 mM KCl, 1.2 mM KH2PO4, 2

mM MgCl2, pH 7.4 (Kuo and Miki, 1980)] with CaCl2 added in the desired

concenfrations. The tissue was recentrifuged, resuspended in the

aforementioned sodium-bicarbonate buffer and gendy homogenized with a

glass-to-glass homogenizer. For some experiments, Krebs-Ringer-

bicarbonate (KRB) buffer [118 mM NaCl, 25 mM NaHCOj, 5 mM glucose,

1,2 KH2PO4, 4.7 KCl, 1.17 mM MgS04, with the desired amount of CaCl2

added] was used in place of the sodium-bicarbonate buffer (Militante and

Lombardini, 1999ab). KRB buffer was aerated with 5%I95% oxygen for 15

minutes and the pH of the soludon adjusted to 7.4 with concenfrated HCl

For the isoladon of ROS, 0,3 mM mannitol was used instead of 0,32

mM sucrose, Rednal tissue was dissected out as before and the ROS were

22

removed by vortex-mixing the tissue for 10-20 seconds, allowing the tissue

to setde, and the decanting the supernatant which contained the ROS in

suspension. The procedure was sometimes repeated with the pellet to

maximize ROS yield. The supernatant was then centrifiiged for 15 minutes

at 16,000 X g (4°C) and the pellet was then suspended in sodium-

bicarbonate buffer. The remaining tissue components were discarded. For

some experiments, KRB buffer was used instead of sodium-bicarbonate

buffer.

Protein assay

The amount of protein used was assayed using the bicinchoninic acid

(BCA) method (Militante and Lombardini, 1998ab), Briefly, standards and

samples were mixed with a soludon of BCA protein assay reagent and 4%

copper II sulfate (50:1). The mixdire was incubated in a 37°C water bath for

30 minutes and absorbance was read at 560 nm. Protein content was used to

correct and standardize all the data measured. Commonly, 100-300 fig for

the whole retinal homogenate or 50-150 |ig for the ROS was used for each

reaction.

Calcium uptake assav

The assay was performed with either the sodium-bicarbonate or KRB

buffer (Militante and Lombardini, 1998a, 1999b), Reagents such as taurine

and ATP were added to the reaction dibe in the appropriate amounts and

kept on ice until the start of the reacdon, Idendcal amounts of'^CaCl2

(400,000-500,000 dpm) were added to the dibes in the presence of varying

23

amounts of CaCl2. The reaction tubes were then preincubated in a 37°C

water bath for 2 minutes. Whole retinal homogenate or ROS samples were

added in equal amounts to start the reacdon. The reacdon was terminated

by adding 3 ml of the chilled buffer and by immediate filtering through a

glass-fiber filter in a Millipore apparadis. The filter was washed three dmes

with 3 ml chilled buffer and then counted for radioactivity in a scintillation

counter. The amount of' ^Ca' taken up by the tissue sample was

determined by subfracting the counts retained on the filter after a zero-time

incubation with the retinal preparation,

ATPase assay

The assay was adapted from commonly used procedures (Ottlecz et

al , 1993) and activity was measured in whole retinal homogenate in

sodium-bicarbonate buffer only. Briefly, equal amounts of [y-^^PjATP

(400,000-500,000 dpm) were added to each incubadon mixture and the

reaction tubes were preincubated in a 37°C water bath for 2 minutes. The

retinal dssue sample was added to start the reaction with a final total volume

of 250 |il, and the tubes were incubated for an additional 2 minutes. The

reacdon was terminated by adding 250 |il of perchloric acid, and the

radioactivity was exfracted into «-butanol/benzene (1:1) from which an

aliquot was taken and counted in a scindllation counter, Confrol dibes were

incubated for the same length of time, and radioacdvity was counted the

same way, but the perchloric acid was added prior to preincubadon and the

addidon of the rednal dssue, ATPase acdvity was determined by

subfracdng experimental values from confrol counts.

24

Taurine uptake assay

The assay used in measuring taurine uptake was modified from

previous procedures (Lake and Cocker, 1983; Quesada et al, 1984; Salceda,

1980). Briefly, the reacdon was done with whole retinal homogenate or

with isolated ROS in KRB buffer. The appropriate reagents was added to

the buffered solution in the reaction dibes with varying concenfrations of

unlabeled taurine (10 |iM - 10 mM) or CaCl2 (0-1000 |iM) present. Equal

amounts of [^Hjtaurine were added to each test dibe (-1 |iCi) and incubated

in a 37°C water bath for 2 minutes. The reacdon was initiated by the

addition of equal amounts of tissue sample (200-300 |ig for the whole

retinal homogenate and 50-150 |ig for the ROS) to each dibe in a final

volume of 250 |il. The reaction was allowed to proceed for 7 minutes

before the reaction was terminated with the addidon of 3 ml ice-cold buffer

and filfration through a Millipore apparatus. Each filter paper was then

washed three times with 3 ml ice-cold buffer and the remaining radioactivity

was measured in a scintillation counter. Blanks were determined by letting

the reaction proceed exactly as the experimental tubes except the reaction

was kept on ice.

Calcium binding assav

The calcium binding assay was adapted from previous studies

(Lombardini and Prien, 1983; Sebring and Huxtable, 1985), and

experiments were done with either lysed whole retinal homogenate or lysed

ROS in KRB buffer. Reagents such as taurine and ATP were added to the

reaction tube in the appropriate amounts and kept on ice until the end of the

25

reacdon. Idendcal amounts of ^ CaCl2 (400,000-500,000 dpm) were added

to the dibes in the presence of 10 |iM CaCl2, In preliminary binding

experiments, calcium binding increased slowly and equilibrium was reached

at around 60 minutes and maintained for an additional 60 minutes. Tissue

samples were added in equal amounts to start the reaction, and the reacdon

was terminated after 90 minutes for whole rednal samples. The reacdon

was terminated by adding 3 ml of the chilled buffer and by immediate

filtering through a glass-fiber filter in a Millipore apparatus. The filter was

washed three times with 3 ml of the chilled buffer and then counted for

radioactivity in a scintillation counter. The amount of " Ca" taken up by the

tissue sample was determined by subfracting the counts from confrol

reactions that were freated with 6N HCl,

Statisdcal analvsis

Each datum point was a measurement derived from an independent

experiment. Analyses were performed on the means ofdata pooled from

several independent experiments unless otherwise stated. Statisdcal

analyses were performed using the GraphPad Prism software (GraphPad

Software, Inc., San Diego, CA). Data were analyzed using the one-way

analysis of variance (ANOVA), Linear and non-linear regression analysis

were also performed for some of the data to esdmate various kinedc

constants like IC50 (antagonist concenfradon which produces half-maximal

inhibidon). Km (subsfrate concenfrations which produces half-maximal

activity), and Vmax (maximal activity). Post-hoc analysis was

accomplished using Dunnett's multiple comparison test, a test that

26

specifically compares experimental groups only with the confrol group.

Otherwise, the Bonferroni multiple comparison test was performed to

compare all pairs of groups. On certain experiments, unpaired t-test was

performed to determine significance between the means of specific

experimental groups.

For the taurine uptake experiments, raw data were corrected for

diffusion by graphically calculating a diffusion constant (K^) at high taurine

concenfradons. This factor was then subfracted from each measure of

taurine uptake concenfrations (K^S; S = total taurine). Non-linear

regression analysis was then performed on uptake data following the

method described by Neal and White (1978). Mathemadcal equadons for a

single and a double saturable hyperbola were used to test for a one- and a

two-component uptake system, respecdvely. Iterative optimization and best

fit analysis were done with GraphPad Prism software. Because the two

equations have different numbers of variables, the best equadon fitdng the

data was determined by the program as follows: the simpler equadon (single

saturable hyperbola) was deemed best if the corresponding sum-of-squares

were lower, but if the more complicated equadon (double saturable

hyperbola) had the lower sum-of-squares, then a F-test was calculated. The

simpler equadon was deemed best if the P value is > 0,05, and the more

complication best if the P value is < 0,05, Maximum velocity (Vmax) and

Michaelis-Menten constants (Km) were then calculated using the

appropriate equations. Linear regression analyses were also performed on

transformed data (Eadie-Hofstee plots) to provide graphic representation

and qualitadve descriptions of the uptake system or systems observed. The

27

lack of a single line fit was taken as an indication that more than one uptake

system was present.

To estimate the antagonist concenfration that produces half-maximal

inhibition (IC50), the Dixon plot methods was also employed, specifically in

cases where the use of non-linear regression analysis was ruled out by

insufficient numbers of inhibitor concentrations (Dixon and Webb, 1964).

The procedure calls for the fransformadon of enzyme or uptake activity data

into fraction of confrol values (experimental/confrol). The reciprocal of the

fraction of confrol values were then plotted against the inhibitor

concenfration and linear regression analysis was performed. The inhibitor

concenfration corresponding to the value of 2, which denotes 50%)

inhibidon, was taken as the IC50. Only data points found between the

threshold concenfrations producing minimal and maximal inhibition were

used as exfreme concenfradons producing 100%) or 0%) inhibidon can skew

the linear regression analysis of the Dixon plot, IC50 values were taken from

independent experiments and the mean ± SEM was calculated.

28

CHAPTER III

RESULTS

Preliminary data

Calcium uptake was measured in the presence of 10 \xM CaCl2 in

whole retinal homogenate by measuring the uptake of radiolabeled ' ^CaClj

in tissue that was filtered over glass fiber filters in a Millipore filfration

apparatus. The appropriate reagents were added to the reaction tube before

the reaction was started. The reaction was carried out in a 37°C water bath

and initiated with the addition of the retinal tissue. Taurine (32 mM) had no

effect on calcium uptake in the absence of ATP (Figure 1), However, ATP

alone (1,2 mM) produced a significant increase compared to confrol (p <

0.05). Moreover, the addition of taurine and ATP increased calcium uptake

above that produced by ATP alone (Figure 2),

0,125

0.000 0 32 mM TAURINE

Figure 1: Calcium uptake in whole retinal homogenate in the presence of taurine alone (mean ± SEM, N = 3),

29

O)

O

CONTROL ATP ATP+TAU TREATMENT

Figure 2: Calcium uptake in whole retinal homogenate in the presence of 1.2 mM ATP ± 32 mM taurine (mean ± SEM, N = 5-7). A different letter represents a statisdcal difference from the other groups (p < 0,05).

Chelerythrine (CHT) is a benzophenanthridine alkaloid that exhibits

several biological effects including the inhibition of protein kinase C

(PKC)(Herbert et al , 1990) and ATPase (Cohen et al , 1978). CHT has

inhibitory activity against Na" , K" -ATPase, producing 81.7% inhibition at

100 |lM concentration. CHT was also demonsfrated to modulate the

phosphorylation of a specific retinal protein that is also affected by both

taurine depledon and by taurine freatment in vitro (Lombardini, 1995;

Lombardini et al , 1996). Given these data and the dependence of the

stimulatory effects of taurine on ATP, the effect of CHT on calcium uptake

was examined in the whole retinal homogenate.

30

Figure 3 shows that basal calcium uptake in the absence of any

sdmulation was not affected by CHT up to 100 flM. CHT freatment

resulted in the inhibition of calcium uptake with ATP only and with ATP

and taurine together, but had no effect in the absence of ATP and taurine.

Non-linear regression analysis revealed that the concenfradon of CHT that

produced half-maximal inhibition (IC50) were approximately 65.05 and

46.59 |iM for 0 taurine and 32 mM taurine-induced calcium uptake,

respectively (Table 1). This represented a marginally significant increase in

CHT potency with the addidon of taurine (P < 0,07, paired t-test), and

suggested the existence some kind of drug interacdon. At 100 [J-M CHT,

both ATP and [ATP + taurine] treatments had completely lost their

sdmulatory effects as compared to basal calcium uptake.

0.75

+

*« 0,50 LJJ _ l

o S 0.25

° ° ° 0 1 3 10 30 50 100

jiMCHT

(A) Raw uptake data (mean ± SEM, N = 5-7),

Figure 3: The inhibitory effects of CHT on calcium uptake in whole retinal homogenate in the absence and presence of 1.2 mM ATP ± 32 mM taurine.

31

UJ ^ ^ 1 0 0

%S 76 s z =) o « 3 o 50 - 1 ^ < 2-O 25

0

I

i ^ B' i

1

1 0

'

1

1

T

1

2

'

• ATP

^

-

3 4

LOG \M CHT

(B) Calcium uptake in the whole retinal homogenate in the presence of 1,2 mM ATP presented in Figure 3(A) converted to %> confrol, and best-fit non-linear regression curve (N = 6-7),

1 2

LOG jiM CHT

(C) Calcium uptake in the whole retinal homogenate in the presence of 1,2 mM ATP and 32 mM taurine presented in Figure 3(A) converted to Vo confrol, and best-fit non­linear regression curve (N = 6-7),

Figure 3: Continued,

32

Table 1: Inhibitory potency (IC50) of CHT against calcium uptake and ATPase acdvity in rat retinal homogenate ±32 mM taurine, as shown in Figures 3B and C, and in Figure 8B, respecdvely. Experiments were performed in the presence of 1,2 mM ATP, and the means of esdmated IC50 values are presented with the Hill coefficient (n^) and the 95%) confidence values in parentheses. For these experiments, IC50 values were calculated from non-linear regression analysis of data converted to Vo confrol and combined from several independent experiments.

