3H-dopamine uptake by synaptic storage vesicles of rat whole brain and brain regions

Life Sciences, Vol . 22, pp . 823-830 Printed in the U .S .A .

Pergamon Press

3 H-DOPAMINE UPTAKE BY SYNAPTIC STORAGE VESICLES OF RAT WHOLE

BRAIN AND BRAIN REGIONS

T .A . Slotkin, M . Salvaggio, C . Lau and D.F . Kirksey Department of Pharmacology Duke University Medical Center Durham, N C 27710 (Received in final form January 19, 1978)

A crude preparation of neurotransmitter storage vesicles was obtained by differential centrifugation and the ability to take up 3H-dopamine was evaluated in vitro . The uptake was highly dependent on tem?eraTure, had an absolute requirement for ATP and Mg + and was inhibited totally by reserpine . The uptake displayed saturation kinetics, with a Km of 0 .26 pM at 20° . 9 H-dopamine uptake was inhibited competitively by norepinephrine, with a Ki of 0 .69 vM . Vesicles derived from a primarily dopaminergic region (corpus striatum) exhibited the same ratio of uptakes of 9H-dopamine/ sHnorepinephrine as did those from a primarily noradrenergic region (cerebral cortex) . These results indicate that viable rat brain storage vesicles can be readily prepared and used for evaluation of pharmacologic effects on 9 H-dopamine uptake, and that dopaminergic and noradrenergic storage vesicles exhibit identical uptake properties . Biogenic amine uptake into neurons is a two-step process : amines are first transported from the synaptic cleft to the neuronal cytoplasm and then moved from the cytoplasm to the in terior of the storage vesicle . Uptake from the synaptic cleft utilizes a sodium-potassium-activated ATPase, is inhibited by ouabain, cocaine and tricyclic antidepressants, and is responsible primarily for termination of the actions of the released neurotransmitter (1, 2) . In contrast, vesicular uptake is activated by magnesium and ATP and inhibited by reserpine, and is responsible for maintenance of neurotransmitter levels and for packaging of transmitters for release during neuronal stimulation (3-6) . The transport of amines in vitro into synaptic vesicles derived from a variety of neuronâl tissues appears to be relatively non-specific in that numerous phenethylamine and indolethyl amine derivatives can act as uptake substrates or competitive inhibitors in catecholamine-containing vesicles (6-12) ; this is in marked contrast to synaptosomal uptake mechanisms which are much more specific (13-15) . Most studies of vesicle uptake have been performed in peripheral tissues where a single transmitter is usually involved . The more recent development of techniques to isolate and characterize vesicles from rat brain (11) enables determinations to be made with small amounts of tissue . However, because the vesicles obtained from the brain clearly constitute a 0300-9653/78-0313-0855$02 .00/0 Copyright (D Pergamon Press

