Involvement of Lactic Acidosis in Anoxia-Induced Perturbations of Synaptosomal Function

L-Lactate (4-32 mM) added exogenously to resting or depolarised rat forebrain synaptosomes led to a significant decrease in intrasynaptosomal pH. Similarly depolarisation-induced increases in intrasynaptosomal calcium, calcium uptake, and acetylcholine release were all inhibited. These effects mimicked those previously observed in synaptosomes under anoxic conditions and suggest that lactate may be involved in limiting the damage due to calcium accumulation occurring during ischaemia. D-Lactate (added exogenously up to 32 mM) did not produce similar effects on these parameters even though the concentrations of intrasynaptosomal D-lactate reached levels comparable to those obtained with L-lactate (at 8-16 mM exogenous concentration). The results suggest that the mechanism of action of lactate on these parameters is stereospecific for the L-enantiomer. The effect of glucose availability on lactate production was assessed to explore the role of substrate availability on ischaemia/anoxic events. When exogenous glucose was increased (10-60 mM), there was no further increase in lactate production in normoxic synaptosomes, which suggests that glucose is not limiting under these conditions. When glucose was removed, as may occur in complete ischaemia, there was a significant decrease in lactate production after 60 min under anoxic or normoxic conditions. It would seem likely therefore that the mechanism underlying the changes observed in synaptosomes incubated under conditions reflecting complete ischaemia does not involve lactate.     

Protection of Ischaemic Synaptosomes from Calcium Overload by Addition of Exogenous Lactate

In depolarised anoxic synaptosomes, in which lactate production was significantly raised compared with normoxic conditions, calcium uptake, net acetylcholine release, and the intrasynaptosomal calcium concentration were all significantly lowered. In contrast, lactate production in synaptosomes incubated under aglycaemic- and ischaemic-type conditions was significantly lower and basal calcium uptake, acetylcholine release, and intrasynaptosomal calcium concentration were elevated compared with normoxia. In addition, the increase in intrasynaptosomal calcium concentration under the ischaemic-type condition appeared to be greater than could be accounted for by the rise in calcium uptake alone. Intrasynaptosomal pH reflected the lactate production under each condition investigated. Addition of exogenous lactate to normoxic synaptosomes mimicked the effects observed in anoxia, suggesting that lactate itself may have blocked the calcium uptake, inhibiting the rise in intrasynaptosomal calcium and acetylcholine release occurring in depolarised anoxic synaptosomes. When lactate was added to ischaemic synaptosomes, the large rise in intrasynaptosomal calcium concentration, calcium uptake, and acetylcholine release were decreased, suggesting that lactate may have a protective role in preventing cell death by calcium overload under ischaemic-type conditions. Evidence is presented to suggest that the effect of L-lactate was due to the lactate moiety itself rather than the associated acidosis.     

Menadione-treated synaptosomes as a model for post-ischaemic neuronal damage.

Menadione bisulphite increased endogenous oxygen-radical production by rat brain synaptosomes, as indicated by H2O2 generation. Increased oxygen-radical production was also demonstrated in synaptosomes prepared from menadione-treated rats and synaptosomes reoxygenated after an anoxic insult. Acetylcholine synthesis de novo was inhibited in synaptosomes incubated with menadione in vitro, in synaptosomes prepared from menadione-treated animals in vivo, and in depolarized post-anoxic synaptosomes. Intrasynaptosomal free Ca2+ was increased by menadione in vitro (50 microM), but this increase was not due to stimulation of Ca2+ entry into the nerve terminals. Acetylcholine release was stimulated by menadione in vitro, possibly as a consequence of the elevated intrasynaptosomal Ca2+ content. The Ca2+ contents of synaptosomes prepared from menadione (10 mg/kg)-treated animals in vivo and synaptosomes reoxygenated after anoxia were unchanged. In synaptosomes prepared from menadione-treated animals, acetylcholine release was no longer significantly stimulated by K+, whereas it was unchanged from control (normoxic) values in synaptosomes reoxygenated after anoxia. None of these treatments caused any measurable damage to the synaptic plasma membrane (as judged by the release of lactate dehydrogenase), or to synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Synaptosomes prepared from menadione-treated rats were found to be a good model for the study of post-anoxic damage to nerve-terminal function.     

Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

Calcium-Dependent and -Independent Acetylcholine Release from Electric Organ Synaptosomes by Pardaxin: Evidence of a Biphasic Action of an Excitatory Neurotoxin

Abstract: The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 × 10⁻⁷ M ), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 × 10⁻⁷ M to 10⁻⁵ M , the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10⁻⁶ M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations ( 10⁻⁵ M ), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.     

Large-Scale Purification of Torpedo Electric Organ Synaptosomes

Abstract: A procedure for the large-scale purification of Torpedo electric organ synaptosomes is described. The synaptosomal fraction obtained is very pure as judged from biochemical and morphological data. In addition, acetylcholine (ACh) release was demonstrated after KCl depolarization of synaptosomes in the presence of calcium. Two hundred grams of electric organ can be fractionated in a single run, allowing biochemical studies on presynaptic membrane constituents.     

Conditions Restricting Depolarization-Dependent Calcium Influx in Synaptosomes Reveal a Graded Response of P96 Dephosphorylation and a Transient Dephosphorylation of P65

Temporal changes in the phosphorylation level of synaptosomal phosphoproteins following depolarization of synaptosomes were investigated under conditions restricting calcium influx. High-K+ depolarization in media of low [Na+]o (32 mM during preincubation and depolarization) at pH 6.5 resulted in a pronounced fall in the cytosolic free calcium concentration transient, and in a reduction in the initial K(+)-stimulated 45Ca2+ uptake and endogenous acetylcholine release relative to the values obtained with control synaptosomes (preincubated and depolarized in Na(+)-based media). This reduction was paralleled by a decrease in the rate of dephosphorylation of the synaptosomal protein P96. A slower dephosphorylation of P96 also was observed on exposure to 20 microM veratridine at 0.5 mM external calcium. Our results indicate that, similar to synapsin I phosphorylation, P96 dephosphorylation shows a graded response to the amount of calcium entering the presynaptic terminal. Depolarization of synaptosomes under conditions restricting the influx of calcium revealed a transient dephosphorylation (reversed within 10 s) of the phosphoprotein P65. The possible significance of this finding to the process of neurotransmitter release is discussed.     

Solubilization and Partial Purification of a Presynaptic Membrane Protein Ensuring Calcium-Dependent Acetylcholine Release from Proteoliposomes

In previous work, it was shown that cytoplasmic acetylcholine decreased on stimulation of Torpedo electric organ or synaptosomes in a strictly calcium-dependent manner. This led to the hypothesis that the presynaptic membrane contained an element translocating acetylcholine when activated by calcium. To test this hypothesis, the presynaptic membrane constituents were incorporated into the membranes of liposomes filled with acetylcholine. The proteoliposomes thus obtained released the transmitter in response to a calcium influx. The kinetics and calcium dependency of acetylcholine release were comparable for proteoliposomes and synaptosomes. The presynaptic membrane element ensuring calcium-dependent acetylcholine release is most probably a protein, since it was susceptible to Pronase, but only when the protease had access to the intracellular face of the presynaptic membrane. Postsynaptic membrane fractions contained very low amounts of this protein. It was extracted from the presynaptic membrane under alkaline conditions in the form of a protein-lipid complex of large size and low density which was partially purified. The specificity of the calcium-dependent release for acetylcholine was tested with proteoliposomes filled with equal amounts of acetylcholine and choline or acetylcholine and ATP. In both cases, acetylcholine was released preferentially. After cholate solubilization and gel filtration, the protein ensuring the calcium-dependent acetylcholine release was recovered at a high apparent molecular weight (between 600,000 and 200,000 daltons), its apparent sedimentation coefficient being 17S after cholate elimination. This protein is probably an essential coin of the transmitter release mechanism. We propose to name it mediatophore.     

