Interrelationship between secretion, protein phosphorylation and intracellular Ca2+ concentration in platelets stimulated by thrombin or thromboxane A2 analogue

The interrelationship between ATP-secretion, protein phosphorylation and intracellular Ca²⁺ concentration ([Ca²⁺]i) was studied in both ³²P and quin 2 loaded human platelets stimulated by thrombin or thromboxane A₂ analogue (STA₂). In platelets stimulated by thrombin, the degree of 47,000 dalton polypeptides (P47) phosphorylation was observed in completely dose-related manner, regardless of the amount of [Ca²⁺]i. In the same condition, the degree of myosin light chain (P20) phosphorylation, however, was well correlated with ATP secretion and [Ca²⁺]i, when platelets were stimulated by lower dose of thrombin. The similar results were obtained in platelets stimulated by STA₂. These findings suggested that P20, but not P47, phosphorylation in activated platelets is mediated by a rise of [Ca²⁺]i and is well correlated with the secretory reaction. It was unlikely that P47 phosphorylation plays any role in promoting platelet activation.     

Elevation of platelet cyclic AMP level by thromboxane A2/prostaglandin H2 receptor agonists.

Arachidonic acid (AA)-or thromboxane A₂/prostaglandin H₂ (TXA₂/ PGH₂) analog (STA₂ and U-46619)-induced aggregations yielded a bell-shaped dose-response curve. The inhibitory mechanism by high concentrations of the agonists was examined. STA₂ elevated cAMP level of platelet in a dose-dependent manner. And the aggregation was affected by metabolic inhibitors of cAMP. AA also rised cAMP level, and the rise was suppressed by indomethacin. These results indicate that the reduction of aggregation by high dose of the agonists is through cAMP elevation. The cAMP elevation was not suppressed by ruling out phospholipase C effects by chelation of cytoplasmic Ca²⁺ and inhibition of protein kinase C (PKC). These results suggest that the cAMP elevation is not due to activation of phospholipase C-linked TXA₂/PGH₂ receptor. 13-APA, an antagonist of TXA₂/PGH₂ receptor, suppressed the cAMP elevation, although ONO-3708, another antagonist, had no effect. As to be expected from this result, inhibitory effect of 13-APA on high STA₂ level-induced aggregation was weaker than that of ONO-3708. The antagonists did not inhibit PGE,- or PGD₂-induced cAMP elevation. These findings suggest that platelet has adenylate cyclase-linked TXA₂/PGH₂ receptor.     

Intracellular Ca activates phospholipase C

It is well established that a receptor-mediated mechanism, perhaps involving a guanine nucleotide binding protein, directly activates polyphosphoinositide-specific phospholipase C. Recent evidence indicates that in excitable tissues a rise in cytosolic Ca²⁺ can also activate the phospholipase C. The activation of phospholipase C by Ca²⁺ can be a direct effect rather than a result of the Ca²⁺-dependent release of neurotransmitters which activate phospholipase C through a receptor-mediated mechanism. Ca²⁺-activated phospholipase C may represent a positive feedback system for Ca²⁺: small increases in cytosolic Ca²⁺ induced by Ca²⁺ influx across the plasma membrane may result in higher cytosolic Ca²⁺ concentrations due to IP₃-induced release of Ca²⁺ from intracellular stores. The activation of phospholipase C by Ca²⁺ may also provide a mechanism for diacylglycerol generation and protein kinase C activation following Ca²⁺ influx. Thus, the regulation of phospholipase C activity by Ca²⁺ may be physiologically important in regulating cytosolic Ca²⁺ and protein kinase C in excitable tissues.     

