Intrasynaptosomal Ca2 Research Articles

Brain synaptosomes display a diadenosine tetraphosphate (Ap4A)‐mediated Ca2 influx distinct from ATP‐mediated influx

Studies undertaken to compare the effects of Ap4A and ATP on altering intrasynaptosomal Ca2+ levels from deermouse brain reveal that both ligands induce a rapid influx of extracellular Ca2+. The Ca2+ profile elicited by 167 μM Ap4A is “spike-like” (half-time for decline to baseline, 19.1 ± 1.2 sec), in contrast to the gradual decline observed with ATP (104.0 ± 7.4 sec). DIDS (4-4′-diisothiocyano-2,2′-disulfonic acid stilbene) and suramin preincubation alter only the ATP-induced Ca2+ profile. Cross-desensitization studies indicate that prior application of ATP does not significantly affect the Ca2+ influx elicited by Ap4A, and that prior application of Ap4A does not affect the Ca2+ influx elicited by ATP. These results demonstrate that extracellular Ap4A and ATP elicit distinct intrasynaptosomal Ca2+ influx profiles, and suggest that these two nucleotides may be interacting with distinct purinoceptor subclasses or purinoceptor-effector complexes. Subjecting the synaptosomes simultaneously to depolarization and Ap4A, or to depolarization and ATP, induces an additive effect on Ca2+ influx. Preincubation with verapamil negates the effects of depolarization without modifying the ligand-elicited Ca2+ fluxes. These results indicate the presence of Ap4A and ATP ligand-gated channels that may function as modulators of neuronal activity. © 1996 Wiley-Liss, Inc.

Diadenosine polyphosphates selectively potentiate N-type Ca2+ channels in rat central neurons.

The action of diadenosine polyphosphates on Ca2+ channels was studied in two preparations: isolated hippocampal neurons and synaptosomes, both from the rat brain. High-voltage-activated Ca2+ channels were recorded in freshly isolated CA3 neurons using a whole-cell patch-clamp technique. Current-voltage relationships were measured in the control and after incubation in 5 microM diadenosine pentaphosphate. In the majority of tested pyramidal neurons, the latter procedure led to a reversible increase in the high-voltage-activated current through Ca2+ channels when measured at the holding potential of -100 mV but not at -40 mV. In experiments on synaptosomes from the whole brain, diadenosine pentaphosphate taken at a concentration of 100 microM increased the intrasynaptosomal calcium level measured by means of spectrofluorimetry for 26 +/- 1.8 nM (by 24 +/- 2%). Nifedipine failed to block this effect both in synaptosomes and hippocampal neurons. Potentiation of the current through Ca2+ channels in hippocampal neurons as well as the increase in intrasynaptosomal Ca2+ were irreversibly blocked by 5 microM omega-conotoxin, but not by 200 nM omega-Agatoxin-IVA. These data indicate that diadenosine polyphosphates enhance the activity of N-type Ca2+ channels in many central neurons of the rat brain.

A novel receptor for diadenosine polyphosphates coupled to calcium increase in rat midbrain synaptosomes

