Calcimycin potentiates responses of rat hippocampal neurons to N-methyl-D-aspartate

We examined the effect of elevating intracellular calcium ([Ca2+]i) on responses to iontophoretically applied N-methyl-D-aspartate (NMDA), and quisqualate in CA1 neurons of the hippocampal slice. Topical application of calcimycin (A23187), a calcium ionophore, potentiated responses to NMDA but not to quisqualate. This potentiation was prevented by loading cells with the calcium chelator, BAPTA, suggesting that the action of calcimycin on NMDA receptors was mediated by an elevation of [Ca2+]i in the recorded cell. The potentiation was also recorded in voltage-clamped and in cesium-loaded cells, suggesting that it was not mediated by non-specific changes in voltage or input resistance of the cell that may have resulted from the rise in [Ca2+]i. We propose that intracellular calcium plays a crucial role in regulating the activity of the NMDA subtype of L-glutamate receptor.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

Regulation of Intracellular Ca2+ in Response to Muscarinic and Glutamate Receptor Agonists During the Differentiation of NTERA2 Human Embryonal Carcinoma Cells into Neurons

Single cell microfluorimetry was used to study intracellular calcium ion signals ([Ca²⁺]_i) evoked by acetylcholine (ACh), glutamate receptor agonists and by KCI-induced membrane depolarization, during neuronal differentiation of the human embryonal carcinoma (EC) cell line, NTERA2. In undifferentiated NTERA2 EC cells, [Ca²⁺]_i) was elevated in response to ACh, but not to the glutamate receptor agonists NMDA, kainate or AMPA. The ACh-induced rise in [Ca²⁺]_i) was dependent upon both Ca²⁺ influx and Ca²⁺ mobilization from cytoplasmic calcium stores. Three other human EC cell lines responded similarly to ACh but not to glutamate or KCI-induced depolarization. In neurons derived from NTERA2 cells by retinoic acid induction, [Ca²⁺]_i) signals were evoked by ACh, NMDA, kainate and by an elevation of the extracellular KCI concentration. As in undifferentiated EC cells, the ACh-mediated increases in [Ca²⁺li were governed by both Ca²⁺ influx and Ca²⁺ mobilization. In contrast, the effects of NMDA, kainate and KCI did not involve intracellular Ca²⁺ mobilization. The appearance of glutamate and KCI responsiveness was not detected in non-neuronal differentiated derivatives of NTERA2 cells. Using a number of pharmacologically defined muscarinic receptor antagonists we found that NTERA2 EC cells express M₁, M₃, M₄ and possibly M₅ receptor subtypes linked to changes in [Ca²⁺]_i), whilst only M₃ and M₅ are present in NTERA2-derived neurons. The results were supported by PCR analysis of the muscarinic mRNA species expressed in the cells. The data demonstrate that differentiation of NTERA2 EC cells into neurons involves the induction of functional glutamate receptors coupled to rises in [Ca²⁺]_i), and changes in the expression of muscarinic ACh receptor subtypes.     

Calcium Signalling in Mouse Bergmann Glial Cells Mediated by α1-adrenoreceptors and H1 Histamine - Receptors

