62 Brain Research. 426 / 1987) 62 - Og Elsevier

BRE 13047

Effects of caffeine and cyclic adenosine 3',5'-monophosphate on adenosine triphosphate-dependent calcium uptake by lysed brain

synaptosomes

Kamilia Mekhail-Ishak, Pierre-Andr6 Lavoie and Mahmoud Sharkawi DOpartement de pharmacologie. FacultO de mddecine, UniversitO de MontrOal, MontrOal, Que. (Canada)

(Accepted 28 April 1987)

Key words': Adenosine triphosphate-dependent calcium uptake; Neuronal endoplasmic reticulum; Lysed brain synaptosome; Caffeine; Cyclic adenosine 3' ,5'-monophosphate

The adenosine triphosphate-dependent calcium uptake by endoplasmic reticutum elements of lysed synaptosomes from rat brain cortex was studied. Caffeine exhibited a biphasic effect on this calcium uptake activity: concentrations of 1, 2, 5, t 0 or 30 mM caffeine stimulated calcium uptake by 62, 111, 73, 88 and 60% respectively, whereas calcium uptake was inhibited by 55% at a 60-mM concen- tration of caffeine. Calcium release from endoplasmic reticulum elements of lysed brain synaptosomes was stimulated by 10 mM caf- feine. Cyclic adenosine 3' ,5'-monophosphate stimulated calcium uptake in the lysed synaptosome preparationi exogenous concentra- tions of 0.05, 0.5, 5, 50, or 500 ktM stimulated uptake by 67, 67, 95, 38 or 67% respectively. To explore the possibility that caffeine stimulated calcium uptake through inhibition of phosphodiesterase and consequent preservation of cyclic adenosine 3',5'-monophos- phate, we have tested whether caffeine retained its ability to stimulate calcium uptake under conditions of maximal stimulation by cy- clic adenosine 3',5'-monophosphate. The combined presence of 10 mM caffeine and 5/~M cyclic adenosine 3',5'-monophosphate re- sulted in an approximate doubling of the calcium uptake as compared to the uptake in the presence of the cyclic nucleotide alone, indi- cating that the stimulation due to caffeine does not occur via cyclic adenosine 3' ,5'-monophosphate.

INTRODUCTION

On both biochemical and morphologica l grounds,

an adenosine t r iphosphate (ATP) -dependen t calci-

um uptake activity in lysed bra in synaptosomes has

been a t t r ibuted to the neuronal smooth endoplasmic

reticulum elements 2'3'2°. This calcium-sequester ing

role of the neuronal endoplasmic ret iculum is also

suppor ted by the presence of calcium deposi ts in axo-

nal endoplasmic ret iculum 6,n and by the presence of

an A T P - d e p e n d e n t calcium uptake activity in micro- somal prepara t ions from neural tissue 7'22'25'3°.

The A T P - d e p e n d e n t calcium uptake activity of

neuronal endoplasmic ret iculum can be modif ied by

several drugs, such as local anesthetics, e thanol , pen-

tobarbi ta l and caffeine. Ethanol and pentobarb i ta l

inhibit A T P - d e p e n d e n t calcium uptake by lysed

brain synaptosomes w. Local anesthetics exhibit a bi-

phasic effect on A T P-de pe nde n t calcium uptake in

brain microsomes, whereby a s t imulat ion of calcium

uptake occurs at low concentrat ions of local anes-

thetics and an inhibit ion of calcium uptake occurs at

high drug concentrat ions 32. Local anesthetics also

produce a biphasic effect on A T P - d e p e n d e n t calcium

uptake by lysed brain synaptosomes 15, indicating that

the endoptasmic ret iculum elements of a synaptic ter-

minal p repara t ion respond to local anesthet ics in a

fashion very similar to that of whole brain micro-

somes. The effect of caffeine on A T P - d e p e n d e n t cal-

cium uptake has previously been s tudied in brain mi-

crosomes, where caffeine inhibi ted calcium uptake at

concentrat ions ranging from 5 to 15 m M 31. W e repor t

here that , depending on the concentra t ion , caffeine

can st imulate or inhibit A T P - d e p e n d e n t calcium up-

Correspondence: P.-A. Lavoie, D6partement de pharmacologic, Facult6 de m6decine, Universit6 de Montr6al, C.P. 6128, Succ. A ' . Montr6al, Que., Canada H3C 3J7.

