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Calcium-dependent and -independent release of endogenous dopamine from rat striatal synaptosomes
Brain Research, 473 (1988) 91-98 Elsevier
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Calcium-dependent and-independent release of endogenous dopamine from rat striatal synaptosomes John J. Woodward, L. Judson Chandler* and Steven W. Leslie Division of Pharmacology and Toxicology, College of Pharmacy and Institute for Neurological Sciences Research, University of Texas at Austin, Austin, TX 78712 (U.S.A.) (Accepted 24 May 1986) Key words: Dopamine release; Synaptosome; Calcium; Fura-2
We examined the role of calcium in the stimulus-secretion coupling process of brain neurons by measuring the potassium-stimulated release of endogenous dopamine from striatal synaptosomes in the presence and absence of extracellular calcium. Intracellular free calcium levels were also monitored under these conditions using the fluorescent calcium chelator, fura-2. The fast-phase (<3 s) of potassium-stimulated dopamine release was completely blocked by removing calcium from the external medium. Elimination of calcium from the medium with EGTA only partially blocked the slow phase (60 s) of K+-stimulated dopamine release. Depolarization of synaptosomes in the presence of extracellular calcium significantly increased intracellular calcium levels as measured by fura-2. No changes in intracellular calcium were observed during depolarization in calcium free-medium. Reductions in the sodium concentration of the extracellular medium produced a significant increase in the basal release of dopamine under calcium-free conditions. Depolarization of synaptosomes under these conditions markedly enhanced the release of dopamine. These results suggest that the slow-phase of dopamine release from synaptosomes does not require calcium but may be mediated via the reversal of the sodium-linked dopamine transport system.
INTRODUCTION The exocytotic release of many neurotransmitters is linked to an increase in intracellular calcium following activation of voltage-dependent calcium channels. This process, known as stimulus-secretion coupling, underlies the propagation of neuronal conduction in the central nervous system 5. Synaptosomes (pinched-off nerve terminals) are an excellent preparation for studying the relationship between calcium channel activation and endogenous transmitter release at central nervous system synapses. Calcium influx and the subsequent release of neurotransmitters from rat brain synaptosomes occur in two distinct phases following potassium depolarization 6"11A6. The fast-phase of calcium uptake and neurotransmitter release terminates within 1-3 s and
is thought to reflect the opening and closing of voltage-dependent calcium channels in the neuronal membrane. Removing calcium from the media with E G T A completely abolishes the fast component of potassium stimulated release of neurotransmitter H. The slow phase of uptake and release begins about 3 5 s following depolarization and is characterized by a reduced rate of ion flux and neurotransmitter secretion. Neurotransmitter release from several cell types has been reported to be independent of extracellular calcium under certain conditions. In isolated animal retinal cells for example, electrical depolarization or elevated potassium both increased the efflux of the amino acid G A B A in the absence of calcium z14,15. Calcium-independent release of glutamate, G A B A , and acetylcholine has also been reported following
* Present address: Department of Pharmacology, College of Medicine, Box J-267, JHMHC, University of Florida, Gainesville, FL 32610, U.S.A. Correspondence: J.J. Woodward, Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
92 depolarization of rodent brain slices and synaptosomes 1"~'12. In contrast, [3Hlnorepinephrine release
from cortical synaptosomes was eliminated in the absence of extracellular calcium s. These results suggest that the release of some transmitters from brain neurons can occur in the absence of extracellular calcium. Release of intracellular calcium from cellular stores during prolonged depolarization may be sufficient, however, to stimulate a calcium-dependent transmitter release process. We have examined this possibility by monitoring intracellular free calcium levels with fura-2 and the release of endogenous dopamine by HPLC following potassium stimulation of rat brain synaptosomes. MATERIALSAND METHODS
Materials Fura-2 AM (acetoxy methylester) was obtained from Molecular Probes. It was stored in aliquots frozen over nitrogen as a 10-~M solution in DMSO. A fresh aliquot was used for each day's experiment. Radioisotopes were obtained from New England Nuclear. All other reagents were purchased from commercial sources.
Synaptosomal preparation Male Sprague-Dawley rats (200-250 g) were killed by decapitation. Synaptosomes were prepared from the corpus striatum according to Cotman 3. Briefly, one pair of striata were homogenized in 10 vols. of ice-cold isotonic sucrose for a total of 8 up and down strokes. The homogenate was centrifuged at 3000 g for 5 rain. The resulting supernatant was centrifuged at 17,000 g for 12 rain. The pellet was resuspended in calcium-free incubation medium to give a protein concentration between 1.8 and 2.0 mg/ml. Calcium-free incubation medium consisted of (in mM): NaCI 136, KCl 5, MgCI2 1.3, EGTA 0.1, glucose 10, and Tris base 20 adjusted to a pH of 7.4 at 37 °C. In some experiments, calcium chloride was added to the medium to give a final free calcium concentration of 100 ~M. Protein concentrations were measured according to Oyama and Eagle L~.
Fura-2 measurements lntracellular free calcium concentrations ([Ca]i)
were measured according to the method of Komulainen and Bondy with slight modifications i''. A synaptosomal P2 pellet was obtained as described above and resuspended in 4 ml of incubation meditim to provide a protein concentration of 6-8 mg/ml. Aliquots of the tissue suspension were incubated with 10 ILM fura-2/AM in DMSO (final DMSO concentration of 1.[)%) at 37 °C in a Dubnoff metabolic shaking waterbath. After 45 min, 10 ml of warm incubation inedium was added to each vial and incubated for an additional 15 min. The suspensions were decanted into centrifuge tubes and centrifuged at 10,001) g for 10 min. A 100-/~1 aliquot of the synaptosomal suspension was pipetted into a conical microfuge tube containing 500 i~1 of ice-cold incubation medium and spun at 7000 g for 1 min. The resulting pellet was resuspended in 1.425 ml of the appropriate incubation medium. The suspension was transferred to a plastic cuvette, gently vortexed and incubated for 10 min at 35 °C. After this period, the baseline fluorescence was measured at 340 and 380 nm excitation (emission set at 510 rim). Fluorescence measurements were made in a water jacketed (35 °C) Aminco Bowman spectrofluorometer equipped with a strip chart recorder. The fluorescence of indicator in the absence of calcium (Fmi~) was determined by adding 50 ktl of sodium dodecyl sulfate (SDS; 0.1% final concentration) and 30 ~1 of [).75 M EGTA (in 3 M Tris base) to the cuvette. The maximum fluorescence emitted by fura-2 (Fm~0 was determined by adding 5[) ul of SDS and 30~1 of 200 mM CaCI 2 to a separate aliquot of loaded synaptosomes. A correction factor for leakage of fura-2 out of the synaptosomes during the course of an experiment was determined by periodically adding 15 I~1of 4 t~M MnCI 2 to separate aliquots of loaded tissue and reading the fluorescence immediately. The Mn correction factor was calculated by subtracting the fluorescence in the presence of Mn from the fluorescence in the absence of Mn at both 340 and 380 nm. To correct for tissue autofluorescence, several control experiments were performed with unloaded tissue and a standard curve of tissue autofluorescence versus protein concentration was obtained. The [Ca]i was calculated according to the method of Grynkiewicz et al. using a dissociation constant of 224 nM 7.
