Lila Sciantes Vol. 16, pp " 315-322Printed in t2te U.S.A .

NSÜLY SYNTHSSIZSD NDRSPIIiSPHBIt~ ACCUMQI1iTI0N IIi THE RSADILYRßIFASABLB POOL OF A POBIFISD A

IC VSSICLS FRACTIONl

Richard L. Rinin and T. Randall HardentDepartment of Pharmacology and Tosi.cology

IIniveraity of Mississippi Medical Center, Jackson, ~ 39216

(Received is final form December 19, 1974)

Pergamon Press

A highly purified fraction of large dense core adrenergicvesicles sons studied after isolation from bovine splenic nervechilled within 10 to 12 minutes

et mortem .

In a standard medinm containing 5 mM each of

and ATP and 6 ~Ti norapinaphrine(NS), this vesicle fraction contained NS in a readily releasablecad a more stable pool. When vesicle dopamine ß-hydrozylasa seasactivated with 1.33 mM ascorbic acid using 6 Fù! l4C-dopamine assubstrate at 30°C, 1~+C-N8 sloes synthesised at a linear rate duringthe 45 mim><te incubation. Hat accsmmulation of NS ~ < 0.01) anda proportional net retention of nmoly synthesised ~-NS occurredonly sohen the readily releasable pool could still be demonstrated.The halftime for the fast release pool awes doubled from 3 to 6minutes (p < 0.01) with no effect oa the slaver released, ATP-facilitated uptake pool . Tfws, both during azoplasmic transportand induced NS synthesis in vitro , there is evidence that newlysynthesised NB preferentially accumulates in the readily releas-able pool, a property also characteristic of the physiologicallyactive pool in viva.

A fraction containing 80-90x pure large dense core vesicles can be rou-tinely prepared from bovine splenic nerve (1-4) .

When the nerves are obtain-ed and chilled within 10 to 12 minutes poet mortem, the vesicle NE content isrelatively high (3,5) and essentially all vesicles contain a dense stainingmstriz or core (4,6) . A portion of the high NS content is difficult to main-

tain during incubation at 30 to 37°C (3,7) and ie held differently from thethe ATP-facilitated uptake pool (8,9) .

Ia fact,rapidly loot after addition of Mgt and ATP (3,

more stable fraction of NB inNE in the labile pool is moreYen et al., unpublished) .

Purified large dense coretial segmente of the nerve (7), allowing comparison of net release curves ae a

vesicles can be prepared identically from sequen-

1This study alas supported by grants from thé National Institute of GeneralMedical Sciences, GM 15490 and GM 00359, U.S.P.H.S .. The authors are alsoindebted to the Jackson Packing Co. for bovine splenic nerves.

tPresent address : Department of Pharmacology, The University of ColoradoMedical Center, Deiner, CO 80220

315

316

Accumulation of Newly Synthesized Norepinephrine

Vol. 16, No . 2

function of vesicle maturation or of axoplasmic transport. The vesicle frac-

tion increases its particulate NE content during transport . Vesicles isolated

from a proximal nerve segment near the cceliac ganglion consistently show an

initial small net uptake of NE upon incubation in standard medium, which is

followed by a single exponential release from the ATP-facilitated uptake pool .

Vesicles from the midportion of the nerve begin to demonstrate a fast compo-

nent of NE release in addition to the slw component. Vesicles from the intra-

splenic segment of the nerve possess an even larger fast release component

comprising 40~ of the total NE content as estimated by graphical analysis .

Thus, the NE content of the ATP-facilitated uptake pool remains unchanged in

the three segments, while the NE content of the more labile, readily releas-

able pool is established during axoplasmic transport. It is reasonable to

assume that in vivo the fast release pool fills with newly synthesized NE dur-

ing axoplasmic transport, and that this accumulation is reflected by the in-

crease in NE content per mg vesicle protein.

One of the advantages of the purified vesicle preparation is that it ser-

ves as a simplified model for the study of the action of dopamine ß-hydroxyl-

ase (DßH ; EC 1.14 .17.1) .