0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

take by lysed brain synaptosomes. We have also ex-

plored the possible relationship of stimulation of cal-

cium uptake by caffeine in lysed brain synaptosomes to the ability of caffeine to inhibit the enzyme phos- phodiesterase. Preliminary accounts of some of these findings have been presented 16'17.

MATERIALS AND METHODS

Chemicals The special chemicals used in this study were:

4-(2-hydroxyetbyl)- 1-piperazine-ethanesulfonic acid (HEPES) and cyclic adenosine 3' ,5 '-monophosphate

(cAMP), from Boehringer Mannheim Canada, Montrdal, Que.; Tris-base (J.T. Baker Chemicals, Phillipsburg, N J); magnesium salt of ATP, 2,4-dini- trophenol, sodium azide, oligomycin, caffeine and

ethyleneglycol-bis-(fl-aminoethyl ether)N,N'-tetraa- cetic acid (EGTA), all from Sigma Chemicals, St. Louis, MO; 45CAC12, spec. act. 4-50 Ci/g calcium (New England Nuclear, Boston, MA) or 5-30 Ci/g

calcium (ICN Radiochemicals, Irvine, CA).

Preparation of the lysed synaptosomes Male Sprague-Dawley rats (175-225 g; Charles

River Canada, St-Constant, Que.) were used as a source of brain tissue in all experiments. Synapto- somes were prepared as described in Blaustein et al. 2. This method involves the homogenization of the grey matter from rat brain cortex in 0.32 M sucrose to obtain a 10% w/v tissue homogenate. The bomoge- nate underwent several centrifugations to obtain syn- aptosomal pellets, which were resuspended in the hy- potonic lysis solution described in Blaustein et al. 2.

The lysis solution also contained 3 mitochondrial poi- sons in the following concentrations: sodium azide,

0.1 raM; 2,4-dinitrophenol, 0.1 mM; and oligomycin 0.7/~g/ml. Ten ml of lysis solution were used for each gram wet weight of brain tissue, and the lysis proce- dure involved a 3-rain incubation at 30 °C. The lysed synaptosome preparation was then kept on ice. Ali- quots (0.9 ml each) of this lysed synaptosomes prepa- ration were used for calcium uptake assays, these ex- periments being completed within 2.5 h after the os- motic lysis of the synaptosomes. One or two of the synaptosomal pellets were resuspended in distilled water, and used for protein determination by the method of Lowry et al. Is with bovine serum albumin

63

as a standard.

45 Ca2 + uptake by lysed synaptosomes A 45Ca2+ uptake experiment generally consisted of

4 incubation conditions: uptake in the absence of ATP, uptake in the presence of ATP, uptake in the

absence of ATP but in the presence of drug, and up- take in the presence of ATP and drug. To achieve these 4 different conditions, 4 different media of the appropriate composition were prepared, and a 0.9- ml aliquot of each medium was mixed with a 0.9-ml

aliquot of the lysed synaptosome suspension. In one instance, namely the experiment testing the effect of

a combination of caffeine and cAMP, two more incu- bation conditions were needed: uptake in the ab- sence of ATP but in the presence of caffeine and cAMP, and uptake in the presence of ATP, caffeine and cAMP. Calcium uptake was started by the addi- tion of 0.2 ml of EGTA-buffered calcium solution to each tube. The pH of the above solutions was brought to 7.5 with Tris-base.