The presence of adrenergic and histaminergic receptors in Bergmann glial cells from cerebellar slices from mice aged 20–25 days was determined using fura-2 Ca²⁺ microfluorimetry. To measure the cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_i), either individual cells were loaded with the Ca²⁺-sensitive probe fura-2 using the whole-cell patch-clamp technique or slices were incubated with a membrane-permeable form of the dye (fura-2/AM) and the microfluorimetric system was focused on individual cells. The monoamines adrenalin and noradrenalin (0.1-10 μM) and histamine (10-100 μM) triggered a transient increase in [Ca²⁺]_i. The involvement of the α₁-adrenoreceptor was inferred from the observations that monoamine-triggered [Ca²⁺]_i responses were blocked by the selective α¹-adreno-antagonist prazosin and were mimicked by the α¹-adreno-agonist phenylephrine. The monoamine-induced [Ca²⁺]_i signals were not affected by β- and α₂-adrenoreceptor antagonists (propranolol and yohimbine), and were not mimicked by β- and α₂-adrenoreceptor agonists (isoproterenol and clonidine). Histamine-induced [Ca²⁺]_i responses demonstrated specific sensitivity to only H₁ histamine receptor modulators. [Ca²⁺]_i responses to monoamines and histamine did not require the presence of extracellular Ca²⁺ and they were blocked by preincubation of slices with thapsigargin (500 nM), indicating that the [Ca²⁺]_i increase is due to release from intracellular pools. No [Ca²⁺]_i responses were recorded after application of aspartate, bradykinin, dopamine, GABA, glycine, oxytocin, serotonin, somatostatin, substance P, taurine or vasopressin. We conclude that cerebellar Bergmann glial cells are endowed with α₁ -adrenoreceptors and H₁ histamine receptors which induce the generation of intracellular [Ca²⁺]_i signals via activation of Ca²⁺ release from inositol-l,4,5-trisphosphate-sensitive intracellular stores.     

Halothane-induced intracellular calcium release in cholinergic cells

Low concentrations of halothane and isoflurane can release acetylcholine in an extracellular Ca²⁺-independent manner. In the present study, a cholinergic cell line (SN56) was used to examine whether release of calcium from intracellular stores occurs in the presence of halothane. Changes in intracellular calcium concentration ([Ca²⁺]_i) were measured using fluo-3, a fluorescent calcium-sensitive dye and laser scanning confocal microscopy. Halothane, at sub-anesthetic concentrations (14, 28, 40 and 56 μM), increased [Ca²⁺]_i in SN56 cells. This effect remained even when the cells were perfused with medium lacking extracellular calcium, suggesting the involvement of intracellular Ca²⁺ sources. SN56 cells responded to ryanodine by increasing [Ca²⁺]_i and this effect was blocked by dantrolene, an inhibitor of Ca²⁺-release from ryanodine-sensitive stores. The effect of halothane was attenuated after the increase in [Ca²⁺]_i induced by ryanodine and it was suppressed by dantrolene, suggesting the participation of ryanodine-sensitive stores. Using cyclopiazonic acid, a Ca²⁺-ATPase inhibitor, we investigated whether the depletion of intracellular Ca²⁺ stores interfered with the effect of halothane. Cyclopiazonic acid significantly decreased the increase in [Ca²⁺]_i induced by the volatile anesthetic. It is suggested that sub-anesthetic concentrations of halothane may increase [Ca²⁺]_i by releasing Ca²⁺ from intracellular stores in cholinergic cells.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

The effect of sevoflurane on intracellular calcium concentration from cholinergic cells

The mechanism of action of volatile anesthetics is not completely understood. Calcium release from internal stores may alter signaling pathways that influence neurotransmission. Abnormalities of the regulation of intracellular calcium concentration ([Ca²⁺]_i) from patients with malignant hyperthermia is a hallmark of this syndrome indicating the potential of these agents to interact with proteins involved in Ca²⁺ signaling. In the present study, a cholinergic cell line (SN56) was used to examine whether the release of calcium from intracellular stores occurs in the presence of sevoflurane. Changes in [Ca²⁺]_i were measured using fluo-4, a fluorescent calcium sensitive dye and laser scanning confocal microscopy. Sevoflurane induced an increase on [Ca²⁺]_i from SN56 cells. The sevoflurane-induced increase on [Ca²⁺]_i remained even when the cells were perfused with medium lacking extracellular calcium. However, this effect was abolished by BAPTA-AM, a chelator of intracellular calcium, suggesting the involvement of intracellular Ca²⁺ stores. Using cyclopiazonic acid, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase, we investigated whether the depletion of intracellular Ca²⁺ stores interfered with the effect of sevoflurane. In the presence of this agent, sevoflurane caused a small but not significant rise on [Ca²⁺]_i of the SN56 cells. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores did not modify the sevoflurane increase on [Ca²⁺]_i. Carbachol, a drug that releases Ca²⁺ from the IP₃ pool, abolished the effect of sevoflurane. In addition, xestospongin D, a cell-permeant IP₃ receptor antagonist, decreased significantly the sevoflurane increase on [Ca²⁺]_i. Our data suggest that the sevoflurane-induced increase on [Ca²⁺]_i from SN56 cells occurs through the release of calcium from IP₃-sensitive calcium stores.     