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Measurementof endogenous dopamine release Release of endogenous dopamine from rat striatal synaptosomes was measured as previously described 18. Briefly, a 200 ktl aliquot of either incubation or depolarizing medium (NaCI replaced iso-osmotically with KCI) was added to the P2 synaptosomal aliquot to give a final potassium concentration of 5 or 30 mM. Dopamine release was terminated at the appropriate time by the rapid addition of one ml of ice-cold stopping solution (in mM: NaCI 136, KCI 5, MgCI 2 1.3, EGTA 3, glucose 10, Tris base 20, pH adjusted to 7.00 at room temperature with 1 M maleic acid). The diluted synaptosomal preparation was immediately filtered through Whatman GF/B filters using a Hoefer manifold (vacuum 25 cm). Filters were washed twice with two ml portions of ice cold incubation medium. The dopamine containing filtrate was collected in a test tube (mounted under the filter) which contained 500 pg of the internal standard dihydroxybenzylamine (DHBA) and 200 ~1 of 1 M perchloric acid. In some experiments, 45Ca2+ (75 //Ci 45Ca2+//~mol4°Ca2+) was added to the synaptosomal aliquot during depolarization to measure the flux of calcium under normal calcium conditions. The acidified filtrate containing the released dopamine was extracted with acid-washed aluminum oxide prior to analysis by high-performance liquid chromatography with electrochemical detection (LCEC). Recovery of the internal standard averaged 65%. The LCEC system consisted of a Bioanalytical Systems 200 LCEC system equipped with a Rheodyne 7010 injector valve (40/d loop) and a Beckman ODS reverse-phase column (3 /~m particle size). Peaks were integrated and compared to external standards by the BAS chromatography software using an IBM personal computer. The mobile phase consisted of 0.15 M monochloracetate, 2 mM disodium ethylenediamine-tetraacetic acid (EDTA), 2.5 mM sodium octyl sulfate and 2.5% grade acetonitrile (final pH 4.0). Retention times for D H B A and dopamine averaged 5 and 9 min, respectively. RESULTS Fig. 1 shows the net voltage-dependent influx of calcium and the release of endogenous dopamine from rodent striatal synaptosomes during potassium depolarization in the presence of 100/~M extracellu-
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Fig. 1. Net voltage-dependent calcium uptake and release of endogenous dopamine from rat striatal synaptosomes. Synaptosomes resuspended in incubation medium containing 100/,M free calcium were exposed to resting (5 raM) or depolarizing (30 raM) concentrations of KCI for the indicated time periods. Net voltage-dependent calcium uptake and dopamine release were calculated for each time point by subtracting the magnitude of uptake or release which occurred under resting conditions from that which occurred during potassium depolarization. Data represent the mean (+ S,E.M.) from 6 experiments performed in duplicate. Note the differences in the scale of the y-axes.
lar calcium. Voltage-dependent uptake and release occurred in two distinct phases during the 60 s depolarization. A fast-phase of uptake and release occurred during the first 3 s of depolarization. Between 55 and 65% of the total amount of calcium influx and dopamine release which occurred during the 60-s depolarization took place within this time. A slower phase of uptake and release was evident following 5-15 s of potassium depolarization. The net influx of calcium and release of dopamine reached a plateau after 30 s of depolarization. These data are consistent with our previous studies which suggested that the fast-phase (<3 s) of dopamine release from synaptosomes is calcium-dependent and is linked to the activation of voltage-dependent calcium channels II. This calcium-dependency is illustrated in Fig. 2 in which synaptosomes were depolarized for 3 s with potassium in the absence or presence of extracellular calcium. As expected, the removal of calcium from the medium did not significantly alter basal dopamine release but totally abolished release in response to 30 mM KCI. Fig. 2 shows that removal of calcium from the medium again did not alter basal dopamine release but only partially reduced the potassium-stimulated release of dopamine over a 60 s depolarization period. The amount of dopamine release which was
94
e~
5
30
5
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[KC]] Fig. 2. Calcium dependency of the fast and slow phases of dopamine release from rat striatal synaptosomes. Synaptosomes were exposed to resting (5 mM) or depolarizing (30 mM) concentrations of KCI for 3 (A) or 60 (B) s in the absence or presence of 100/~M extracellular calcium. Note difference in the scale of the y-axes for A and B. Data are expressed as the mean (_+ S.E.M.) from 6 experiments performed in duplicate. *, significantly different from corresponding 5 mM KCI value, P < 0.05, paired t-test. calcium-dependent during the 60 s depolarization period (approximately 50%) was roughly equal to that released during the first 3 - 5 s of depolarization in the presence of calcium (see Fig. 1). These results suggested that the slow-phase release of dopamine may be independent of extracellular calcium. This does not rule out the possibility that calcium was recruited from intracellular sites. 600
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Fig. 3. Changes m the intracellular free calcium ([Ca]i) concentration of rat striatal synaptosomes upon depolarization. Synaptosomes loaded with fura-2 (see Methods) in a calcium-free medium were exposed to resting (5 mM) or depolarizing (30 mM) concentrations of KC1 in the absence or presence of 100 ~M free calcium. [Ca]i was calculated at l0 and 60 s following addition of the appropriate medium to the synaptosomes. Data represent the mean (+ S.E.M.) from two experiments performed in duplicate. *, significantly different from corresponding 5 mM KCI value, P < 0.05, paired t-test.