The final incubation medium in which 4SCa2+ up-

take by lysed synaptosomes was studied had the fol- lowing composition: lysed synaptosomal protein, ap- proximately 0.4 mg/ml; KC1, 145 mM; NaC1, 5 raM;

MgCI 2, 1.4 mM; KH2PO 4, 2 mM; HEPES, 20 mM; EGTA, 70/2M; CaCI 2, 50/2M (containing approxi- mately 50 uCi 45Ca2+/j~mol calcium): sodium azide,

0.1 mM; oligomycin, 0.7 j2g/ml; 2,4-dinitrophenol, 0.1 mM; ATP (when present), 1 raM; drug (when present), variable. The combination of 701~M EGTA and 50 j2M calcium produced a free ionized calcium concentration of 0.55 ,uM 2. The mitochondrial poi-

sons were present to prevent calcium uptake by the mitochondria in the lysed synaptosome preparation.

Calcium uptake was allowed to proceed for 4 rain at 30 °C; this time period was chosen since Blaustein et al. 2 have shown that calcium uptake in lysed synap-

tosomes from rat brain increases linearly between 1 and 9 rain of incubation. At the end of the 4-min period, aliquots of 0.5 ml from each incubation con- dition were then filtered on Millipore filters of cellu- lose esters (type PH, 0.3/~m pore size, Millipore, Bedford, MA). The filters were immediately rinsed with three 5-ml aliquots of the wash solution de- scribed in Blaustein et al. 2. The radioactivity re- tained by the filters was determined by liquid scintil- lation spectrometry, each filter being immersed in 15

~4

ml Aquasol (New England Nuclear, Boston, MA)

and kept overnight at 4 °C prior to the counting pro-

cedure. The ATP-dependent calcium uptake was

considered to be the difference between uptake in

the absence of ATP and uptake in the presence of

ATP (this uptake is expressed in pmol calcium/rag protein); the non-specific retention of ~5Ca2+ by fil-

ters in the absence of tissue is part of the uptake in the

absence of ATP, and was thus automatically sub-

tracted in the calculation used to obtain the ATP-de-

pendent calcium uptake. A Student's t-test or analy-

sis of variance for paired samples was used to evalu- ate the statistical significance of the difference ob-

served between the ATP-dependent uptake in the presence and absence of drug.

45 Ca 2+ release from lysed synaptosomes

Lysed synaptosomes were first incubated for 4 rain at 30 °C with 4~Ca2+ and ATP as described above, in

a total incubation volume of 8 ml instead of the usual

2 ml. At the end of this incubation period, the incuba-

tion mixture was centrifuged at 15,000 g for 6 rain,

and the pellet was used as the source of endoplasmic reticulum elements from which 45Ca2+ release was

studied ~-. The effect of caffeine on the release of 45Ca2+ was

studied as follows. The pellet was resuspended in 8

ml of a solution identical to the one used in the calci-

um uptake period, except that it contained only unla-

beled calcium. Three aliquots of 2 ml each were tak-

en from the resuspended pellet solution: one aliquot

was immediately filtered and washed on a ().3-~m

pore size Millipore filter, and the radioactivity re-

tained by the filter served as a measure of the total amount of sequestered 4~Ca2+; the two other aliquots

were reincubated at 30 °C for 4 min, one aliquot in

the absence of caffeine and the other in the presence

of 10 mM caffeine, and then filtered and washed as above to determine the quantity of 45Ca 2+ still se-

questered by the organelles after the 4-min reincuba- tion period. The difference between total seques- tered 45CaZ+ and 45Ca2+ remaining in the organelles

after the 4-min reincubation period served to assess the release of 45Ca 2+. A Student's t-test for paired

samples was used to evaluate the statistical signifi- cance of the difference observed between the release of 45Ca 2+ in the presence or in the absence of caf-

feine.