Assay

calcium uptake (N = 6-7)

ATPase activity (N = 3)

0 mM taurine

65,05 \1M CHT nH = -1.16

(50,61-83.62)

88.29 |iM CHT nH = -1.58

(75,96 - 102,6)

32 mM taurine

46,59 jlM CHT nH = -1,56

(27.98 - 77.56)

165.6 flM CHT nH = -2,13

(128,9-212,9)

33

The effects of CHT were measured in the presence of increasing

concentrations of ATP and taurine to ascertain if the effects of CHT were

competitive or non-competitive. In theory, a competidve inhibitor could be

competed out by increasing the concentradon of the agonist, and at a high

enough concenfradon, the inhibition can be totally overcome. Calcium

uptake was measured in the presence of increasing concenfrations of ATP in

the absence of taurine ± 100 flM CHT (Figure 4). One-way ANOVA

coupled with posttests were performed (Figure 4), With the use of non­

linear regression analysis, half-maximal stimulation was estimated at around

0.286 mM ATP. The inhibidon of calcium uptake by CHT appeared to be

non-competitive as increasing agonist (ATP) concenfradons did not

overcome the effects of CHT,

OfiM CHT T 1

-lOOjiMCHT

1 2 3 4 5

mM ATP (+ 0 mM TAURINE)

Figure 4: Calcium uptake in whole retinal homogenate in the presence of increasing concentradons of ATP ± 100 |iM CHT (mean ± SEM, N = 4). (*p < 0,05 vs, 0 |iM CHT, Dunned's posdest)

34

The effect of taurine in the presence of 1,2 mM ATP was observed to

be dose-dependent also (Figure 5), Again, unpaired t-tests revealed

significant differences which one-way ANOVA coupled with posttests did

not (Figure 5). With one-way ANOVA and Dunned's posttest, significant

differences were observed with 32 mM and 80 concenfration. However,

unpaired t-test revealed a significant increase with 16 and 48 mM taurine

also. Non-linear regression analysis revealed that half-maximal stimulation

was around 18 mM taurine. As in the absence of taurine (Figure 4), the

effect of CHT was found to be non-compedtive in nature. Stadstical

analyses indicated that ATP (Figure 4) and taurine (Figure 5) produced

maximal effects at 0.6 mM and 32 mM concenfrations, respecdvely.

25 50 75 100

mM TAURINE

Figure 5: Calcium uptake in whole rednal homogenate in the presence of increasing concenfradons of taurine and 1,2 mM ATP ± 100 |iM CHT (mean ± SEM, N = 4), (*p < 0,05 vs,. 0 mM taurine, Dunned's post-test).

35

Given the sfrong inhibitory effects of CHT, the agent may be a usefiil

tool in studying the mechanism behind calcium uptake in the retina in

general, and the mechanism behind the stimulation produced by taurine

specifically. The effect of CHT to antagonize the stimulatory effects of ATP

and taurine may have been mediated by the inhibition of PKC activity, i,e.,

ATP and taurine may have activated PKC which in dim sdmulated calcium

uptake. Another inhibitor of PKC was used to verify this possibility,

K252b, a natural mold product, is an inhibitor of PKC (Nakanishi et al,

1986) and was used to antagonize the effects of ATP and taurine. In condast

to CHT, K252b was found not to affect calcium uptake, either in the

presence of ATP only or in the presence of ATP and taurine together (Figure

6), The effect of K252b alone was not studied. Regardless, the data in effect

preclude the involvement of PKC modulation in the mechanism of acdon of

taurine.

0.00 1000 0 20 100

nM K252b

Figure 6: The effect of the PKC inhibitor K252b on calcium uptake in whole retinal homogenate in the presence of 1.2 mM ATP ± 32 mM taurine (mean ± SEM, N = 3).

36

Hypothesis 1

The preliminary data left the possibility that CHT antagonism of the

effects of ATP and taurine on calcium uptake may be dependent on the

modulation of ATPase activity by taurine. The first hypothesis deals with

the involvement of ATPase activity in the stimulatory effects of taurine on

retinal calcium uptake. Total ATPase activity in whole retinal homogenate

was thus studied. As was expected with an enzymatic reaction, ATPase

acdvity increased as the ATP substrate was increased (Figure 7). The

maximal enzymatic velocity (Vmax) was not achieved with 4.2 mM

concenfradon of ATP and higher concentrations were not tested.

100

UJ 50

Q. 25

0,3 0,6 1.2 1.8 2.4 4.2 mMATP

Figure 7: ATPase activity in whole retinal homogenate in the presence of increasing concenfrations of ATP (mean ± SEM, N = 8),

37

The effect of increasing concenfradons of CHT was sdidied in the

presence of 1.2 mM ATP ± 32 mM taurine. It was found that 100 |iM

concenfration or greater of CHT decreased ATPase activity significandy in

the absence of taurine (Figure 8 A, a: p < 0.05 vs. corresponding control at 0

jiM CHT). In the presence of taurine, only 170 |J-M CHT produced

significant inhibidon (Figure 8A, b: p < 0.05 vs. corresponding control at 0

|iM CHT), Taurine in the presence of ATP produced no effect on ATPase

activity (ATP vs, ATP+TAU, 0 \XU CHT), suggesting that the modulation of

ATPase activity may not be a mechanism of action for taurine. However, the

inhibitory effect of CHT was found to be slighdy diminished with taurine

(Figure 8A, a: p < 0.05 vs. corresponding ATP+TAU ) which suggested an

interacdon between the drugs. Non-linear regression revealed that in the

absence of taurine, IC50 was esdmated at around 88.29 |iM CHT

concenfradon, while in the presence of 32 mM taurine the value was

produced was significandy higher at around 165.6 |iM CHT (Table 1, page

33).

38

3 10 30 50 100 170 [iMCHT

(A) Raw ATPase data (mean ± SEM, N = 3), (See text for significance of a and b).

1 2 3 LOG \iM CHT

(B) Data presented in Figure 8(A) expressed in terms of % confrol, and best-fit non-linear regression curve.

Figure 8: ATPase activity in whole rednal homogenate in the presence of 1,2 mM ± 32 mM taurine and of increasing concenfrations of CHT.

39

Characterization of the total ATPase activity was performed in an

effort to identify the specific ATPase involved. ATPase activity was sdidied

using pharmacological agents instead of alteradon of the ionic composidon

of the buffer, as is usually done (Berman et al, 1977), as this would result in

acdvity that might not be present in the original buffer condidons under

which calcium uptake had been measured. Ouabain and thapsigargin were

used to inhibit Na^ K^-ATPase and SERCA (sarcoplasmic/endoplasmic

redculum calcium ATPase), respecdvely (Kijima et al, 1991; Lytton et al,

1991;Ottclezetal, 1993).

Na^, K^-ATPase was first studied because of the known effects of

CHT to inhibit its acdvity. Ouabain had been used to inhibit Na" ,

K" -ATPase in the rat retina using a bicarbonate-free system and was found to

cause half-maximal inhibition at around 20 jiM (Otdecz et al , 1993). In our

system, ouabain at concenfrations ranging from 20-3000 \XM did not cause a

decrease in total ATPase activity in the presence of 1.2 mM ATP, with or

without 32 mM taurine present (Figure 9). CHT (in the absence of taurine)

consistently produced inhibidon greater than 1 mM ouabain (Figure 10) and

so is shown, for the first time, to inhibit rednal ATPase activity other than

ouabain-sensidve Na^, K^-ATPase. Because all of the components of the

whole cell were present in the sample, it can be assumed that the ouabain-

sensidve Na^, K^-ATPase enzyme was present. Ouabain caused a slight but

insignificant decrease in total ATPase, suggesting that the enzyme may be

minimally funcdonal, if at all, under the ionic condidons of the bicarbonate

buffer used.

40

I ATP I ATP + TAU

h i r nl ^i ^i ^i x nl

10

0 20 100 200 1000 2000 3000 ^M OUABAIN

Figure 9: ATPase acdvity in whole retinal homogenate in the presence of increasing concenfradons of ouabain (mean ± SEM, N = 3-7). Activity measured in the presence of 1,2 mM ATP ±32 mM taurine.

CON CHT OUA CHT+OUA TREATMENT

Figure 10: ATPase activity in the presence of 100 |iM CHT, 1 mM ouabain (OUA), 1.2 mM ATP ± 32 mM taurine (mean ± SEM, N = 4). (unpaired t-tests: *p < 0.05 vs, respective confrols, **p < 0,05 vs, CHT+ATP)

41

Thapsigargin, an inhibitor of SERCA (sarcoplasmic/endoplasmic

redculum calcium ATPase) (Kijima et al , 1991; Lytton et al, 1991) was also

used to treat whole retinal homogenate (Figure 11). Microsomes are present

in the whole rednal homogenate and SERCA activity may account for some

calcium uptake acdvity, Thapsigargin has been used to inhibit SERCA

activity in brain microsomes, but in our system it was found to have no effect

on total ATPase acdvity in the rat retina. It is possible that while the SERCA

protein is present in the whole cell preparation, it may not be active under the

buffer condidons used in the experiment. Similar to Na" , K""-ATPase,

SERCA activity probably is barely funcdonal, if at all, and thus may make

up only a small component of the total ATPase activity that is observed.

0 4 10 jiM THAPSIGARGIN

Figure 11: ATPase activity in whole retinal homogenate in the presence of increasing concentrations of thapsigargin (mean ± SEM, N = 3-4). Acdvity was measured in the presence of 1.2 mM ATP ± 32 mM taurine.

42

Hvpothesis 2

The second sub-hypothesis involves the question of whether taurine

uptake is necessary for its stimulatory effects. If it is so, then the specific

inhibition of taurine uptake would abolish or attenuate its effects. The

kinedcs of taurine uptake were sdidied using both whole retinal homogenate

and isolated ROS, initially by measuring the uptake of labeled [^Hjtaurine in

the presence of increasing concenfradons of total unlabeled taurine. In

Figure 12, taurine uptake in whole retinal homogenate is shown in the

presence of 10-10,000 |iM taurine. As expected, uptake increased as total

taurine increased and was found to be saturable.

2500 5000 7500 10000 12500 \i.M TAURINE

Figure 12 : Taurine uptake in whole retinal homogenate in the presence of increasing concenfrations of total taurine (mean ± SEM, N = 3-6),

43

Non-linear regression analysis of the data in Figure 12 revealed two

saturable components, one of high-affinity and the other of low-affinity. The

Michaelis-Menten constant (Km) is the estimated concenfradon of the

subsfrate which results in half-maximal uptake, and values of 133 ± 47 |J,M

and 2.74 ± 1.25 mM were calculated for the high- and the low-affinity

systems, respectively. The corresponding maximum velocity (Vmax) values

were estimated at 0,73 ± 0.27 and 3.15 ± 0.42 pmol/|ig. Eadie-Hofstee

fransformation of the data allowed for the visualization and confirmation of

the existence of the two different uptake systems (Figure 13). Velocity was

plotted against velocity/subsfrate and linear regression analysis was

performed. A two-line plot was found to be a better fit than a single line

plot, suggesting a two-component system.

o o _ l m >

O.'OOO 0,002 0,004 0.006

VELOCITY/SUBSTRATE

Figure 13 : Eadie Hofstee plot ofdata from Figure 12,

44

The same experiments were performed with isolated ROS, Taurine

uptake was measured in ROS in the presence of 10-3500 jlM taurine (Figure

14), Non-linear regression analysis revealed a saturable single component

uptake system with an apparent Km value of 140 ± 8 ^iM and a Vmax value

of 2,46 ± 0.08 pmol/|ig. The linear regression analysis of the Eadie-Hofstee

conversion of the uptake data confirmed the existence of one uptake system

(Figure 15). All the uptake kinetic values are summarized in Table 2.

"0 1000 2000 3000 4000 [iM TAURINE

Figure 14: Taurine uptake in isolated ROS in the presence of increasing concenfrations of total taurine (mean ± SEM, N = 3),

45

0.005 0,010 0.015

VELOCITY / SUBSTRATE 0,020

Figure 15: Eadie Hofstee plot of the data in Figure 14,

Table 2: Kinetic constants for taurine uptake calculated from data shown in Figures 12 and 14. Experiments were performed in the presence of increasing concenfrations of taurine, and values are presented as mean ± SEM,

Taurine uptake

high-affinity uptake (in whole retinal

homogenate)

low-affinity uptake (in whole retinal

homogenate)

high-affinity uptake (in isolated ROS)

Michaelis-Menten constant (Km)

133±47|iM

2.74 ± 1,25 mM

140±8| iM

maximum velocity (Vmax)

0.73 ± 0.27 pmol/|ag

3.15 ± 0.42 pmol/|ig

2.46 ± 0.08 pmol/|ig

46

The Km values in Table 2 were used to determine the taurine

concentrations to use in the inhibition experiments. Taurine uptake was

measured in the presence of 50 [IM and 1,5 mM taurine to study high- and

low-affinity uptake, respectively, CHT effecdvely inhibited both uptake

systems in whole retina (Figures 16A and 17A) and high-affinity uptake in

the ROS (Figure 18A), CHT may, thus, inhibit the effects of taurine on

calcium uptake by blocking taurine uptake. Half-maximal inhibition

constants were calculated by normalizing the data to % control which would

correspond to uptake in the absence of CHT (Figures 16B, 17B and 18B).