Species differences in platelet responses to thrombin and SFLLRN. receptor-mediated calcium mobilization and aggregation, and regulation by protein kinases

The thrombin receptor on human platelets is activated by thrombin to stimulate platelet aggregation through the tethered ligand SFLLRN. This study examined the effects of thrombin and SFLLRN on aggregation and calcium mobilization ([Ca²⁺]_i) in rat, guinea pig, rabbit, dog, monkey, and human platelets, and the role of protein kinases in regulating these functions. Thrombin induced platelet aggregation and [Ca²⁺]_i in all species studied; however, only guinea pig, monkey and human platelets were responsive to SFLLRN. Similar species specific effects were obtained with [Ca²⁺]_i studies. The kinetic profile for [Ca²⁺]_i differed among species, suggesting that regulatory mechanisms for calcium differed between agonists and among species. Staurosporine, a non-selective inhibitor of protein kinases, inhibited platelet aggregation induced by thrombin or SFLLRN in all species. Staurosporine inhibited thrombin-induced [Ca²⁺]_i in guinea pigs, had no effect in rat, and increased [Ca²⁺]_i in all other species. Staurosporine inhibited SFLLRN-induced [Ca²⁺]_i in guinea pig, yet had no effect in monkey or human. Tyrphostin 23, a specific inhibitor of tyrosine protein kinases, inhibited thrombin-induced aggregation of rabbit, monkey, dog and human platelets. SFLLRN-induced aggregation was also inhibited by tyrphostin 23. Tyrphostin 23 inhibited [Ca²⁺]_i induced by either thrombin or SFLLRN in all species. Based on the differential response to agonist stimulation, we propose that thrombin can activate platelets via SFLLRN-dependent and independent mechanisms, which could involve yet unrecognized subtypes of the thrombin receptor or distinct cellular activating mechanisms. Furthermore, differential regulation of calcium mobilization and aggregation was observed in those platelets responding to either thrombin or SFLLRN.     

-Receptor and cholinergic receptor-linked changes in cytosolic Ca and membrane potential in primary rat astrocytes

Both phenylephrine and carbachol caused a sustained increase in Ca²⁺ influx and intracellular free Ca²⁺ of primary astrocytes as measured with ⁴⁵Ca²⁺ and fura-2. The responses to phenylephrine and carbachol were additive, suggesting that they use different releasable pools of Ca²⁺. If extracellular Ca²⁺ was removed by EGTA only a transient rise in cytosolic Ca²⁺ was seen upon application of the agonists. Both compounds caused depolarization of the astrocyte membrane as determined with the optical probe 3,3-diethylthiadicarboxyamineiodide. Activation of protein kinase C with 12-tetradecanoylphorbol myristate acetate (TPA) or the diacylglycerol analogue dioctanoylglycerol (DiC₈) also depolarized the cells. A prior activation of protein kinase C with TPA or DiC₈ abolished the depolarizing effect of phenylephrine suggesting that they act through the same mediators. If the cells were made ideally permeable to K⁺ with the ionophre valinomycin, or the K⁺ channels had been blocked with Ba²⁺, neither TPA nor phenylephrine had any significant effect on the membrane potential. Neither TPA nor phenylephrine had any effect on the ⁸⁶Rb⁺ equilibrium potential across the cell membrane. The results suggest that the depolarizing effect of these substances could be through a blocking of K⁺ channels.     

Characterization of phospholipase A2 secretion from human platelets

Human platelets secreted phospholipase A₂ in a dose- and time-dependent manner when challenged with thrombin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or collagen. Enzyme release was maximal at concentrations of 0.1 units/ml of thrombin, 100 nM TPA, or 2 μg/ml of collagen; and complete by 2 min in platelets treated with thrombin or TPA. Cells challenged with collagen required up to 5 min for maximal secretion. Besides dose and time functions, phospholipase A₂ secretion was also dependent on platelet concentration and the levels of bovine serum albumin in the incubation medium. The secreted enzyme was soluble and exhibited substrate and Ca²⁺ requirements similar to a detergent-solubilized, partially purified phospholipase A₂ from whole platelets [Kramer et al., Biochim. Biophys. Acta (1988) 959, 269–279]. The pH optimum of the secreted enzyme, however, was 1–2 units lower than the pH optimum of the phospholipase A₂ from whole cells. Secreted phospholipase A₂ hydrolyzed phosphatidylethanolamine at 5–12 times the rate of phosphatidylcholine when the substrates were present in pure form. These apparent differences in activity were greatly diminished, though, when 1:1 molar mixtures of the two substrates were employed. Because phospholipase A₂ catalyzes a key reaction during the formation of bioactive arachidonate metabolites, the secretion of this enzyme from platelets may be important in the regulation of thrombosis.     