RESULTS AND DISCUSSION

Caffeine exhibited a biphasic effect on ATP-de-

pendent calcium uptake by lysed brain synaptosomes (Fig. 1): caffeine concentrations of 10 and 30 mM

stimulated calcium uptake by 88% and 60% respec-

tively, whereas 60 mM caffeine inhibited calcium up-

take by 55%. Concentrations of caffeine lower than 10 mM were also tested; as shown in Table I, 1, 2 and

5 mM caffeine all stimulated calcium uptake. This is

unlike the effect of caffeine in brain microsomes,

where no biphasic effect of caffeine was reported and

where inhibition of calcium uptake occurred with 5-15 mM caffeine 3~. Thus, the lysed synaptosomes

preparation appears relatively insensitive to the in-

hibition of calcium uptake by caffeine, as compared to brain microsomes.

In sarcoplasmic reticulum vesicies derived from skeletal muscle, caffeine inhibits calcium uptake at

concentrations of 5-20 mMl'~'~"33 ~: the mechanism

of this effect of caffeine involves a decreased ability

1000

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E 6oo

~ 4OO

I"- I~. 3OO

: ~ 200

...1

O 100

~k

:i!iiii i:i:~:~

i!!!ii

iiiiiii l O m M

:5::::: ,.,,.,..

:i:~:~:i ....,...

iiiiiii ! . . , .

- . + :

~:~:~:~:

~<!2iI

3 0 r a M

CONTROL

CAFFEINE

I

i* J

Fig. 1. Biphasic effect of caffeine on the ATP-dependent up- take of calcium by lysed synaptosomes from rat brain cortex. Lysed synaptosomes were incubated with 0.55 pM free calci- um, containing 45Ca 2+. for 4 min at 30 °C; the incubation pro- ceeded either in the absence of caffeine, or in the presence of 10, 30 or 60 mM caffeine. Each column represents the mean + S.E.M. of 7 separate determinations. The asterisk indicates a statistically significant difference (P < 0.05) between uptake in the presence of drug and uptake in the corresponding control~

6 0 m M

TABLE I

Stimulation of the A TP-dependent uptake of calcium in lysed brain synaptosomes by various low concentrations of caffeine

Each value is the mean _+ S.E.M. of 6 or 7 separate determina- tions; * P < 0.05. Lysed synaptosomes were incubated with 0.55/LM free calcium, containing 45Ca2+, for 4 min at 30 °C.

Caffeine A TP-dependent calcium uptake (pmol/mg protein)

Drug-treated Control % Change

5 mM 1030 + 129" 595 _+ 83 + 73% 2mM 836 _+ ll0* 396 + 68 + 111% 1 mM 858_+ 85* 529_+ 67 + 62%

of the vesicles to retain the transported calcium, i.e. a

stimulation of calcium effiux from the vesicles s'

12,27,34. The caffeine-induced enhancement of calci-

um effiux seems to be produced through an action of

caffeine on specific calcium gates located in sarco-

plasmic reticulum membranes 27'35. Neering and Mc-

Burney 21 have shown that 10 mM caffeine releases

calcium from microsomal stores in rat dorsal root

ganglion neurons, and Gill and Chueh 9 ment ion that

5 mM caffeine releases calcium sequestered by endo-

plasmic reticulum elements of saponin-permeabi-

lized neuroblastoma cells. We have explored wheth-

er a 10-raM concentrat ion of caffeine has a calcium-

releasing effect in the lysed brain synaptosomes

preparation by determining whether caffeine mod-

ifies the release of 45Ca 2+ taken up by lysed brain syn-

aptosomes. As shown in Fig. 2, virtually no 45Ca2+

was lost from the organelles during a 4-min incuba-

tion at 30 °C in the absence of caffeine; however, ap-

proximately 30% of the original 45Ca2+ content was

released during a 4-min exposure of the organelles to

10 mM caffeine (Fig. 2). This result clearly shows

that 10 mM caffeine is causing a significant release of

sequestered calcium under the same conditions used

for our calcium uptake assays. Calcium uptake is the

resultant of calcium inflow and calcium outflow:

since calcium uptake is stimulated by 10 mM caffeine

(Fig. 1), the calcium-releasing effect of this concen-

tration of caffeine (Fig. 2) was obviously masked by

the presence of a separate pathway for stimulation of

calcium inflow by caffeine.