Effects of calcium ionophore A23187 and calcium antagonists on 32Pi incorporation into polyphosphoinositides of rat cortical synaptosomes

The role of Ca2+ on 32Pi incorporation into polyphosphoinositides (PPI) of rat cortical synaptosomes was studied. Stimulation of muscarinic receptor by carbachol (1 mM) resulted in a decrease in 32Pi incorporation into phosphatidylinositol-4,5-bisphophaphate (TPI) and phosphatidylinositol-4-phosphate (DPI), and an increase in 32Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA), whereas no significant effect on other membrane phospholipids was found. This response could be blocked by atropine (1 microM). The stimulatory effect of carbachol required Ca2+ in the medium; the presence of 0.5 mM EGTA blocked the effect of carbachol on PPI turnover completely. Calcium ionophore A23187, at 1 microM, had a similar effect on PPI turnover by carbachol (1 mM). At higher concentrations (10-100 microM) of A23187, the PPI turnover rate was much enhanced. Depolarization of the membrane by high potassium (60 mM) in the presence of calcium resulted in an enhanced PPI turnover, which was similar to the results of the carbachol (1 mM) effect but to a lesser extent. Calcium antagonists, diltiazem and trifluoperazine, at 10 microM could block the carbachol effect on 32Pi incorporation into PPI in this preparation. Our results suggest that the enhancement of PPI turnover in rat cortical synaptosomes by carbachol, calcium ionophore or high potassium requires Ca2+, and it can be blocked by compounds which interfere with the availability of this ion, such as EGTA or calcium antagonists.     

Effect of dichloroacetate on acetyl-CoA content and acetylcholine synthesis in rat brain synaptosomes

In potassium-depolarized synaptosomes Ca²⁺ inhibited oxidation of pyruvate (30%) and decreased the level of acetyl-CoA in intrasynaptosomal mitochondria (32%). On the other hand, Ca²⁺ facilitated provision of acetyl-CoA to synaptoplasm, since under these condition no change of synaptoplasmic acetyl-CoA and twofold stimulation of acetylcholine synthesis were found. However, in Ca²⁺-activated synaptosomes both synaptoplasmic acetyl-CoA and acetylcholine synthesis were suppressed by 1 mM (–)hydroxycitrate by 27 and 29%, respectively. It was not the case in resting synaptosomes. Dichloroacetate (0.05 mM) partially reversed the inhibitory effect of Ca²⁺ on pyruvate metabolism in synaptosomes and whole brain mitochondria. In Ca²⁺-stimulated synaptosomes, the dichloroacetate overcame suppressive effects of (–)hydroxycitrate on the level of synaptoplasmic acetyl-CoA and acetylcholine synthesis, but not on citrate clevage. It is concluded that dichloroacetate may improve the metabolism of acetylcholine in activated cholinergic terminals by increasing the production of acetyl-CoA in mitochondria and increasing its provision through the mitochondrial membrane to synaptoplasm by the transport system, independent of the ATP-citrate lyase pathway.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Somatostatin Release from Rat Cerebral Cortex Synaptosomes

Rat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin-like immunoreactivity (SRIF-LI) was measured by means of a radioimmunoassay procedure. High KCl (9-50 mM) concentration dependently evoked SRIF-LI release; the evoked overflow reached a plateau at 25 mM KCl and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KCl used. The 15 mM K(+)-evoked release of SRIF-LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K(+)-evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 microM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF-LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1-50 microM) induced release of SRIF-LI in a concentration-dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF-LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium-deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that greater than 90% of the SRIF-LI released either during the spontaneous outflow or by 15 mM KCl was represented by SRIF-14 (SRIF-28(14-28]. These values reflected the ratio SRIF-14/SRIF-28 found in synaptosomes at the end of the experiments.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Effects of chemical ischemia in cerebral cortex slices. Focus on nitric oxide