DßH activity was found to be almost entirely latent

in the presence of 6 ~aM substrate without addition of exogenous activator (10;

also see 11) . This was to some degree dependent on the substrate chosen .

With 6 ~M tyramine, there was negligible conversion to octopamine ; with 6 ~I

dopamine (1~), a low level of ß-hydroxylation occurred to form NE, apparently

because DA served as an electron donor to reduce the enzyme (12) . With DA as

substrate, maximal activation of DßH required only the addition of ascorbic

acid . Various concentrations of fumaric acid and catalase were either without

effect or inhibitory . The latency of DßH in the purified vesicles was not due

to the presence of endogenous inhibitors . Lagercrantz et al . (5) using the

method of Molinoff et al . (13) demonstrated that progressively less inhibitor

remained as the vesicles were purified . Negligible inhibitor remained in the

purified fraction, FIIIM-15, as tested by a wide concentration range of copper

ions . Similarly, N-ethylmaleimide over a wide concentration range failed to

enhance ß-hydroxylation but could serve to inhibit the enzyme (10) .

The present investigation was undertaken to determine if newly synthesized

NE could augment the amine content of the purified vesicle fraction during in-

cubation in vitro and, if eo, in which pool s) this occurred .

Materials and Methods

Purified vesicles were prepared from whole bovine splenic nerve trunk ob-

tained at the slaughterhouse, crudely dissected and chilled within 10 to 12

minutes post mortem (3) . Vesicles were incubated in a standard medium

Vol . 16, No . 2

Accumulation of Newlp Synthesized Norepiaephrine

317

containing the following:

0.1 M sucrose, 0.1 M potassismm phosphate buffer at

pH 7.2-7.4, 5 mM eech of Mgt and ATP, 6 u?i each of 14C-DA and 3H-1-NS (final

concentration includes NS carried over from the gradient and average NH re-

leseed during incubation) and 50 ~!1 pargyliae.HCl . A concentration of 1 .33 mM

ascorbic acid was chosen to activate DßH, ae it produced approximately half-

mszimal activity without accelerating vesicle N8 depletion in the absence of

substrate (10) .

3H-1-NS was included to measure thn effect of DßR activation

on NB exchange .

After 0 .4 M perchloric acid extraction of vesicle pallets, NS was analyzed

using alumina purification, the trihydroxylndole method and an Aminco Bowman

Spectrophotofluorometer.

Protein was determined using the Folin phenol rea

gent with Armour crystalline bovine eenim albumen se standard.

Complete re-

covery of internal ataadard NS indicated negligible spontaneous or enzymatic

inactivation during the ezperimantal procedures . The stability of DA was de-

pendent on the concentration employed. At b.uM DA with 50 uM pargyline pre-

sent, ~ 95x of the DA internal standard was recovered unchanged after 30 min-

utes incubation at 30°C (corrected for any ß-hydroxylation) ; stability was

checked both by direct analysis (14) and by chromatographic separation of la-

belled amines .

Others reported similar AA recovery using partially purified

bovine epleaic nerve vesicles (15) .

Isotope exchange was followed during the DßH activation experiments in a

temperature regulated agitating water bath at 30oC. Vesicle separation from

the incubation medium was accomplished either by a Millipore filter method

(16) or by rapid chilling of samples and centrifugation (3) . In NE synthesis

experiments, vesicle and supernatant amines were extracted in 0.4 M perchloric

acid and separated by paper chromatography using n-butanol-acetic acid-water,

150:30:48. These were compared with controls without ascorbic acid and with

known specific activity standards for estimation of recovery. Radioactivity

woes measured in a Beckman IS-250 3-chapel scintillation system with automatic

quench compensation . The scintillation fluid contained 50 ml BBS-3, 8 g

butyl-PBD and 0.5 g PBBO (Beckman Co . products), diluted to one liter with

scintillation grade toluene. Counts were corrected for efficiency and samples

containing both 14C- and 3R- labels were adjusted for spillaver after dual

chancel counting.

Results

Effect of DßR activation on NS content sad net release rates.