The stimulation of calcium uptake which we have

observed in the lysed synaptosomes preparation with

low concentrations of caffeine is a finding without

65

[ ] CONTROL

~' [] CAFFEINE --- 1 0 0 ( l O m M )

.E

.___ 80

E

+ 60 (kl

0 ,~- 40

"5

c 2 0 (3

Fig. 2. Effect of 10 mM caffeine on the release of 45Ca2+ from endoplasmic reticulum elements of lysed synaptosomes from rat brain cortex. Lysed synaptosomes were first incubated with 0.55/~M free calcium, containing 45Ca2+, and ATP for 4 rain at 30 °C. After centrifugation of the incubation mixture at 15,000 g for 6 rain, the pellet was resuspended in solution containing only unlabeled calcium and the 45CaZ+ content of organelles was determined immediately in one aliquot, and after a reincu- bation of the suspension at 30 °C for 4 rain in the presence or in the absence of caffeine in two other aliquots; the ~SCa2+ re- maining in the organelles after the 4-rain reincubation is ex- pressed as a percentage of the ~SCa 2+ content in the sample which was not reincubated. Each column represents the mean _+ S.E.M. of 9 separate determinations. The asterisk indicates a statistically significant difference (P < I).05) between the 45Ca2+ content of organelles incubated in the presence of caf- feine and that of organelles incubated in the absence of caf- feine, i.e. between the 45Ca 2+ released in the presence or in the absence of caffeine.

precedent in either brain microsomes or muscle sar-

coplasmic reticulum. An attempt to elucidate the

mechanism of this stimulation was made. The ability

of caffeine to inhibit the enzyme phosphodiesterase 24

emerged as a prime candidate for the mechanism of

stimulation of calcium uptake by caffeine for several

reasons: (1) cAMP, which is inactivated by phospho-

diesterase, is known to stimulate calcium uptake by

sarcoplasmic reticulum vesicles from cardiac or ske-

letal musclely14"26"29; this effect of cAMP usually re-

quires the addition of exogenous cAMP-activated

protein kinase (but see La Raia and Morkinl4), and

results from the phosphorylation of a specific sarco-

plasmic reticulum protein involved in the regulation

of calcium transport 2~. (2) Several groups have

shown that the enzymes responsible for the synthesis

and degradation of cAMP, as well as cAMP-depen-

dent protein kinases are abundant in the synaptosom-

al subfraction of rat cerebrum 45'1'~. Appreciable

C

,,¢

0

IJJ

I-- 0..

< 0

500

I_~CONTROL

~cAMP

0 05uM 05JJM 5pM 50uM 500uM

Fig. 3. Effect of various concentrations of cAMP on the ATP- dependent uptake of calcium by lysed synaptosomes from rat brain cortex. Lysed synaptosomes were incubated with 0.55 jiM free calcium, containing 4SCa2+, for 4 min at 30 °C; the in- cubation proceeded either in the absence of cAMP, or in the presence of 0.05, 0.5, 5, 50, or 500 aM cAMP. Each column represents the mean _+ S.E.M. of 9 separate determinations, except for the set of experiments with 500#M cAMP where n = 6. The asterisk indicates a statistically significant difference (P < 0.05) between uptake in the presence of cAMP and uptake in the corresponding control.

quantities of these various enzymes should therefore

be present in our final incubation medium which is

made up in part of lysed synaptosomes.

Blaustein et al. 3 have examined a variety of nu-

cleotides for their ability to promote calcium uptake

in lysed brain synaptosomes, and they found that re-

placement of ATP by cAMP reduced the calcium up-

take to below the nucleotide-free control level.