The means normalized values were calculated and non-linear regression

analyses were done to esdmate the concenfradon of CHT that produces half-

maximal inhibition (IC50).

47

D) •4. LU Z

a: D < 1 -UJ _ l o Q-

0.5

04

0.3

0.2

0.1

0,0 0.0 0,1 0.2 1,0 2.0 10 20 100

jiMCHT

(A) Raw uptake data (mean ± SEM, N = 3, *p < 0,05 vs. 0 iM CHT, Dunned's post-test).

UJ ii:_100

a a: ^ I - 75-

50-

25-

i

+ 50 ^M TAURINE

-1 0 1 2 LOG jiM CHT

(B) Data presented in Figure 16(A) expressed in terms of %) confrol, and best-fit non-linear regression curve (N = 3),

Figure 16: High-affinity taurine uptake in whole retinal homogenate in the presence of increasing concenfrations of CHT, Uptake was measured in the presence of 50 |iM taurine.

48

0,0 0,1 0,2 1.0 2.0 10 20 100 HMCHT

(A) Raw uptake data (mean ± SEM, N = 3, *p < 0.05 vs. 0 [XM CHT, Dunned's post-test).

- 2 - 1 0 1 2 3 LOG \iM CHT

(B) Data presented in Figure 17(A) expressed in terms of % confrol, and best-fit non-linear regression curve (N = 3).

Figure 17: Low-affinity taurine uptake in whole retinal homogenate in the presence of increasing concenfrations of CHT, Uptake was measured in the presence of 1.5 mM taurine.

49

0,0 0.1 0.2 1.0 2.0 10 20 100 jiMCHT

(A) Raw uptake data (mean ± SEM, N = 3, *p< 0.05 vs. 0 |iM CHT, Dunned's post-test).

LU is: _ 100 < -1 t -o ^f^ 75 U j Z z o ^ g O 50 = ) ^ < ^ ^^ P 25

n

1

-

I

— 1 1 1

• + 50 iiM TAURINE 1 _

( ,

• » » _

" 2 - 1 0 1 2 3

LOG \LM CHT

(B) Data presented in Figure 18(A) expressed in terms of %> confrol, and best-fit non-linear regression curve (N = 3),

Figure 18: High-affinity taurine uptake in isolated ROS in the presence of increasing concenfradons of CHT. Uptake was measured in the presence of 50 [iM taurine.

50

Table 3: Inhibitory potency (IC50) of CHT against high- and low-affinity uptake in whole rednal homogenate, and of high-affinity taurine uptake in isolated ROS, as shown in Figures 16B and 17B, and in Figure 18B, respectively. The means of estimated IC50 values are presented with the Hill coefficient (nH) and 95% confidence values in parentheses. For these experiments, IC50 values were calculated from non-linear regression analysis of data converted to %> confrol and combined from several independent experiments.

Final taurine concenfration

50| iM (for high-affinity uptake)

1,5 mM (for low-affinity uptake)

Whole rednal homogenate

7,92 flM nH = -1.88

(6.16-10.20)

4.37 |iM nH = -0.84

(3.17-6.03)

ROS

2.66 [IM nH = -1.35

(2.04 - 3.47)

51

Submaximal inhibition of high-affinity uptake in the ROS were

studied to determine if the inhibition is competitive or not. Taurine uptake

was measured in the presence of 25-750 \xM total taurine ± 5 jiM CHT. The

data were then transformed and velocity was plotted against

velocity/subsfrate in an Eadie-Hostee plot. Linear regression analysis of the

Eadie-Hofstee fransformation of the data indicated that while Vmax values

changed (Y-intercept) with CHT freatment. Km values (slope) did not,

suggesting non-competidve inhibition (Figure 19).

• 0 [iM CHT A 5 iiM CHT

0,000 0.005 0.010 0.015 0.020 0.025

VELOCITY/SUBSTRATE

Figure 19: Eadie-Hofstee fransformadon of taurine uptake data as measured in the presence of 25-750 |iM total taurine ± 5 |lM CHT (N= 3). Linear regression analysis was performed and the slopes of the two lines were not significandy different from each other.

52

The involvement of PKC in the inhibitory effect of CHT on taurine

uptake was also sdidied. The PKC activator, phorbol-myristate acetate

(PMA), and the PKC inhibitor, staurosporine (STAU), were found to be

ineffective in modulating high-affinity taurine uptake in whole retinal

homogenate (Figure 20). CHT is known to half-maximally inhibit PKC

acdvity at around 0.66 [XM (Herbert et al , 1990), about an order of

magnitude less than what is observed with CHT inhibition of taurine uptake

(Table 3, page 51). Along with the absence of PKC reguladon, these data

suggest that CHT inhibition involves a PKC-independent regulatory system.

The inhibition of taurine uptake was further studied to allow for the

verificadon of the hypothesis that taurine uptake is necessary in the

stimulation of calcium uptake.

CON 0.8 8 16 2 20 40 [iM TREATMENT

Figure 20: High-affinity taurine uptake in whole retinal homogenate in the presence of phorbol myristate acetate (PMA) and staurosporine (STAU)(mean ± SEM, N = 4-5). Uptake was measured in the presence of 50 l-lM taurine.

53

An inhibitor of taurine uptake in whole retinal homogenate was

required to study the necessity for taurine uptake in its stimulatory effects.

GES has been sdidied as a compeddve inhibitor of taurine uptake in the rat

retina (Quesada et al , 1984) and was used in these experiments. To study

the high- and low-affinity systems, uptake was measured in the presence of

50 |iM and 1.5 mM taurine. Taurine uptake in whole retinal preparations in

the presence of 50 |iM taurine was atfributed mostly to the activity of the

high-affinity fransporter, though a minority component could still be due to

the low-affinity fransporter. The converse applies for taurine uptake in the

presence of 1.5 mM taurine. GES inhibited taurine uptake in whole retinal

homogenate under both condidons, suggesting that GES inhibits both high-

and the low-affinity taurine uptake (Figures 21 and 22). IC50 values were

calculated using the Dixon plot method and presented in Table 4.

50 100 500

[iMGES

Figure 21: High-affinity taurine uptake in whole retinal homogenate inhibited by GES (mean ± SEM, N = 3-4). Uptake was measured in the presence of 50 flM taurine(*p < 0,05 vs. 0 |iM GES)

54

+ 1,5 mM TAURINE

0.8 1,5 5.0 mMGES

20.0

Figure 22: Low-affinity taurine uptake in whole retinal homogenate inhibited by GES (mean ± SEM, N = 3-4), Uptake was measured in the presence of 1.5 mM taurine (*p < 0.05 vs. 0 mM GES)

Table 4: Half-maximal inhibidon constant values (IC50) for GES inhibidon of high- and low-affinity uptake in whole retinal homogenate, and of high-affinity taurine uptake in isolated ROS. IC50 values are presented as mean ± SEM of 3 independent values calculated from individual experiments using the Dixon plot (see Methods).

Final taurine concenfration

50| iM

1.5 mM

Whole retinal homogenate

280 ± 64 |iM

0.93 ± 0.18 mM

ROS

352±89| iM

-

55

When measured in the presence of 50 jiM taurine, taurine uptake in

the ROS was found to be inhibited by GES freatment (Figure 23). The IC50

value was estimated to be 352 ± 89 |iM, which is approximately the same

potency as with the high-affinity uptake system in whole retinal preparations

(Figure 21 and Table 4). The data suggest that the high-affinity uptake

observed in both systems is probably identical. Inhibition was observed with

500 [IM GES but was not significant because of the wide standard error.

iJ 0.75 z Of < 0.50 liJ _i

I 0.25

0.00

1 + 50 ^M TAURINE _

0 50 100 500 750 tiMGES

Figure 23: High-affinity taurine uptake in isolated ROS inhibited by GES (mean ± SEM, N = 3-4). (*p < 0.05 vs. 0 jlM GES)

56

The effect of taurine on calcium uptake was sdidied in the presence of

GES to inhibit taurine uptake. Calcium uptake was measured in whole

retinal homogenate (Figure 24) and isolated ROS (Figure 25) in the presence

of increasing concenfradons of taurine and 32 mM GES. Given the

calculated IC50 values for GES (Table 4), the uptake of taurine should have

been inhibited significandy by the GES freatment. However, 32 mM GES

did not reverse the effects of taurine, suggesting that taurine uptake is not

essential in the stimulation of calcium uptake by taurine, either in the whole

retinal homogenate or in isolated ROS.

mM TAURINE (+ 1.2 mM ATP)

Figure 24: Calcium uptake in whole retinal homogenate in the presence of GES, a taurine uptake inhibitor (mean ± SEM, N = 3). (*p < 0.05 vs. 0 mM taurine)

57

0,0 8,0 16,0 32.0 mM TAURINE (+ 1.2 mM ATP)

Figure 25: Calcium uptake in isolated ROS in the presence of GES, a taurine uptake inhibitor (mean ± SEM, N = 5). (*p < 0.05 vs. 0 mM taurine)

58

Hvpothesis 3

Calcium uptake may be increased by the acdvadon of ion channels

that allow for the influx of calcium ions. Cadmium, a non-specific inhibitor

of calcium currents in neuronal type cells, was used to inhibit the effects of

taurine on calcium uptake in whole retinal homogenate. The effect of taurine

to potentiate ATP-dependent calcium uptake was effectively inhibited by 5

|lM cadmium while ATP-dependent uptake was not (Figure 26). However,

100 |lM cadmium inhibited both types of uptake significantly. This latter

effect is probably due to non-specific displacement of calcium from its

binding sites as calcium is present only in 10 flM concenfration, which is an

order of magnitude less than 100 |iM cadmium.

0.75

0,00 0,0 100.0 5,0

^M CADMIUM

Figure 26: Calcium uptake in whole rednal homogenate in the presence of 1,2 mM ATP ±32 mM taurine, and cadmium, a non­specific cation channel inhibitor (mean ± SEM, N = 4-5). (*p < 0-05 vs. respective 0 |iM cadmium)

59

L-type calcium channels may be present in the whole retinal samples

and may account for some calcium uptake activity. However, nifedipine, an

inhibitor of L-type calcium channels, was not effecdve in inhibiting calcium

uptake (Figure 27). LY83583 (6-anilino-5,8-quinolinedione) inhibits

cGMP-dependent currents in concenfradons as low as 1 |iM. (Leinders-

Zufall and Zufall, 1995), and acts both directly and indirectly on the cGMP-

gated cation channel. This inhibitor of cGMP-gated channels inhibited the

effects of taurine to potentiate the effects of ATP while ATP-dependent

calcium uptake per se was not inhibited (Figure 28).

1.00

0.00 0.0 40,0 10,0 20,0

|xM NIFEDIPINE

Figure 27: Calcium uptake in whole rednal homogenate in presence of 1,2 mM ATP ± 32 mM taurine, and of increasing concenfrations of nifedipine, a blocker of voltage-gated L-type calcium channels (mean±SEM,N = 3).

60

Ol

+

o UJ _J

o

1.5 IZIDATP IATP+TAU

1 oi i oi oil ^m nm r

0,0 80.0 20.0 40,0 60,0 IiM LY 83583

Figure 28: Calcium uptake in whole retinal homogenate in the presence of 1,2 mM ATP ± 32 mM taurine, and of LY83583, a blocker of cGMP-gated channels (mean ± SEM, N = 5-8). (*p < 0,05 vs, respective 0 [iU LY83583).

61

In the process of photofransducdon, the acdvadon of cGMP-gated

channels in the ROS is a crucial step. The data suggested that taurine may

activate this channel to increase calcium influx in the redna. Calcium uptake

in the isolated ROS was then studied. As expected, LY83583 also inhibited

the specific effects of taurine in the isolated ROS, and not those of ATP

alone (Figure 29), A cell permeant analogue of cGMP (Rp-8-Br-PET-

cGMPS) that acts as a specific blocker of cGMP-gated channels inhibited the

effects of taurine in the ROS in the same way (Figure 30), Inhibition was

observed with 100 |lM Rp-8-Br-PET-cGMPS, but this inhibition was not

statistically significant because of the wide standard error of the controls.

Thus, it seems that taurine enhances the activadon of cGMP-gated channels

in the retina, specifically in the ROS.

0.0 80.0 40,0 JiM LY83583

Figure 29: Calcium uptake in the isolated ROS in the presence of 1,2 mM ATP ± 32 mM taurine, and LY83583, a blocker of cGMP-gated channels (mean ± SEM, N = 4-7). (*p<0,05 vs. Op,MLY83583).

