An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.     

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells

Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.     

Ca2+-Independent Release of Glutamate During In Vitro Anoxia in Isolated Nerve Terminals

The effects of in vitro anoxia on the release of glutamate in isolated nerve terminals were studied. The extra-synaptosomal concentration of glutamate ([Glu]ext) under aerobic conditions was 2.3 microM and increased to 4.9 microM after 10 min of anoxia. However, when synaptosomes were incubated in the presence of lactate plus pyruvate instead of glucose, to prevent anaerobic glycolysis, anoxia induced an eightfold increase in the [Glu]ext. The accumulation of glutamate in the external medium during anoxia was Ca2+ independent and insensitive to a significant reduction of the Ca(2+)-dependent release of the amino acid. These results indicate that a Ca(2+)-independent efflux of cytoplasmic glutamate occurs during in vitro anoxia in isolated nerve terminals.     

Facilitatory effect of aspirin on glutamate release from rat hippocampal nerve terminals: involvement of protein kinase C pathway

The effect of aspirin on glutamate release from isolated nerve terminals (synaptosomes) from rat hippocampus was examined. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4AP) was facilitated by aspirin in a concentration-dependent manner, but the 4AP-evoked Ca(2+)-independent release was not modified. Also, aspirin-mediated facilitation of glutamate release was completely inhibited by bafilomycin A1, which depletes vesicle content by inhibiting the synaptic vesicle H(+)-ATPase that drives glutamate uptake, not by l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a excitatory amino acid (EAA) transporter inhibitor, suggesting that the facilitation of glutamate release produced by aspirin originates from synaptic vesicle exocytosis rather than reversal of the plasma membrane glutamate transporter. In addition, aspirin did not alter either 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release, but significantly increased in 4AP-evoked Ca(2+) influx. A possible effect of aspirin on synaptosomal Ca(2+) channels was confirmed in experiments where synaptosomes pretreated with a combination of the N- and P/Q-type Ca(2+) channel blockers, which abolished the aspirin-mediated facilitation of glutamate release. The facilitatory action by aspirin observed in glutamate release was mimicked and occluded by arachidonic acid (AA) and eicosatetraynoic acid (ETYA), an analogue of AA that mimics the effect of AA but cannot be metabolized. Furthermore, this aspirin-mediated facilitation of glutamate release may depend on activation of protein kinase C (PKC), because PKC activator and PKC inhibitor, respectively, superseding or suppressing the facilitatory effect of aspirin. Together, these results suggest that aspirin exerts their presynaptic facilitatory effect, likely through AA directly to induce the activation of PKC, which subsequently enhances the Ca(2+) influx through voltage-dependent N- and P/Q-type Ca(2+) channels to cause an increase in evoked glutamate release from rat hippocampal nerve terminals.     

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.     

Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

Intracellular Ca2+ mobilization by arachidonic acid. Comparison with myo-inositol 1,4,5-trisphosphate in isolated pancreatic islets

Previous studies have demonstrated that myo-inositol 1,4,5-trisphosphate (IP3) mobilizes Ca2+ from the endoplasmic reticulum (ER) of digitonin-permeabilized islets and that an increase in intracellular free Ca2+ stimulates insulin release. Furthermore, glucose stimulates arachidonic acid metabolism in islets. In digitonin-permeabilized islets, exogenous arachidonic acid at concentrations between 1.25 to 10 microM elicited significant Ca2+ release from the ER at a free Ca2+ concentration of 0.1 microM. Arachidonic acid-induced Ca2+ release was not due to the metabolites of arachidonic acid. Arachidonic acid induced a rapid release of Ca2+ within 2 min. Comparison of arachidonic acid-induced Ca2+ release with IP3-induced Ca2+ release revealed a similar molar potency of arachidonic acid and IP3. The combination of both arachidonic acid and IP3 resulted in a greater effect on Ca2+ mobilization from the ER than either compound alone. The mass of endogenous arachidonic acid released by islets incubated with 28 mM glucose was measured by mass spectrometric methods and was found to be sufficient to achieve arachidonic acid concentrations equal to or exceeding those required to induce release of Ca2+ sequestered in the ER. These observations indicate that glucose-induced arachidonic acid release could participate in glucose-induced Ca2+ mobilization and insulin secretion by pancreatic islets, possibly in cooperation with IP3.     