5-Hydroxytryptamine2A receptors on cultured rat Schwann cells

Intracellular calcium responses of cultured rat Schwann cells to 5-hydroxytryptamine (5-HT) were examined using the calcium indicator dye fluo-3. Consistent changes in [Ca²⁺]_i were observed with bath application of 5-HT and the basis of these responses was characterized. Application of 5-HT elicited a transient increase in intracellular calcium in a subpopulation of cultured Schwann cells. In many responding cells, the response recurred at approximately regular intervals following the initial transient. In some cases, these oscillations lasted for hours following removal of 5-HT from the bath. The increase in intracellular calcium evoked by 5-HT still occurred in the absence of extracellular calcium, suggesting that 5-HT induces calcium release from intracellular stores. Consistent with this hypothesis, the response to 5-HT was prevented by depletion of inositol trisphosphate-sensitive intracellular calcium stores with thapsigargin. Bath application of caffeine, known to activate Ca²⁺ release from ryanodine receptor-mediated stores, did not elicit an increase in [Ca²⁺]_i. These results also suggested that 5-HT acted by stimulating a member of the 5-HT₂ receptor family since this family employs inositol trisphosphate as a second messenger. In agreement with this interpretation, it was found that the 5-HT-induced intracellular calcium transients could be reversibly blocked by both ketanserin and spiperone, suggesting that the transients are mediated by 5-HT_2A receptors. Additional support for this conclusion was obtained by immunocytochemistry using an anti-idiotypic antibody that recognizes a subset of 5-HT receptors. © 1996 Wiley-Liss, Inc.     

Taxol stabilizes [Ca]i and protects hippocampal neurons against excitotoxicity

Elevation of intracellular calcium levels [Ca²⁺]_i induces microtubule depolymerization, a process which plays roles in regulation of cell motility and axonal transport. However, excessive Ca²⁺ influx, as occurs in neurons subjected to excitotoxic conditions, can kill neurons. We now provide evidence that the polymerization state of microtubules influences neuronal [Ca²⁺]_i homeostasis and vulnerability to excitotoxicity. The microtubule-stabilizing agent taxol significantly attenuated glutamate neurotoxicity in cultured rat hippocampal neurons. Experiments in which [Ca²⁺]_i was monitored using the Ca²⁺ indicator dye fura-2 showed that the elevation of [Ca²⁺]_i induced by glutamate was significantly attenuated in neurons pretreated with taxol. Experiments using selective glutamate receptor agonists suggested that taxol suppressed Ca²⁺ influx through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, but not through N-methyl-D-aspartate (NMDA) receptors. Taxol attenuated the neurotoxicity of the microtubule-depolymerizing agent colchicine; colchicine neurotoxicity was, in part, dependent on Ca²⁺ influx. These findings suggest that microtobules play a role in the mechanism of excitotoxicity and suggest that taxol and related compounds may be useful as antiexcitotoxic agents.     

Flow-cytometric estimation on glutamate- and kainate-induced increases in intracellular Ca of brain neurons: a technical aspect

Effects of glutamate and kainate on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a large population (several thousand) of dissociated cerebellar granule cell neurons were evaluated using a flow-cytometer and a combination of two fluorescent dyes, fluo-3-AM for estimating [Ca²⁺]_i and ethidium bromide for removing neurons that had compromised membranes from the cell population examined. The number of neurons responding to glutamate or kainate in augmenting the fluo-3 fluorescence increased in a dose-dependent manner. The number of neurons responding to kainate was much greater than that to glutamate. CNQX, a blocker of non-NMDA receptors, completely blocked the response elicited by kainate while the complete blockade of this glutamate-induced response was made by a combination of MK-801, a NMDA receptor blocker, and CNQX. Nicardipine, a calcium antagonist, decreased the number of neurons responding to glutamate and kainate, suggesting involvement of voltage-dependent calcium channels. These results indicate that the flow-cytometric measurement of glutamate and kainate responses has the potential to provide answers to such questions as what percentage of the population of neurons respond to these amino acids and what is the resulting distribution of [Ca²⁺]_i.     