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mixed population containing different transmitters, it is important to distinguish whether a given vesicle type displays specificity for its natural substrate . In previous studies of vesicles derived from rat whole brain a single Km value was found for uptake of 9 H-norepinephrine and the uptake was inhibited competitively by dopamine (11) . Determinations in preparations from brain regions specifically rich in dopamine or norepinephrine indicated that, while 9H-norepinephrine entered both dopaminergic and noradrenergic vesicles in vitro, the Km for norepinephrine was the same in both types, prodúcTng what appeared to be a single uptake characteristic (12) . Furthermore, destruction of catecholaminergic nerve endings with 6-hydroxydopamine or of serotonergic nerve endings with 5,6-dihydroxytryptamine demonstrated that in the whole brain preparation 9 H-norepinephrine uptake was restricted to catecholaminergic vesicles (11, 12) . In the present study, the uptake of 9H-dopamine into preparations from whole brain and from brain regions has been compared to that of 9 H-norepinephrine ; the results suggest that dop amine is an uptake substrate for vesicles from noradrenergic as well as dopaminergic neurons, and that the uptake of dopamine is probably the same in both vesicle types . Methods Preparation of synaptic vesicles . Subcellular fractions containing brain synaptic vesicles were prepared by the method of Philippu and Beyer (16) as modified by Seidler et al . (11) . Male Sprague-Dawley rats (Zivic-Miller), weighing 200-300 g. were decapitated and brains removed and used either as a whole tissue, or dissected into three regions : corpus striatum, cerebral cortex, and rest of brain (brainstem + midbrain + cerebellum) . Tissues were homogenized in 4 volumes of 300 mM sucrose containing 25 mM Tris (pH 7 .4) and 10 UM iproniazid (irreversible monoamine oxidase inhibitor), using 5 up-down strokes in a Dual ground-glass homogenizer . The homogenate was centrifuged at 1000 x g for 15 min and the supernatant recentrifuged at 20,000 x g for 30 min . The supernatant of the latter centrifugation was sedimented at 100,000 x g for 30 min in the No . 40 rotor of a Beckman L5-50 ultracentrifuge and the supernatant solution was discarded . The crude, vesicle containing pellet was resuspended gently in a volume of 130 mM potassium Phosphate (pH 7 .4) equal to that of the original homogenate using 2 up-down strokes in a Teflon-to-glass homogenizer, and this suspension was used for subsequent incubations . Although this microsomal fraction contains many particles and organelles, uptake of amines in vitro appears to occur primarily into the synaptic vesicles present in the preparation (11, 12) . Determinations of amine uptake . For determinations of vesicular uptake, incubations contained 0 .67 ml of the vesicle preparation (corresponding to 133 mg of original brain tissue), 0 .83 ml of 1 or 2 mM ATP-Mg 2+ in phosphate buffer, 17 ul of 1 mM ascorbic acid, 8 .3 ul of 1 mM iproniazid, either 0 .38 Ul of 220 uM 3H-norepinephrine or 1 .25 pi of 67 uM 3H-dopamine and phosphate buffer to make a final incubation volume of 1 .7 ml . In some experiments, concentrations of ATP and Mg t+ were varied, or other substances added to the incubation medium (EDTA, reserpine,

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825

unlabeled norepinephrine or dopamine) . Unless otherwise noted, samples with 3 H-norepinephrine were incubated for 4 min at 30 0 and those with 9 H-dopamine at 20 0 ; in both cases, duplicate tubes were kept on ice to serve as blanks . Uptake was stopped by the addition of 1 .7 ml of ice-cold phosphate buffer and the labeled vesicles trapped on cellulose acetate filter paper (Millipore type EG, pore size 0 .2 um, or equivalent Gelman paper) by rapid vacuum filtration . The filtration apparatus contained 12 2 .5 cm ports connected in parallel, with vacuum adjusted such that filtration took approximately 20 sec . The paper was washed three times with ice-cold buffer, placed in a scintillation vial containing 10 ml of Aquaflour (New England Nuclear), and counted at an efficiency of 40% . Uptake was determined by subtracting the 00 blank from the 30 0 or 20 0 sample and expressed as pmols of norepinephrine or dopamine taken up per gram of original tissue . Data analysis . Results are reported as means and standard errors w th levels of significance calculated by the two-tailed Student's t-test . Kinetic studies are represented as double reciprocal plots, with slopes, intercepts and standard errors calculated by least-squares analysis ; intercepts are compared using the t-test . Materials . (-) Norepinephrine-7- 9H (2 .20 Ci/mmol) and dopamineHH (IT4 .6 Ci/mmol) were obtained from New England Nuclear Corp . (-) Norepinephrine HC1, dopamine HC1, iproniazid phosphate and ATP were purchased from Sigma Chemical Co . Reserpine phosphate was obtained from Ciba Pharmaceuticals . Results and Discussion There was a marked temperature dependence for uptake of 3 Hdopamine by the subcellular fraction which contains synaptic storage vesicles (Fig . 1) . While no uptake was observed at 00 , Figure 1 Effects of incubation time and temperature on 3H-dopamine uptake into rat whole brain storage vesicles . Each point is the average of 5 or 6 determinations . Concentration of ATP-Mg 2+ was 1 mM and of 3H-dopamine 0 .05 PM .

