Differential effect of protein kinase inhibitors on calcium-dependent and calcium-independent [14C]GABA release from rat brain synaptosomes

Pergamon

PII:

Neuroscience Vol. 85, No. 3, pp. 989–997, 1998 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00599-X

DIFFERENTIAL EFFECT OF PROTEIN KINASE INHIBITORS ON CALCIUM-DEPENDENT AND CALCIUM-INDEPENDENT [14C]GABA RELEASE FROM RAT BRAIN SYNAPTOSOMES L. G. STORCHAK, N. G. POZDNYAKOVA and N. H. HIMMELREICH* Department of Neurochemistry, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiev 252030, St Leontovich 9, Ukraine Abstract––Rat brain synaptosomes were isolated to study the effects of protein kinase inhibitors (sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, N-(6-aminohexyl)-5-chloro1-naphtalenesulfonamide, staurosporine) on Ca2+-dependent and Ca2+-independent [14C]GABA release. The Ca2+-dependent [14C]GABA release was stimulated by depolarization with a K+-channel blocker, 4-aminopyridine, or high K+ concentration. It has been shown that 4-aminopyridine-evoked [14C]GABA release strongly depends on extracellular Ca2+ while K+-evoked [14C]GABA release only partly decreases in the absence of calcium. The substitution of sodium by choline in Ca2+-free medium completely abolished Ca2+-independent part of K+-evoked [14C]GABA release. So the main effect of 4-aminopyridine is the Ca2+-dependent one while high K+ is able to evoke [14C]GABA release in both a Ca2+-dependent and Na+-dependent manner. In experiments with protein kinase inhibitors, 4-aminopyridine and high K+ concentration were used to study the Ca2+-dependent and the Ca2+-independent [14C]GABA release, respectively. In addition, the Ca2+-independent [14C]GABA release. was studied using á-latrotoxin as a tool. Pretreatment of synaptosomes with protein kinase inhibitors tested, except of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, resulted in a marked inhibition of 4-aminopyridine-stimulated Ca2+-dependent [14C]GABA release. The inhibitory effects of N-(6aminohexyl)-5-chloro-1-naphtalenesulfonamide and staurosporine on [14C]GABA release were not due to their effects on 4-aminopyridine-promoted 45Ca2+ influx into synaptosomes. Only sphingosine (100 µM) reduced the 45Ca2+ influx. All the inhibitors investigated were absolutely ineffective in blocking the Ca2+-independent [14C]GABA release stimulated by á-latrotoxin. Three of them, except for sphingosine, did not affect the Ca2+-independent [14C]GABA release stimulated by high potassium. The inhibitory effect of sphingosine was equal to 30%. Thus, if [14C]GABA release occurred in a Ca2+-independent manner irrespective of whether á-latrotoxin or high K+ stimulated this process, it was not inhibited by the drugs decreased the Ca2+-dependent [14C] GABA release. Given the above points it is therefore not unreasonable to assume that the absence of Ca2+ in the extracellular medium created the conditions in which the activation of neurotransmitter release was not accompanied by Ca2+-dependent dephosphorylation of neuronal phosphoproteins, and as a consequence the regulation of exocytotic process was modulated so that the inhibition of protein kinases did not disturb the exocytosis.  1998 IBRO. Published by Elsevier Science Ltd. Key words: 4-aminopyridine, [14C]GABA release, á-latrotoxin, protein kinase inhibitors, synaptosome.

A great body of evidence has been accumulated lately that in in vitro experiments the release of GABA can occur both in the presence and absence of extracellular calcium. Depolarization with different agents (KCl, veratridine, ouabain) in Ca2+containing or Ca2+-free media was taken as a main tool to study the Ca2+-dependent and the Ca2+independent release of the neurotransmitter.11,27,32 In accordance with these data, it was proposed that two *To whom correspondence should be addressed. Abbreviations: 4-AP, 4-aminopyridine; EDTA, ethylenediaminetetra-acetate; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; H-7, 1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride; HEPES, N-2hydroxyethylpiperazine-N -2-ethanesulphonic acid; PKC, protein kinase C; SDS, sodium dodecyl sulphate; W-7, N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide. 989

different GABA pools, vesicular and cytoplasmic, which have different Ca2+-sensitivity, may exist in synaptosomes. The release of vesicular pool of GABA can be induced by plasma membrane depolarization in the presence of Ca2+, while the release of cytoplasmic pool of GABA is thought to occur in a Ca2+-independent manner. It has also been shown that depolarization-evoked Ca2+-independent release of GABA is completely abolished if Na+ is removed from the external medium and, as a result, Na+ electrochemical gradient across the plasma membrane is declined.11 These data have been interpreted as arguments for the hypothesis that Na+ electrochemical gradient favours the transport of neurotransmitter amino acids in the outward direction by reversal of the Na+ co-transport pathway at the plasma membrane.