Superfused rat cerebral cortex slices were submitted to a continuous electrical (5 Hz) stimulation and treated with sodium azide (1-10 mM) in the presence of 2 mM 2-deoxyglucose ("chemical ischemia"). Presynaptic cholinergic activity, evaluated as acetylcholine release, was inhibited depending on the sodium azide concentrations and on the length of application (5-30 min). Following a 5-min treatment with 10 mM sodium azide, acetylcholine release was reduced to 45+/-2.3%; simultaneously, there was a 15- and 10-fold increase in glutamate and nitric oxide effluxes, respectively. After restoring normal superfusion conditions, acetylcholine release recovered to 70+/-3.1% of the controls; the N-methyl-D-aspartate receptor antagonist MK-801 (10 microM) as well as the nitric oxide scavengers, haemoglobin (20 microM) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (150 microM), improved the recovery in presynaptic activity, showing that both glutamate and nitric oxide play detrimental roles in chemical ischemia. On the other hand, the post-ischemic recovery was worsened by the guanylylcyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (10 microM), suggesting that the activation of such a pathway plays a neuroprotective role and that the nitric oxide-induced harmful effects depend on different mechanisms. Chemical ischemia-evoked nitric oxide efflux partly derived from its calcium-dependent endogenous synthesis, since both the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (1 mM), and the nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 microM), substantially prevented sodium azide effects. Nitric oxide efflux was only weakly reduced by MK-801 and was not modified by either the L-type calcium channel blocker, nifedipine (10 microM) or the N-type calcium channel blocker omega-conotoxin (0.5 microM), thus suggesting a prevailing intracellular calcium-dependence of nitric oxide production, although a partial extracellular calcium source cannot be ruled out. These findings show that sodium azide plus 2-deoxyglucose treatment is a useful protocol to induce brain ischemia in vitro and underline the involvement of nitric oxide in the complex events following the ischemic insult.     

Phosphatidic acid metabolism,calcium ions and transmitter release from electrically stimulated synaptosomes

Synaptosomes isolated from guinea pig brain cortex were stimulated electrically in a medium containing [32P]-orthophosphate. The electrical stimulation caused increased labelling of phosphatidic acid in a synaptic vesicle fraction prepared by osmotic shock of the incubated synaptosomes. Electrical stimulation also provokes transmitter release from the synaptosomes. Both increased phosphatidate labelling and transmitter release required calcium ions in the medium. The effects are discussed in relation to earlier work with acetylcholine and the possible involvement of membrane phosphatidic acid in transmitter release by exocytosis.     

The effect of oxidative stress on levels of cytosolic calcium within and uptake of calcium by synaptosomes

The ability of synaptosomes subjected to oxidative stress, to maintain homeostasis has been evaluated using various indices of cellular integrity. These include levels of cytosolic calcium and leakiness of the plasma membrane. The status of a neural characteristic; depolarization-induced calcium entry into the cytoplasm, has also been studied. The presence of 5 μM FeSO₄ and 0.1 mM ascorbic acid increased peroxidative activity as judged by the rate of thiobarbituric acid reactive material production, and depressed levels of free ionic calcium [Ca²⁺]_i as determined using the calcium-sensitive flouorescent indicator dye fura-2. Depolarization-induced influx of ⁴⁵Ca²⁺ was greatly depressed under these conditions, while basal calcium uptake was inhibited to a much lesser degree. The efflux of fura-2 from synaptosomes was enhanced in the oxidizing environment, suggesting increased permeability of the synaptosomal outer limiting membrane.

The treatment of synaptosomes with 25 μM -tocopherol succinate before and during exposure to the Fe²⁺/ascorbate mixture prevented many of the changes otherwise induced by the oxidizing system. Similar pretreatment with β-carotene or superoxide dismutase did not have any protective effect. Ganglioside GM₁ pre-exposure did not alter the Fe²⁺/ascorbate-induced changes in calcium-related parameters, but mitigated synaptosomal plasma membrane damage as judged by fura-2 leakage. Thus exogenous agents may be capable of reducing the severity of oxidative stress in nervous tissue.     