In order to assess this possibility, intracellular calcium levels were monitored in striatal synaptosomes using the calcium indicator fura-2. Fig. 3 shows that synaptosomes maintained in calcium-free buffer maintained a [Ca]i of approximately 115 nM under resting potassium conditions. Addition of 100/~M calcium to these synaptosomes increased resting [Ca]i to approximately 250 nM within 10 s and to 450 nM following 60 s of exposure to calcium. When 100 /~M calcium and 30 mM KC1 were added simultaneously to a separate aliquot of synaptosomes incubated in calcium-free medium, [Ca]i increased to 350 nM within 10 s and to 550 nM within 60 s. The net increase in [Ca]i produced by potassium depolarization at the two time periods monitored was calculated as the difference in [Ca]i during potassium stimulation and that observed at rest. This resulted in a net voltage-dependent change in [Ca]i of approximately 100 nM which was observed during the first 10 s. A prolonged period of depolarization (60 s) did not significantly increase the net voltage-dependent change in [Ca]i observed at 10 s. These data are consistent with those shown in Fig. 1 in which the net voltage-dependent influx of calcium (as measured by 45Ca2+) appeared to plateau after 15-30 s of depolarization. Longer periods of depolarization produced no further increases in calcium uptake. Exposure of synaptosomes to 30 mM KCI in the absence of extracellular calcium produced no changes in [Ca]i even after 60 s of depolarization (Fig. 3). These are the same conditions that stimulated a large release of dopamine from the striatal synaptosomes (Fig. 2). These results indicated that elevations in the intracellular levels of calcium may not be necessary for the release of dopamine during prolonged potassium depolarizaton. In a previous study, we reported that potassium depolarization of striatal synaptosomes reduces the specific uptake of labelled dopamine into the synaptosomes ~8. Taken together with the current results, this observation suggested that alterations in the synaptosomal dopamine uptake system might underlie the calcium-independent release of dopamine observed during potassium stimulation. Depolarization of synaptosomes with a high potassium medium requires an equivalent reduction in the sodium content of that medium to maintain the osmolarity of the solution. The final concentration of sodium produced
95 200
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Ionic Conditions Fig. 4. Basal and stimulated release of endogenous dopamine from rat striatal synaptosomes under various ionic conditions. Synaptosomes were exposed to medium containing different concentrations of sodium and potassium for 60 s. The solid bar shows the release of dopamine under resting sodium and potassium conditions. The hatched bar shows the effects of reducing the sodium concentration to 110 mM by isotonic substitution with choline chloride on the basal release of dopamine. The light colored bar shows the release of dopamine following depolarization of the synaptosomes with potassium. Data represent the mean (+ S.E.M.) from 4 experiments performed in triplicate. *, significantly different from 5 mM K, 136 mM Na value, P < 0.05, paired t-test; **, significantly different from 5 mM K, 110 mM Na value, P < 0.05, paired t-test.
during depolarization with 30 mM potassium was 110 mM. The increase in dopamine levels observed during depolarization might simply be due to an increased basal efflux of dopamine due to the reduced sodium levels. This change in sodium produced during potassium depolarization was mimicked by substituting choline for a portion of the sodium in the non-depolarizing medium. This was added to the synaptosomes for the 60-s incubation period to give sodium and potassium concentrations of 110 mM and 5 mM, respectively. Fig. 4 shows that even under resting conditions and in the absence of calcium, synaptosomes released a small amount of dopamine. This basal release is constant over incubation times as long as 20-30 min at 37 °C (data not shown). Synaptosomes exposed to the choline-substituted medium increased their basal (5 mM KC1) release of dopamine by approximately 50% during the 60 s incubation period as compared to controls incubated in normal sodium medium (Fig. 5). Release which occurred under these conditions, however, was only 50% of that observed with potassium depolarization. The importance of sodium in regulating the basal release of dopamine from synaptosomes was further in-
-Ca, +Na
+Ca, -Na
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Fig. 5. Basal release of endogenous dopamine from rat striatal synaptosomes under various ionic conditions. Dopamine release was measured from synaptosomes resuspended in medium containing either: (1) 100/~M calcium and 136 mM sodium; (2) 10/zM EGTA and 136 mM sodium; (3) 100/tM calcium and 136 mM choline; or (4) 100/~M EGTA and 136 mM choline for 14 min. Data represent the mean (+ S.E.M.) from 3 experiments performed in triplicate. *, significantly different from basal release under normal calcium and sodium conditions, P < 0.05, paired t-test; **, significantly different from release under normal calcium, no sodium conditions; P < 0.05, paired t-test.
vestigated by exposing synaptosomes to a totally sodium-free medium for various time periods. Fig. 5 shows the basal release of dopamine following a 14min incubation of synaptosomes in non-depolarizing medium with different ionic compositions. Basal 200 -
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Time, minutes Fig. 6. Time-dependent release of dopamine from rat striatal synaptosomes resuspended in sodium-free medium. Basal dopamine release was measured at the indicated time points from synaptosomes incubated in sodium-free, normal calcium medium (136 mM choline chloride, 100,uM free calcium) or sodium-free, calcium-free medium (136 choline chloride, 100/tM EGTA). Data represent the mean (+ S.E.M.) from 3 experiments performed in triplicate. Elimination of calcium from the medium significantly reduced the amount of basal dopamine release at all time points tested; P < 0.05, Student's t-test.
96 dopamine release from synaptosomes resuspended in medium containing 136 mM NaCI and 100 ~M calcium represented approximately 1.5% of the total dopamine content of the synaptosomes. Removal of calcium from the medium had no significant effect on this basal release. Synaptosomes exposed for 14 min to a sodium-free medium with 100 ~M calcium present released significant amounts of dopamine. This release occurred in a time-dependent fashion and reached its peak after approximately 8 rain of incubation (Fig. 6). The dopamine released over this period represented approximately 50% of the total dopamine content of the synaptosomes. Potassium (15-60 mM final concentration) added to the synaptosomes following this incubation did not produce any further release of dopamine (data not shown). Elimination of extracellular calcium and addition of E G T A to the medium reduced the basal release of dopamine during the 14-min incubation in sodium-free medium by approximately 50% (Figs. 5 and 6). DISCUSSION The results from this study indicate that potassiumstimulated dopamine release from rodent striatal synaptosomes occurs via calcium-dependent and -independent processes. Dopamine released during the fast-phase of potassium stimulation required extracellular calcium. This is consistent with our previous findings which showed that both voltage-dependent calcium entry and endogenous dopamine release show similar kinetics during potassium stimulation 1~. These previous results suggested that calcium entry through voltage-sensitive calcium channels was linked to the fast-phase release of dopamine. Eliminating calcium from the medium reduced the 60 s release of dopamine by approximately 50%. This is approximately the magnitude of dopamine release that occurred during the first 3-5 s of depolarization in the presence of calcium. This suggests that the slow phase of dopamine release is not dependent on extracellular calcium. This finding is in agreement with other studies which have demonstrated a potassium stimulated calcium-independent release of G A B A from retinal neurons and release of glutamate, G A B A , and acetylcholine from rat brain slices and synaptosomes 2,8.is. Although dopamine and other transmitters can be