990

L. G. Storchak et al.

There are, however, other proofs concerning the Ca2+-independent neurotransmitter release stimulated by á-latrotoxin, a presynaptic neurotoxin from the black widow spider venom, suggesting, that the toxin-stimulated release may proceed without the involvement of a Na+-coupled carrier. Our earlier experiments showed that á-latrotoxin can stimulate the [14C]GABA release from synaptosomes in Na+free, bivalent cation-free medium.31 Studies on the Ca2+-independent effect of á-latrotoxin with application of immunogold technique to ultrathin frozen sections from neuromuscular junctions established that á-latrotoxin provokes translocation of the synaptic vesicle proteins, synaptophysins and synapsins I from the vesicle compartment to the axolemma.36 Incorporation of synaptic vesicle membrane proteins into the axolemma is thought to indicate that in Ca2+-free medium á-latrotoxin stimulates the fusion of synaptic vesicle with the presynaptic membrane. Similar results were obtained at Drosophila neuromuscular junctions treated with black widow spider venom. The optical monitoring of the exocytotic process and synaptic vesicle recycling with the fluorescent dye FM 1-43 and application of immunogold technique showed that in synapse treated with the venom the plasma membrane is highly enriched with synaptotagmin, another synaptic vesicle protein, and synaptic boutons swell dramatically as would be expected if vesicle recycling was blocked.25 Thus, all these results are consistent with the notion that the release of neurotransmitter stimulated by á-latrotoxin in Ca2+-free medium is a result of activation of the exocytosis, i.e. the release of neurotransmitters from vesicular pool can occur in the Ca2+-independent manner. In the studies of the mechanism of neurotransmitter release the main efforts were directed to the comprehension of the Ca2+-dependent exocytotic process. It is widely accepted that release of neurotransmitter stores from synaptic vesicles is triggered by membrane depolarization that causes Ca2+ influx into the nerve terminal. The increase in terminal [Ca2+]i provokes changes in the phosporylation levels of some synaptosomal proteins and thus stimulates synaptic vesicle traffic, docking them to the active zones of presynaptic membrane and finally fusion with the presynaptic membrane.2,8,33 The mechanism by which the release of vesicular pool of neurotransmitters is stimulated in the Ca2+-independent manner has not been ascertained. Our recent investigation shows that á-latrotoxinstimulated Ca2+-independent neurotransmitter release is accompanied by changes in phosphorylation level of synaptic vesicle proteins which are somewhat distinct from those typical of the Ca2+stimulated process of exocytosis.24 Based on these data we would like to propose that some phosphorylation steps control the realization of neurotransmitter release in both cases, but different protein kinases are involved in the phosphorylation process.

The question we now raise is how the protein kinase inhibitors (sphingosine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride [H-7], N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide [W-7] and staurosporine) affect the Ca2+-dependent and Ca2+-independent release of GABA from rat brain synaptosomes. We aimed at studying the Ca2+dependent release of GABA from synaptosomes evoked by plasma membrane depolarization with a K+-channel blocker, 4-aminopyridine, and Ca2+independent release evoked by high KCl and á-latrotoxin in Ca2+-free medium. We also focused our attention on comparative estimation of the effects of protein kinase inhibitors on Ca2+independent release of GABA depending on whether KCl or á-latrotoxin stimulates the process of neurotransmitter release. EXPERIMENTAL PROCEDURES

Purification of á-latrotoxin á-Latrotoxin was isolated from Latrodecrus mactans tredecimguttatus venom by fast performance liquid chromatography (FPLC) using a Mono Q column.14 Purification of synaptosomes Synaptosomes were purified from cortical homogenates of rats (100–120 g, male) by differential and density-gradient centrifugation as described by Cotman5 with small modifications: sucrose solution for Ficoll gradient contained 5 mM HEPES–NaOH, pH 7.4, and 0.2 mM EDTA. The synaptosomal fraction obtained from the density gradient was diluted with 10 volumes of 0.32 M sucrose with 5 mM HEPES (pH 7.4) and was centrifuged at 20,000 g for 20 min. The pellet was then slowly resuspended in an ice-cold standard salt solution containing (in mM): NaCl 126, KCl 5, MgCl2 1.4, NaH2PO4 1.0, EGTA 0.5, HEPES 20, pH 7.4, and -glucose 10. The standard salt solution was used as a Ca2+-free medium. The Ca2+-supplemented medium contained 3 mM CaCl2. Salt solution was oxygenated by bubbling with a flow of O2 before use. Treatment of synaptosomes with protein kinase inhibitors [14C]GABA preloaded synaptosomes (1 mg of protein/ml) for [14C]GABA assay were incubated for 10 min at 37C in the absence or in the presence of protein kinase inhibitors, sphingosine, H-7, W-7 and staurosporine, before the agents inducing [14C]GABA release (45 mM KCl or 2 mM 4-aminopyridine or 3 nM á-latrotoxin) were added. Synaptosomes (4 mg of protein/ml) were treated with protein kinase inhibitors under the same conditions for 45 Ca2+ influx assay. [14C]GABA release from synaptosomes The suspension was preloaded with [14C]GABA according to the method described in 15. [14C]GABA release from synaptosomes was performed using one of the following methods. Method 1. [14C]GABA preloaded synaptosomes (1 mg of protein/ml) untreated or treated with protein kinase inhibitors were immediately used for measuring of the initial level of [14C]GABA. That moment was taken as zero point. Depolarizing agents or á-latrotoxin were added at zero point. If 45 mM KCl was used as a depolarizing agent, the Na+ concentration was decreased by an equivalent amount to maintain isotonicity. The solution used for [14C]GABA