The abso-

lute net accumulation of NE by direct analysis of pellet eztracts after var-

ious incubation times in the standard Mg++ and ATP-containing medium is given

in Figure 1.

These values were determined by subtracting the I78 content of

318

Accumulation of Newly Synthesized Norepinephrine

Vol. 16, No . 2

control vesicles incubated without ascorbic acid from those incubated with

ascorbic acid in eight paired experiments . Cold control vesicles at zero time

contained 9.75 + SEM 0.49 (n = 16) ~.g NE/mg protein. A statistically signifi-

cant net accumulation of NE occurred after 4 and 8 minutes of incubation (p <

0.01 by paired analysis of the estimated Standard Error of the Mean Difference,

SEd), after which most of this NE was lost into the medium. The coincident

release of newly synthesized 14C-NE into the medium, expressed as ~.g 14C-NE/mg

protein is also given in Figure 1 ; note that the scale of the right ordinate

is ten times that of the left ordinate .

The net increase in vesicle NE con-

tent compared to the total vesicle NE content of the control (Pig . 2) amounted

to 13.07 at 4 minutes and 14.57 at 8 minutes.

By 12 minutes of incubation the

increase in NE content had decreased to 77 which was no longer significantly

different. Thus, it is only during the initial augmentation of vesicle NE

content at 4 and 8 minutes that newly synthesized 14C-NE does not appear to be

lost by the vesicles into the medium .

FIG. 1

~v0 ~,0 10 20 30 40

Minutaa Incubaflon 30~

2.0 _

Increased vesicle NE contest with DßH activation .

Solid-line curve iescribed by left ordinate, dashed-line curve by right ordinate . Valuesdifferences between samples incubated with and without ascorbic acid .equal 1 SEM; 8 paired experiments.

de-ar~Barn

s L0-I3 p<0.01

vlo.o

a v t .G0 ô Q6~ 1 T

8.0

û ~~a

iii S0~Q6-

i

~i6.0

i E

.S z 0.4iii 4.0 ~ w~

zo '

Vol. 16, No . 2 Accumulation of Newly Synthesized Norepinephrine

FIG. 2

Nat NE release rates from vesiclesduring DßH activation . N8 contentat zero time was 9.75 ± 0.49 kg/mgprotein (n ~ 16) .

Open circles arewithout and triangles with ascorbicacid . Solid symbols are resolvedfast components included in curvi-linear portion of respective curve.Parentheses show halftimes in min.± SEM. Bars ~ SEM; 8 paired expte.

c.d

mTi.ônm

wW2U

N.pF

3

O

FIG. 3

Newly synthesized NE retained byvesicles (triangles) and total syn-thesis (circles). Dashed-linecurve is calculated portion of ves-icle 14C-NE which could result fromexchange (reuptaka) . Bars ~ 1 SEM;8 paired azpte.

319

The NB content of the cold control vesicles at zero time was set equal to

unity sad the NE content of the vesicles during incubation was expressed se a

fraction of this initial content (Fig . 2) .

The data were plotted on a semi

logarithmic scale in order to determine the rate of net NE release during in-

cubation.

In the control without ascorbic acid, the vesicle NE content de-

creased rapidly and in a curvilinear fashion during the initial 15 minutes of

incubation .

The subsequent net release of NE occurred more slowly and was

described by a single exponential component.

Vesicles to which ascorbic acid

was added to activate D¢H lost NE less rapidly during the initial period ;

thereafter, N8 ass lost at as exponential rate paralleling the control . The

320

Accumulation of Newly Synthesized Norepinephrine

Vol. 16, No . 2

SEà between respective points on the two curves was 0.028 at 4 minutes and0.021 at 8 minutes, both being significant at p < 0.01 ; all other differences

were not significant (p > 0.05) .The slower exponential component of each curve was extrapolated to zero

time, where similar ordinate intercepts were obtained . The halftimes of 42.6

minutes without ascorbic acid and 46 .8 minutes with ascorbic acid were notsignificantly different (SEd 5.66, p > 0.2) .

The halftimea for the fast re-

lease components were 2 .96 minutes3 without ascorbic acid and 5.95 minuteswith ascorbic acid . These were significantly different (SEâ 0 .80, p < 0 .01) .