substantial incorporation was seen at 20 0 , 30 0 or 37 0 . The initial uptake was greater at 30 0 or 37 0 than at 20 0 , but only at 20 0 was the uptake linear with time over a time course of several minutes ; hence, a 4 min incubation at 20 0 was utilized in the subsequent experiments to ensure a linear relationship with time and stability of the preparation . Similar time and temperature

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Brain Synaptic Vesicle Dopamine Uptake

relationships have been reported for uptake of serotonin by vesicles isolated from rat whole brain (12) ; on the other hand, norepinephrine uptake exhibits linearity even at 30*, possibly resulting from the generally lower uptake velocity of this amine (11) . 9H-Dopamine uptake exhibited an absolute requirement for both ATP and Mg t + ; between 0 .005 and 3 mM ATP-Mg + there was a 10-fold increase in uptake (Fig . 2) . No statistically significant increment was obtained above 0 .5 mM ATP-Mg 2 + ; since high Figure 2

6

Effects of ATP-Mg 2+ concentration on 3H-dopamine uptake into rat whole brain storage vesicles . Points and bars represent the means and standard errors of 3-4 determinaIncubations lasted 4 tions . min at 20 0 and contained 0 .05 uM 9H-dopamine .

°4 W

0

IH 1 Q005 0D5 05 * [ATP-Mp 2 ] (mM)

ATP-Mg 2+ concentrations tended to increase the 00 blank or decrease the uptake in some preparations (11, 12), 0 .5 or 1 mM was utilized in the remaining studies . Addition to the incubation medium of Mg2+ without ATP or of ATP without Mg2+ (with EDTA added to chelate endogenous Mg t+) failed to stimulate 9 H-dopamine uptake (Table 1), indicating that both ATP and Mg2+ were required, Table 1 Uptake of Dopamine by Rat Whole Brain Synaptic Storage Vesicles ATP (1mm)

Addit ions*

Mg2+

(1mm)

EDTA (1mm)

Reserpine (0 .lum)

Uptake

I

(pmols/g) 5 .16 t 0 .34 o .84 ± o .o9** 0 .83 t 0 .10** 0 .51 t 0 .10**

Data represent means and standard errors of 4 determinations . Samples contained 0 .05 uM 3H-dopamine and were incubated for 4 min . at 20 0 . *Final concentrations **p<0 .001 vs . ATP + Mg2+

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Incubation in the presence of reserpine, which inhibits vesicular uptake systems (3, 4, 6), resulted in total inhibition of 3Hdopamine uptake (Table 1) . Thus, the uptake of 9H-dopamine by the rat whole brain preparation exhibits those properties which typify catecholamine uptake into storage vesicles from a wide-variety of central and peripheral neural tissues (6) . These include marked temperature dependence, an absolute requirement for ATP and magnesium, complete inhibition of uptake by reserpine and thermolability of the preparation . It is therefore extremely likely that, although this is a crude microsomal preparation, ° H-dopamine is being taken up almost solely by the storage vesicles present in the preparation, as is the case for 3H-norepinephrine and 3H-serotonin (11, 12) . Vesicular amine uptake in central and peripheral tissues exhibits saturation kinetics (3, 6, 11, 12, 17, 18) . A similar result was obtained for 3 H-dopamine uptake by rat whole brain storage vesicles (Fig . 3) . The uptake exhibited a single Km value of 0 .26 t 0 .01 uM, with a maximal uptake of 34 t 2 pmols/g Figure 3