Increase of [Ca(2+)]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophageal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachidonic acid interact synergistically to activate protein kinase C. We have therefore investigated the relationship between cytosolic Ca(2+) and activation of phospholipase A(2) in response to acetylcholine-induced stimulation, by measuring the intracellular free Ca(2+) ([Ca(2+)]i), muscle tension, and [3H] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca(2+)]i and arachidonic acid release in a dose-dependent manner. In Ca(2+)-free medium, acetylcholine did not produce contraction, [Ca(2+)]i increase, and arachidonic acid release. In contrast, after depletion of Ca(2+) stores by thapsigargin (3 microM), acetylcholine caused a normal contraction, [Ca(2+)]i increase and arachidonic acid release. The increase in [Ca(2+)]i and arachidonic acid release were attenuated by the M2 receptor antagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hexahydro siladifenidol. Increase in [Ca(2+)]i and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findings indicate that contraction and arachidonic acid release are mediated through muscarinic M2 coupled to Gi or rho protein activation and Ca(2+) influx. Acetylcholine-induced contraction and the associated increase in [Ca(2+)]i and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid and a phospholipase D inhibitor pCMB. They increased by the action of the inhibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal circular muscle acetylcholine-induced [Ca(2+)]i increase and arachidonic acid release are mediated through activation of M2 receptor coupled to Gi or rho protein, resulting in the activation of phospholipase A(2) and phospholipase D to activate protein kinase C.     

Phenylarsine oxide inhibits alpha-latrotoxin-stimulated [3H]GABA release from rat brain synaptosomes

Phosphatidylinositol 4,5-biphosphate has been implicated in a variety of membrane-trafficking processes, including exocytosis of neurotransmitters. However, there are contradictory findings concerned ability of phenylarsine oxide (PAO), an inhibitor of phosphatidylinositol 4-kinase, to affect exocytotic release of different types of neurotransmitters. We bent our efforts to a detailed analysis of action of PAO on Ca(2+)-dependent and Ca(2+)-independent [3H]GABA release produced by exposure of rat brain synaptosomes to different concentrations of alpha-latrotoxin. We also compared PAO action on alpha-latrotoxin- and 4-aminopyridine (4-AP)-evoked [3H]GABA release. The experiments have shown that release of [3H]GABA evoked by the depolarization with 4-AP was decreased by 80% as a result of action of 3 microM PAO and the complete inhibition of release was observed with 10 microM PAO. When alpha-latrotoxin as a stimulant was applied, release of [3H]GABA was increased as toxin concentration used was elevated from 0.5 to 3.0 nM, however, concomitantly, the response of the toxin-induced [3H]GABA release to PAO became attenuated: 10 microM PAO led to almost complete inhibition of the effect of 0.5 nM alpha-latrotoxin and only partly decreased (by 40%) the response to 3.0 nM alpha-latrotoxin. To test whether the efficacy of PAO depended on the toxin-induced outflow of cytosolic [3H]GABA, synaptosomes with depleted cytosolic [3H]GABA pool were also exploited. Depletion was performed by means of heteroexchange of cytosolic [3H]GABA with nipecotic acid. The experiments have shown that treatment of loaded synaptosomes with nipecotic acid resulted in some increase of [3H]GABA release evoked by 0.5 nM alpha-latrotoxin, but in the two-fold decrease of the response to 3.0 nM alpha-latrotoxin. PAO essentially inhibited [3H]GABA release from depleted synaptosomes irrespective of alpha-latrotoxin concentration used. Therefore, the amount of [3H]GABA released from cytosolic pool determined, in considerable degree, the insensitivity of alpha-latrotoxin action to PAO. Thus, our data show that subnanomolar concentrations of alpha-latrotoxin may be used for stimulation of exocytotic release of [3H]GABA. Exposure of synaptosomes with nanomolar toxin concentrations leads not only to stimulation of exocytosis, but also to leakage of [3H]GABA from cytosolic pool. PAO potently inhibits exocytotic release of [3H]GABA and its inhibitory effectiveness is diminished as far as the outflow of [3H]GABA is elevated.     