Nonetheless, this result did not eliminate the possi-

bility of stimulation of ATP-dependen t calcium up-

take by cAMP in lysed synaptosomes when both ATP

and cAMP would be present in the incubation medi-

um. As a first step to test whether the st imulation of

calcium uptake by caffeine in our preparat ion oc-

curred through a cAMP-related mechanism (a pres-

ervation of cAMP through phosphodiesterase in-

hibition), we have first studied whether cAMP could

1100

IOO0

"E 9 0 0

~-, 8 0 0 p c~

0'} 700 E "......

600 0 E

500 Lt.I

I-- 400 13_

300

. J ,,~ 200 0

100

[ ] CONTROL

[ ] c A M P (5 uM)

[ ] c A M P ( 5 1 J M ) + C A F F E I N E ( 1 0 r a M )

F . . . . . . . . . . . . . . .

f . . . . . . . .

[

I

~ =

Fig. 4. Effect of cAMP alone, or in combination with caffeine, on the ATP-dependent uptake of calcium by lysed synapto- somes from rat brain cortex. Lysed synaptosomes were incu- bated with 0.55 uM free calcium, containing 4SCa2+, for 4 rain at 30 °C: the incubation proceeded either in the absence of cAMP and caffeine, in the presence of 5 uM cAMP, or in the presence of 5 #M cAMP and l0 mM caffeine. Each column rep- resents the mean + S.E.M. of 9 separate determinations. The asterisks indicate a statistically significant difference (P < 0.05) between uptake in the absence Of cAMP and uptake in the presence of cAMP, and between uptake in the presence of cAMP and uptake in the presence of both cAMP and caffeine.

stimulate ATP-dependen t calcium uptake in the

lysed synaptosomes preparation. Exogenous cAMP

did indeed stimulate calcium uptake at concentra-

tions which were in the same range as those which

stimulate calcium uptake in muscle sarcoplasmic reti-

culum vesicles (Fig. 3): 0.05, 0.5, 5, 50, or 500 #M

cAMP-stimulated calcium uptake by 67, 67, 95, 38

and 67% respectively. The next step was to establish

whether caffeine retained its ability to stimulate cal-

cium uptake in the presence of the maximal stimula-

tion of the calcium uptake system which can be pro-

duced by cAMP; 5 # M cAMP was chosen as the con-

centration of cAMP which produced maximal stimu-

lation, and the effect of 10 mM caffeine was thus

studied in the presence of 5 #M cAMPI Each agent

alone causes an approximate doubling of the ATP-

67

dependent calcium uptake by lysed brain synapto-

somes (Figs. 1 and 3), and Fig. 4 demonstrates that 10

mM caffeine still causes an approximate doubling of

calcium uptake even in the presence of the usual in-

crease in calcium uptake due to cAMP. It thus seems

that the stimulation of calcium uptake in lysed brain

synaptosomes by caffeine does not occur via cAMP.

The sequestration of calcium by endoplasmic reti-

culum elements of synaptic terminals is believed to

play an important role in the regulation of intratermi-

nal free calcium concentrations 2, and could influence

calcium-dependent processes such as transmitter re-

lease. Pedata et al. 23 have recently described an in-

hibition of acetylcholine release from electrically

stimulated brain slices by 0.5 mM caffeine. A modifi-

cation of the mobilization of intracellular calcium by

caffeine was suggested by these authors as one of the

mechanisms which could potentially be involved in

the inhibition of acetylcholine release by caffeine.

The present demonstrat ion of a stimulation of calci-

um uptake in the endoplasmic reticulum elements of

a brain synaptic terminal preparation by caffeine

would be consistent with the hypothesis of an in-

volvement of an enhancement of intracellular calci-

um buffering in the inhibition of acetylcholine release

by caffeine: if more calcium was taken up by the en-

doplasmic reticulum elements of brain synaptic ter-

minals, less calcium might then be available for trans-

mitter release.

ACKNOWLEDGEMENTS

This study was made possible by grants from the

Medical Research Council and the Muscular Dystro-

phy Association of Canada. We thank Lucie H~roux

for her excellent technical assistance, and Suzanne

Philippe for secretarial help.

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