0.3 ó E a

0.3 uM NOREPINEPHRINE

0.2 W ]L Q H

NO NOREPINEPHRINE

-2.5

0

2.5

5

10

15

20

1 / [DOPAMINE] (»M-1 ) Double-reciprocal plots for uptake of 3H-dopamine into rat whole brain storage vesicles in the presence (o) and absence (0) of 0 .3 UM norepinephrine . Points and bars represent means and standard errors of 4 determinations . Lines are drawn by least-squares analysis . Intercepts on the abscissa are significantly different from each other (p<0 .005) while those on the ordinate are not . Incubations lasted 4 min at 20° and contained 0 .5 mM ATP-Mg 2 + . of brain . The Km for dopamine reported here is approximately 6 times lower than that obtained in pig striatal vesicles (16) ; a similar relationship has been found for 3 H-norepinephrine uptake

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in rat vs . pig brain preparations (11, 19), which may reflect either species differences or the use of slightly different preparative or incubation procedures . Since the whole brain vesicle preparation undoubtedly contains populations of vesicles with a variety of transmitters, it may seem somewhat surprising that a single Km value was found . This result indicates that the uptake is either entirely specific (i .e . - 3 H-dopamine enters dopaminergic vesicles only) or else the uptake characteristics are similar in the different populations . In view of the general lack of specificity of vesicle uptake systems (6-8), absolute specificity for dopaminergic vesicles seems unlikely . Furthermore, earlier studies in the rat whole brain preparation indicated that 3 H-norepinephrine is taken up readily by dopaminergic storage vesicles with the same Km as in noradrenergic vesicles (12) . In the present experiments, norepinephrine inhibited 3 H-dopamine uptake in a purely competitive fashion (Fig . 3) ; in the presence of 0 .3 pM norepinephrine, the Km for dopamine was increased significantly to 0 .37 t 3 pinols/g of brain . The calculated value of the Ki for norepinephrine was 0 .69 t 0 .17 pM (p<0 .02 vs . the Km for dopamine), and thus the vesicular uptake system displayed an affinity for dopamine slightly more than twice that for norepinephrine . This is the same as the affinity ratio for dopamine inhibition of 3 H-norepinephrine uptake (11), strongly suggesting that 3 H-dopamine and 3 H-norepinephrine enter the same vesicle populations . Further support for this hypothesis is evidenced by the value for the maximal uptake of H-dopamine, which is nearly identical to that of 3 H-norepinephrine (11, 12) . Table 2 Uptake of Dopamine and Norepinephrine by Storage Vesicles from Rat Brain Regions Region Corpus Striatum

3H-Dopamine (pmols/g) 11 .7 t 0 .3

(4)

Cerebral Cortex

1.99 t 0 .10 (15)

Midbrain Brainstem Cerebellum

5 .18 t q .20 (15)

Whole Brain

5 .00 t 0 .29 (14)

3 H-Norepinephrine DAME (pmola/g) Uptake Ratio 17 .6 t 0 .2

(4)

3 .12 t 0 .25 (14)

10 . .2 t 0 .7 (14) 8. .36 t 0 .44. (14)

o .66 0 .64

0 .51 0 .6o

Data represent means and standard errors of the number of determinations in parentheses . Samples contained 0 .5 mM ATP-Mg t+ and 0 .05 uM 3H-dopamine or 3 H-norepinephrine and were incubated for 4 min at 20 0 (dopamine) or 30 0 (norepinephrine) .