Suppression of protein kinase C is associated with inhibition of PYK2 tyrosine phosphorylation and enhancement of PYK2 interaction with Src in thrombin-activated platelets

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca²⁺ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130^Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130^Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca²⁺ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130^Cas involvement in platelets.     

Porcine pancreatic group IB secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels

Secretory phospholipase A₂ (sPLA₂) exhibits neurotoxicity in the central nervous system. There are high-affinity binding sites of the porcine pancreatic group IB sPLA₂ (sPLA₂-IB) in the brain. sPLA₂-IB causes neuronal cell death via apoptosis in the rat cerebral cortex. Although apoptosis is triggered by an influx of Ca²⁺ into neurons, it has not yet been ascertained whether the Ca²⁺ influx is associated with the neurotoxicity of sPLA₂-IB. We thus examined the possible involvement of Ca²⁺ in the neurotoxicity of sPLA₂-IB in the primary culture of rat cortical neurons. sPLA₂-IB induced neuronal cell death in a concentration- and time-dependent manner. This death was accompanied by condensed chromatin and fragmented DNA, exhibiting apoptotic features. Before apoptosis, sPLA₂-IB markedly enhanced the influx of Ca²⁺ into neurons. A calcium chelator suppressed neurons from sPLA₂-IB-induced neuronal cell death in a concentration-dependent manner. An L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blocker significantly protected the sPLA₂-IB-potentiated influx of Ca²⁺. On the other hand, blockers of N-VSCC and P/Q-VSCC did not. An L-VSCC blocker protected neurons from sPLA₂-IB-induced neuronal cell death. In addition, the L-VSCC blocker ameliorated the apoptotic features of sPLA₂-IB-treated neurons. Neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of the enzyme. In conclusion, these findings demonstrate that the influx of Ca²⁺ into neurons play an important role in the neurotoxicity of sPLA₂-IB. Furthermore, the present study suggests that L-VSCC contribute to the sPLA₂-IB-potentiated influx of Ca²⁺ into neurons.     

Adenosine A1 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 P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-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 A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ 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 Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Chloroquine inhibits stimulated platelets at the arachidonic acid pathway

Chloroquine inhibited arachidonic acid liberation from membrane phospholipids of thrombin and A23187- stimulated platelets. In addition, it dose-dependently inhibited stimulated malondialdehyde formation and thromboxane B₂ generation in the same platelets. The linear correlation between the inhibition of arachidonic acid liberation and malondialdehyde formation indicated that chloroquine inhibited activated phospholipase A₂ in thrombin-stimulated platelets, similarly as it does in different cells and tissues. Yet, the nonlinear relationship between arachidonic acid liberation along with malondialdehyde formation and thromboxane generation as well as aggregation suggest that phospholipase A₂ does not seem to be the only site of chloroquine action. Rather, it may affect platelets either at other levels of the arachidonic acid cascade too, or at some different stimulatory pathways, like intraplatelet calcium mobilisation, phosphoinositide cycle, calmodulin and protein kinase C activation.     