Effects of protein kinase inhibitors on GABA release

991

efflux experiments contained 10
5 M amino oxyacetate. At definite time 0.5 ml aliquots were removed and filtrated through the Whatman GF/C filters (prewashed by 126 mM NaCl, 5 mM KCl, 20 mM HEPES pH 7.4). The filters were washed twice with 4 ml of ice-cold washing solution, pellets on the filters were solubilized in 1% sodium dodecyl sulphate (SDS). The radioactivity was determined by scintillation counting. The protein recovery of synaptosomal suspension was 93–96%. Method 2. [14C]GABA preloaded synaptosomes (1 mg of protein/ml) untreated or treated with protein kinase inhibitors were immediately used for [14C]GABA release assay. 0.5 ml aliquots of synaptosomal suspension were centrifuged for 20 s at 10,000 g and [14C]GABA was determined in the supernatants and in the SDS-solubilized pellets by liquid scintillation counting. [14C]GABA in supernatant was expressed as a percentage of total synaptosomal [14C]GABA. This initial level of [14C]GABA was taken as basal level. [14C]GABA release started just after the treatment of synaptosomes with protein kinase inhibitors by addition of 45 mM KCl, 2 mM 4-aminopyridine or 3 nM á-latrotoxin. Incubation lasted 5 min in experiments with KCl and 4-aminopyridine (4-AP) and 2 min in experiments with á-latrotoxin. When stimulation was over, 0.5 ml aliquots were centrifuged and [14C]GABA was determined as mentioned above. [14C]GABA release was evaluated by subtracting the percentage of supernatant [14C]GABA at initial point (basal level) from that calculated at the end of stimulation. Calcium influx Normally all steps were performed at 37C immediately after obtaining synaptosomal preparations. The synaptosomes were resuspended at 4 mg of protein/ml in Ca2+supplemented solution. 45Ca2+ (0.5 µCi/ml) was added after 15 min pre-incubation to ensure a return to steady-state conditions. KCl or 4-AP were added to synaptosomal suspension simultaneously with 45Ca2+. To quench 45Ca2+ uptake at definite time-intervals, 100 µl aliquots were taken and layered on to Sephadex C-50 columns (1 ml bed volume) pre-equilibrated with 2 mM CaCl2, 50 mM Tris–HCl, pH 7.4. The elution buffer contained 100 mM NaCl, 10 mM Tris–HCl at pH 7.4 and no Ca2+ added. Synaptosomes were eluted from the column with 0.8 ml of this buffer. After elution synaptosomes were solubilized in SDS (final concentration 1%), synaptosomal 45Ca2+ was quantified by scintillation counting in Delta 300 counter (Tracor Analytic, U.S.A.). Materials The following reagents were purchased from Sigma (U.S.A.): -sphingosine, H-7, W-7, staurosporine (antibiotic AM-2282) and EGTA. Ficoll 400 was from Pharmacia LKB Biotechnology Inc. (Switzerland), HEPES was from Fluka (Sweden), [14C]GABA (232 mCi/mmol) and 45CaCl2 (40 mCi/mg) were from Amersham (U.K.), 4-AP was from RBI (U.S.A.), GF/C filters were from Whatman (U.K.). Analytical grade salts were from Reachim (Russia). RESULTS

Comparison of calcium-dependent and calciumindependent effects of KCl and 4-AP on [14C]GABA release To study the effect of protein kinase inhibitors on [14C]GABA release stimulated by plasma membrane depolarization, we used high KCl concentration (45 mM) and a K+-channel blocker (2 mM). It was

Fig. 1. 4-AP-stimulated 45Ca2+ influx and [14C]GABA release. (A) 45Ca2+ influx into synaptosomes. (1) Baseline 45 Ca2+ influx; (2) 45Ca2+ influx induced by 4-AP. Results are expressed in counts/min. (B) [14C]GABA release from synaptosomes. (1) Baseline [14C]GABA efflux; (2) [14C]GABA efflux induced by 4-AP. Results are expressed as a percentages of total synaptosomal [14C]GABA at zero time. Each point represents mean value of triplicate assay. The figure is a representative of four independent experiments.

suggested that the effect of 4-AP on neurotransmitter release most closely mimicked physiological stimulation.20 In our experiments with 4-AP we showed that 4-AP augments the entrance of 45Ca2+ into synaptosomes and stimulates [14C]GABA release from synaptosomes (Fig. 1). The kinetics of both processes are similar. The next experiment was aimed at comparing the abilities of the two depolarizing agents (KCl and 4-AP) to stimulate [14C]GABA release in a Ca2+dependent and Ca2+-independent manner. Figure 2 illustrates outflux of [14C]GABA from synaptosomes in the Ca2+-free medium during resting conditions—the baseline outflux (curve 1), [14C]GABA release stimulated by 45 mM KCl in the same Ca2+-free medium (curve 2) and [14C]GABA release at depolarization induced with 45 mM KCl in Ca2+-supplemented medium (curve 3). Comparing curves 2 and 3, i.e. the release of [14C]GABA evoked by KCl in the Ca2+-free medium and in the Ca2+supplemented medium, it can be seen that in both cases the effect of KCl is considerable. Figure 2 (inset) shows that the values of [14C]GABA release evoked by KCl in the Ca2+-independent and Ca2+dependent manner are almost equal, since in the presence of Ca2+ we observed [14C]GABA release as both dependent on and independent of calcium. But we did not observe any [14C]GABA release in

992

L. G. Storchak et al.

2+

2+

+

Fig. 2. Ca -dependent and Ca -independent high K evoked [14C]GABA release from synaptosomes. (1) Baseline outflux of [14C]GABA from synaptosomes; (2) K+-evoked [14C]GABA release in Ca2+-free medium; (3) K+-evoked [14C]GABA release in the presence of 3 mM CaCl2. Each point represents the mean of triplicate assay. The figure is representative of four independent experiments. Results are expressed in counts/min. In the inset, bar graph presentation of the value of the Ca2+-independent and the Ca2+dependent high K+-evoked [14C]GABA release for 5 min. Results are expressed as a percentage of total synaptosomal [14C]GABA. The values represent the meanS.E.M. of duplicate experiments each performed in triplicate.