Thus, the halftime for net release from the fast release pool was doubled by

the increase in NE content at 4 and 8 minutes resulting from DßH activation

(Fig . 1), whereas there was no significant effect on the slow release pool .

Retention of newly synthesized l4C-NE . The previous data were based pri-

marily on direct apectrophotofluorometric determinations of NE content . The

production of newly synthesized 14C-NE ie expressed in ~g/mg protein (Fig . 3) .

There was a linear rate (~ 0.21 pg l4C-NE.min-l .mg-l) of NE synthesis by the

vesicles during the entire incubation period . In spite of a constant rate of

synthesis, the vesicles showed only a significant net retention of newly sya-

theeized l4C-NE between 0-4 minutes (100 retained) and 4-8 minutes (70-807retained), after which essentially all l4C-NE was lost into the medium .

The

set retention of newly synthesized l4C-NE at 4 and 8 minutes was coincidentwith the net increase in vesicle NE content shown by direct analysis (Fig . 1)

and the doubling of the halftime for net NE release from the readily releasable

pool (Fig . 2) .Estimated vesicle content of newly synthesized 14C-NE reaultin~3 from re-

uptake of 14C-NE previously released into the medium. It ie known that ieo-topically labelled exogenous NE will readily exchange with endogenous vesicle

NE under the present conditions and that the exchangeability is essentially1007 (16) . Therefore, the portion of vesicle 14C-NE content resulting from

exchange with newly synthesized l4C-NE lost by the vesicles into the mediumwas calculated (Fig . 3) . The latter was estimated on the basis of 3A-1-NE

uptake measured siunltaneously during l4C-NE synthesis, which indicated nosignificant difference between 3H-1-NE exchange with and without 1.33 ascorbic

acid (data not presented ; 10) . At any time 't', the estimated proportional re-uptake of newly synthesized l4C-NS which had been released into the mediumwas calculated as follows :

14C-NE-medium/2 . 3H-NE taken up/3H-NE-medium.

3The halftime of the fast release pool ie decreased from 4-5 minutes with 6 ~.MNE alone (3) since DA acts like tyramine to hasten NE loss from this pool,but unlike tyramine, has no effect on the slower release pool .

Vol . 16, No . 2

Accumulation of Newly Synthesised Norepinephrine

321

Correction was made for any specific activity differences between the two iso-

topes . During the initial period of 10 to 15 minutes, the retention of newly

synthesised 14C-NE could entirely account for the net increase in vesicle NE

content.

Later, the exchange of vesicle NE with 14C-NE lost into the medium

progressively became a more dominant factor .

By 45 minutes of incubation,

nearly all vehicle 14C-NE could have resulted from exchange diffusion.

Dihcuseion

The earlier work of Euler and Lishajko (8,9) showed an inability to en-

hance the NE content of the slow released pool above that in the cold control

during incubation of the vesicles under similar conditions . Also very perti

nent is the report of Stjärne et al . (15) in which NE synthesis from DA wan

studied in a partially purified fraction of bovine splenic nerve vesicles.

These authors are to be complimented for their insight and difficult interpre-

tations of data obtained from a vesicle preparation produced after a post mor-

tem delay of sufficient length to preclude demonstration of a fast release

pool (see Ref. 3 for poet mortem effects) .

Stjärne et al. (15) suggested

"the existence of a separate compartment for storage of newly formed NE,

possibly with rapid turnover, inside the NS storage Vesicle or in separate

synthesis vesicles." üe suggest that their 'wash' fraction or 'loosely bound'

NE, which was also found to be less saturable with NE than their wash resis-

tant fraction (ATP-facilitated uptake pool) and to be inhibited by reserpine,

was probably a measure of a residual fast release pool .