Independent regulation of activation and inactivation phases in non-contractile Ca transients by nicotinic receptor at the mouse neuromuscular junction

Non-contractile Ca²⁺ mobilization (not accompanied by muscle contraction) occurs by the prolonged activation of nicotinic acetylcholine receptor in mouse diaphragm muscles treated with anticholinesterase. To elucidate the regulation properties of non-contractile Ca²⁺ mobilization by nicotinic receptor, the modes of action of competitive and depolarizing neurmuscular blockers were investigated. (+)-Tubocurarine (0.07–0.1 μM), pancuronium (0.05 μM) and -bungarotoxin (0.03–0.06 μM) decreased decay time (T₂, duration of inactivation phase) without changes in rise time (T₁, duration of activation phase) of non-contractile Ca²⁺ transients. These competitive antagonists also suppressed their peak amplitude at higher concentrations than those affectingT₂. Contractile Ca²⁺ transients were not inhibited by these antagonists at the concentrations used. Decamethonium (1 μM), a depolarizing blocker, suppressed the peak amplitude of non-contractile Ca²⁺ transients without affecting their duration. In contrast, succinylcholine (0.3 μM) suppressed both peak amplitude andT₁ without changingT₂, presumably via the receptor desentization. Succinylcholine but not decamthonium inhibited contractile Ca²⁺ transients at the concentrations used. These results demonstrate that the activation and inactivation phase in non-contractile Ca²⁺ transients are independently regulated by nicotinic acetylcholine receptor.     

Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex

Elevated levels of glutamate and aspartate have been implicated in the pathogenesis of neural injury and death induced by ischemia. The mechanism(s) whereby they escape into the extracellular environment have been a subject of controversy. This study evaluated the contribution of phospholipases and protein kinases to ischemia-evoked glutamate and aspartate release from the ischemic/reperfused rat cerebral cortex. Changes in the extracellular levels of these amino acids during four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked amino acid release was compared in control vs. drug treated animals, in which selective inhibitors of phospholipases and protein kinases were applied topically onto the cerebral cortex. The phospholipase inhibitors tested included 4-bromophenacyl bromide, a non-selective inhibitor; 7,7-dimethyleicosadienoic (DEDA), an inhibitor of secretory type phospholipase A₂ (PLA₂); AACOCF₃, an inhibitor of the Ca²⁺-dependent cytoplasmic form of PLA₂, HELSS, which inhibits a Ca²⁺-independent cytoplasmic PLA₂, and U73122, a selective inhibitor of phospholipase C (PLC). All five phospholipase inhibitors significantly attenuated glutamate and aspartate release into the extracellular milieu, indicating the possibility that several forms of the enzyme are likely to be involved. The protein kinase C (PKC) inhibitor, chelerythrine chloride, also reduced excitatory amino acid efflux, whereas the PKC activator phorbol 12-myristate 13-acetate (PMA) enhanced their release. The non-selective kinase inhibitor, staurosporine, and H-89, which selectively inhibits protein kinase A, did not reduce ischemia-evoked amino acid efflux. These results suggest that ischemia-evoked release of the excitatory transmitters amino acids is a result, in part, of the activation of phospholipases A₂ and C, with PKC involvement in the transduction process. Destabilization and deterioration of the plasma membrane, as a consequence of phospholipid hydrolysis, may allow these transmitter amino acids to diffuse down their concentration gradients into the extracellular fluid.     

Modulation of Neuropeptide-lnduced Membrane Currents by Protein Kinase C in Xenopus Oocytes Injected with GH3 Pituitary Cell Poly(A)+ RNA

Protein kinase C was activated in Xenopus laevis oocytes by phorbol ester treatment and its effects on the inositol trisphosphate/Ca²⁺ transmembrane signalling pathway analysed. Induction of the pathway was achieved by ligand stimulation of TRH receptors translated from GH₃ pituitary cell mRNA. In voltage-clamped oocytes bath application of peptide, injection of guanosine 5'-(3-O-thio) triphosphate (GTPγS), inositol trisphosphate or Ca²⁺ all elicited inward membrane currents. Treatment of oocytes with tumour-promoting phorbol esters for 35 min almost completely abolished the ligand and GTPγS-induced responses. In contrast, phorbol ester treatment enhanced inositol trisphosphate-generated membrane currents. Ca²⁺-mediated responses remained unaffected by tumour promoters. The data indicate a dual role for protein kinase C in the modulation of transmembrane signalling: a feedback mechanism prevents phosphoinositide turnover whereas a feedforward reaction triggers the effect of intracellular inositol trisphosphate on the Ca²⁺ release.     