62

0.0 100.0 400.0 JiM Rp-8-Br-PET-cGMPS

Figure 30: Calcium uptake in isolated ROS in the presence of 1,2 mM ATP ± 32 mM taurine, and Rp-8-Br-PET-cGMPS, a specific blocker of cGMP-gated charmels (mean ± SEM,N = 4). (*p<0.05 vs, 0|iMRp-8-Br-PET-cGMPS)

63

To verify the specific involvement of cGMP-gated channels in the

calcium uptake measured in the retina and in the ROS, activating agents of

the channel were used to stimulate uptake. In these experiments, the isolated

ROS are supposed to be intact as no homogenization was done on the

samples, and the cGMP binding site is not readily accessed by compounds

added in the buffer. The acdvating agents were specifically chosen because

of their ability to cross the plasma membrane and bind to the agonist binding

site of the cGMP-gated channel. Dibutyryl cGMP is a membrane-permeant

analogue of cGMP, and it was found to stimulate ATP-dependent calcium

uptake, but not taurine potendated uptake (Figure 31), This effect was less

potent than what was expected as cGMP acdvates the receptor with a

dissociation constant of 17-30 |iM in patch clamp experiments (Pugh and

Lamb, 1990). The effects of another membrane-permeant analogue of cGMP

(Rp-cGMP) on ATP-dependent calcium uptake were studied, but no

significant effects were observed (Figure 32). The reason behind the

discrepancies with both potency and efficacy observed in these two

experiments may involve a slow rate of diffusion of the cGMP analogues

through the plasma membrane. Incubation was done for only 2 minutes,

during which time the cGMP analogue must diffiise through the plasma

membrane, bind to its receptor and acdvate the channel. A longer

incubadon time may have been necessary for the compounds to access the

appropriate binding site.

64

1.00

50.0 100.0 200,0 400.0

JiM DIBUTYRYL cGMP

Figure 31: Calcium uptake in isolated ROS in the presence of 1.2 mM ATP ±32 mM taurine, and of increasing concenfrations of dibutyryl cGMP, a membrane-permeant analogue of cGMP (mean ± SEM, N = 6-7). (*p < 0,05 vs, 0 |iM dibutyryl cGMP)

0.75

D)

*« 0,50 O UJ —I O S 0.25

0.00 0.000 100.000 400.000

JiM Rp-cGMP

Figure 32: Calcium uptake in the isolated ROS in the presence of 1.2 mM ATP and increasing concenfrations of Rp-cGMP, an analogue of cGMP (mean ± SEM, N = 3).

65

The cGMP-gated channels may be activated by increasing the

endogenous levels of cGMP inside the ROS. Zaprinast, otherwise known as

M&B 22,948, is a potent inhibitor (IC50 = 160 nM) of the cGMP-specific

phosphodiesterase (PDE) found in the rod photoreceptor (Gillespie and

Beavo, 1989), and it was used to prevent the degradadon of endogenous

cGMP. However, zaprinast did not have any significant effect on ATP-

dependent calcium uptake in the ROS either in the presence or absence of

taurine (Figure 33). The lack of effects in this experiment and in the

preceding experiment may be due to the fact that the total time of

preincubation and incubation was only 4 minutes which may not be

sufficient time for cGMP analogues and zaprinast to diffuse into the tissue

and produce their effects. It is entirely possible that longer incubation

periods would result in significant stimulatory effects for these. An

important consideration, however, was the maximization or saturation of

calcium uptake that occurs with longer incubation periods, at which point the

observadon of sdmulation would be impossible. Regardless, fiall dme course

studies need to be done to verify the results presented here.

66

0,0 50,0 100,0 200.0 400,0 JiM ZAPRINAST

Figure 33: Calcium uptake in the isolated ROS in the presence of 1.2 mM ATP ±32 mM taurine, and of increasing concentradons of zaprinast, an inhibitor of cGMP-specific phosphodiesterase (mean ± SEM, N = 6-7).

67

Hvpothesis 4

The effects of taurine may involve calcium binding to membranes and

not calcium uptake exclusively. Experiments were performed to determine if

taurine affects calcium binding to retinal membranes. Calcium binding

experiments were performed in retinal tissue and the effects of taurine were

tested. The retinal tissue was osmodcally lysed with water to denadire the

membranes and incubated with radiolabeled calcium on ice. These

procedures were followed to inhibit active calcium uptake as much as

possible. In preliminary experiments, taurine (32 mM) and ATP (1.2 mM)

individually did not produce any significant effect, but together both

increased calcium binding (Figure 34), However, the effect could not be

replicated in succeeding experiments in which increasing concenfrations of

ATP and taurine were used to stimulate binding (Figure 35 and 36), There

appears to be a slight increase in calcium binding with ATP and taurine

freatment, but statistical significance could not be established. The

inconsistency may be due to the rather small magnitude of the effect

originally observed. Perhaps, increasing the N value for the experiments

would result in significance. Further studies were, thus, done to evaluate the

involvement of calcium binding in the stimulatory effects of taurine on

calcium uptake through the use of A23187, a calcium ionophore.

68

CON ATP TAU ATP+TAU TREATMENT

(1.2 mM ATP/32 mM TAURINE)

Figure 34: Calcium binding in whole retinal homogenate in the presence of 1.2 mM ATP and/or 32 mM taurine (mean ± SEM, N = 6), (*p < 0.05 vs. confrol, unpaired t-test)

0.4

;5- 0.3

o UJ 0.2

O

Q- 0.1

0.0

atO°C

llllU 0.0 0.4 1.2 2.4 3.6 4.8 mM ATP (+ 0 mM TAURINE)

Figure 35: Calcium binding in whole retinal homogenate in the presence of increasing concentradons of ATP (mean ± SEM, N =

5).

69

0.5

S 0.4 +

O 0.3 m d 0.2

0.1

0.0

atO°C

+1.2 mMATP

hll l l l l CON 0 8 16 32 48 80 mM TAURINE

Figure 36: Calcium binding in whole retinal homogenate in the presence of ATP and increasing concenfrations of taurine (mean ± SEM, N = 4-3).

The data suggest that taurine does not affect calcium binding and that

the stimulatory effects of taurine are specific to calcium uptake. This idea

was verified with the use of A23187, a calcium ionophore that dissipates the

calcium gradient across the cell membrane and abolishes the accumulation of

calcium due to acdve calcium uptake. The rednal tissue was also lysed in

water to denature the membranes and inhibit calcium uptake activity.

A23187 was effecdve in abolishing the sdmuladon produced by ATP and

taurine, in lysed whole retinal homogenate (Figure 37) and in lysed ROS

(Figure 38). Stimulation was observed in dssue that was osmodcally

denatured, indicadng that denaturadon was not complete or effective. In

addition, A23187 produced no effect on basal calcium uptake (Figures 37

and 38), i,e,, in the absence of ATP and taurine, suggesdng that basal

calcium uptake may indeed correspond to calcium binding to membranes.

70

0.6

J . + o m _i o

0.4

0.2

0.0

IZZJ CONTROL 1.2 mMATP ^ A 2 3 1 8 7 *

i . -IL

n n n i l l l 32 0 32 0

mM TAURINE

Figure 37: Calcium uptake in whole retinal lysate in the presence of A23187, a calcium ionophore (mean ± SEM, N = 4-5). (*p 0.05 vs. 0 mM taurine + 0 mM ATP).

<

D) 1.00

5 0.75 UJ ^ 0.50-

^ 0.25

0.00

CONTROL A23187

1.2 mMATP

Ol 32 0 mM TAURINE

32

Figure 38: Calcium uptake in lysed ROS in the presence of A23187, a calcium ionophore (mean ± SEM, N = 5). (*p < 0.05 vs. 0 mM taurine).

71

CHAPTER IV

DISCUSSION

Taurine effects on calcium uptake

Taurine is abundant in the retina, comprising 40-50%o of the total free

amino acids (Macaione et al , 1974) and reaching concentrations as high as

79 mM (Voaden et al , 1977). Retinal cells, as well as other cell types,

possess rather well-regulated uptake systems for taurine, systems that

function to maintain a very steep concenfration gradient for taurine across

the plasma cell membrane. In comparison, only small osmotic ions like

chloride and potassium are handled as carefully and as efficiendy. Taurine is

not incorporated into protein molecules and therefore is not important in

building the cytoskeletal or organellar structure of the cell. Taurine exists

freely in solution, and so if we are to assume that physiologic form follows

physiologic function, then we can surmise that taurine can act as an

osmolyte, and even more significantly, as a ligand of some kind which binds

with low millimolar affinity to receptor molecules to produce specific

infracellular effects.

A good illusfration of the potendal of taurine to funcdon as a regulator

of infracellular events is in vitro phosphoryladon experiments that make use

of retinal subfractions (reviewed in Militante and Lombardini, 2002). In

these experiments, millimolar concenfradons of taurine produced what could

only be described as direct sdmulatory effects on the activity of kinases to

phosphorylate specific proteins. Similar concenfrations of taurine analogues

either did not produce any effects or produced effects opposite that of

72

taurine, suggesting that the effects of taurine are not osmotic in nature.

While the mechanism is unclear, we may assume that taurine binds with low

affinity to either the kinase or the protein substrate to be phosphorylated as a

necessary biochemical mechanism, ft is also reasonable to assume that

within the living cell, taurine is regulating kinase activity in much the same

way. The fact that taurine acts with millimolar potency, as opposed to

neurofransmitter or hormonal ligand molecules which usually act with

micromolar or even nanomolar potency to produce their effects, gives

physiologic relevance to the unusually high concenfradons of taurine

retained within the cell, at least in reladon to the funcdon of protein kinases.

The effects of taurine on calcium uptake present an even more

significant physiologic possibility for taurine. Dwarfing the signaling

possibilides of direct moduladon of specific proteins, moduladon of

intracellular calcium levels could potentially result in calcium-dependent

cascade events involving muldple proteins and multiple cellular systems.

Truly, taurine modulation of calcium flux in the redna could be

physiologically relevant in many ways. For example, in a model of

experimental regeneration of goldfish retina, taurine was demonsfrated to

sdmulate neuridc growth (Lima et al , 1988). This is a system commonly

used for the study of cenfral nervous system regeneration (Landreth and

Agranoff, 1979). In these experiments, taurine appeared to promote growth

by increasing calcium influx (Lima et al , 1993). The exact pathway for this

effect is unclear, but it is almost certain that the stimulation of growth is

dependent on a calcium signal spreading in multiple cellular compartments

and coordinating diverse biochemical processes in its wake.

73

The experiments in this thesis address a specific phenomenon long

observed with taurine and the retina. Taurine is known to produce

stimulatory effects on in vitro calcium uptake in whole retinal homogenate,

specifically, taurine potentiates ATP-enhanced calcium uptake increase

under condidons of low calcium (<500 |iM)(reviewed in Lombardini, 1991).

This effect was also observed with various subcellular fractions: PI fractions

(photoreceptor synaptosomes), P2 fractions (mitochondria and microsomes),

mitochondrial fracdons and ROS fractions (Lombardini, 1985a; Lombardini,

1988). Taurine requires the presence of ATP this stimulatory effect on

calcium uptake, and this phenomenological condngency is yet to be fully

understood.

In the experiments presented in this thesis, it is reported that taurine

produced half-maximal stimuladon at around -18 mM concenfradon, while

previous studies report this value at 8-10 mM (Liebowitz et al, 1987;

Liebowitz et al , 1988) or at -30 mM (Lombardini et al, 1989). While the

potency with which taurine acts may seem to be too low to be

physiologically relevant, the range of effecdve concenfrations, in fact,

coincides with reported levels of taurine in the retina. Additionally, the

active sequesfration of taurine inside the cells where the levels of calcium are

low, and the presence of ATP within the cell gives credence to the nodon

that this specific ATP-dependent sdmulation of calcium uptake in the retina

by taurine may be regarded as material to normal rednal function.

The experiments in this thesis studied the effects of taurine in the

presence of low calcium levels instead of high calcium levels which would

correspond to exfracellular levels of calcium. This condition was chosen

74

mainly to mimic the low intracellular levels of calcium so as to study the

effects of taurine under conditions comparable to conditions found inside the

cell Calcium uptake in the retinal tissue samples increases in almost direct

relation to the level of calcium in the buffer (Militante and Lombardini,

2000), and so it can be assumed that the use of high calcium levels would

result in tissue calcium levels that are higher than physiologic levels of

infracellular calcium. In all types of dssues, calcium flucdiadons occur

inside the cell and taurine may have different effects at different calcium

levels.

Isolated ROS, as opposed to whole retinal homogenate, were also used

in these experiments to discem more closely the site of action of taurine. In

many of the experiments, the stimulatory effects of taurine were

recapitulated in the ROS, mirroring previous reports on calcium uptake in

ROS (Lombardini, 1985a). The ROS is the focal point in the excitation and

recovery of photoreceptor cells with light stimuladon (Baylor, 1996). In

ROS, closure of cGMP-gated cation channels, net exfrusion of calcium, and

the decrease in infracellular calcium that occur during photoexcitadon lead to

the activation of a calcium-sensitive guanylyl cyclase and to an increase in

cGMP. Acdvation of cGMP-gated channels allows for the increased entry of

calcium. Increased calcium levels result in the inhibition of guanylyl cyclase

and in the breakdown of cGMP by phosphodiesterase, allowing for recovery

from the hyperpolarized state of the photoreceptor. The fast and efficient

uptake of calcium is, thus, key to the continued funcdon of the retina. The

data in this thesis suggest that taurine assists in this recovery, specifically in

light of the diminished levels of infracellular calcium that serve as the signal

75

for recovery and that at the same time satisfies the low calcium prerequisite

for stimulatory effects of taurine to occur. The involvement of taurine in

this recovery while unproven, makes kinetic sense as taurine would assist in

the faster uptake of calcium. Indeed, we can speculate that taurine is a vital

component in the immediate recovery of the depolarized state of the

photoreceptor cell during photofransduction. While this idea is largely

untested, it is supported by the report of electrical deficits found in the retina

after taurine depletion in the rat (Cocker and Lake, 1987) or with nufritional

taurine deficiency in human children (Geggel et al , 1985).