1. Diadenosine polyphosphates, Ap4A and Ap5A, as well as ATP, alpha,beta-MeATP and ADP-beta-S, were able to elicit variable intrasynaptosomal Ca2+ increases in rat midbrain synaptic terminals. The origin of the Ca2+ increment was the extra synaptosomal space since the elimination of extracellular Ca2+ abolished the effect of all the agonists. 2. The P2-purinoceptor antagonist, suramin, did not affect the Ca(2+)-increase evoked by diadenosine polyphosphates but dramatically blocked the Ca2+ entry induced by ATP and its synthetic analogues. 3. The actions of Ap5A and ATP on the intrasynaptosomal Ca2+ increase did not cross-desensitize. 4. Concentration-response studies for diadenosine polyphosphates showed pD2 values of 54.5 +/- 4.2 microM and 55.6 +/- 3.8 microM for Ap4A and Ap5A, respectively. 5. The entry of calcium induced by diadenosine polyphosphates could be separated into two components. The first represented a selective voltage-independent Ca2+ entry; the second, a sustained phase which was voltage-dependent. 6. Studies on the voltage-dependent Ca(2+)-channels involved in the effects of the diadenosine polyphosphates, demonstrated that omega-conotoxin G-VI-A inhibited the sustained Ca(2+)-entry, suggesting the participation of an N-type Ca(2+)-channel. This toxin was unable to abolish the initial cation entry induced by Ap4A or Ap5A. omega-Agatoxin IV-A, tetrodotoxin, or nifedipine did not inhibit the effects of the diadenosine polyphosphates. 7. The effect of ATP on Ca(2+)-entry was abolished by nifedipine and omega-conotoxin G-VI-A, suggesting the participation of L- and N-type Ca(2+)-channels in the response to ATP. 8. These data suggest that Ap4A, Ap5A and ATP activate the same intracellular Ca2+ signal through different receptors and different mechanisms. Ap4A and Ap5A induce a more selective Ca2+-entry in a voltage-independent process. This is the first time that a selective action of diadenosine polyphosphate through receptors other than P1 and P2-purinoceptors has been described.

Adenosine A1 receptors in rat brain synaptosomes: Transductional mechanisms, effects on glutamate release, and preservation after metabolic inhibition

AbstractSynaptosomes from various rat brain areas show saturable and specific binding to an adenosine A1 receptor ligand [Dagani et al. (1993): Drug Dev Res 28:359–363]. In this study, the functional correlates of these receptors were characterized in rat hippocampal synaptosomes by evaluating the ability of the adenosine analogue cyclo‐pentyl‐adenosine (CPA) to modulate KCI‐ and veratridine‐induced glutamate outflow. CPA concentration‐dependently reduced both depolarization‐induced glutamate release and the associated increases of intrasynaptosomal Ca2+ concentrations. Maximal reduction by 100 μM CPA (30% and 40% with respect to control for veratridine‐ and KCI‐induced release, respectively) was completely antagonized by the xanthine adenosine receptor blocker bamyfilline, confirming the involvement of adenosine A1 receptors. CPA selectively affected outflow of glutamate, with no significant influence on release of aspartate or GABA. Experiments performed in the absence of extrasynaptosomal Ca2+ ions suggested that adenosine selectively modulates glutamate Ca2+ ‐dependent vesicular pool. Transductional studies showed that mobilization of Ca2+ from intrasynaptosomal inositol‐phosphate‐sensitive stores does not contribute to adenosine effects on release, therefore implying that CPA reduction of Ca2+ influx is due to direct effects on membrane Ca2+ conductance. Conversely, activation of A1 receptors resulted in inhibition of forskolin‐stimulated cAMP production in the same agonist concentration range effective on modulation of release, suggesting that this second messenger may play a role in the presynaptic effects of adenosine analogues. Finally, CPA reduced both glutamate efflux and the increases of internal Ca2+ concentrations associated with block of synaptosomal energy metabolism with rotenone and iodoacetic acid, suggesting that presynaptic A1 receptors represent a strategic site of action for adenosine also under experimental conditions resembling brain ischemia and hypoxia. © 1995 Wiley‐Liss, Inc.

Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome.

To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium.