Platelet aggregation and endogenous 5-ht secretion in presence of ca, sr and ba. effects of calcium antagonists

Washed rat platelets aggregation and endogenous serotonin release were studied after thrombin stimulation in the presence of different concentrations of Ca²⁺, Sr²⁺ or Ba²⁺. The extent of platelet aggregation and release was found to depend upon the external concentration of these cations. For all of them, an optimum concentration could be defined. Higher concentrations were shown to inhibit both aggregation and release. Efficiency to support thrombin-induced aggregation was in the order Ca²⁺>Sr²⁺>Ba²⁺. Complete inhibition of aggregation and release induced by thrombin was obtained after a 30 second preincubation with 38 uM nitrendipine, 1 nM Cd^2- or 1 mM Mn²⁺. Inhibition was obtained in the presence of Ca²⁺, Sr²⁺ or Ba²⁺. These results are consistent with the hypothesis that Sr²⁺ and Ba²⁺ are able to support platelet activation acting as Ca²⁺ substitutes. Following thrombin stimulation, they could penetrate the platelets and mimick a rise in cytoplasmic Ca²⁺.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

Activin exerts a neurotrophic effect on cultured hippocampal neurons

Activin is a member of the transforming growth factor (TGF)-β superfamily, which comprises a growing list of multifunctional proteins that serve as regulators of cell proliferation and differentiation. Recently, activin was shown to regulate the neurotransmitter phenotype in peripheral neurons. It is also a potent survival factor for neurogenic clonal cell lines, retinal neurons and midbrain dopaminergic neurons. We have studied the effect of activin on hippocampal cells which show abundant expression of activin receptors or binding sites. Exposure of primary cultures of rat hippocampal neurons to activin supported neuronal survival. This neurotrophic action of activin was blocked by treatment with the tyrosine kinase inhibitor genistein or the protein kinase C inhibitor calphostin C. However, the Ca²⁺/calmodulin kinase inhibitor KN-62 had no effect. Nicardipine, a blocker of the

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-type Ca²⁺ channel, also inhibited the neurotrophic effect of activin. Furthermore, activin potentiated the depolarization-induced elevation in intracellular Ca²⁺ concentration ([Ca²⁺]_i). The neurotrophic effect and the potentiation of depolarization-induced increase of [Ca²⁺]_i caused by activin were completely abolished by the protein synthesis inhibitor cycloheximide. These results suggest that activin supports neuronal survival by increasing the expression of voltage-dependent Ca²⁺ channel through the action of a tyrosine kinase and of protein kinase C, but not of Ca²⁺/calmodulin kinase.     

Calcium homeostasis in rat septal neurons in tissue culture

Septal neutons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca²⁺ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca²⁺]_i) in the neurons was low (50–100 nM). Depolarization of the neurons with 50 mM K⁺ resulted in rapid elevation of [Ca²⁺]_i to 500–1,000 nM showing recovery to baseline [Ca²⁺]_i over several minutes. The increases in [Ca²⁺]_i caused by K⁺ depolarization were completely abolished by the removal of extracellular [Ca²⁺], and were reduced by 80% by the ‘L-type’ Ca²⁺ channel blocker, nimodipine (1 μM). [Ca²⁺]_i was also increased by the excitatory amino andl-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNOX and DNOX. In the absence of extracellular Mg²⁺, large fluctuations in [Ca²⁺]_i were observed that were blocked by removal of extracellular Ca²⁺, by tetrodotoxin (TTX), or by antagonists ofN-methyld-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg²⁺ and TTX, NMDA caused dose-dependent increases in [Ca²⁺]_i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca²⁺]_i in the absence of extracellular Ca²⁺, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca²⁺ stores. Thapsigargin (2 μM) had little effect on [Ca²⁺]_i, or on the recovery from K⁺ depolarization. Removal of extracellular Na⁺ had little effect on basal [Ca²⁺]_i or on responses to high K⁺, suggesting that Na⁺/Ca²⁺ exchange mechanisms do not play a significant role in the short-term control of [Ca²⁺]_i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca²⁺]_i; however, [Ca²⁺]_i recovered as normal from high K⁺ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca²⁺]_i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca²⁺]_i associated with action potential activity was measured. The amplitude of the [Ca²⁺]_i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca²⁺]_i from the modest Ca²⁺ loads imposed on the neuron by action potential trains follows a simple exponential decay (τ = 3–5s).     