PnTx3-6 a spider neurotoxin inhibits K+-evoked increase in [Ca2+](i) and Ca2+-dependent glutamate release in synaptosomes

The present experiments investigated the effect of a neurotoxin purified from the venom of the spider Phoneutria nigriventer. This toxic component, P. nigriventer toxin 3-6 (PnTx3-6), abolished Ca(2+)-dependent glutamate release with an IC(50) of 74.4nM but did not alter Ca(2+)-independent secretion of glutamate when brain cortical synaptosomes were depolarized by KCl (33mM). This effect was most likely due to interference with the entry of calcium through voltage activated calcium channels (VACC), reducing the increase in the intrasynaptosomal free calcium induced by membrane depolarization with an IC(50) of 9.5nM. We compared the alterations induced by PnTx3-6 with the actions of toxins known to block calcium channels coupled to exocytosis. Our results indicate that PnTx3-6 inhibition of glutamate release and intrasynaptosomal calcium involves P/Q type calcium channels and this toxin can be a valuable tool in the investigation of calcium channels.     

Src family tyrosine kinases differentially modulate exocytosis from rat brain nerve terminals

We have studied the role of src family tyrosine kinases in regulating synaptic transmitter release from rat brain synaptosomes by using two assays that measure different aspects of synaptic vesicle exocytosis: glutamate release (that directly measures exocytosis of vesicle contents) and release of FM 2-10 styryl dye (that is proportional to the time the synaptic vesicle is fused to the plasma membrane). Depolarisation was induced by KCl (30 mM) or 4-aminopyridine (4AP: 0.3mM) to induce release by full fusion (FF) exocytosis, or by 1 mM 4AP to induce release by both FF and kiss-and-run (KR)-like exocytosis. The src family selective inhibitor, PP1 (10 microM), increased KCl and 0.3 mM 4AP-evoked Ca2+ -dependent release of glutamate, but had little effect upon exocytosis evoked by 1mM 4AP. PP1 did not affect the release of FM 2-10 under any of the depolarisation conditions used. PP1 also had no effect on overall intracellular calcium levels, as measured by FURA2, suggesting that the effects of the inhibitor are downstream of calcium entry. At the same concentration the inactive analogue of this compound, PP3, had no effect on any measure. Immunoblotting with an antibody to phosphotyrosine revealed that phosphorylation of many synaptosomal proteins was reduced by PP1. The immunoreactivity of three protein bands increased upon depolarisation and this increase was blocked by PP1. Phosphorylation of src at tyrosine-416 was reduced by PP1 but changes in its phosphorylation did not correlate with the effects of PP1 on release. These results suggest one or more members of the src family of tyrosine kinases is a negative regulator of the KR mode of exocytosis in synaptosomes, perhaps by tonically inhibiting KR under normal stimulation conditions.     

Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B

The inhibitors okadaic acid (OA), fostriecin (FOS) and cyclosporin A (CsA), were used to investigate the roles of protein phosphatases in regulating exocytosis in rat brain synaptosomes by measuring glutamate release and the release of the styryl dye FM 2-10. Depolarization was induced by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA and FOS produced a similar partial inhibition of KCl- and 0.3 mM 4AP- evoked exocytosis in both assays, but had little effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA had no effect upon KCl- and 0.3 mM 4AP-evoked exocytosis, but significantly enhanced glutamate release but not FM 2-10 dye release evoked by 1 mM 4AP. None of the phosphatase inhibitors changed calcium signals from FURA-2-loaded synaptosomes either before or after depolarization. Pretreatment with 100 nM phorbol 12-myristate 13-acetate abolished the inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP. Taken together, these results show that exocytosis from synaptosomes has a phosphatase-sensitive and phosphatase-insensitive component, and that there are two modes of phosphatase-sensitive exocytosis that can be elicited by different depolarization conditions. Moreover, these two modes are differentially sensitive to phosphatase 2A and 2B.