released from synaptosomes in the absence of external calcium, amounts of calcium sufficient to support a calcium-dependent release process may be liberated from internal stores of calcium during depolarization. The calcium ionophore A23187 has been reported to stimulate the release of acetylcholine from rat brain synaptosomes even in the absence of extracellular calcium 1. These authors suggested that internal stores of calcium were released by A23187 and stimulated the observed transmitter release. Synaptosomes resuspended in calcium-free medium with 100/~M EGTA in the current study maintained intracellular free calcium levels of approximately 115 nM. Depolarization of these synaptosomes with potassium produced a large release of dopamine but had no effect on intracellular calcium levels as measured by fura-2. These results are similar to those reported by Schwartz who monitored intracellular free calcium levels with fura-2 during electrical depolarization of catfish retinal neurons 15. Endogenous transmitter released from retinal cells suspended in calcium-free medium during electrical depolarization elicited a large outward current from juxtaposed 'postsynaptic' catfish bipolar cells that were sensitive to the presence of GABA. This outward current occurred in the absence of any observable increase in intracellular free calcium levels of depolarized 'presynaptic' retinal cells. In the current study, it is possible that transient and local changes in [Ca]i occurred during depolarization which were not detected by fura-2. However, this seems unlikely since changes in extracellutar ionic conditions which were expected to produce changes in [Ca]i (e.g. altered Na +, K + and Ca 2+ levels in the medium) resulted in rapid and significant changes in the fluorescence of the indicator. For example, addition of potassium to fura-2 loaded synaptosomes in the presence of calcium produced significant changes in [Ca]i which were apparent during the initial 10 s of depolarization. Current studies in our laboratory using a dual beam spectrofluorometer equipped with a motorized beam splitter indicate that this change in [Ca]i is apparent within 60-100 ms following potassium stimulation of fura-2 loaded synaptosomes (personal observation). Other studies have shown that fura-2 is also sensitive to changes in [Ca], produced by liberation of calcium from internal stores by agents such as caffeine r. Thus, the calciumindependent release of dopamine observed during
97 potassium depolarization of synaptosomes probably occurs through a mechanism which does not require elevations in intracellular calcium. It is well known that certain experimental conditions can increase the apparent release of dopamine through blockade of the high-affinity presynaptic uptake system. This ATP-dependent process is capable of translocating dopamine and sodium ions into the cell against a steep concentration gradient via a carrier-mediated symporter 4"9. Alterations in the normal sodium concentration in the medium have been shown to reduce the uptake of dopamine into synaptosomes and therefore increase the extraceUular concentration of dopamine 9'18. In the present study, the total substitution of choline for sodium in the calcium-free resting medium resulted in a significant loss of intracellular dopamine over an 8-10-min incubation period. In synaptosomes depolarized with potassium and a slightly reduced level of sodium (110 mM), transport of dopamine out of the cell may have resulted not from depolarization per se but via the symporter due to alterations in the sodium gradient. This was tested by exposing the synaptosomes to resting medium with the same lowered external sodium concentrations found in the depolarizing medium. As expected, synaptosomes maintained under these conditions did release significant amounts of dopamine. However, the amount released accounted for only approximately 50% of the total dopamine released during potassium depolarization. This suggests that the membrane depolarization produced by potassium in addition to changes in intracellular sodium concentrations may contribute to the relative direction of the sodium-linked symporter. Increased sodium inside the cell following sodium channel activation during depolarization would favor the outward movement of sodium and dopamine. Prolonged depolarization of the membrane would also establish a transmembrane electrical gradient which would tend to drive positively charged sodium ions and the carrier-mediated transport of dopamine out of the cell. Both of these mechanisms may account for the
calcium-independent release of dopamine observed during prolonged depolarization in the current study. It has been observed that many types of transmitters including dopamine can be released from nerve cells by calcium-dependent and -independent processes. The role of calcium-dependent transmitter release is well established in brain neurons. Synaptic transmission requires transmitter to be released into the synapse within milliseconds of an action potential in order to stimulate a post-synaptic response. Studies with synaptosomes have demonstrated that the calcium-dependent release of dopamine and other transmitters is inextricably linked with voltage-sensitive calcium channels and occurs only during the first few seconds of depolarization. This time division is largely artefactual due to methodological limitations in quantitating release of endogenous transmitter from brain neurons over millisecond time periods. A slower calcium-independent release of transmitter from synaptosomes becomes apparent only after prolonged periods of depolarization. This relatively slow process would seem to preclude a physiologically important role in many brain synapses where action potentials and release of transmitter are rapid and regenerative events. Transmitter release during prolonged tetanic-like nerve stimulation in which calcium channels are likely to be inactivated or during other processes involved in the regulation of intracellular processes may involve this second calcium-independent process. Finally, the results of this study and others indicate the importance of choosing experimental conditions judiciously in order to fully and accurately quantitate the relative importance of these two processes as they relate to the regulation of neuronal activity.
REFERENCES
2 Ayoub, G.S. and Lam, D.M., The release of gammaaminobutyric acid from horizontal cells of the goldfish (Carassius auratus) retina, J. Physiol. (Lond.), 355 (1984) 191-214. 3 Cotman, C.W., Isolation of synaptosomal and synaptic plasma membrane fractions. In L. Fleischer and L. Packer
1 Adam-Vizi, V. and Ligeti, E., Release of acetylcholine from rat brain synaptosomes by various agents in the absence of external calcium ions, J. Physiol. (Lond.), 353 (1984) 505-521.
ACKNOWLEDGEMENTS The authors would like to express their gratitude to M. Piccolo and S. Tipton for their excellent technical assistance. This work was supported by grants (S.W.L.) from the National Institute of Alcohol Abuse and Alcoholism (AA05809 and RSDA 00044).