Nitric oxide stimulates Ca 2+-independent synaptic vesicle release

A new fluorescence method using the dye FM1-43 was used to examine exocytotic release from hippocampal synaptosomes. Nitric oxide caused a marked transient stimulation of vesicle release. Several structurally unrelated nitric oxide donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, 3-morpholino-sydnonimine, and acidified sodium nitrite, were effective. Release stimulated by nitric oxide and KCI were comparable in time course, using both the fluorescence assay and [ 3H] l-glutamate to monitor neurotransmitter release. Activation of guanylyl cyclase was not responsible for nitric oxide-stimulated release. Unlike release stimulated by KCl or A23187, nitric oxide-stimulated release was found to be independent of a rise in intrasynaptosomal Ca 2+. Indo-1/AM measurements indicated that nitric oxide actually decreased intracellular Ca 2+, and the Ca 2+ channel blocker Cd 2+ did not affect nitric oxide-stimulated vesicle release. Nitric oxide does, however, appear to act on the Ca 2+-sensitive pool of vesicles. Nitric oxide may be the first physiological mediator that induces vesicle exocytosis without raising Ca 2+ and may provide an interesting new tool for the study of molecules involved in vesicle exocytosis.

Aniracetam improves behavioural responses and facilitates signal transduction in the rat brain

The effect of aniracetam (10, 50, 100 mg/kg i.p. daily for 15 days) on both behavioural and biochemical parameters was investigated in the adult rat. Animals given aniracetam (50 mg/kg 1 h before the trial) showed a significant increase in the percentage of conditioned active avoidance responses and a reduction of latency times. Aniracetam significantly counteracted the scopolamine-induced memory failure at the passive avoidance (step down) test, while it did not modify the locomotion of the animals. In purified frontocortical and hippocampal synaptic membranes of rats treated with aniracetam (50 mg/kg i.p. daily for 15 days) a potentiation of basal, carbamylcholine-, dopamine- and norepinephrine-stimulated adenylyl cyclase activity was observed, while forskolin-stimulated enzyme activity was not modified. With regard to inositol phosphate production measured in fronto-cortical synaptoneurosomes, aniracetam potentiated the stimulation by angiotensin II, while the stimulation by carbamylcholine, not affected by 10 and 50 mg/kg aniracetam, was notably, although not significantly, decreased by 100 mg/kg aniracetam. Furthermore, in synaptosomes derived from hippocampus, aniracetam (50 mg/kg i.p. daily for 15 days) caused an increase of both basal and K(+)-stimulated intrasynaptosomal Ca(2+) concentration. In conclusion, a correlation between the improvement of behavioural performance and the modulation of transducing systems by aniracetam seems to take place in brain areas, such as frontal cortex and hippocampus, known to play a major role in the control of cognitive functions.

Adenosine A 1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [ 3H]cyclohexyladenosine (CHA) to the cellular fractions and P 2 subfractions of the goldfish brain was studied. The A 1 receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P 2), 30 mM K + stimulated glutamate, taurine and GABA release in a Ca 2+-dependent fashion, whereas the aspartate release was Ca 2+-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K +-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A 1 receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K + (30 mM) stimulated Ca 2+ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca 2+ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A 1 receptor inhibition of glutamate release and inhibition of calcium influx is discussed.

Action and binding of omega-conotoxin on the putative calcium channel of synaptosomal plasma membrane from electric organ of Japanese electric ray, Narke japonica

Actions of ω-conotoxin GVIA on synaptosomes isolated from a Japanese electric ray, Narke japonica, were investigated. ω-Conotoxin inhibited, in a dose-dependent manner, both increases in free calcium concentration in, and acetylcholine release from synaptosomes depolarized with a high concentration of potassium ions. The concentrations of ω-conotoxin required for half-maximal inhibition ( ic 50) of increase in intrasynaptosomal Ca and acetylcholine release were 8 and 7 μM, respectively. Assay using radioiodinated toxin derivative revealed a specific binding site with a dissociation constant ( K D) of 2.8 μM and a density ( B max) of 45 pmol/mg protein of synaptosome. Binding assay with synaptosomal plasma membrane showed a K D = 7μ M and a B max = 200pmol/mg protein. Autoradiography with sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after covalent cross-linking of the toxin, using disuccinimidyl suberate, revealed the 170,000 mol. wt peptide to be an ω-conotoxin receptor. The present study has directly and clearly shown that ω-conotoxin inhibits acetylcholine release by blocking Ca influx into nerve terminals. The 170,000 mol. wt peptide identified as a receptor of the toxin exists in high density in the plasma membrane of the presynaptic nerve terminal and is likely to be a component of a voltage-dependent Ca channel responsible for the neurotransmitter release.