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If vesicular uptakes of 3 H-dopamine and 9H-norepinephrine are occurring indiscriminately in dopaminergic and noradrenergic vesicles, then the preference for dopamine vs norepinephrine should be identical in vesicles from primarily dopaminergic or primarily noradrenergic brain regions . The regions chosen were corpus striatum (predominantly dopamine), cerebral cortex (predominantly norepinephrine) and the remainder of the brain (midbrain + brainstem + cerebellum) . Uptake of 3 H-dopamine and 8 Hnorepinephrine occurred in vesicles from all three regions ; for both amines the magnitude of uptake was greatest in corpus striatum and lowest in cerebral cortex (Table 2) . Despite the fact that cerebral cortex contains almost no dopamine, the ratio of all-dopamine uptake to 3 H-norepinephrine uptake was the same as in corpus striatum . These studies indicate that both dopamine- and norepinephrine-containing vesicles take up dopamine in vitro . In conclusion, a subcellular fraction containing synaptic storage vesicles can be prepared from rat whole brain or brain regions ; the fraction readily takes up 9 H-dopamine by a process which is stimulated by ATP-Mg 2 + and inhibited by reserpine, and which exhibits properties indicative of uptake into the vesicles present in the preparation . The uptake appears to occur equally well in both dopaminergic and noradrenergic vesicles . Acknowledgements Supported by USPHS HD-09713 and DA-00465 . Theodore A . Slotkin is recipient of Research Scientist Development Award DA-00006 from the National Institute on Drug Abuse . References 1. 2. 3. 4.

5.

6. 7. 8.

9. 10 . 11 . 12 .

G .B . KOELLE, in "The Pharmacological Basis of Therapeutics," 5th edition (Ed . L .S . Goodman and A . Gilman) pp . 404-444, MacMillan, New York (1975) . D .M . PATON, in "The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines" (Ed . D .M . Paton) pp . 49-66, Raven Press, New York (1976) . A . CARLSSON, Handb . Ex p . Pharmacol . 19 : 529-592 (1965) . T .A . SLOTKIN, n Neuropo sons : heir Pathophysiological Actions" vol . 2 (Ed . L .L . Simpson and D .R . Curtis) pp . 1-60, Plenum, New York (1974) . R .E . STITZEL, Pharmacol . Rev . 28 : 179-205 (1976) . A . PHILIPPU, in he echanismof Neuronal and Extraneuronal Transport of Catecholamines" (Ed . D .M . Paton) pp . 215-246, Raven Press, New York (1976) . T .A . SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 7 : 308-316 (1971) . T .A . SLOTKIN and N . KIRSHNER, Mol . Pharmacol . 7 : 581-592 (1971) . A . PHILIPPU and H . MATTHAEINaun n-Schmiedeber 's Arch . Pharmacol . 287: 191-204 (19i5) . T .A . SL KIN, T .R . ANDERSON, F.J . SEIDLER and C . LAU, Biochem. Pharmacol . 24 : 1413-1419 (1975) . F .J . SEIDL R, D .F . KMSEY, C . LAU, W.L . WHITMORE AND T .A . SLOTKIN, Life Sci . 21 : 1075-1086 (1977) . T .A . SLOTKK NP_ . 7. M`IDLER, W .L . WHITMORE, C . LAU, M . SALVAGGIO and D .F . KIRKSEY, J . Neurochem ., submitted .

830

13 . 14 . 15 " 16 .

17 . 18 . 19 .

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K.FUXE and U . UNGERSTEDT, Histochemie 13 : 16-28 (1968) . L .L . IVERSEN, in "Advances n B ochemical Psychopharmacology" vol . 2 (Ed . E . Costa and E. Giacobini) pp . 109-132, Raven Press, New York (1969) . M.J . KUHAR, E .G . SHASKAN and S .H . SNYDER, J . Neurochem . 18 :

333-343 (1971) .

A . PHILIPPU and J . BEYER, Naun n-Schmiedeber 's Arch . Pharmacol . 278 : 387-402 (1-91-7-3-T .

J . JONASSON, E . ROSENGREN and B . WALDECK, Acta Physiol . Scand . 6o : 136-14o (1964) . =. SLOTKIN, Biochem . Pharmacol . 24 : 89-97 (1975) . A . PHILIPPU, H . BEC and A . URGER, Eur . J . Pharmacol . _6 :

96-101 (1969) .