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(Eds.), Methods in Enzymology, Vol. 31A, Academic, New York, 1974, pp. 445-452. Coyle, J.T. and Snyder, S.H., Catecholamine uptake by synaptosomes in homogenates of rat brain: stereospecificity in different areas. J. Pharmacol. Exp. Ther., 170 (1969) 221-231. Douglas, W.W. and Rubin, R.P., The role of calcium in the secretory response of the adrenal medulla to acetylcholine, J. Physiol. (Lond.), 159 (1961) 40-57. Drapeau, P. and Blaustein, M.P., Initial release of [3H]dopamine from rat striatal synaptosomes: correlation with calcium entry, J. Neurosci., 3 (1983) 703-713. Grynkiewicz, G., Poenie, M. and Tsien, R., A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440-3450. Haycock, J.W., Levy, W.B., Denner, L.A. and Cotman, C.W., Effects of elevated [K+]0 on the release of neurotransmitters from cortical synaptosomes: Efflux or secretion?, J. Neurochem., 30 (1978) 1113-1125. Holz, R.W. and Coyle, J.T., The effects of various salts, temperature, and the alkaloids veratridine and batrachotoxin on the uptake of 3H-dopamine into synaptosomes from rat striatum, Mol. Pharmacol., 10 (1974) 746-758. Komulainen, H. and Bondy, S.C., The estimation of free calcium within synaptosomes and mitochondria with fura-2; comparison to quin-2, Neurochem. Int., 10 (1987) 55-64. Leslie, S.W., Woodward, J.J. and Wilcox, R.E., Correlation of rates of calcium entry and endogenous dopamine re-
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lease in mouse striatal synaptosomes, Brain Research. 325 (1985) 99-105. Nicholls, D.G., Sihra, T.S. and Prieto, J.S., Calcium-dependent and -independent release of glutamate from synaptosomes monitored by continuous fluorometry, J. Neurochem., 49 (1987) 50-57. Oyama, V.I. and Eagle, H., Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau), Proc. Soc. Exp. Biol., 91 (1956) 305-307. Schwartz, E.A., Calcium independent release of GABA from isolated horizontal cells of the toad retina, J. Physiol. (Lond.), 323 (1982) 211-227. Schwartz, E.A., Depolarization without calcium can release gamma-aminobutyric acid from a retinal neuron, Science, 238 (1987) 350-355. Suszkiw, J.B. and O'Leary, M.E., Temporal characteristics of potassium-stimulated acetylcholine release and inactivation of calcium influx in rat brain synaptosomes, J. Neurochem., 41 (1983) 868-873. Thayer, S.A., Hirning, L.D., Harris, K.M. and Miller, R.J., Distribution of multiple Ca > channel types and intracellular Ca 2+ stores in single central and peripheral neurons, Soc. Neurosci. Abstr., 13 (1987) 281-282. Woodward, J.J., Wilcox, R.E., Leslie, S.W. and Riffee, W.H., Dopamine uptake during fast-phase endogenous dopamine release from mouse striatal synaptosomes, Neurosci. Lett., 71 (1986) 106-112.
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Calcium-dependent and-independent release of endogenous dopamine from rat striatal synaptosomes John J. Woodward, L. Judson Chandler* and Steven W. Leslie Division of Pharmacology and Toxicology, College of Pharmacy and Institute for Neurological Sciences Research, University of Texas at Austin, Austin, TX 78712 (U.S.A.) (Accepted 24 May 1986) Key words: Dopamine release; Synaptosome; Calcium; Fura-2
We examined the role of calcium in the stimulus-secretion coupling process of brain neurons by measuring the potassium-stimulated release of endogenous dopamine from striatal synaptosomes in the presence and absence of extracellular calcium. Intracellular free calcium levels were also monitored under these conditions using the fluorescent calcium chelator, fura-2. The fast-phase (<3 s) of potassium-stimulated dopamine release was completely blocked by removing calcium from the external medium. Elimination of calcium from the medium with EGTA only partially blocked the slow phase (60 s) of K+-stimulated dopamine release. Depolarization of synaptosomes in the presence of extracellular calcium significantly increased intracellular calcium levels as measured by fura-2. No changes in intracellular calcium were observed during depolarization in calcium free-medium. Reductions in the sodium concentration of the extracellular medium produced a significant increase in the basal release of dopamine under calcium-free conditions. Depolarization of synaptosomes under these conditions markedly enhanced the release of dopamine. These results suggest that the slow-phase of dopamine release from synaptosomes does not require calcium but may be mediated via the reversal of the sodium-linked dopamine transport system.
INTRODUCTION The exocytotic release of many neurotransmitters is linked to an increase in intracellular calcium following activation of voltage-dependent calcium channels. This process, known as stimulus-secretion coupling, underlies the propagation of neuronal conduction in the central nervous system 5. Synaptosomes (pinched-off nerve terminals) are an excellent preparation for studying the relationship between calcium channel activation and endogenous transmitter release at central nervous system synapses. Calcium influx and the subsequent release of neurotransmitters from rat brain synaptosomes occur in two distinct phases following potassium depolarization 6"11A6. The fast-phase of calcium uptake and neurotransmitter release terminates within 1-3 s and
is thought to reflect the opening and closing of voltage-dependent calcium channels in the neuronal membrane. Removing calcium from the media with E G T A completely abolishes the fast component of potassium stimulated release of neurotransmitter H. The slow phase of uptake and release begins about 3 5 s following depolarization and is characterized by a reduced rate of ion flux and neurotransmitter secretion. Neurotransmitter release from several cell types has been reported to be independent of extracellular calcium under certain conditions. In isolated animal retinal cells for example, electrical depolarization or elevated potassium both increased the efflux of the amino acid G A B A in the absence of calcium z14,15. Calcium-independent release of glutamate, G A B A , and acetylcholine has also been reported following
* Present address: Department of Pharmacology, College of Medicine, Box J-267, JHMHC, University of Florida, Gainesville, FL 32610, U.S.A. Correspondence: J.J. Woodward, Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, TX 78712, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
92 depolarization of rodent brain slices and synaptosomes 1"~'12. In contrast, [3Hlnorepinephrine release
from cortical synaptosomes was eliminated in the absence of extracellular calcium s. These results suggest that the release of some transmitters from brain neurons can occur in the absence of extracellular calcium. Release of intracellular calcium from cellular stores during prolonged depolarization may be sufficient, however, to stimulate a calcium-dependent transmitter release process. We have examined this possibility by monitoring intracellular free calcium levels with fura-2 and the release of endogenous dopamine by HPLC following potassium stimulation of rat brain synaptosomes. MATERIALSAND METHODS
Materials Fura-2 AM (acetoxy methylester) was obtained from Molecular Probes. It was stored in aliquots frozen over nitrogen as a 10-~M solution in DMSO. A fresh aliquot was used for each day's experiment. Radioisotopes were obtained from New England Nuclear. All other reagents were purchased from commercial sources.
Synaptosomal preparation Male Sprague-Dawley rats (200-250 g) were killed by decapitation. Synaptosomes were prepared from the corpus striatum according to Cotman 3. Briefly, one pair of striata were homogenized in 10 vols. of ice-cold isotonic sucrose for a total of 8 up and down strokes. The homogenate was centrifuged at 3000 g for 5 rain. The resulting supernatant was centrifuged at 17,000 g for 12 rain. The pellet was resuspended in calcium-free incubation medium to give a protein concentration between 1.8 and 2.0 mg/ml. Calcium-free incubation medium consisted of (in mM): NaCI 136, KCl 5, MgCI2 1.3, EGTA 0.1, glucose 10, and Tris base 20 adjusted to a pH of 7.4 at 37 °C. In some experiments, calcium chloride was added to the medium to give a final free calcium concentration of 100 ~M. Protein concentrations were measured according to Oyama and Eagle L~.