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Lead-dependent effects on arachidonic acid accumulation and the proliferation of vascular smooth muscle

Lead (Pb(2+)) has been implicated in the development of hypertension and atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) is a central feature of both conditions and there is evidence that Pb(2+) potentiates serum-dependent cell growth. The aim of this work was to examine the role of phospholipase A(2) in mitogen-dependent VSMC proliferation and determine if Pb(2+) interacts with this system in order to potentiate mitotic events. It was observed that cell proliferation induced by angiotensin II, or fetal bovine serum, required the activation of a Ca(2+)-dependent cytosolic phospholipase A(2) and the subsequent release of unesterified arachidonic acid. This path was affected by Pb(2+) as the metal increased the amount of arachidonic acid accumulation induced by either mitogen. In addition, Pb(2+) potentiated mitogen-induced DNA synthesis when present at lower doses (0.02 or 0.2 mg%), but had no effect on DNA synthesis, or cell numbers, in unstimulated cells. However, a high dose (2 mg%) of Pb(2+) attenuated the DNA synthesis stimulated by angiotensin II, or serum, but induced the accumulation of unesterified arachidonic acid in unstimulated cells. A biphasic effect of Pb(2+) on cell numbers and viability was also observed as 0.02 or 0.2 mg% Pb(2+) did not affect cell numbers or trypan blue exclusion in unstimulated cells, while 2 mg% Pb(2+) reduced cell numbers and viability. It appeared, therefore, that the lower concentrations of Pb(2+) increased arachidonic acid release and DNA synthesis only in stimulated VSMC, perhaps due to further activation of a Ca(2+)-dependent processes. In contrast, the high dose of Pb(2+) reduced DNA synthesis in stimulated cells and reduced cell numbers and viability in unstimulated cells, which may relate to the noted increase in unesterified arachidonic acid.     

Arachidonic acid-induced calcium influx in human platelets. Comparison with the effect of thrombin.

The effects of arachidonic acid and thrombin on calcium movements have been studied in fura-2-loaded platelets by a procedure which allows simultaneous monitoring of the uptake of manganese, a calcium surrogate for Ca2+ channels, and the release of Ca2+ from intracellular stores. Arachidonic acid induced both Ca2+ (Mn2+) entry through the plasma membrane and Ca2+ release from the intracellular stores. The release of Ca2+ was prevented by cyclo-oxygenase inhibitors and mimicked by the prostaglandin H2/thromboxane A2 receptor agonist U46619. Ca2+ (Mn2+) entry required higher concentrations of arachidonic acid and was not prevented by either cyclo-oxygenase or lipoxygenase inhibitors. Several polyunsaturated fatty acids reproduced the effect of arachidonic acid on Ca2+ (Mn2+) entry, but higher concentrations were required. The effects of maximal concentrations of arachidonic acid and thrombin on the uptake of Mn2+ were not additive. Both agonists induced the entry of Ca2+, Mn2+, Co2+ and Ba2+, but not Ni2+, which, in addition, blocked the entry of the other divalent cations. However, arachidonic acid, but not thrombin, increased a Ni2(+)-sensitive permeability to Mg2+. The effect of thrombin but not that of arachidonic acid was prevented either by pretreatment with phorbol ester or by an increase in cyclic-AMP levels. Arachidonic acid also accelerated the uptake of Mn2+ by human neutrophils, rat thymocytes and Ehrlich ascites-tumour cells.     

Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes

The objective of this study was to get a more understandable picture of the mechanism underlying the anticonvulsant action of vinpocetine. The question of how the cerebral excitability is affected was investigated by determining the effect of vinpocetine on the changes on the internal concentrations of Na(+) (Na(i)) and Ca(2+) (Ca(i)) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Na(i) and Ca(i) were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na(+) channel blocker, tetrodotoxin, abolished the increase in Na(i) induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Na(i) induced by 1mM 4-AP. In contrast with the different sensitivity of the rise in Na(i) induced by 0.1 and 1mM 4-AP to vinpocetine and tetrodotoxin, the rise in Ca(i) induced by the two concentrations of 4-AP was markedly inhibited by vinpocetine (and tetrodotoxin), indicating that only the voltage-sensitive sodium channels (VSSC)-mediated fraction of the rise in Na(i) induced by 4-AP is linked with the activation of pre-synaptic Ca(2+) channels. The elevation of Ca(2+) induced by high K(+) (30 mM) does not require a Na(+) gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Ca(i) induced by 4-AP, requires a physiological (out/in) Na(+) gradient and is vinpocetine and tetrodotoxin-sensitive. It is concluded that by blocking the tetrodotoxin-sensitive fraction of the rise in Na(i) induced by 4-AP, vinpocetine inhibits the concomitant rise in Ca(i) induced by 4-AP. The inhibitory effect of vinpocetine on pre-synaptic voltage-sensitive sodium channels may underlie the in vivo anticonvulsant action of vinpocetine.     