ATPase activity in the redna

A major requirement for the stimulatory effects of taurine is ATP.

Early studies reported that the stimulatory effects of taurine appear to be

dependent not only on the presence but also the hydrolysis of ATP

(Pasantes-Morales, 1982). This specific requirement has not been sdidied

much, owing perhaps to the default and seemingly obvious assumption that

adenosinefriphosphatase (ATPase) activity is necessary. Indeed, there have

been very few experiments reported on the interacdon between ATPase and

taurine in the retina.

In the retina, the total ATPase activity, as with other cell types, is

comprised of the activity of many different species of ATP-hydrolyzing

enzymes. Among the subtypes found in the retina are Na^ K^-ATPase, Ca'^-

acdvated, Mg^"-dependent ATPase (Ca^^ Mg^^-ATPase) and Mg^^-ATPase

(Berman et a l , 1977; Chambers et al , 1990). There is also evidence of

bicarbonate-stimulated ATPase activity (Winkler and Riley, 1977) but

76

almost certainly other types of ATPase activity are present. One eariy sdidy

done in this lab suggested that 20 mM taurine had no effect on 4 types of

retinal ATPase activity: NaVK" dependent, Mg^" dependent, Ca'^ dependent,

and Mg^VCa^^ dependent. However, the experiments required that different

buffers be used to delineate ATPase types. As the buffer conditions were

significandy different from that used with calcium uptake assays, it is

difficult to relate the findings to the ATP-dependence of the effects of

taurine. During experimentation, the activity of these enzymes and the other

ATPases that may be present may change depending on the specific ions

present in the assay buffer, e.g., without bicarbonate in the buffer,

bicarbonate-stimulated ATPase would be inactive, and vice versa.

In our experiments, total ATPase acdvity was measured under the

same buffer conditions as with the calcium uptake assay, to make certain that

the same types of ATPase activity are functional in both assays. Moreover,

ATPase activity was measured in the same time interval as the calcium

uptake assay was conducted. Taurine did not affect total ATPase activity,

sfrongly suggesting that the mechanism of action behind the sdmulatory

effects of taurine on calcium uptake does not involve the moduladon of

ATPase activity. However, there is a possibility that the assay is not

sensitive enough to measure the changes in ATPase activity taurine may

produce. Clearly, more accurate experimental techniques need to be used to

verify these findings in the future.

What type or types of ATPase activity acdially fiincdon in our system

and whether this activity is involved in calcium uptake is unknown and

constitutes another interesting field of sdidy. Ouabain did not inhibit the

77

ATPase acdvity to a significant degree, suggesting that Na^, K^ATPase is not

active under the experimental conditions or that it consddites a very small

percentage of the total ATPase activity in the retina. Also, the preparation

of the membranes from the whole cell could have resulted in the formation of

sealed vesicles that do not allow for opdmal binding of sodium and

potassium to the ATPase enzyme, thereby decreasing ATPase activity. In

any case, these results differ from other reports that show measurable levels

of Na" , K"^ATPase activity in the rat redna, a difference probably home out

of the variation in the buffer composition or experimental procedure.

A non-mitochondrial Ca " , Mg^" -ATPase has also been examined in the

bovine retina (Chambers et al , 1990), and CHT might be inhibiting this type

of ATPase in the rat retina. SERCA probably comprises a part, if not all, of

this specific ATPase acdvity, though present literadire does not allow for any

certainty on the matter. SERCA is potentially an important component in the

mechanism of action of taurine in calcium uptake because it is involved in the

acdve uptake of calcium into the endoplasmic redculum. However,

experiments with thapsigargin suggest that SERCA is not a major component

of the total ATPase activity studied.

As mentioned before, some form of bicarbonate-acdvated ATPase is

fimctional with a 25 mM bicarbonate concenfration in the retina (Winkler and

Riley, 1977). Our experiments use 25-50 mM bicarbonate (see Methods),

which possibly activates the enzyme even more. It is possible that this is the

ouabain-insensidve ATPase that is inhibited by CHT. It is infriguing to

correlate the bicarbonate-dependency of this enzyme with the

78

bicarbonate-dependency of the potendadng effects of taurine on calcium

uptake, but our data are not extensive enough to support this concept.

An interesting set of experiments that could be done to verify the data

here reported would be to use subcellular retinal fractions in the ATPase

assays. The whole retinal homogenate samples used would almost certainly

possess several different ATPase types which would be pharmacologically

difficult to differentiate from each other. Perhaps, specific ATPase types

colocalize with different membrane fractions or subcellular organelles, and

the effect of taurine would be more easily demonsfrated. In this light,

ATPase activity in the isolated ROS should be studied as a priority.

Taurine uptake versus taurine binding

Taurine produces its effects on calcium uptake only in the millimolar

range. Thus, the relatively high infracellular concenfradon of taurine appears

to be a funcdonal necessity, at least with respect to its effects on calcium

uptake. Although some degree of endogenous biosynthesis exists, most of

the taurine present in mammalian dssues is exogenous in its source, requiring

a very efficient transport system to achieve high dssue concenfrations

(reviewed in Lombardini, 1991). In the redna of several different species,

taurine uptake is made up of two sadirable components, a high- and a

low-affinity system, and a non-sadirable diffiasion component (reviewed in

Huxtable, 1989). In experiments that use rats with hereditary retinal

degeneradon, the high-affinity system has been shown to be dependent on the

normal ftmcdon of the photoreceptor cells (Schmidt and Berson, 1978).

However, high-affinity taurine uptake may also occur in retinal pigment

79

epithelium (RPE) cells (Salceda and Saldana, 1993). Regardless, high-

affinity taurine uptake seems to disappear with the loss of photoreceptor cells

(Schmidt and Berson, 1978), suggesting that only high-affinity uptake occurs

in the photoreceptor cells and only low-affinity is found in the rest of the

retina.

It is of great interest that the inhibidon of taurine uptake does not

significantly diminish the stimulatory effects of taurine on calcium uptake.

Careful analysis of kinetic data would reveal that taurine uptake cannot be

correlated directed with taurine stimulation of calcium uptake. Half-maximal

low-affinity uptake (Km) in the retina occurs at -2,7 mM taurine, while high-

affinity taurine uptake is saturated at taurine concenfrations below 1 mM. In

confrast, previous studies report that taurine produced half-maximal

stimuladon (EC50) of calcium uptake at 8-10 mM concenfradons (Liebowitz

et al , 1987, 1988), In the experiments presented in this thesis, the

potendadng effect of taurine on ATP-dependent calcium uptake in whole

retinal homogenate was half-maximal at around 18 mM. Indeed, the half-

maximal values for Km for taurine uptake and EC50 for taurine stimulation of

calcium uptake do not correspond and so perhaps may not interact.

Theoredcally speaking, the mechanism behind the sdmulatory effects

of taurine on calcium uptake should exhibit affinity lower than what is

observed with taurine uptake systems. A mechanistic altemadve may be

taurine binding, i,e,, taurine may exert its effects by binding to and modifying

phospholipid membranes (Huxtable and Sebring, 1986), thereby moduladng

the ftinction of membrane-bound proteins. The biological consequences of

the binding of taurine to membranes is unclear. However, early sdidies in this

80

lab which measured calcium uptake while varying incubation temperadire

suggested that taurine acts to induce a conformadonal change within the lipid

membrane (Lombardini, 1985b), Calcium uptake in the retina is temperadire-

dependent, increasing as temperadire increases (Pasantes-Morales, 1982;

Lombardini, 1985b). Experiments were performed which correlated the

temperadire of incubation and retinal calcium uptake. Arrhenius plots for the

ATP-dependent calcium ion uptake in the presence and absence of 20 mM

taurine were constructed from the data and the activadon energy for calcium

uptake was calculated. Taurine produced a decrease in the activadon energy

for calcium uptake, an effect which is consistent with observed increase in

uptake seen with taurine. Moreover, the decrease in activation energy could

be interpreted as an alteration in the crystalline-liquid crystalline phase

fransition of the membrane. These studies suggest that the modificadon of

the cell membrane is a possible mechanism of action for the effects of taurine.

Taurine is known to increasingly bind to membranes in a dose-

dependent manner up to 30 mM concenfradon (for review, see Huxtable,

1989). Huxtable reviewed sodium-dependent taurine binding in neuronal-

type membranes (1989) and showed Kd measurements within the

corresponding range of taurine concenfrations used in experiments with rat

synaptosomal P2B fracdons, and rat glial, brain and synaptic membranes. In

these sdidies that resolved a low-affinity type of taurine binding, the Kd was

estimated to be higher than the highest concenfradon of taurine used. The

taurine binding sdidies of Lombardini and Prien (1983) observed that in the

redna, the calculated Kd of the low-affinity site (-2738 \iU) was higher than

the highest taurine concenfradon used (1000 |iM). By definidon, Kd values

81

determine the ligand concenfration that results in half-maximal binding, and

thus, in these experiments, the reported Kd values imply that maximal ligand

binding is achieved at taurine concentrations that are significantly higher than

taurine concentrations used and that, logically, maximal binding was not

achieved. As half-maximal binding cannot be determined if maximal binding

was not determined, these Kd values were probably significandy

underesdmated, suggesdng that ligand binding affinity is much lower than

expected. The apparent consistency may also represent a non-sadirable type

of binding that manifested itself as a dissociadon constant that would float

outside the range of total ligand used.

That the binding of taurine to neuronal type membrane is non-saturable

is not unreasonable, given that taurine is for the most part assumed to interact

with phospholipids in membranes (Huxtable and Sebring, 1986). High-

affinity binding interactions are associated with minute amounts of

endogenous ligands attaching to very specific protein receptor molecules. If

taurine were to act in this manner, then the vast amount of taurine actively

sequestered by the cell would be pharmacologically useless. The massive

binding of taurine to phospholipids was postulated to affect, among others,

the binding of calcium to membranes, the operadon of ion channels and

protein phosphoryladon processes. This nodon is of great importance in the

redna because of the high levels of taurine and the possible reguladon of ion

channels demonsfrated in the experiments in this thesis.

The binding of taurine to the retinal membrane should be sdidied in

relation to the effects of taurine to calcium uptake. Preliminary binding

sdidies showed that frifluoperazine (TFP), a calmodulin antagonist,

82

antagonized the effects of taurine on calcium uptake (Militante and

Lombardini, 1999b), TFP was also found to inhibit the binding of taurine to

retinal membrane, indicating that TFP, and other inhibitors of the effects of

taurine may interfere with taurine binding as a mechanism of action. The

experiments of this thesis suggest that taurine binding to membranes is a

major mechanism behind the effects of taurine, not only in the retina but also

in other cell types, A new approach to the study of taurine function may be to

relate modulators of taurine effects to the modulation of taurine binding to

membrane, i,e,, inhibitors of the effects of taurine may decrease taurine

binding. An interesting experiment would be to test the effect of CHT on

taurine binding. CHT may antagonize the effects of taurine by inhibidng the

binding of taurine to cell membranes.

The experimental use of chelervthrine (CHT)

The exact mechanism of acdon responsible for the PKC-independent

CHT inhibidon of calcium uptake is unknown. There are several interacdons

between CHT and taurine which are independent of PKC acdvity. For

example, CHT has been shown to exhibit taurine-related effects in other

rednal systems, namely, in the phosphorylation of a -20 kDa mitochondrial

protein (Lombardini, 1995). ft was reported that CHT stimulates

phosphorylation of a -20 kDa protein, a process that taurine inhibits. When

present together, it was found that taurine antagonized this specific effect of

CHT in a non-competitive manner. As shown in the experiments in this

thesis, CHT antagonized taurine uptake and the stimulatory effect of taurine

on ATP-dependent calcium uptake in a non-competitive manner. Both these

83

effects of CHT were also demonsfrated to be independent of PKC acdvity and

both effects exhibited similar potencies for CHT. A possible mechanism may

have CHT acting at one specific site to first affect the phosphorylation of the

-20 kDa protein which, in dim, affects ATP-dependent calcium uptake. It is,

thus, reasonable to speculate that the specific site of CHT action may also

regulate taurine uptake aside from regulating the phosphorylation of the -20

kDa protein and calcium uptake. As CHT and taurine produce effects on the

phosphorylation of the -20 kDa protein and on ATP-dependent calcium

uptake in the absence of the other agent, it is clear that the effects of CHT in

the redna are not totally dependent on its inhibitory effect on taurine uptake.