Fig. 4. Ca2+-dependent and Ca2+-independent 4-AP-evoked [14C]GABA release from synaptosomes. (1) Baseline outflux of [14C]GABA from synaptosomes; (2) 4-AP-evoked [14C]GABA release in Ca2+-free medium; (3) 4-AP-evoked [14C]GABA release in the presence of 3 mM CaCl2. Each point represents the mean of triplicate assay. The figure is representative of four independent experiments. Results are expressed in counts/min. In the inset, bar graph presentation of the value of Ca2+-independent and Ca2+-dependent 4-AP-evoked [14C]GABA release for 5 min. Results are expressed as a percentage of total synaptosomal [14C]GABA. The values represent the meanS.E.M. of duplicate experiments each performed in triplicate.

than the effect of KCl under the same conditions. It is also obvious that the main effect of 4-AP is the Ca2+-dependent one. Effects of protein kinase inhibitors on the calciumdependent and calcium-independent release of [14C]GABA

Fig. 3. Cationic dependence of high K+-evoked [14C]GABA release from synaptosomes. The synaptosomal suspension is loaded with [14C]GABA. Then the aliquots of suspension are washed three times with either standard salt solution without Ca2+ or the same solution with choline chloride instead of NaCl. The final pellets are resuspended in appropriate media. (1) Baseline outflux of [14C]GABA in Na+ medium; (2) K+-evoked [14C]GABA release in Na+ medium; (3) baseline outflux of [14C]GABA in Na+-free medium; (4) K+-evoked [14C]GABA release in Na+-free medium. Each point represents the mean of triplicate assay. The figure is representative of two independent experiments. Results are expressed as a percentages of total synaptosomal [14C]GABA.

Ca2+-free medium if Na+ is substituted for choline (Fig. 3). It should be noted that in the cholinecontaining medium the baseline outflux of [14C]GABA was somewhat increased. We carried out similar experiments with 4-AP. The outflux of [14C]GABA from synaptosomes in the Ca2+-free medium (baseline outflux), release of [14C]GABA stimulated by 4-AP in the same medium and in the Ca2+-supplemented medium are illustrated by curves 1, 2 and 3, respectively (Fig. 4). Comparing Figs 2 and 4 we can see that the Ca2+-independent effect of 4-AP on [14C]GABA release is by far less

Based on the above stated results we used 4-AP to study the Ca2+-dependent release of [14C]GABA and high K+ to study the Ca2+-independent process. Investigation of the Ca2+-independent release of [14C]GABA hase also been performed with á-latrotoxin. In these experiments we used commonly employed protein kinase inhibitors: sphingosine that inhibits protein kinase C (PKC) only by preventing the formation of an active lipid-enzyme complex;9 H-7 that inhibits PKC and cyclic nucleotide-dependent protein kinases by binding enzymes at the nucleotidesubstrate binding sites;22 W-7 that inhibits Ca2+/ calmodulin regulated activation of protein kinases13 and staurosporine that potently inhibits PKC and other kinases including c-AMP-dependent kinase and tyrosine kinase by binding near the nucleotide-substrate binding site.6,39 Figure 5 shows the effects of pretreatment of synaptosomes with the protein kinase inhibitors on the Ca2+-dependent release of [14C]GABA from synaptosomes. Column 1 reflects the release of [14C]GABA evoked by 4-AP at a 5 min interval from control synaptosomes. Columns 2 and 3 illustrate the effect of sphingosine. As can be seen, sphingosine inhibits the neurotransmitter release and the elevation of the sphingosine concentration from 10 to 100 µM leads to an increase of inhibitory effect up to 82% as compared to control. Similar concentrations

Effects of protein kinase inhibitors on GABA release

Fig. 5. Effect of protein kinase inhibitors on Ca2+dependent 4-AP-evoked [14C]GABA release. Before 4-AP stimulation synaptosomes were exposed to inhibitors of protein kinases for 10 min at 37C. (1) Untreated synaptosomes; (2,3) synaptosomes treated with 10 µM and 100 µM sphingosine, respectively; (4,5) synaptosomes treated with 10 µM and 100 µM H-7, respectively; (6,7,8) synaptosomes treated with 0.1 µM, 1.0 µM and 10 µM staurosporine, respectively; (9,10,11) synaptosomes treated with 0.1 µM, 1.0 µM and 10 µM W-7, respectively. Results are expressed as a percentages of total synaptosomal [14C]GABA. Each bar is the meanS.E.M. of three independent experiments each performed in triplicate.

of H-7 (columns 4 and 5) have only a small inhibitory effect on [14C]GABA release. However, staurosporine that acts similarly to H-7 within the catalytic domain of protein kinases almost completely inactivates the Ca2+-dependent release of [14C]GABA. Even at a concentration of 100 nM it reduces the release of [14C]GABA (column 6). This effect increases with the elevation of staurosporine concentration (effects of 1 µM and 10 µM staurosporine are illustrated in Fig. 5 by columns 7 and 8). The extent of inhibition of [14C]GABA release by 10 µM staurosporine is more pronounced than the 100 µM sphingosine effect and is equal to 92% of control. Using W-7 at the concentration range from 100 nM to 10 µM, as in the case of staurosporine, we observed that its inhibitory effect on the 4-AP-evoked [14C]GABA release was more potent (Fig. 5, columns 9–11). Ten micromolar W-7 completely abolished the 4-AP-evoked [14C]GABA release. It should be noted that the Ca2+-independent effect of á-latrotoxin on [14C]GABA release is much more potent than the effect of KCl under the same conditions. High K+-evoked release of [14C]GABA for 5 min contributes nearly 4% of total initial label, while á-latrotoxin-induced [14C]GABA release for 2 min is equal to 22% of it. In experiments with á-latrotoxin we treated synaptosomes with the same concentrations of protein kinase inhibitors as in the experiments with 4-AP. But in all cases we could find no differences in the ability of á-latrotoxin to stimulate [14C]GABA release from treated and untreated synaptosomes (data not shown). In order to determine whether the lack of sensitivity to protein kinase inhibitors is a characteristic for the Ca2+-independent [14C]GABA release, we also studied the effects of these inhibitors on the

993

Fig. 6. Effect of protein kinase inhibitors on Ca2+independent high K+-evoked [14C]GABA release. Before high K+ stimulation synaptosomes were exposed to inhibitors of protein kinases for 10 min at 37C. (1) Untreated synaptosomes; (2) synaptosomes treated with 100 µM sphingosine; (3) synaptosomes treated with 100 µM H-7; (4) synaptosomes treated with 10 µM staurosporine; (5) synaptosomes treated with 10 µM W-7. Results are expressed as a percentages of total synaptosomal [14C]GABA. Each bar is the meanS.E.M. of four independent experiments each performed in triplicate.