Parameters not influenced by vesamicol: membrane potential, calcium uptake, and internal calcium concentration of synaptosomes.

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca(2+)-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca(2+)-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca(2+)-uptake, and intracellular Ca2+ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 microM veratridine (from 67 +/- 2.3 mV resting membrane potential to 50.7 +/- 2.5 mV) was not significantly influenced by vesamicol (1-20 microM). Vesamicol (1-20 microM) had no effect on either the overall curve of the veratridine-evoked 45Ca2+ uptake or the amount of Ca2+ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca2+ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 microM). The K(+)-evoked (40 mM) increase of Ca2+ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 microM) vesamicol inhibited both the fall in membrane potential and the elevated Ca2+ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na+ channels at this concentration. Vesamicol, in lower concentration (20 microM) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [14C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [14C]choline.(ABSTRACT TRUNCATED AT 250 WORDS)

The facilitating and suppressing effects of Δ9-tetrahydrocannabinol on the rise in intrasynaptosomal Ca 2+ concentration in rats

The effects of Δ 9-tetrahydrocannabinol ( Δ 9-THC) on the rise in intracellular Ca 2+ concentrations ([Ca 2+]i) after stimulation with 15 mM or 29 mM K + in rat whole brain synaptosomes were examined. A fluorescent chelating agent, Rhod-2, was employed to monitor any alterations of K +-evoked [Ca 2+] i. Pretreatment with 10 −10 M Δ 9-THC for 3 min enhanced K +-evoked [Ca 2+] i significantly, while 10 −9, 10 −8 or 5 × 10 −8 M Δ 9-THC significantly inhibited the K +-evoked [Ca 2+] i. These results suggest that Δ 9-THC had a biphasic effect on the K +-evoked Ca 2+ response in rat brain synaptosomes.

Advances in the use of the fluorescent probe fura-2 for the estimation of intrasynaptosomal calcium.

Fura-2 has been used to measure intracellular Ca2+ with great success in a variety of cell and subcellular preparations, including synaptosomes. There is, however, a great deal of variability in the reported estimates of resting intrasynaptosomal Ca2+ ([Ca2+]i). Fura-2 AM is highly lipophilic and passes readily across the plasma membrane into the cytoplasm, where it is de-esterified and trapped. The lipophilicity of fura-2, however, promotes the formation of micelles in aqueous media, which may impede the passage of the probe across cell membranes. Our results suggest that some of the variability in the reported [Ca2+]i estimates may be related to fura-2 de-esterification and loading efficiencies. The use of the nonionic detergent pluronic F-127 is recommended to prevent the formation of fura-2 micelles. The use of a detergent is not always an acceptable practice, however, especially in studies in which detergent-lipid interactions may influence membrane parameters. We found that fatty acid free bovine serum albumin (BSA) (0.25%) greatly increases the intrasynaptosomal concentration of the probe, resulting in a significant increase in the signal-to-noise (S/N) ratio. The mechanism appears to be independent of effects of BSA on synaptosomal integrity and directly related to the prevention of fura-2 micelle formation, as evidenced by light spectroscopic scattering measurements. Thus, BSA appears to keep the probe in a form that crosses the synaptic plasma membrane more readily. The effectiveness of BSA in improving the loading of fura-2 into synaptosomes was comparable to the detergent pluronic F-127, making it possible to measure [Ca2+]i without compromising membrane integrity.