The data presented in this thesis suggest that taurine and CHT may

interact at the membrane sfructures of the cell. Both agents individually may

bind to the membrane to produce their effects, whether alone or together. This

notion would explain their effects on protein phosphorylation for this process

probably involve membrane-bound protein kinases or protein kinase

substrates. Even more relevant, the binding of taurine to membranes would

explain its effects on ion channels that by definition are always membrane-

bound. Regardless, understanding the effects of CHT will add gready to the

understanding of the effects of taurine.

Modulation of calcium channels

To test for the involvement of calcium channels in the observed taurine

effects, cadmium was used to inhibit calcium uptake. In neurons, cadmium

causes a non-selecdve block of calcium currents at micromolar concenfrations

(2-20 laM) (Carbone and Swandulla, 1990). In Figure 26, the increase in

84

ATP-dependent calcium uptake due to exogenous taurine is shown to be

inhibited by 5 xM cadmium while ATP-dependent calcium uptake (in the

absence of taurine) is not affected. At very high cadmium concentration (100

^M), both ATP-dependent and taurine-stimulated ATP-dependent calcium

uptake were inhibited. These data suggest that the effects of taurine on ATP-

dependent calcium uptake are dependent on the opening of a calcium channel

that cadmium blocks. Exacdy what this channel, or channels, may be is

uncertain as the dssue preparadon contains all the cell types found in the

redna.

In terms of the role taurine may play in the visual signaling process, the

possible effect of taurine on the cGMP-gated channel, among all the other

types of calcium channels, holds the most importance. LY83583 (6-anilino-5,

8-quinolinedione) has been shown to potently block cGMP-gated channels in

olfactory receptor neurons, causing inhibidon of cGMP-dependent currents at

concenfradons as low as 1 |iM (Leinders-Zufall and Zufall, 1995), LY83583

appears to act both directly on the channel and on soluble guanylyl cyclase,

the enzyme that produces cGMP in olfactory receptor neurons. This

compound was thus used to inhibit the increase of calcium uptake due to

taurine. Figure 28 shows the effect of LY83583 on calcium uptake in retinal

membrane preparations, LY83583 has no effect on ATP-dependent calcium

uptake but has a significant inhibitory effect on taurine-potentiated calcium

uptake, albeit much less potendy when compared to patch recording

experiments in olfactory receptor neurons that directly measured calcium

currents (Leinders-Zufall and Zufall, 1995). Though there is no direct way to

correlate patch recordings with the calcium uptake measured in these

85

experiments, the data suggest that the effect of taurine is at least partially

dependent on the open state of the cGMP-gated channel which is allowing

calcium flow into the cell.

In theory, the effect of LY83583 with retinal membrane preparations

should also be observed in isolated ROS. Figure 29 shows ATP-dependent

calcium uptake measured in isolated ROS and the inhibitory effect of

LY83583 on taurine-stimulated ATP-dependent calcium uptake, similar to the

effects seen in retinal membrane preparations. The inhibition is not complete,

indicadng the involvement of other uptake systems or, perhaps, inefficient

drug delivery or effect. As in the homogenate preparadon of the rednal

membranes, LY83583 had no effect on ATP-dependent calcium uptake in the

absence of taurine.

To verify the involvement of the cGMP-gated channel with the effects

of taurine, a competitive antagonist of the channel was used to inhibit ATP-

dependent calcium uptake in the ROS. Rp-8-Br-PET-cGMPS is a

cell-permeant cGMP derivative that has been found to inhibit cGMP-induced

current with an IC50 of 25 |iM in excised patches (Wei et al , 1996).

ATP-dependent calcium uptake was not affected by Rp-8-Br-PET-cGMPS

but taurine-stimulated uptake was inhibited (Figure 30), demonsfradng a

certain level of specificity in the interacdon of taurine and cGMP-gated

channels.

It is important to note that in the absence of taurine, ATP-dependent

calcium uptake in the retina, specifically in the ROS, does not seem to

involve the opening of the cGMP-gated channel, while in the presence of

taurine it does. Thus, it is reasonable to assume that while ATP-dependent

86

calcium uptake in the absence of taurine probably involves a variety of

different systems, taurine may specifically stimulate cGMP-gated channel

opening to induce calcium uptake in the ROS, Taurine could modulate

channel opening by increasing the levels of cGMP, thereby increasing

channel activation, or by increasing the affinity of the channel for its agonist.

In our experimental system, the cGMP-gated channels are assumed to

be preferentially closed as the retinal sample is exposed to ambient light. In

theory, the opening of these channels should result in increased ATP-

dependent calcium uptake, an effect best seen in isolated ROS, as the cGMP-

gated channels are found in the ROS. In patch clamp experiments, cGMP is

known to activate the channel with a dissociation constant Kd (for channel

opening) of 17-30 |iM (Pugh and Lamb, 1990), Dibutyryl-cGMP, a

cell-permeant analogue of cGMP, sdmulated ATP-dependent calcium uptake,

but the increase was minimal for all concenfradons of the agonist below 400

|iM (Figure 31), Another cGMP analogue, Rp-cGMP, did not produce any

effects at all, probably due to limited diffiision and disfribution into the dssue.

There is an obvious difference in potency when these effects of

dibutyryl-cGMP are compared to its effects in patch clamp sdidies, but this is

probably another manifestadon of the lack of direct correladon between

channel sdidies involving patch clamp techniques and acdial calcium uptake

measurements in ROS isolates. It is also possible that, within the

experimental dme period of 2 minutes, dibutyryl-cGMP did not diffiise

quickly enough through the cell membrane to raise the intemal cGMP level to

a level that would result in adequate channel opening and, in dim, stimulation

of ATP-dependent calcium uptake.

87

In confrast, no effect was observed when taurine-stimulated ATP-

dependent calcium uptake was measured in the presence of dibutyryl-cGMP,

However, it is known that without proper depletion protocols, some level of

endogenous cGMP remains in experimentally prepared dissociated ROS

(Cote and Brunnock, 1993), providing low levels of endogenous agonist for

channel activation. Thus, it is possible that cGMP-gated channels were

maximally opened in the presence of taurine, through a mechanism of action

that makes use of endogenously present cGMP, making ineffecdial the

addition of exogenous agonist.

Zaprinast, otherwise known as M&B 22,948, is a potent inhibitor (IC50

= 160 nM) of the cGMP-specific phosphodiesterase (PDE) found in the rod

photoreceptor (Gillespie and Beavo, 1989). This compound was used to

inhibit the degradation of endogenous cGMP in the ROS, potentially

elevadng, or at least maintaining, cGMP levels and theoredcally causing

greater acdvation of the cGMP-gated channels. Linear regression analyses of

the data indicated a significant increasing frend (P < O.Ol) in ATP-dependent

calcium uptake with zaprinast freatment (Figure 33), although the absolute

change above confrol (0 |iM zaprinast) was found to be not significant using

the one-way ANOVA, The data suggest that endogenous cGMP levels were

sufficiendy maintained to cause significant but not maximal channel opening.

Perhaps, there was not enough time for endogenous cGMP to accumulate or

that endogenous cGMP can only accumulate to a limited degree under these

experimental condidons. Similar to the effects of dibutyryl-cGMP, zaprinast

had no significant effect on taurine-sdmulated ATP-dependent calcium, also

suggesting that cGMP-gated channels have been maximally sdmulated

88

already in the presence of taurine. Though the effects of dibutyryl-cGMP and

zaprinast do not suggest an exact mechanism of acdon of taurine, the data still

suggest that the stimuladon of cGMP-gated channels is somehow involved in

calcium uptake measured in ROS,

Experiments that would verify the interaction between taurine and

cGMP-gated channels are primarily of the electrophysiological type. Patches

of ROS could be excised using the patch clamp pipede and cGMP-dependent

conductance through the patch sdidied. Then taurine could be applied to the

patch and any changes in conductance noted. How taurine may increase the

flow of calcium through the channel is unclear, but moduladon of cGMP-

gated channels could at least be verified. Another experiment which would

be more physiological in significance would be to use intact, dark-adapted

ROS with the calcium sensitive dye Indo-1 through a patch-clamp pipette

(Gray-Keller and Detwiler, 1994) and to sdidy light-dependent calcium flux.

Taurine can be dialyzed through the pipette also into the ROS and its effects

on the calcium response inside the intact ROS could be observed.

The idea that taurine modulates the opening of the cGMP-gated

channel is a novel one and may present a very important function for taurine

in the retina, specifically in the ROS. The decrease in calcium levels within

the ROS is a crucial step in the process of photoexcitation. During this period

of low calcium concenfration, the expected effect of taurine would be to

stimulate calcium uptake into the ROS, a potentially beneficial effect in the

process of reestablishing the standing dark current. Taurine, in theory,

stimulates the acdvation of the cGMP-gated channel by endogenous cGMP

during this recovery period. Afterwards, as infracellular calcium levels rise.

89

taurine would lose this stimulatory effect through some feedback mechanism

and would acdially inhibit calcium uptake, preventing calcium overioad. This

inhibitory effect of taurine may or may not involve the function of

cGMP-gated channels, and provides another interesting field for inquiry. In

any case, this type of biphasic modulation of calcium uptake by taurine could

become a very significant consideration in the understanding of

photofransduction in the ROS.

Calcium uptake versus calcium binding

Many similarities can be observed in cardiac sarcolemma calcium

binding and retinal calcium uptake. Taurine has long been considered as a

positive modulator of calcium binding in cardiac sarcolemma (Sebring and

Huxtable, 1985). Specifically, taurine increased calcium binding in

"infracellular" buffers (high sodium-low potassium) but did not affect low-

affinity calcium binding in "extracellular" (low sodium-high potassium)

buffers. When ATP was added, taurine increased calcium binding, regardless

of the sodium concenfration. The findings mirror the stimulatory effects of

ATP and taurine observed in retinal calcium uptake. Two calcium binding

sites were identified in the cardiac sarcolemma, one with dissociation

constant (Kd) about a hundredfold greater than the other (-3.94 mM vs. -0.03

mM). Similarly, in the whole rat retina, two putative "uptake" sites have been

described for calcium, exhibiting Km values of 2076 and 35 jiM

(Lombardini, 1983). Furthermore, previous sdidies with bovine ROS that

used A23187 suggested that more than 50%o of maximal calcium binding

capacity is adained after only one minute incubadon while active ATP uptake

90

condnues for up to 30 minutes after the start of the incubation (Hemminki,

1975). The current experiments measured uptake after only two minutes of

incubadon and may in fact be composed mosdy of calcium binding. It is thus

a valid suspicion that the calcium uptake measured in retinal membranes in

these experiments may have a significant binding component, and that the

stimulatory effects of taurine may also be directed toward this binding

component, as it is with the cardiac sarcolemma. The data in this thesis

which demonsfrated the effects of the calcium ionophore A23187 on calcium

uptake may support this notion. In theory, A23187 should not affect calcium

binding to membranes but only the flux of calcium through the membrane.

Differences should be noted between sarcolemmal calcium binding and

rednal calcium uptake. The calcium uptake components in the retina were

observed with experiments performed at 37''C, a temperature that allows for

the function of acdve calcium fransport systems, with a very short incubadon

time. In confrast, the calcium binding experiments with cardiac sarcolemma

were performed at 24°C for 45 minutes with a special equilibrium dialysis

system that displayed an equilibrium dme beginning at 30 minutes and lasting

for at least two hours. The low temperature and delayed equilibrium are

classic experimental parameters in binding experiments. In order to answer

the question of whether calcium binding or uptake is involved in the effects

of taurine in the retina, conditions were modified to allow for the inhibition of

active calcium uptake,

ATP sdmulated calcium uptake in frog ROS (Pasantes-Morales, 1982),

The ROS membranes were found to be osmodcally lysed, probably an ardfact

of dssue processing. While the osmodc lysis of bovine ROS membrane in

91

water inhibited ATP stimuladon of calcium uptake at 37°C temperadires,

some level of stimuladon could still be observed (Hemminki, 1975).

Similariy, the calcium uptake in both lysed whole retinal homogenate and

ROS (Figures 37 and 38), also performed at 37°C, exhibited some level of

ATP sdmulation and in tum taurine potentiation. It is most interesdng to note

that the osmotic lysis of cell membranes does not completely obliterate the

active fransport of calcium, although clearly ATP-dependent uptake is

compromised. The calcium ionophore A23187 was employed to distinguish

between calcium binding and calcium uptake. A23187 (0.9 |iM) obliterated

the sdmulatory effects of ATP on calcium uptake in both intact and lysed

bovine ROS (Hemminki, 1975). Calcium ionophores A23187 and X537A

also completely inhibited ATP-dependent calcium uptake in frog ROS

(Pasantes-Morales, 1982). In the same manner, A23187 (10 flM) inhibited

the effects of both ATP and taurine in rat retinal samples (Figure 37 and 38).

It can be assumed that calcium uptake measured in the presence of the

calcium ionophore corresponds to the calcium binding to membranes. Given

that A23187 did not decrease uptake below that of confrol levels, the data

suggest that in the absence of ATP and taurine, calcium uptake is almost

totally composed of calcium binding. It is clear that ATP and taurine do not

affect this calcium binding. Conversely, stimuladon by ATP and taurine

corresponds to calcium uptake and not to binding. Experiments on calcium

binding at 0°C support this nodon in that ATP and taurine did not appear to

have any stimulatory effect.