Ca2+-independent K+-evoked [14C]GABA release. In Fig. 6 the results obtained are illustrated. Only sphingosine diminishes [14C]GABA release stimulated by high K+, averaging 34% of control. The treatment of synaptosomes with H-7, staurosporine and W-7 do not lead to inhibition of the effect of high K+-induced depolarization on [14C]GABA release if it occurs in a Ca2+-independent manner. Thus, all inhibitors tested with the exception of sphingosine have no inhibitory effects on the Ca2+-independent [14C]GABA release irrespective of either á-latrotoxin or high K+ induces this process. Effect of protein kinase inhibitors on 4-aminopyridinestimulated 45Ca2+ influx To understand whether the action of the protein kinase inhibitors on the Ca2+-dependent 4-APevoked [14C]GABA release is due to inactivation of potential-dependent calcium channels by these agents, we investigated the effects of the protein kinase inhibitors on 45Ca2+ influx into synaptosomes stimulated by 4-AP. Results of our experiments show that all inhibitors do not affect basal unstimulated 45 Ca2+ influx into synaptosomes (data not shown). Increase of 45Ca2+ influx induced by plasma membrane depolarization with 4-AP is not prevented by either staurosporine or W-7 (Fig. 7, curves 4 and 5). The inhibition of 45Ca2+ influx can be observed only as a result of the pretreatment of synaptosomes with sphingosine (at 100 µM) (Fig. 7, curve 3). DISCUSSION

Calcium-dependent [14C]GABA release

and

calcium-independent

It is well known that depolarization of nerve terminal plasma membrane with high K+ concentration

994

L. G. Storchak et al.

Fig. 7. Effect of protein kinase inhibitors on 4-APstimulated 45Ca2+-influx into synaptosomes. Before 4-AP stimulation synaptosomes were exposed to inhibitors of protein kinases for 10 min at 37C. 2 mM 4-AP (final concentration) was added at zero time. (1) Baseline 45Ca2+ influx into synaptosomes; (2) 4-AP-stimulated influx of 45 Ca2+; (3) 4-AP-stimulated influx of 45Ca2+ into synaptosomes pretreated with 100 µM sphingosine; (4) 4-APstimulated influx of 45Ca2+ into synaptosomes pretreated with 10 µM staurosporine; (5) 4-AP-stimulated influx of 45 Ca2+ into synaptosomes pretreated with 10 µM W-7. Each point represents the mean of triplicate assay. The figure is representative of four independent experiments. Results are expressed in counts/min.

or with the K+-channel blocker 4-AP leads to elevation of cytosolic free Ca2+ concentration, and the enhancing effect of depolarizing agents on the release of neurotransmitters depends on the entry of extracellular Ca2+.34,38 But at present there are not enough data available about the mechanism of the Ca2+independent release of neurotransmitters. One of the findings of the present work is a distinct ability of the two agents, high K+ and 4-AP, to induce the Ca2+independent release of [14C]GABA from synaptosomes. When Ca2+ is not present in the extracellular medium, the effect of 4-AP on [14C]GABA release from synaptosomes is negligible or even absent while high K+ at the same conditions causes the release of half of the total quantity of [14C]GABA that can be released by high K+ in the presence of Ca2+. The Ca2+-independent release of [14C]GABA from synaptosomes can be eliminated if Na+ is substituted by choline in the medium. In contrast to 4-AP, high K+ can induce both the Ca2+-dependent [14C]GABA release and the Na+-dependent Ca2+-independent one. Therefore we used 4-AP to study the Ca2+dependent [14C]GABA release and high K+ concentration and á-latrotoxin to study the Ca2+independent one. It has been suggested that the mechanism of the Ca2+-dependent release of neurotransmitter markedly differs from the Na+-dependent release process and only the former is due to exocytotic process.1 However, recently evidence has been obtained that the Ca2+-independent release of neurotransmitters may be induced by agents that do not cause depolarization of the plasma membrane, so their effects are not coupled with declining of Na+ electrochemical gradient across the plasma membrane and can not be explained by the transport of neurotransmitter amino

acids in the outward direction.17,30,31 In in vitro experiments nitric oxide has been shown to have a stimulatory effect on [3H]dopamine release that is independent of calcium and this effect was not due to cellular damage.30 The massive Ca2+-independent neurotransmitter release induced by á-latrotoxin is independent of Na+,31 and if the medium does not contain other bivalent cations is also not accompanied by plasma membrane depolarization.17 The morphological studies of neuromuscular junctions treated with black widow venom show that neurotransmitter release stimulated by á-latrotoxin in the absence of Ca2+ is a result of fusion of synaptic vesicles with the presynaptic membrane.25 Recently it has been established that the Ca2+-independent outflow of [3H]noradrenaline from permeabilized synaptosomes can be inhibited by tetanus toxin and botulinum B toxin. The analysis of the data on neurotransmitter outflow and on degradation of synaptobrevin, one of the known targets for clostridial neurotoxins, has revealed the correlation between the two processes and led to the conclusion that the clostridial neurotoxins interfere with a process common to Ca2+-dependent and Ca2+-independent exocytosis.10 These data together with the results concerning the Ca2+-independent effect of álatrotoxin on neurotransmitter release support the view that the vesicular release apparatus may be involved in the Ca2+-independent neurotransmitter release. Effects of inhibitors of protein kinases on calciumdependent and calcium-independent [14C]GABA release. To obtain information about the process of the Ca2+-independent release of the neurotransmitter we wanted to resolve the following questions: (1) how do the protein kinase inhibitors affect the Ca2+dependent [14C]GABA release stimulated by 4-AP? (2) Are the effects of inhibitors due to preventing the increase in 45Ca2+ influx induced by 4-AP? (3) Does the process of the Ca2+-independent [14C]GABA release have the same sensitivity to the inhibitors of protein kinases as the Ca2+-dependent one? (4) Does the sensitivity of Ca2+-independent [14C]GABA release to protein kinase inhibitors depend on the type of agents that stimulated the release process or high K+-stimulated [14C]GABA release and á-latrotoxin-stimulated [14C]GABA release have the same sensitivity to the inhibitors tested? The experimental results show that all the inhibitors tested, except for H-7, markedly decrease the 4-AP evoked Ca2+-dependent [14C]GABA release. At 10 µM staurosporine or W-7, [14C]GABA release is almost totally abolished. In this regard data concerning the effect of staurosporine on frog motor nerve terminals should be noted.12 Using optical monitoring of fluorescent dye FM1-43 destaining, the method that allows the precise measurement of transmitter