Rapid kinetics of depolarization-induced changes in intrasynaptosomal calcium concentrations

The rapid kinetics of depolarization-evoked calcium influxes in isolated nerve terminals from rat cortex were monitored by stopped-flow spectrofluorimetry using specific indicators (Fluo-3, Indo-1). A very rapid increase in the intrasynaptosomal Ca 2+-level was detected within the subsecond time range after depolarizing synaptosomes by mixing with physiological saline containing elevated K +-concentrations. About 15 mM [K +] o was determined as threshold concentration for inducing Ca 2+-influx, which increased with higher concentration and saturated at [K +] o-concentrations of about 40 mM [K +] o.

Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel.

Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca2+ regulated events, we have studied its effects on the compartmentation of free Ca2+ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca2+ concentration ([Ca2]i) to micromolar levels. The increase in [Ca2]i was not transient and a steady rise was observed with time. Omission of Ca2+ from the external medium prevented the AM- and DEA-induced rise in [Ca2+]i suggesting that AM and DEA increased the intracellular [Ca2+]i due to increased influx of Ca2+ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca2+]i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na+ channel blocker, tetrodotoxin. However, the blockade of [Ca2+]i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca2+]i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca2+]i through receptor mediated channel. 45Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca2+ release from these intracellular organelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ethanol on phospholipid turnover and calcium mobilization in chick embryos

Effects of ethanol on [ 3H]inositol and [ 14C]choline incorporation into phosphatidylinositol (PI) and phosphatidylcholine (PC), free intrasynaptosomal Ca 2+ ([Ca 2+] i) and synaptosomal 45Ca 2+ uptake, were investigated in the brain and heart of 17-day-old chick embryos to which a 10% ethanol solution had been injected on the 3rd day of embryogenesis. In brain synaptosomes, ethanol increased the incorporation of [ 3H]inositol and [ 14C]choline into PI and PC, increased [Ca 2+] i, and decreased 45Ca 2+ uptake. On the other hand, in heart synaptosomal membrane, ethanol decreased the incorporation of [ 3H]inositol and [ 14C]choline into PI and PC, decreased [Ca 2+] i, and increased 45Ca 2+ uptake. Ethanol stimulated in vitro [ 3H]inositol and [ 14C]choline incorporation into PI and PC in the brain and heart in born the control and ethanol-treated groups. However, addition of ethanol did not affect the release of 45Ca 2+ from the synaptosomal membrane of either organ in either group. Addition of ethanol inhibited 45Ca 2+ uptake in a dose-dependent manner in the brain but not in the heart. In both organs, there was a relationship between phospholipid turnover and [Ca 2+] i after ethanol.

A comparative study of histamine and K + effects on (Ca 2+Mg 2+)-ATPase activity in synaptosomes

Histamine (10 −4M) and 60 mM K +, but not 60 mM Na + or 60 mM choline +, increased the maximal synaptosomal (Ca 2+-Mg 2+)-ATPase activity by 15 and 36% respectively and decreased the extrasynaptosomal Ca 2+ concentration necessary to reach it. Histamine and K + enhanced the synaptosomal (Ca 2+-Mg 2+)-ATPase activity in a concentration-dependent manner. In synaptic plasma membranes histamine (10 −4M) and 60 mM choline + were not able to alter the enzymatic activity, however 60 mM K + and 60 mM Na + elevated (Ca 2+-Mg 2+)-ATPase activity by 20 and 15%, repectively, without altering the affinity for Ca 2+. Histamine effects in synaptosomes were mediated by H 2 receptor stimulation. 3-Isobutyl-1-methyl-xanthine (10 −4M) potentiated (15%) the maximal histamine effect. The slow Ca 2+ channel antagonists verapamil and diltiazem, both at 10 −6M, completely inhibited K + effects in synaptosomes, however histamine effects were only blocked by verapamil. The data suggest that K + and histamine effects on synaptosomal (Ca 2+-Mg 2+)-ATPase activity are mediated by increases of intrasynaptosomal Ca 2+ levels. Moreover, histamine effects on synaptosomal enzyme activity were mediated by cAMP.