Fura-2 measurements lntracellular free calcium concentrations ([Ca]i)
were measured according to the method of Komulainen and Bondy with slight modifications i''. A synaptosomal P2 pellet was obtained as described above and resuspended in 4 ml of incubation meditim to provide a protein concentration of 6-8 mg/ml. Aliquots of the tissue suspension were incubated with 10 ILM fura-2/AM in DMSO (final DMSO concentration of 1.[)%) at 37 °C in a Dubnoff metabolic shaking waterbath. After 45 min, 10 ml of warm incubation inedium was added to each vial and incubated for an additional 15 min. The suspensions were decanted into centrifuge tubes and centrifuged at 10,001) g for 10 min. A 100-/~1 aliquot of the synaptosomal suspension was pipetted into a conical microfuge tube containing 500 i~1 of ice-cold incubation medium and spun at 7000 g for 1 min. The resulting pellet was resuspended in 1.425 ml of the appropriate incubation medium. The suspension was transferred to a plastic cuvette, gently vortexed and incubated for 10 min at 35 °C. After this period, the baseline fluorescence was measured at 340 and 380 nm excitation (emission set at 510 rim). Fluorescence measurements were made in a water jacketed (35 °C) Aminco Bowman spectrofluorometer equipped with a strip chart recorder. The fluorescence of indicator in the absence of calcium (Fmi~) was determined by adding 50 ktl of sodium dodecyl sulfate (SDS; 0.1% final concentration) and 30 ~1 of [).75 M EGTA (in 3 M Tris base) to the cuvette. The maximum fluorescence emitted by fura-2 (Fm~0 was determined by adding 5[) ul of SDS and 30~1 of 200 mM CaCI 2 to a separate aliquot of loaded synaptosomes. A correction factor for leakage of fura-2 out of the synaptosomes during the course of an experiment was determined by periodically adding 15 I~1of 4 t~M MnCI 2 to separate aliquots of loaded tissue and reading the fluorescence immediately. The Mn correction factor was calculated by subtracting the fluorescence in the presence of Mn from the fluorescence in the absence of Mn at both 340 and 380 nm. To correct for tissue autofluorescence, several control experiments were performed with unloaded tissue and a standard curve of tissue autofluorescence versus protein concentration was obtained. The [Ca]i was calculated according to the method of Grynkiewicz et al. using a dissociation constant of 224 nM 7.
93
Measurementof endogenous dopamine release Release of endogenous dopamine from rat striatal synaptosomes was measured as previously described 18. Briefly, a 200 ktl aliquot of either incubation or depolarizing medium (NaCI replaced iso-osmotically with KCI) was added to the P2 synaptosomal aliquot to give a final potassium concentration of 5 or 30 mM. Dopamine release was terminated at the appropriate time by the rapid addition of one ml of ice-cold stopping solution (in mM: NaCI 136, KCI 5, MgCI 2 1.3, EGTA 3, glucose 10, Tris base 20, pH adjusted to 7.00 at room temperature with 1 M maleic acid). The diluted synaptosomal preparation was immediately filtered through Whatman GF/B filters using a Hoefer manifold (vacuum 25 cm). Filters were washed twice with two ml portions of ice cold incubation medium. The dopamine containing filtrate was collected in a test tube (mounted under the filter) which contained 500 pg of the internal standard dihydroxybenzylamine (DHBA) and 200 ~1 of 1 M perchloric acid. In some experiments, 45Ca2+ (75 //Ci 45Ca2+//~mol4°Ca2+) was added to the synaptosomal aliquot during depolarization to measure the flux of calcium under normal calcium conditions. The acidified filtrate containing the released dopamine was extracted with acid-washed aluminum oxide prior to analysis by high-performance liquid chromatography with electrochemical detection (LCEC). Recovery of the internal standard averaged 65%. The LCEC system consisted of a Bioanalytical Systems 200 LCEC system equipped with a Rheodyne 7010 injector valve (40/d loop) and a Beckman ODS reverse-phase column (3 /~m particle size). Peaks were integrated and compared to external standards by the BAS chromatography software using an IBM personal computer. The mobile phase consisted of 0.15 M monochloracetate, 2 mM disodium ethylenediamine-tetraacetic acid (EDTA), 2.5 mM sodium octyl sulfate and 2.5% grade acetonitrile (final pH 4.0). Retention times for D H B A and dopamine averaged 5 and 9 min, respectively. RESULTS Fig. 1 shows the net voltage-dependent influx of calcium and the release of endogenous dopamine from rodent striatal synaptosomes during potassium depolarization in the presence of 100/~M extracellu-
400 14-
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30° i 200 8. -*- C a l c i u m I n f l u x
- 100
~.
6.
i 10
I 20
i 30
i 40
i 50
i 60
70
Time, sec
Fig. 1. Net voltage-dependent calcium uptake and release of endogenous dopamine from rat striatal synaptosomes. Synaptosomes resuspended in incubation medium containing 100/,M free calcium were exposed to resting (5 raM) or depolarizing (30 raM) concentrations of KCI for the indicated time periods. Net voltage-dependent calcium uptake and dopamine release were calculated for each time point by subtracting the magnitude of uptake or release which occurred under resting conditions from that which occurred during potassium depolarization. Data represent the mean (+ S,E.M.) from 6 experiments performed in duplicate. Note the differences in the scale of the y-axes.
lar calcium. Voltage-dependent uptake and release occurred in two distinct phases during the 60 s depolarization. A fast-phase of uptake and release occurred during the first 3 s of depolarization. Between 55 and 65% of the total amount of calcium influx and dopamine release which occurred during the 60-s depolarization took place within this time. A slower phase of uptake and release was evident following 5-15 s of potassium depolarization. The net influx of calcium and release of dopamine reached a plateau after 30 s of depolarization. These data are consistent with our previous studies which suggested that the fast-phase (<3 s) of dopamine release from synaptosomes is calcium-dependent and is linked to the activation of voltage-dependent calcium channels II. This calcium-dependency is illustrated in Fig. 2 in which synaptosomes were depolarized for 3 s with potassium in the absence or presence of extracellular calcium. As expected, the removal of calcium from the medium did not significantly alter basal dopamine release but totally abolished release in response to 30 mM KCI. Fig. 2 shows that removal of calcium from the medium again did not alter basal dopamine release but only partially reduced the potassium-stimulated release of dopamine over a 60 s depolarization period. The amount of dopamine release which was
94
e~
5
30
5
30
[KC]] Fig. 2. Calcium dependency of the fast and slow phases of dopamine release from rat striatal synaptosomes. Synaptosomes were exposed to resting (5 mM) or depolarizing (30 mM) concentrations of KCI for 3 (A) or 60 (B) s in the absence or presence of 100/~M extracellular calcium. Note difference in the scale of the y-axes for A and B. Data are expressed as the mean (_+ S.E.M.) from 6 experiments performed in duplicate. *, significantly different from corresponding 5 mM KCI value, P < 0.05, paired t-test. calcium-dependent during the 60 s depolarization period (approximately 50%) was roughly equal to that released during the first 3 - 5 s of depolarization in the presence of calcium (see Fig. 1). These results suggested that the slow-phase release of dopamine may be independent of extracellular calcium. This does not rule out the possibility that calcium was recruited from intracellular sites. 600
500
400
300
200
100
5
30
5
30
[KCI] raM
Fig. 3. Changes m the intracellular free calcium ([Ca]i) concentration of rat striatal synaptosomes upon depolarization. Synaptosomes loaded with fura-2 (see Methods) in a calcium-free medium were exposed to resting (5 mM) or depolarizing (30 mM) concentrations of KC1 in the absence or presence of 100 ~M free calcium. [Ca]i was calculated at l0 and 60 s following addition of the appropriate medium to the synaptosomes. Data represent the mean (+ S.E.M.) from two experiments performed in duplicate. *, significantly different from corresponding 5 mM KCI value, P < 0.05, paired t-test.