92

Conclusions

Taurine appears to bind to the retinal cell membrane and to increase the

activadon of calcium channels, allowing greater influx of calcium into the

cell. While the effects of taurine are dependent on the presence of ATP, the

activity of ATPase enzymes is probably not involved in the mechanism of

acdon of taurine. The specific inhibidon of taurine uptake did not affect the

stimulatory effects of taurine on calcium uptake, and it was concluded that

taurine uptake is also not involved in the mechanism of acdon of taurine. The

only other altemative was the binding of taurine to the cell membrane and the

moduladon of membrane-bound systems like ion channels. Indeed, the

sdmulatory effects of taurine on retinal calcium uptake was specifically

correlated to the activation of a specific calcium channel for the first time.

The identity of the calcium channels which taurine affects was established

through the use of specific channel inhibitors. Taurine appears to modulate

the activadon of cGMP-gated channels, the channels which confrol the flux of

calcium into the ROS and the function of which is crucial in the

photofransducdon process. The involvement of ion channel activadon was

complemented by the finding that the stimulatory effects of taurine on

calcium uptake are dependent on calcium flux and not on calcium binding to

membrane.

The set of data presented and interpreted in this thesis serves to clarify

some quesdons regarding the effects of taurine on calcium uptake in the

redna, but moreover, it serves to expand the same set of quesdons. If taurine

is acting on the membrane to produce its effects, in what way is it affecdng

the membrane? Does it change the fluidity of the membrane, as early studies

93

using Arrhenius plots seem to indicate (Lombardini, 1985b)? It is an

infriguing possibility as the mechanism would provide an almost blanket

explanadon for the various effects of taurine on cellular processes,

specifically those that can be considered membrane-limited or membrane-

dependent processes. For example, protein phosphorylation can be

modulated by taurine if the kinase and/or the substrate protein involved are

membrane-bound. The function of membrane-bound ion channels should be

easily affected by taurine with this theory in mind. The data truly expands on

the physiological possibilities of taurine beyond the realm of the retina, as the

data can be readily applied to any cell type that expresses funcdonal ion

channels on its cell membrane and that retains millimolar levels of taurine

infracellulariy. In fact, it is entirely possible that the physiological funcdon of

most ion channels is dependent on taurine because it is more the rule that high

levels of taurine are found within a cell than it is the excepdon. In the case of

the retina, the sdmulation of cGMP-gated channels by taurine would serve an

obvious role in the fact and efficient elecfrophysiological recovery of the

photoreceptor cell after light stimulus. This possibility warrants greater

study.

94

REFERENCES

Baylor, D, (1996) How photons start vision. Proceedings of the National Academy of Sciences of the United States of America, 93, 560-5.

Berman, A.L., Azimova, A.M., and Gribakin, F,G, (1977) Localization of Na , K^-ATPase and Ca^^-activated Mg^-dependent ATPase in retinal rods. Vision Research, 17:527-36.

Berson, E.L., Hayes, K.C, Rabin, A,R., Schmidt, S.Y., and Watson, G. (1976) Retinal degeneradon in cats fed casein. II. Supplementadon with methionine, cysteine, or taurine. Investigative Ophthalmology and Visual Science, 15, 52-8,

Carbone, E, and Swandulla, D, (1989) Neuronal calcium channels: kinetics, blockade and modulation. Progress in Biophysics and Molecular Biology, S4,'i\-5%.

Chambers, J,P,, Kumar, P,, Tsin, A,T,C., and Valdez, J,J, (1990) Pardal characterization of a high-affinity [Ca ^ + Mg^^]-dependent adenosinefriphosphatase from bovine retina. Experimental Eye Research, 50, 127-34.

Cocker, S.E. and Lake, N, (1987) Elecfrorednographic alteradons and their reversal in rats freated with guanidinoethyl sulfonate, a taurine depletor. Experimental Eye Research, 45, 977-87,

Cocker, S.E, and Lake, N, (1989) Effects of dark maintenance on retinal biochemistry and function during taurine depletion in the adult rat. Visual Neuroscience, 3, 33-8,

Cohen, H,G., Seifen, E.E., Sfraub, K.D., Tiefenback, C , and Stermitz, F,R. (1978) Strucdiral specificity of the NaK-ATPase inhibidon by sanquinarine, an isoquinolone benzophenanthridine alkaloid. Biochemical Toxicology, 27, 2555-8,

95

Cote, R,H„ and Brunnock, M,A. (1993) Intracellular cGMP concentradon in rod photoreceptors is regulated by binding to high and moderate affinity cGMP binding sites. Journal of Biological Chemistry, 268, 1/1 yu-o.

Fletcher, E,L„ and Kalloniatis, M. (1996) Neurochemical architecdire of the normal and degenerating rat retina. Journal of Comparative Neurology, 376, 343-60,

Gray-Keller, M,P., and Setwiler, P.B. (1994) Teh calcium feedback signal in the photofransducdon cascade of vertebrate rods. Neuron 13 849-61,

Geggel, H,S,, Ament, M,E,, Heckenlively, J,R,, Mardn, D,A., and Kopple, J,D, (1985) Nufridonal requirement for taurine in padents receiving long-term parenteral nutrition. New England Journal of Medicine, 312, 142-6.

Gillespie, P,G., and Beavo, J,A. (1989) Inhibidon and stimuladon of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948. Molecular Pharmacology, 36, 773-81,

Gray-Keller, M.P,, and Detwiler, P,B, (1994) The calcium feedback signal in the phoofransduction cascade of vertebrate rods. Neuron, 13, 849-61,

Heller-Sdlb, B,, van Roeyen, C, Rascher, K., Hartwig, H,G,, Huth, A,, Seeliger, M,W,, Warskulat, U,, and Haussinger, D, (2002) Disrupdon of the taurine fransporter gene (taut) leads to rednal degeneradon in mice, FASEB Journal, 16: 231-3.

Hemminki, K. (1975) Accumuladon of calcium by retinal outer segments. Acta Physiologica Scandinavica, 95, 117-25.

Herbert, J,M,, Augereau, J,M,, Gleye, J,, and Maffrand, J.P, (1990) Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochemical and Biophysical Research Communications, 173, 993-9.

96

Huxtable, R.J, (1989) Taurine in the central nervous system and the maimnalian actions of taurine. Progress in Neurobiology, 32,

Huxtable, R,J. (1992) Physiological actions of taurine. Physiological Reviews, 12, \0\-6?>.

Huxtable, R.J. and Sebring L.A. (1986) Towards a unifying theory for the acdons of taurine. Trends in Pharmacology, 1, 481-5.

Imaki, H„ Jacobson, S,G., Kemp, CM,, Knighton, R.W., Neuringer, M., and Sturman, J, (1993) Retinal morphology and visual pigment levels in 6- and 12-month-old rhesus monkeys fed a taurine-free human infant formula. Journal of Neuroscience Research, 36, 290-304.

Jacobson, S,G,, Kemp, CM,, Borruat, F,X,, Chaitin, M,H,, and Faulkner D.J, (1987) Rhodopsin topography and rod-mediated function in cats with the rednal degeneration of taurine deficiency. Experimental Eye Research, AS, A%\-9^.

Jhiang, S,M., Fithian, L., Smanik, P., McGill, J., Tong, Q,, and Mazzaferri, E,L, (1993) Cloning of the human taurine fransporter and characterizadon of taurine uptake in thyroid cells. FEBS Letters, 318, 139-44,

Kalloniads, M,, Marc, R,E,, and Murry, R.F. (1996) Amino acid signadires in the primate retina. Journal of Neuroscience, 16, 6807-29.

Kijima, Y,, Ogunbunmi, E,, and Fleischer, S. (1991) Dmg acdon of thapsigargin on the Ca^^ pump protein of sarcoplasmic reticulum. Journal of Biological Chemistry, 266, 22912-8.

Kuo, C-H., and Miki, N. (1980) Stimulatory effect of taurine on Ca-uptake by disk membranes from photoreceptor cell outer segments. Biochemical Biophysical Research Communications, 94, 646-51.

97

Lake, N. (1981) Depledon of retinal taurine by treatment with guanidinoethyl sulfonate. Life Sciences, 29, 445-8.

Lake, N. (1982) Depletion of taurine in the adult rat retina. Neurochemical Research,!, 1385-90.

Lake, N. (1986) Electroretinographic deficits in rats freated with guanidinoethyl sulfonate, a depletor of taurine. Experimental Eye Research, 42, S7-9\.

Lake, N. and Cocker, S.E. (1983) In vifro studies of guanidinoethyl sulfonate and taurine fransport in the rat retina. Neurochemical Research, 8, 1557-63,

Lake, N. and Malik, N, (1987) Retinal morphology in rats freated with a taurine fransport antagonist. Experimental Eye Research, 44, 331-46.

Lake, N. and De Marte, L. (1988) Effects of beta-alanine freatment on the taurine and DNA content of the rat heart and retina. Neurochemical Research, 13, 1003-6.

Lake, N,, Marshall, J, and Voaden, M.J. (1978) High affinity uptake sites for taurine in the retina. Experimental Eye Research, 27, 713-8,

Lake, N, and Verdone-Smith, C (1989) Immunocytochemical localization of taurine in the mammalian retina. Current Eye Research, 8, 163-73,

Landreth, G.E. and Agranoff, B,W, (1979) Explant culdire of adult goldfish retina: A model for the sdidy of CNS regeneradon. Brain Research, 161,39-55.

Leinders-Zufall, T. and Zufall, F. (1995) Block of cyclic nucleodde-gated channels in salamander olfactory receptor neurons by guanylyl cyclase inhibitor LY 83583. Journal of Neurophysiology, 74, 2759-62.

98

Leon, A., Levick, W.R., and Sarossy, M.G, (1995) Lesion topography and new histological features in feline taurine deficiency retinopathy. Experimental Eye Research, 61, 731-41.

Liebowitz, S,M., Lombardini, J,B,, and Salva, P, (1987) Cyclic taurine analogs: Synthesis and effects on ATP-dependent Ca2+ uptake in rat retina. Biochemical Pharmacology, 36, 2109-14.

Liebowitz, S.M., Lombardini, J.B., and Allen, CI. (1988) Effects of aminocycloalkanesulfonic acid analogs of taurine on ATP-dependent calcium ion uptake and protein phosphorylation. Biochemical Pharmacology, "M, 1303-9.

Liebowitz, S.M,, Lombardini, J.B,, and Allen, CI. (1989) Sulfone analogues of taurine as modifiers of calcium uptake and protein phosphorylation in rat retina. Biochemical Pharmacology, 38, 399-406.

Lima, L, (1999) Taurine and its frophic effects in the retina. Neurochemical Research, 24, 1333-8,

Lima, L,, Madis, P,, and Drujan, B, (1988) Taurine effects on neuridc growth from goldfish retinal explants. International Journal of Developmental Neuroscience, 6, 417-24,

Lima, L„ Madis, P,, and Drujan, B, (1993) Taurine-induced regeneradon of ' goldfish retina in culture may involve a calcium-mediated mechanism. Journal ofNeurochemistry, 60, 2153-8.

Liu, Q,_R„ Lopez-Corcuera, B., Nelson, H,, Mandiyan, S„ and Nelson, N, (1992) Cloning and expression of a cDNA encoding the fransporter of taurine and P-alanine in mouse brain. Biochemistry, 89, 12145-9,

Lombardini J B (1983) Effects of ATP and taurine on calcium uptake by

membrane preparadons of the rat redna. Journal ofNeurochemistry,

40, 402-6,

99

Lombardini, J,B. (1985a) Effects of taurine on calcium ion uptake and protein phosphorylation in rat retinal membrane preparations. Journal ofNeurochemistry, 45, 268-75.

Lombardini, J.B. (1985b) Taurine effects on the transition temperadire in Arrhenius plots of ATP-dependent calcium ion uptake in rat rednal membrane preparadons. Biochemical Pharmacology, 34,3741-5.

Lombardini, J.B. (1983) Effects of taurine and mitochondrial metabolic inhibitors on ATP-dependet Ca ^ uptake in synaptosomal and mitochondrial subcellular fractions of rat retina. Journal of Neurochemistry, 51, 200-5.

Lombardini, J.B. (1991) Taurine: retinal ftinction. Brain Research Brain Research Review, 16, 151-69. Review.

Lombardini, J.B. (1992) Effects of taurine on the phosphoryladon of specific proteins in subcellular fractions of the rat retina. Neurochemical Research, 17,821-4.

Lombardini, J.B. (1993) Partial characterizadon of an approximately 20 K M(r) retinal protein whose phosphorylation is inhibited by taurine. Biochemical Pharmacology, 46, 1445-51,

Lombardini, J,B, (1995) Paradoxical stimulatory effect of the kinase inhibitor chelerythrine on the phosphoryladon of a approximately 20 K M(r) protein present in the mitochondrial fracdon of rat redna. Brain Research, 673, 194-8,

Lombardini, J,B, (1996) Stimuladon by chelerythrine of the phosphorylation of the amino acid serine in an -20 kDa protein present in the mitochondrial fracdon of the rat retina. Biochemical Pharmacology, 52, 253-7.