Effects of protein kinase inhibitors on GABA release

release during nerve stimulation, Henkel and Betz12 have demonstrated that staurosporine irreversibly blocks the FM1-43 destaining. The inhibitory effect of staurosporine was also studied in experiments with cultured rat hippocampal neurons loaded with the same fluorescent dye FM1-43. Staurosporine was shown to greatly limit the mobility of organelles within nerve terminals. It is suggested that this effect of staurosporine is possibly attributable to its influence on the cytoskeleton.16 Sphingosine is less potent at 10 µM, but at 100 µM it reduces [14C]GABA release by 78.4–6.2%. H-7, an effective inhibitor of PKC-mediated phosphorylation of synapsin Ib, stimulated by high K+,29 has a slight effect at 10 µM, but at 100 µM it decreases [14C]GABA release by 20.02.3% of control. No change in the 4-AP-stimulated 45Ca2+ influx is observed with 10 µM staurosporine, 10 µM W-7 or 100 µM H-7. Only sphingosine (100 µM) that interacted with the regulatory domain of PKC reduces the 4-AP stimulated 45Ca2+ influx into synaptosomes. Our results concerning the effects of sphingosine, staurosporine and H-7 on 45Ca2+ influx are in line with the data establishing that sphingosine significantly inhibits the [Ca2+]i response promoted by high K+,29,35 and the two other protein kinase inhibitors, staurosporine and H-7, do not change the [Ca2+]i response.18,29 According to Sitges and Talamo,29 W-7 causes a slight inhibition of the [Ca2+]i response at the concentration of 3 µm and has a more potent effect at 100 µM. We did not observe any changes in the 4-AP- stimulated 45Ca2+ influx at 10 µM W-7. We did not use higher concentration of W-7 (100 µM) because it causes a marked elevation of basal [14C]GABA release. The lack of the effect of protein kinase inhibitors on 45Ca2+ influx is thought to indicate that the inhibition of the Ca2+-dependent [14C]GABA release from synaptosomes is not a result of direct inhibition of plasma membrane Ca2+ channel but is due to alterations in phosphorylation of proteins responsible for traffic and/or docking and fusion of synaptic vesicles with the presynaptic membrane. Recently, in addition to the well known role of synapsins in phosphorylation-dependent interaction of synaptic vesicles with cytoskeletal elements,3 the evidence has been obtained that phosphorylation of synaptobrevin, an integral protein of synaptic vesicle,21 and 25,000 mol. wt synaptosome-associated protein (SNAP-25), a protein of presynaptic membrane,28 plays a role in modulation of the molecular interactions of synaptic vesicles with presynaptic membrane. All the inhibitors investigated are absolutely ineffective in blocking of the Ca2+-independent [14C]GABA release stimulated by á-latrotoxin. Three of them with the exception of sphingosine had also no effect on the Ca2+-independent [14C]GABA release stimulated by high K+. The inhibitory effect of sphingosine is not significant and equals to 30%. Thus, if the process of [14C]GABA release occurs in

995

the Ca2+-independent manner, it is not inhibited by the drugs that potently decreased the Ca2+-dependent [14C]GABA release irrespective of either á-latrotoxin or high K+ which stimulates this process. Insensitivity of á-latrotoxin-stimulated exocytotic process to staurosporine has recently been observed in studies with FM1-43.12 It has been established that the blockade of FM1-43 destaining, as a result of the treatment of the nerve terminals with staurosporine, can be completely abolished by the consequent treatment of nerve terminals with black widow spider venom. The insensitivity of K+-evoked neurotransmitter release to trifluoperazine, a potent inhibitor of Ca2+/calmodulin-dependent protein kinase, like W-7, was observed when Ca2+ in the extracellular medium was substituted by Ba2+.37 The results of these experiments led us to conclude that calmodulin-dependent phosphorylation does not appear to be essential for transmitter release. Our observations, however, show that the sensitivity of [14C]GABA release process to protein kinase inhibitors strongly depends on the mode of activation of the release process, calcium-dependent or calcium-independent. The question arises why is the Ca2+-independent [14C]GABA release insensitive to inhibitors of protein kinases, particularly, to staurosporine which has wide specificity? Several possibilities have to be considered to explain this fact. Recently it was found that some protein kinases specific for either Ser/Thr or Tyr, notably protein kinases CK1 and CK2, mitogen-activated protein kinases and proteintyrosine kinases CSK have low sensitivity to staurosporine. The insensitivity to staurosporine is based on the structure of catalytic site of the kinase.19 It is also shown that calcium-dependent and calciumindependent family of PKC (all of the calciumindependent isozymes occur in rat brain) reveal important differences in substrate specificities.26 It cannot be excluded that calcium-independent forms of PKC responsible for realization of Ca2+independent á-latrotoxin- or high K+-stimulated exocytosis are relatively refractory to staurosporine. However, protein kinases are unlikely to have distinct structure of catalytic sites but can phosphorylate the same proteins in intact synaptosomes in Ca2+dependent or Ca2+-independent manner. The study by Pieribone et al.23 showed that clusters of vesicles at synaptic release sites are composed of two pools, a distal pool containing synapsin and proximal pool devoid of synapsin and located adjacent to the presynaptic membrane. It may be thought that the proximal pool of vesicles is a releasable one, and just the vesicles of this pool are involved in a constitutive mode of exocytosis which is independent of extracellular Ca2+ and occurs at low [Ca2+] concentration. If the effect of staurosporine concerning the inhibition of vesicle mobility is due to the effect of the drug on transition of synaptic vesicles from a reserve to a releasable pool,16 the insensitivity of the