In order to assess this possibility, intracellular calcium levels were monitored in striatal synaptosomes using the calcium indicator fura-2. Fig. 3 shows that synaptosomes maintained in calcium-free buffer maintained a [Ca]i of approximately 115 nM under resting potassium conditions. Addition of 100/~M calcium to these synaptosomes increased resting [Ca]i to approximately 250 nM within 10 s and to 450 nM following 60 s of exposure to calcium. When 100 /~M calcium and 30 mM KC1 were added simultaneously to a separate aliquot of synaptosomes incubated in calcium-free medium, [Ca]i increased to 350 nM within 10 s and to 550 nM within 60 s. The net increase in [Ca]i produced by potassium depolarization at the two time periods monitored was calculated as the difference in [Ca]i during potassium stimulation and that observed at rest. This resulted in a net voltage-dependent change in [Ca]i of approximately 100 nM which was observed during the first 10 s. A prolonged period of depolarization (60 s) did not significantly increase the net voltage-dependent change in [Ca]i observed at 10 s. These data are consistent with those shown in Fig. 1 in which the net voltage-dependent influx of calcium (as measured by 45Ca2+) appeared to plateau after 15-30 s of depolarization. Longer periods of depolarization produced no further increases in calcium uptake. Exposure of synaptosomes to 30 mM KCI in the absence of extracellular calcium produced no changes in [Ca]i even after 60 s of depolarization (Fig. 3). These are the same conditions that stimulated a large release of dopamine from the striatal synaptosomes (Fig. 2). These results indicated that elevations in the intracellular levels of calcium may not be necessary for the release of dopamine during prolonged potassium depolarizaton. In a previous study, we reported that potassium depolarization of striatal synaptosomes reduces the specific uptake of labelled dopamine into the synaptosomes ~8. Taken together with the current results, this observation suggested that alterations in the synaptosomal dopamine uptake system might underlie the calcium-independent release of dopamine observed during potassium stimulation. Depolarization of synaptosomes with a high potassium medium requires an equivalent reduction in the sodium content of that medium to maintain the osmolarity of the solution. The final concentration of sodium produced
95 200
20
.9
£ ¢,0
E
100
0 +Ca, +Na
Ionic Conditions Fig. 4. Basal and stimulated release of endogenous dopamine from rat striatal synaptosomes under various ionic conditions. Synaptosomes were exposed to medium containing different concentrations of sodium and potassium for 60 s. The solid bar shows the release of dopamine under resting sodium and potassium conditions. The hatched bar shows the effects of reducing the sodium concentration to 110 mM by isotonic substitution with choline chloride on the basal release of dopamine. The light colored bar shows the release of dopamine following depolarization of the synaptosomes with potassium. Data represent the mean (+ S.E.M.) from 4 experiments performed in triplicate. *, significantly different from 5 mM K, 136 mM Na value, P < 0.05, paired t-test; **, significantly different from 5 mM K, 110 mM Na value, P < 0.05, paired t-test.
during depolarization with 30 mM potassium was 110 mM. The increase in dopamine levels observed during depolarization might simply be due to an increased basal efflux of dopamine due to the reduced sodium levels. This change in sodium produced during potassium depolarization was mimicked by substituting choline for a portion of the sodium in the non-depolarizing medium. This was added to the synaptosomes for the 60-s incubation period to give sodium and potassium concentrations of 110 mM and 5 mM, respectively. Fig. 4 shows that even under resting conditions and in the absence of calcium, synaptosomes released a small amount of dopamine. This basal release is constant over incubation times as long as 20-30 min at 37 °C (data not shown). Synaptosomes exposed to the choline-substituted medium increased their basal (5 mM KC1) release of dopamine by approximately 50% during the 60 s incubation period as compared to controls incubated in normal sodium medium (Fig. 5). Release which occurred under these conditions, however, was only 50% of that observed with potassium depolarization. The importance of sodium in regulating the basal release of dopamine from synaptosomes was further in-
-Ca, +Na
+Ca, -Na
-Ca, -Na
Fig. 5. Basal release of endogenous dopamine from rat striatal synaptosomes under various ionic conditions. Dopamine release was measured from synaptosomes resuspended in medium containing either: (1) 100/~M calcium and 136 mM sodium; (2) 10/zM EGTA and 136 mM sodium; (3) 100/tM calcium and 136 mM choline; or (4) 100/~M EGTA and 136 mM choline for 14 min. Data represent the mean (+ S.E.M.) from 3 experiments performed in triplicate. *, significantly different from basal release under normal calcium and sodium conditions, P < 0.05, paired t-test; **, significantly different from release under normal calcium, no sodium conditions; P < 0.05, paired t-test.
vestigated by exposing synaptosomes to a totally sodium-free medium for various time periods. Fig. 5 shows the basal release of dopamine following a 14min incubation of synaptosomes in non-depolarizing medium with different ionic compositions. Basal 200 -
~
100 • r~ o
~
E
0
E
'~-*-
G
Sodium-free I Ca
T
A
i
0
10
20
Time, minutes Fig. 6. Time-dependent release of dopamine from rat striatal synaptosomes resuspended in sodium-free medium. Basal dopamine release was measured at the indicated time points from synaptosomes incubated in sodium-free, normal calcium medium (136 mM choline chloride, 100,uM free calcium) or sodium-free, calcium-free medium (136 choline chloride, 100/tM EGTA). Data represent the mean (+ S.E.M.) from 3 experiments performed in triplicate. Elimination of calcium from the medium significantly reduced the amount of basal dopamine release at all time points tested; P < 0.05, Student's t-test.