The present data show that when DßH is activated by ascorbic acid to in-

duce NE synthesis from DA in the purified vesicle fraction, a net increase in

particulate NE occurs only initially, that is, prior to the depletion of NE

from the readily releasable pool, The addition of NS to the feet release pool

doubles its halftime without a significant change in the halftime of the slav-

er released, ATP-facilitated uptake pool . Net accumulation of newly synthe-

sised 14C-NE from 14C-DA occurs only during this initial period. It can

account quantitatively for the augmented NE content at 4 and 8 minutes of in-

cubation, after which all synthesised 14C-NE is lost into the medium, in spite

of a continued linear rate of synthesis by the vesicles, The data are in keep-

ing with the interpretation that newly synthesised NE preferentially enters

the more labile pool . However, the net release of NE from this pool is so

rapid that the addition of amine only serves to prolong the time for depletion

of NE from this pool under the experimental conditions . Although synthesis

continues at a linear rata and newly formed NE may still be entering the fast

release pool, it ie usable to accumulate . The lack of accumulation in the

ATP-facilitated uptake pool indicates there are few free binding sites in thin

322

Accumulation of Newly Synthesized Norepinephrine

Vol. 16, No . 2

pool (15) . Little or no newly synthesized 14C-NS is released initially in the

4 to 8 minute period (Fig . 3) when 14 to 28~ (Fig . 2) of the vesicle NE is

lost into the medium .

This suggests that newly formed NE is initially held in

a 'synthesis' compartment which preferentially supplies NE to the readily re-

leasable pool . This compartment logically coataina the physiologically active

D~H complex and could be the vesicle membrane.

The present data support the interpretation that newly synthesized NE pre-

ferentially accumulated in the readily releasable pool in vitro may be the

source of transmitter proposed to be released by some form of exocytoais in

vivo (17-19) .

1.

2.

3.

4.

5.

6.

7.

8 .

9.

10.

14 .

15 .

16 .

17 .

18 .

References

H. Lagercrantz, R. L. Rlein and L. Stjärne, Life Sci. 9 : 639-650 (1970).

H. Lagercrantz, Acta phyaiol. acand. 82 (Suppl . 366) : 1-44 (1971) .

S .-S. Yen, R. L. Klein end S .-H Chen-Yen, J. Neurocytol . 2 : 1-12 (1973) .

. Thureaon-Klein, R. L. Rlein and H. Lagercraatz, J. Neurocytol . 2 :

13-27 (1973) .

H. Lagercrantz, D. F. Kirksey and R. L. Klein, J. Neurochem. 23 : 769-773

(1974) .

R. L.

R. L.

by E.

U. S .

U. S .

T. K.

Proc .

L. Stjärne and F. Liehajko, Br . J. Pharmac. 27 : 398-404 (1966) .

E. Y. Levin aad S. Raufman, J. biol. Chem. 236: 2043-2049 (1961) .

P. Molinoff, R. Weinshilboum and J. Aaelrod, J. Pharmac. ezp. Ther .

178 : 425-431 (1971) .

C. C. Chang, Int. J. Neuropharmac . 3: 643-649 (1964) .

L. Stjärne, R. H. Roth and F. Liahajko, Biochem. Phax~ac. 16 : 1729-1739

(1%7) .

R. L. Rlein and H. Lagercrantz, Acte phyaiol. stand. 83 : 179-190 (1971) .

S. Udenfriend and P. Zaltzman-Nirenberg, Science 142 : 394-396 (1963) .

I. J. Kopin, G. R. Breese, R. R. Rrauss and V. K. Weise, J. Phermac.

exp. Ther . 161: 271-278 (1968) .

N. Weiner, Ann. Rev. Pharmac . 10 : 273-290 (1970) .

Klein and ~. Thureson-Klein, Fedn . Proc . 33 : 2195-2206 (1974) .

Klein, In Frontiers in CatechoLamine Research . pp . 423-425, edited

Usdin and S . Snyder, Pergamon Prese, New

von Eulen and F. Liahajko, Acta phyaiol.

von Eulen and F. Liahejko, Acta phyaiol.

Harden, D. F. Kirksey, R. L. Klein and ~.

33 : 497A (1974) .

York (1973) .

stand. 59 : 454-461 (1963) .

stand. 71 : 151-162 (1967) .

Thureson-Klein, Fedn.