996

L. G. Storchak et al.

Ca2+-independent release process to staurosporine is well understood. á-latrotoxin-induced release is suggested to be mediated by stimulation of a constitutive mode of exocytotic process.7 It can also be supposed that the release of neurotransmitters in the absence of calcium in extracellular medium occurs in the conditions when cellular calcium-dependent protein phosphatases are inactive. In our recent studies we failed to reveal any differences in the phosphorylation level of protein of 96,000 mol. wt when á-latrotoxin stimulated [14C]GABA release in a Ca2+-independent manner.24 We also observed that Ca2+-independent á-latrotoxinevoked [14C]GABA release was accompanied by an increase in phosphorylation of synapsin I and 65,000 mol. wt protein.24 The level of protein phosphorylation is dynamic and reflects the relative activities of both protein kinases and protein phosphatases. However, to what extent the increased phosphorylation level of phosphoproteins is a result of inactivation of protein phosphatases is unclear. The suggestion that the inhibition of protein phosphatases may modulate the process of neurotransmitter release is supported by the data showing that inhibition of calcineurin-

mediated dephosphorylation of proteins with cyclosporin A leads to a substantial enhancement of glutamate release evoked by 4-AP.20 It is also shown that okadaic acid, a specific inhibitor of phosphatases 1 and 2A, induces release of dopamine in the absence of calcium.4 All these findings revealed a connection between dephosphorylation of proteins and regulation of neurotransmitter release and that the calciumdependent protein dephosphorylation is likely to be a part of direct or indirect mechanism which limits neurotransmitter release.

CONCLUSION

Further studies of the effects of protein kinase inhibitors and protein phosphatases on phosphorylation events in the 4-AP- and á-latrotoxin-stimulated synaptosomes would help us to understand the mechanism of the Ca2+-independent neurotransmitter release. Acknowledgement—This work was supported by Civilian Research and Development Foundation grant UB2-339.

REFERENCES

1. Adam-Vizi V. (1992) External Ca2+-independent release of neurotransmitters. J. Neurochem. 58, 395–405. 2. Bahler M. and Greengard P. (1987) Synapsin I bundles F-actin in a phosphorylation-dependent manner. Nature 326, 704–707. 3. Benfenati F., Valtorta F., Chieregatti E. and Greengard P. (1992) Interaction of free and synaptic vesicle-bound synapsin I with F-actin. Neuron 8, 377–386. 4. Bugnon O., Ofori S. and Schorderet M. (1995) Okadaic acid modulates exocytotic and transporter-dependent release of dopamine in bovine retina in vitro. Naunyn-Schmiedeberg’s Arch. Pharmac. 351, 53–59. 5. Cotman C. W. (1974) Isolation of synaptosomal and synaptic plasma membrane fractions. In Methods in Enzymology (eds Fleisher S. and Packer L.), Vol. 31, pp. 445–452. Academic, New York. 6. Fallon R. J. (1990) Staurosporine inhibits a tyrosine protein kinase in human hepatoma cell membranes. Biochem. biophys. Res. Commun. 170, 1191–1196. 7. Geppert M., Goda Y., Hammer R. E., Li C., Rosahl Th. W., Stevens C. F. and Sudhof Th. C. (1994) Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727. 8. Greengard P., Valtorta F., Czernic A. J. and Benfenati F. (1993) Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 259, 780–784. 9. Hannun J. A., Loomis C. R., Merrill A. H. and Bell R. M. (1986) Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J. biol. Chem. 261, 12,604–12,609. 10. Hausinger A., Volknandt W., Zimmerman H. and Habermann E. (1995) Inhibition by clostridial neurotoxins of calcium-independent [3H]noradrenaline outflow from freeze-thawed synaptosomes: comparison with synaptobrevin hydrolysis. Toxicon 33, 1519–1530. 11. Haycock J. W., Levy W. B., Denner L. A. and Cotman C. W. (1978) Effects of elevated K+ on the release of neurotransmitters from cortical synaptosomes: efflux or secretion? J. Neurochem. 30, 1113–1125. 12. Henkel A. W. and Betz W. J. (1995) Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals. J. Neurosci. 15, 8246–8258. 13. Hidaka H., Sasaki Y., Tanaka T., Endo T., Onho Sh., Fujii Y. and Nagata T. (1981) N-(6-aminohexyl)-5-chloro-1naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc. natn. Acad. Sci. U.S.A. 78, 4354–4359. 14. Himmelreich N. H., Pivneva T. A., Lishko V. K. and Ivanov A. P. (1987) Calcium á-latrotoxin-induced permeability of synaptosomes (in Russian). Ukr. biokhim. Zhurn. 59, 39–44. 15. Himmelreich N. H., Pivneva T. A., Storchak L. G., Nikolishina E. V. and Lishko V. K. (1987) Interaction of á-latrotoxin with synaptosomes; metabolic control of the calcium influx induction (in Russian). Ukr. biokhim. Zhurn. 59, 29–34. 16. Kraszewski K., Daniell L., Mundigl O. and De Camilli P. (1996) Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence. J. Neurosci. 16, 5905–5913. 17. Lishko V. K., Saichenko E. A., Storchak L. G. and Himmelreich N. H. (1990) Latrotoxin channels: permeability for bivalent cations (in Russian). Biokhimiya 55, 1578–1583. 18. Maurer J. A., Wenger B. W. and McKay D. B. (1996) Effects of protein kinase inhibitors on morphology and function of cultured bovine adrenal chromaffin cells: KN-62 inhibits secretory function by blocking stimulated Ca2+ entry. J. Neurochem. 66, 105–113.