96 dopamine release from synaptosomes resuspended in medium containing 136 mM NaCI and 100 ~M calcium represented approximately 1.5% of the total dopamine content of the synaptosomes. Removal of calcium from the medium had no significant effect on this basal release. Synaptosomes exposed for 14 min to a sodium-free medium with 100 ~M calcium present released significant amounts of dopamine. This release occurred in a time-dependent fashion and reached its peak after approximately 8 rain of incubation (Fig. 6). The dopamine released over this period represented approximately 50% of the total dopamine content of the synaptosomes. Potassium (15-60 mM final concentration) added to the synaptosomes following this incubation did not produce any further release of dopamine (data not shown). Elimination of extracellular calcium and addition of E G T A to the medium reduced the basal release of dopamine during the 14-min incubation in sodium-free medium by approximately 50% (Figs. 5 and 6). DISCUSSION The results from this study indicate that potassiumstimulated dopamine release from rodent striatal synaptosomes occurs via calcium-dependent and -independent processes. Dopamine released during the fast-phase of potassium stimulation required extracellular calcium. This is consistent with our previous findings which showed that both voltage-dependent calcium entry and endogenous dopamine release show similar kinetics during potassium stimulation 1~. These previous results suggested that calcium entry through voltage-sensitive calcium channels was linked to the fast-phase release of dopamine. Eliminating calcium from the medium reduced the 60 s release of dopamine by approximately 50%. This is approximately the magnitude of dopamine release that occurred during the first 3-5 s of depolarization in the presence of calcium. This suggests that the slow phase of dopamine release is not dependent on extracellular calcium. This finding is in agreement with other studies which have demonstrated a potassium stimulated calcium-independent release of G A B A from retinal neurons and release of glutamate, G A B A , and acetylcholine from rat brain slices and synaptosomes 2,8.is. Although dopamine and other transmitters can be
released from synaptosomes in the absence of external calcium, amounts of calcium sufficient to support a calcium-dependent release process may be liberated from internal stores of calcium during depolarization. The calcium ionophore A23187 has been reported to stimulate the release of acetylcholine from rat brain synaptosomes even in the absence of extracellular calcium 1. These authors suggested that internal stores of calcium were released by A23187 and stimulated the observed transmitter release. Synaptosomes resuspended in calcium-free medium with 100/~M EGTA in the current study maintained intracellular free calcium levels of approximately 115 nM. Depolarization of these synaptosomes with potassium produced a large release of dopamine but had no effect on intracellular calcium levels as measured by fura-2. These results are similar to those reported by Schwartz who monitored intracellular free calcium levels with fura-2 during electrical depolarization of catfish retinal neurons 15. Endogenous transmitter released from retinal cells suspended in calcium-free medium during electrical depolarization elicited a large outward current from juxtaposed 'postsynaptic' catfish bipolar cells that were sensitive to the presence of GABA. This outward current occurred in the absence of any observable increase in intracellular free calcium levels of depolarized 'presynaptic' retinal cells. In the current study, it is possible that transient and local changes in [Ca]i occurred during depolarization which were not detected by fura-2. However, this seems unlikely since changes in extracellutar ionic conditions which were expected to produce changes in [Ca]i (e.g. altered Na +, K + and Ca 2+ levels in the medium) resulted in rapid and significant changes in the fluorescence of the indicator. For example, addition of potassium to fura-2 loaded synaptosomes in the presence of calcium produced significant changes in [Ca]i which were apparent during the initial 10 s of depolarization. Current studies in our laboratory using a dual beam spectrofluorometer equipped with a motorized beam splitter indicate that this change in [Ca]i is apparent within 60-100 ms following potassium stimulation of fura-2 loaded synaptosomes (personal observation). Other studies have shown that fura-2 is also sensitive to changes in [Ca], produced by liberation of calcium from internal stores by agents such as caffeine r. Thus, the calciumindependent release of dopamine observed during
97 potassium depolarization of synaptosomes probably occurs through a mechanism which does not require elevations in intracellular calcium. It is well known that certain experimental conditions can increase the apparent release of dopamine through blockade of the high-affinity presynaptic uptake system. This ATP-dependent process is capable of translocating dopamine and sodium ions into the cell against a steep concentration gradient via a carrier-mediated symporter 4"9. Alterations in the normal sodium concentration in the medium have been shown to reduce the uptake of dopamine into synaptosomes and therefore increase the extraceUular concentration of dopamine 9'18. In the present study, the total substitution of choline for sodium in the calcium-free resting medium resulted in a significant loss of intracellular dopamine over an 8-10-min incubation period. In synaptosomes depolarized with potassium and a slightly reduced level of sodium (110 mM), transport of dopamine out of the cell may have resulted not from depolarization per se but via the symporter due to alterations in the sodium gradient. This was tested by exposing the synaptosomes to resting medium with the same lowered external sodium concentrations found in the depolarizing medium. As expected, synaptosomes maintained under these conditions did release significant amounts of dopamine. However, the amount released accounted for only approximately 50% of the total dopamine released during potassium depolarization. This suggests that the membrane depolarization produced by potassium in addition to changes in intracellular sodium concentrations may contribute to the relative direction of the sodium-linked symporter. Increased sodium inside the cell following sodium channel activation during depolarization would favor the outward movement of sodium and dopamine. Prolonged depolarization of the membrane would also establish a transmembrane electrical gradient which would tend to drive positively charged sodium ions and the carrier-mediated transport of dopamine out of the cell. Both of these mechanisms may account for the
calcium-independent release of dopamine observed during prolonged depolarization in the current study. It has been observed that many types of transmitters including dopamine can be released from nerve cells by calcium-dependent and -independent processes. The role of calcium-dependent transmitter release is well established in brain neurons. Synaptic transmission requires transmitter to be released into the synapse within milliseconds of an action potential in order to stimulate a post-synaptic response. Studies with synaptosomes have demonstrated that the calcium-dependent release of dopamine and other transmitters is inextricably linked with voltage-sensitive calcium channels and occurs only during the first few seconds of depolarization. This time division is largely artefactual due to methodological limitations in quantitating release of endogenous transmitter from brain neurons over millisecond time periods. A slower calcium-independent release of transmitter from synaptosomes becomes apparent only after prolonged periods of depolarization. This relatively slow process would seem to preclude a physiologically important role in many brain synapses where action potentials and release of transmitter are rapid and regenerative events. Transmitter release during prolonged tetanic-like nerve stimulation in which calcium channels are likely to be inactivated or during other processes involved in the regulation of intracellular processes may involve this second calcium-independent process. Finally, the results of this study and others indicate the importance of choosing experimental conditions judiciously in order to fully and accurately quantitate the relative importance of these two processes as they relate to the regulation of neuronal activity.
REFERENCES
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ACKNOWLEDGEMENTS The authors would like to express their gratitude to M. Piccolo and S. Tipton for their excellent technical assistance. This work was supported by grants (S.W.L.) from the National Institute of Alcohol Abuse and Alcoholism (AA05809 and RSDA 00044).
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