Lombardini, J.B, and Liebowitz, S.M. (1990) Taurine analogues as modifiers of the accumulation of''^calcium ions in a rat rednal membrane preparadon. Current Eye Research, 9, 1147-56,

100

Lombardini, J,B, and Prien, S.D. (1983) Taurine binding by rat rednal membranes. Experimental Eye Research, 37, 239-50.

Lombardini, J.B., Liebowitz, S.M., and Chou, T.-C (1989) Analogues of taurine as sdmulators and inhibitors of ATP-dependem calcium ion uptake in rat retina: Combination kinetics. Molecular Pharmacology, 36, 256-264.

Lombardini, J.B., Young, R,S,L. and Props, CL. (1996) Taurine depledon increases phosphorylation of a specific protein in the rat retina. Amino Acids, 10, 153-165,

Lopez-Colome, A.M, and Pasantes-Morales, H. (1980) Taurine interacdons with chick retinal membranes. Journal of Neurochemistrv 34 1047-52.

Lopez-Colome, A.M,, Fragoso, G,, and Salceda, R, (1991) Taurine receptors in membranes from retinal pigment epithelium cells in culdire. Neuroscience, 41, 791-6.

Lytton, J,, Westin, M., and Hanley, M,R. (1991) Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium ^umps. Journal of Biological Chemistry, 266, 17067-17071,

Macaione, S,, Ruggeri, P,, De Luca, F., and Tucci, G. (1974) Free amino acids in developing retina. Journal ofNeurochemistry, 22, 887-891.

Madsen, S,, Ottersen, 0,P,, and Storm-Mathisen, J. (1985) Immunocytochemical visualizadon of taurine: neuronal localization in the rat cerebellum. Neuroscience Letters, 60, 255-60.

Marc, R,E,, Murray, R.F,, and Basinger, S.F. (1995) Pattem recognidon of amino acids in retinal neurons. The Journal of Neuroscience, 15, 5106-29,

101

Marc, R,E., Murry, R.F., Fisher, S.K., Linberg, K.A., Lewis, G.P., and Kalloniatis, M. (1998) Amino acid signadires in the normal cat redna. Investigative Ophthalmology and Visual Science, 39, 1685-93.

Michel, S., Schoch, K., and Stevenson, P.A. (2000) Amine and amino acid fransmitters in the eye of the mollusc Bulla gouldiana: an immunocytochemical study. Journal of Comparative Neurolo2v 425, 244-56,

Militante, J.D, and Lombardini, J.B. (1998a) Effect of taurine on chelerythrine inhibition of calcium uptake and ATPase acdvity in the rat retina. Biochemical Pharmacology, 55, 557-65.

Militante, J.D. and Lombardini, J.B. (1998b) Pharmacological characterization of the effects of taurine on calcium uptake in the rat retina. Amino Acids, 15, 99-108.

Militante, J.D. and Lombardini, J.B. (1999a) Taurine uptake acdvity in the rat retina: protein kinase C-independent inhibidon by chelerythrine. Brain Research, 818, 368-74,

Militante, J,D, Lombardini, J.B, (1999b) Sdmulatory effect of taurine on calcium ion uptake in rod outer segments of rat retina is independent of taurine uptake. Journal of Pharmacology and Experimental Therapeutics, 291, 383-9,

Militante, J,D. and Lombardini, J.B, (2000) Stabilizadon of calcium uptake in rat rod outer segments by taurine and ATP. Amino Acids, 19, 561-70.

Militante, J.D. and Lombardini, J.B. (2002) Taurine: Evidence of physiological function in the retina. Nutritional Neuroscience, 5, 75-90.

Miyamoto, Y,, Liou, G,I., and Sprinkle, T,J. (1996) Isoladon of a cDNA encoding a taurine fransporter in the human retinal pigment epithelium. Current Eye Research, 15, 345-9,

102

Morimura, H„ Shimada, S., Otori, Y., Saishin, Y., Yamauchi, A., Minami, A., et al. (1997) The differendal osmoreguladon and localization of taurine fransorter mRNA and NaVmyo-inositol cofransporter mRNA in rat eyes. Brain Research Molecular Brain Research, 44, 245-252.

Nag, T.C, Jotwani, G., and Wadhwa, S. (1998) Immunohistochemical localization of taurine in the retina of developing and adult human and adult monkey. Neurochemistry International, 33, 195-200.

Nakahishi, S., Matsuda, Y., Iwahashi, K., and Kase, H. (1986) K-252b, c and d, potent inhibitors of protein kinase C from microbial origin. Journal of Antibiotics (Tokyo), 39, 1066-1071,

Neal, M,J. and White, R.D. (1978) Discriminadon between descriptive models of L-glutamate uptake by the retina using non-linear regression analysis. Journal of Physiology, 277, 387-394.

Neuringer, M, and Sturman, J. (1987) Visual acuity loss in rhesus monkey infants fed a taurine-free human infant formula. Journal of Neuroscience Research, 18, 597-601.

Okada, M,, Okuma, Y,, Osumi, Y., Nishihara, M,, Yokotani, K., and Ueno, H, (2000) Neurofransmitter contents in the redna of RCS rat. Graefe's Archive for Clinical and Experimental Ophthalmology, 238, 998-1001,

Omura, Y, and Inagaki, M. (2000) Immunocytochemical localization of taurine in the fish redna under light and dark adaptations. Amino Acids, 19, 593-604,

Ottclecz, A,, Garcia, C,A„ Eichberg, J., and Fox, D,A, (1993) Alteradons in retinal Na^, K^-ATPase in diabetes: Sfreptozotocin-induced and Zucker diabetic fatty rats. Current Eye Research, 12,1111-1121,

103

Ottersen, 0,P„ Madsen, S,, Meldmm, B.S., and Storm-Mathisen, J. (1985) Taurine m the hippocampal formation of the Senegalese baboon, Papio papio: an immunocytochemical sdidy with an antiserum against conjugated taurine. Experimental Brain Research, 59, 457-62.

Pasantes-Morales, H, (1982) Taurine-calcium interacdons in frog rod outer segments: Taurine effects on an ATP-dependent calcium franslocadon process. Vision Research, 22, 1487-1493.

Pasantes-Morales, H,, and Quesada, O, (1980) Effects of the ionophores X537A and A23187 on GABA, glycine, and taurine release from chick rednal subcellular fracdons. Neurochemical Research, 5, 1077-88,

Pasantes-Morales, H,, and Ordoiiez, A„ (1982) Taurine activadon of a bicarbonate-dependent, ATP-supported calcium uptake in frog rod outer segments. Neurochemical Research, 7, 317-28.

Pasantes-Morales, H., Quesada, O,, Carabez, A., and Huxtable, R.J, Effects of the taurine fransport antagonist, guanidinoethane sulfonate, and P-alanine on the morphology of rat retina, (1983) Journal of Neuroscience Research, 9, 135-43,

Pow, D.V. (1994) Taurine, amino acid fransmitters, and related molecules in the retina of the Ausfralian lungfish Neoceratodus forsteri: a light-microscopic immunocytochemical and elecfron-microscopic sdidy. Cell Tissue Research, 278, 311-26.

Pow, D,V., Sullivan, R., Reye, P., and Hermanussen, S. (2002) Localizadon of taurine fransporters, taurine, and ^H taurine accumulation in the rat retina, pituitary, and brain. Glia, 37, 153-168.

Pugh, E.N. Jr. and Lamb, T.D. (1990) Cyclic GMP and calcium: the intemal messengers of excitation and adaptadon in vertebrate photoreceptors. Vision Research, 30,1923-1948,

104

Quesada, O., Huxtable, R.J., and Pasantes-Morales, H. (1984) Effect of guanidinoethane sulfonate on taurine uptake by rat redna. Journal of Neuroscience Resarch, 11,179-86,

Quesada, O., Picones, A., and Pasantes-Morales, H. (1988) Effect of light deprivation on the ERG responses of taurine-deficient rats. Experimental Eye Research, 46, 13-20.

Qian, X,, Vinnakota, S,, Edwards, C, and Sarkar, H.K. (2000) Molecular characterization of taurine transport in bovine aortic endothelial cells. Biochimica et Biophysica Acta, 1509, 324-34.

Ramamoorthy, S., Leibach, F.H., Mahesh, V.B,, Han, H,, Yang-Feng, T., Blakely, R,D., et al, (1994) Functional characterizadon and chromosomal localizadon of a cloned taurine fransporter from human placenta. Biochemical Journal, 300, 893-900,

Rapp, L.M,, Thum, L.A., and Anderson, R.E. (1988) Synergism between environmental lighting and taurine depletion in causing photoreceptor cell degeneration. Experimental Eye Research, 46, 229-38.

Salceda, R. (1980) High-affinity taurine uptake in developing redna. Neurochemical Research, 5, 561-72,

Salceda, R, (1999) Insulin-sdmulated taurine uptake in rat retina and retinal pigment epithelium. Neurochemistry International, 35, 301-6.

Salceda, R, and Pasantes-Morales, H, (1982) Uptake, release, and binding of taurine in degenerated rat retinas. Journal of Neuroscience Research, S, 631-42.

Salceda, R. and Saldana, M,R. (1993) Glutamate and taurine uptake by retinal pigment epithelium during rat development. Comparative Biochemistry and Physiology C-Pharmacology, Toxicology, and Endocrinology, 104, 311-6,

105

Satoh, H. and Sperelakis, N, (1998) Review of some actions of taurine on ion channels of cardiac muscle cells and others. General Pharmacology, 30, 451-63.

Schafer, S., Bicker, G„ Ottersen, 0,P„ and Storm-Mathisen, J, (1988) Taurine-like immunoreacdvity in the brain of the honeybee. Journal of Comparative Neurology, 268, 60-70.

Schmidt, S.Y. and Berson, E.L. (1978) Taurine uptake in isolated retinas of normal rats and rats with hereditary rednal degeneration. Experimental Eye Research, 27, 191-8.

Schmidt, S.Y., Berson, E.L., and Hayes, K.C. (1976) Retinal degeneradon in cats fed casein. I. Taurine deficiency. Investigative Ophthalmology and Visual Science, 15, 47-52.

Sebring, L.A., and Huxtable, R.J, (1985) Taurine moduladon of calcium binding to cardiac sarcolemma. The Journal of Pharmacology and Experimental Therapeutics, 232, 445-51,

Shimada, C , Tanaka, S,, Hasegawa, M,, Kuroda, S., Isaka, K., Sano, M. et al, (1992) Beneficial effect of infravenous taurine infusion on elecfrorednographic disorder in taurine deficient rats, Japanese Journal of Pharmacology, 59, 43-50,

Smith, K,E., Borden, L,A,, Wang, CH., Hartig, P.R., Branchek, T.A., and Weinshank, R.L, (1992) Cloning and expression of a high affinity taurine fransporter from rat brain. Molecular Pharmacology, 42, 563-9,

Sturman, J.A. (1993) Taurine in development. Physiological Reviews, 73, 119-47,

Timbrell, J,A., Seabra, V., and Waterfield, CJ. (1995) The in vivo and in vitro protecdve properties of taurine. General Pharmacology, 26, 453-62. Review.

106

Uchida, S,, Kwon, H,M,, Yamauchi, A., Preston, A.S., Marumo, F,, and Handler, J,S, (1992) Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine fransporter that is regulated by hypertonicity. Proceedings of the National Academy of the Sciences of the United States of America, 89, 8230-4,

Uchida, S., Kwon, H.M,, Yamauchi, A,, Preston, A.S„ Marumo, F,, Handler, J,S, (1993) Molecular cloning of the cDNA for an MDCK cell Na(+)- and Cl(-)-dependent taurine transporter that is regulated by hypertonicity. Proceedings of the National Academy of the Sciences of the United States of America, 90, 7424.

Vinnakota, S., Qian, X., Egal, H,, Sarthy, V,, and Sarkar, H,K, (1997) Molecular characterizadon and in situ localizadon of a mouse rednal taurine fransporter. Journal ofNeurochemistry, 69, 2238-50.

Voaden, M.J,, Lake, N,, Marshall, J,, and Morjaria, B, (1977) Studies on the distribution of taurine and other neuroacdve amino acids in the redna. Experimental Eye Research, 25, 219-257.

Wei, J.-Y,, Cohen, E,D,, Tan, Y,-Y,, Genieser, H.-G., and Barnstable, C.J. (1996) Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: P-phenyl-1, N^-etheno-substituted cGMP analogues as probes of the cGMP-binding site. Biochemistry, 35, 16815-16823.

Winkler, B.S. and Riley, M.V. (1977) Na^-K^ and HCOj" ATPase activity in the redna: Dependence on calcium and sodium. Investigative Ophthalmology and Visual Science, 16, 1151-1154,

Winkler, B,S,, Riley, M,V, (1980) Influence of calcium on retinal ATPases, Investigative Ophthalmology and Visual Science, 19, 562-564.

Wright, CE,, Tallan, H,H., Lin, Y,Y., and Gaull, G.E, (1986) Taurine: biological update, Annuual Review of Biochemistry, 55,427-53,

107