Effects of protein kinase inhibitors on GABA release 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

997

Meggio F., Deana A. D., Ruzzene M., Brunati A. M., Cesaro L., Guerra B., Meyer T., Mett H., Fabbro D., Furet P., Dobrowolska G. and Pinna L. A. (1995) Different susceptibility of protein kinases to staurosporine: inhibition-kinetic studies and molecular bases for resistance of protein kinase CK2. Eur. J. Biochem. 234, 317–322. Nichols R. A., Suplic G. R. and Brown J. M. (1994) Calcineurin-mediated protein phosphorylation in brain nerve terminals regulates the release of glutamate. J. biol. Chem. 269, 23,817–23,823. Nielander H. B., Onofri F., Valtorta F., Schiavo G., Montecucco C., Greengard P. and Benfenati F. (1995) Phosphorylation of VAMP/synaptobrevin in synaptic vesicles by endogenous protein kinases. J. Neurochem. 65, 1712–1720. Ohta H., Tanaka T. and Hidaka H. (1988) Putative binding site(s) of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on protein kinase C. Biochem. Pharmac. 37, 2704–2706. Pieribone V. A., Shupliakov O., Brodin L., Hilfiker-Rothenfluh S., Czernik A. J. and Greengard P. (1995) Distinct pools of synaptic vesicles in neurotransmitter release. Nature 375, 493–497. Pozdnyakova N. G., Storchak L. G. and Himmelreich N. H. (1996) á-Latrotoxin-stimulated protein phosphorylation in rat brain synaptosomes. Biochemistry (Moscow) 61, 1132–1138. Ramaswami M., Krishnan K. S. and Kelly R. B. (1994) Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junction. Neuron 13, 363–375. Robinson Ph. J. (1992) Differential stimulation of protein kinase C activity by phorbol ester or calcium/ phosphatidylserine in vitro and in intact synaptosomes. J. biol. Chem. 267, 21,637–21,644. Santos M. S., Goncalves P. P. and Carvalho A. P. (1990) Effect of ouabain on the [3H] aminobutyric acid uptake and release in the absence of Ca2+ and K+-depolarization. J. Pharmac. exp. Ther. 253, 620–627. Shimazaki Y., Nishiki T., Omori A., Sekiguchi M., Kamata Y., Kozaki S. and Takahashi M. (1996) Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release. J. biol. Chem. 271, 14,548–14,553. Sitges M. and Talamo B. R. (1993) Sphingosine, W-7, and trifluoperazine inhibit the elevation in cytosolic calcium induced by high K+ depolarization in synaptosomes. J. Neurochem. 61, 443–450. Stewart L. T., Michel A. D., Black M. D. and Humphrey P. P. A. (1996) Evidence that nitric oxide causes calcium-independent release of [3H]dopamine from rat striatum in vitro. J. Neurochem. 66, 131–137. Storchak L. G., Pashkov V. N., Pozdnyakova N. G., Himmelreich N. H. and Grishin E. V. (1994) Latrotoxinstimulated GABA release can occur in Ca2+-free Na+-free medium. Fedn Eur. biochem. Socs Lett. 351, 267–270. Szerb J. C. (1979) Relationship between Ca2+-dependent and-independent release of [3H]GABA evoked by high K+, veratridine, or electrical stimulation from rat cortical slices. J. Neurochem. 32, 1565–1573. Tarelli F. T., Bossi M., Fesce R., Greengard P. and Valtorta F. (1992) Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation. Neuron 9, 1143–1153. Tibbs G. R., Dolly J. O. and Nicholls D. G. (1996) Evidence for the induction of repetitive action potentials in synaptosomes by K+-channel inhibitors: an analysis of plasma membrane ion fluxes. J. Neurochem. 67, 389–397. Toernquist K., Pasternack M. and Kaili K. (1995) Sphingosine derivatives inhibit depolarization-evoked calcium entry in rat GH4C1 cells. Endocrinology 136, 4894–4902. Torri-Tarelli F., Villa A., Valtorta F., DeCamilli P., Greengard P. and Ceccarelli B. (1990) Redistribution of synaptophysin and synapsin I during á-latrotoxin-induced release of neurotransmitter at the neuromuscular junction. J. Cell Biol. 110, 449–459. Verhade M., Hens J. J. H., De Graan P. N. E., Boomsma F., Wiegant V. M., Da Silva F. H. L., Gispen W. H. and Ghijsen W. E. J. M. (1995) Ba2+ replaces Ca2+/calmodulin in the activation of protein phosphatases and in exocytosis of all major transmitters. Eur. J. Pharmac. 291, 387–398. Versteeg D. H. G., Heemskerk F. M. J., Spierenburg H. A., De Graan P. N. E. and Schrama L. H. (1995) 4-Aminopyridine differentially affects the spontaneous release of radiolabelled transmitters from rat brain slices in vitro. Brain Res. 686, 233–238. Ward N. E. and O’Brian C. A. (1992) Kinetic analysis of protein kinase C inhibition by staurosporine: evidence that inhibition entails inhibitor binding at a conserved region of the catalytic domain but not competition with substrates. Molec. Pharmac. 41, 387–392. (Accepted 4 November 1997)