Diazepam enhances intrasynaptosomal free calcium concentrations

Synaptosomes prepared from brains of rats were incubated in different concentrations of diazepam under conditions designed to reduce the action of a reversed Na +/Ca 2+ exchanger. In synaptosomes depolarized in the presence of added Ca 2+, doses of diazepam ranging from 0.1 to 100 μM were found to significantly enhance Ca 2+ levels measured with the fluorescent dye fura-2, compared to control incubations without drug. Furthermore, doses of diazepam as low as 1 μM significantly increased the concentration of Ca 2+ in non-depolarized synaptosomes without added Ca 2+ in the medium. The effects of depolarization and diazepam treatment were synergistic in increasing the levels of intrasynaptosomal Ca 2+.

Effect of maitotoxin on cytosolic Ca 2+ levels and membrane potential in purified rat brain synaptosomes

In this study, the effects of the marine toxin maitotoxin on cytosolic Ca 2+ levels and membrane potential in rat brain synaptosomes were evaluated. Maitotoxin (10 ng/ml) caused a remarkable increase of intrasynaptosomal Ca 2+ levels monitored by the fluorescent probe fura-2. This increase was prevented by the removal of external Ca 2+ ions. Tetrodotoxin, as well as the removal of extracellular Na + ions, failed to affect maitotoxin-induced increase of intrasynaptosomal Ca 2+ levels. Also the complete removal of all monovalent and divalent cations, except Ca 2+ ions, from the incubation medium (0.32 M sucrose substitution), was unable to prevent the effect of maitotoxin on intrasynaptosomal Ca 2+ levels. Maitotoxin (0.3–10 ng/ml), produced a dose-dependent depolarization of synaptosomal membranes, which required the presence of extracellular Ca 2+ ions. The substitution of extracellular Na + with choline or the removal of all cations from the incubation medium and their replacement with an isotonic concentration of sucrose (0.32 M), did not prevent the depolarizing effect exerted by maitotoxin. Also under these two ionic conditions, the effect of maitotoxin on membrane potential was critically dependent on the presence of 1 mM extracellular Ca 2+. The depolarizing effect exerted by maitotoxin on synaptosomal membrane potential was also observed when extracellular Ca 2+ ions were substituted with an equimolar concentration of Ba 2+ or Sr 2+ ions. In summary, these results appear to suggest that, in presence of 1 mM extracellular Ca 2+ ions, maitotoxin depolarizes synaptosomal plasmamembrane by promoting the influx of extracellular Ca 2+ ions. This enhanced influx of Ca 2+ causes an increase of intrasynaptosomal Ca 2+ levels.

Differential Calcium Dependence Between the Release of Endogenous Dopamine and Noradrenaline from Rat Brain Synaptosomes

The characteristics of the release of endogenous dopamine and noradrenaline from rat brain synaptosomes were studied using HPLC with an electrochemical detector. The spontaneous release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. Also, the high-K+ (30 mM)-evoked release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. From these results, the ratio of the Ca2(+)-dependent component to the total release of noradrenaline seemed to be similar to that of dopamine. On the other hand, 20 microM La3+ or 1 microM diltiazem inhibited both the spontaneous and 30 mM K(+)-evoked release of dopamine by approximately 50-60% but inhibited neither the spontaneous nor the 30 mM K(+)-evoked release of noradrenaline. The K(+)-evoked rise in intrasynaptosomal Ca2+ concentration was mostly blocked in Ca2(+)-free medium or 100 microM La3(+)-containing medium but was only partially blocked by 20 microM La3+ or 1 microM diltiazem. These data indicate alternative possibilities in that the Ca2(+)-dependent release of noradrenaline might be less sensitive to a change of intracellular Ca2+ concentration than that of dopamine and that the calcium channels directly involved in the noradrenaline release may be more resistant to diltiazem and La3+ than those involved in the dopamine release.

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.