Presynaptic effects of anandamide and WIN55,212-2 on glutamatergic nerve endings isolated from rat hippocampus

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.     

Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis

Abstract: The effects of arachidonic acid and phorbol esters in the Ca²⁺-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 n M ) of 4β-phorbol dibutyrate (4β-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4β-PDBu (1–50 n M ) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.     

Arachidonic acid enhances intracellular [Ca2+]i increase and mitochondrial depolarization induced by glutamate in cerebellar granule cells

Maturation of primary neuronal cultures is accompanied by an increase in the proportion of cells that exhibit biphasic increase in free cytoplasmic Ca2+ ([Ca2+]i) followed by synchronic decrease in electrical potential difference across the inner mitochondrial membrane (DeltaPsim) in response to stimulation of glutamate receptors. In the present study we have examined whether the appearance of the second phase of [Ca2+]i change can be attributed to arachidonic acid (AA) release in response to the effect of glutamate (Glu) on neurons. Using primary culture of rat cerebellar granule cells we have investigated the effect of AA (1-20 microM) on [Ca2+]i, DeltaPsim, and [ATP] and changes in these parameters induced by neurotoxic concentrations of Glu (100 microM, 10-40 min). At =10 microM, AA caused insignificant decrease in DeltaPsim without any influence on [Ca2+]i. The mitochondrial ATPase inhibitor oligomycin enhanced AA-induced decrease in DeltaPsim; this suggests that AA may inhibit mitochondrial respiration. Addition of AA during the treatment with Glu resulted in more pronounced augmentation of [Ca2+]i and the decrease in DeltaPsim than the changes in these parameters observed during independent action of AA; removal of Glu did not abolish these changes. An inhibitor of the cyclooxygenase and lipoxygenase pathways of AA metabolism, 5,8,11,14-eicosatetraynoic acid, increased the proportion of neurons characterized by Glu-induced biphasic increase in [Ca2+]i and the decrease in DeltaPsim. Palmitic acid (30 microM) did not increase the percentage of neurons exhibiting biphasic response to Glu. Co-administration of AA and Glu caused 2-3 times more pronounced decrease in ATP concentrations than that observed during the independent effect of AA and Glu. The data suggest that AA may influence the functional state of mitochondria, and these changes may promote biphasic [Ca2+]i and DeltaPsim responses of neurons to the neurotoxic effect of Glu.     

Correlation of intracellular and extracellular calcium ion concentrations with synergy between 1,2-dioctanoyl-sn-glycerol and ionomycin in platelet arachidonic acid mobilization

The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites. This hypothesis was confirmed by simultaneous measurements of [Ca2+]i increases, and the production of the arachidonic acid metabolites. With the aequorin method, the optimal concentrations of [Ca2+]i fell to between 10 microM and 20 microM, and with the fura-2 method, it fell to between 800 nM and 1800 nM. Direct measurements of [14C]arachidonic acid release suggested that the DiC8-potentiated production of arachidonic acid metabolites induced by ionomycin was attributable to increased arachidonic acid release. Since ionomycin and DiC8 induced relatively low levels of phosphatidic acid production, an indicator of phospholipase C activation, it was suggested that the increased arachidonic acid release was largely dependent upon phospholipase A2. Synergy between DiC8 and ionomycin was also observed with aggregation and serotonin release. Aggregation was induced by lower concentrations of ionomycin, and appeared to be more dependent upon extracellular Ca2+, while serotonin release required higher concentrations of ionomycin, and variations in extracellular Ca2+ affected the response minimally. These findings suggest that the mechanisms underlying the synergy between protein kinase C activation and Ca2+ mobilization differ among the three functions evaluated in this study.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.