Calcium uptake related to K-depolarization and Na/Ca exchange in sheep brain synaptosomes

The uptake of Ca²⁺ by synaptosomes induced by K⁺-depolarization andby Na⁺/Ca²⁺ exchange was studied in synaptosomes in which the internal Na⁺ and K⁺ contents were varied by prolonged incubation at 30 °C or by inhibiting the Na⁺, K⁺-ATPase with 1 mM ouabain. Increased Na⁺ content of the synaptosomes is associated with an increase in Ca²⁺ uptake when the synaptosomes are placed in depolarizing K⁺ media. Furthermore, reduction in the [Na⁺]_o, when the [K⁺]_o is increased, in substitution for [Na⁺]_o, to depolarize the membrane, further increases the Ca²⁺ uptake. Under these conditions, Ca²⁺ entry probably occurs through voltage-sensitive channels and through the Na⁺/Ca²⁺ exchanger. Destruction of the Na⁺ gradient by monensin, or preloading the synaptosomes with K⁺, completely inhibits the Ca²⁺ uptake in a K⁺-depolarizing medium. It is shown that if the Na⁺ gradient is maintained constant during K⁺-depolarization, the Ca²⁺ uptake is very low and that most of the Ca²⁺ uptake is correlated with the Na⁺ gradient. Evidence is presented that K⁺ may stimulate the Na⁺/Ca²⁺ exchange mechanism. Furthermore, divalent cations, Mg²⁺, Mn²⁺ and Zn²⁺, known to block Ca²⁺ channels, also inhibit Na⁺/Ca²⁺ exchange.     

Aging increases calcium influx at motor nerve terminal

To determine whether increased transmitter release from soleus nerve terminals of old C57BL/6J mice is caused by an altered Ca²⁺ regulation, the time course of post-tetanic potentiation of miniature endplate potential (MEPP) frequency was used as an indicator of the kinetics of Ca²⁺ metabolism in young (10 months) and old (24 months) mice. Post-tetanic potentiation properties were studied in either (1) 0.2 mM Ca²⁺, 5.0 mM Mg²⁺ Krebs; or (2) Ca²⁺-free/EGTA Krebs to eliminate Ca²⁺ influx, and thereby isolated Ca²⁺ buffering. In the 0.2 mM Ca²⁺ Krebs, the time constants of decay of augmentation (T_A) and potentiation (T_P) were longer in old (T_A = 10.3 ± 1.0 sec, T_P = 195.3 ± 5.4 sec) than in young (T_A = 7.0 ± 0.7 sec, T_P = 78.8 ± 6.6 sec) nerve terminals. Evoked transmitter release was measured in 0.4 mM Ca²⁺, 2.75 mM Mg²⁺ Krebs. Quantal content of the endplate potential was positively correlated with T_A (r = 0.95) and with T_P (r = 0.98). In the Ca²⁺-free/EGTA Krebs, there was no difference in post-tetanic potentiation properties between young and old terminals. These results suggest that Ca²⁺ influx into the soleus nerve terminal increases with aging. This may explain, at least in part, the increased quantal content observed at old terminals.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.