Calcium oscillations in a subpopulation of S-antigen-immunoreactive pinealocytes of the rainbow trout (Oncorhynchus mykiss)

By means of the fura-2 technique and image analysis the intracellular concentration of free calcium ions [Ca²⁺]_i was examined in isolated rainbow trout pinealocytes identified by S-antigen immunocytochemistry. Approximately 30% of the pinealocytes exhibited spontaneous [Ca²⁺]_i oscillations whose frequency differed from cell to cell. Neither illumination with bright light nor dark adaptation of the cells had an apparent effect on the oscillations. Removal of extracellular Ca²⁺ or application of 10 μM nifedipine caused a reversible breakdown of the [Ca²⁺]_i oscillations. Application of 60 mM KCl elevated [Ca²⁺]_i in 90% of the oscillating and 50% of the non-oscillating pinealocytes. The effect of KCl was blocked by 50 μM nifedipine. These results suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca²⁺]_i in trout pinealocytes. Experiments with thapsigargin (2 μM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role for regulation of [Ca²⁺]_i remains elusive. Treatment with norepinephrine (100 pM–50 μM), previously shown to induce calcium release from intracellular calcium stores in rat pinealocytes, had no apparent effect on [Ca²⁺]_i in any trout pinealocyte. This finding conforms to the concept that noradrenergic mechanisms are not involved in signal transduction in the directly light-sensitive pineal organ of anamniotic vertebrates.     

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of G_s. Measurements of intracellular free calcium concentration ([Ca²⁺]_i) reveal that mastoparan induces a dramatic elevation of [Ca²⁺]_i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca²⁺]_i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca²⁺]_i is maintained in the absence of extracellular Ca²⁺, suggesting that the rise of [Ca²⁺]_i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca²⁺]_i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca²⁺]_i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca²⁺]_i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca²⁺]_i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca²⁺ release from intracellular stores, probably via activation of PLC and IP₃. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.     

Voltage‐induced Ca2+ release by ryanodine receptors causes neuropeptide secretion from nerve terminals

Depolarisation‐secretion coupling is assumed to be dependent only on extracellular calcium ([Ca²⁺]_o). Ryanodine receptor (RyR)‐sensitive stores in hypothalamic neurohypophysial system (HNS) terminals produce sparks of intracellular calcium ([Ca²⁺]_i) that are voltage‐dependent. We hypothesised that voltage‐elicited increases in intraterminal calcium are crucial for neuropeptide secretion from presynaptic terminals, whether from influx through voltage‐gated calcium channels and/or from such voltage‐sensitive ryanodine‐mediated calcium stores. Increases in [Ca²⁺]_i upon depolarisation in the presence of voltage‐gated calcium channel blockers, or in the absence of [Ca²⁺]_o, still give rise to neuropeptide secretion from HNS terminals. Even in 0 [Ca²⁺]_o, there was nonetheless an increase in capacitance suggesting exocytosis upon depolarisation. This was blocked by antagonist concentrations of ryanodine, as was peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Furthermore, such depolarisations lead to increases in [Ca²⁺]_i. Pre‐incubation with BAPTA‐AM resulted in > 50% inhibition of peptide secretion elicited by high K⁺ in 0 [Ca²⁺]_o. Nifedipine but not nicardipine inhibited both the high K⁺ response for neuropeptide secretion and intraterminal calcium, suggesting the involvement of CaV1.1 type channels as sensors in voltage‐induced calcium release. Importantly, RyR antagonists also modulate neuropeptide release under normal physiological conditions. In conclusion, our results indicate that depolarisation‐induced neuropeptide secretion is present in the absence of external calcium, and calcium release from ryanodine‐sensitive internal stores is a significant physiological contributor to neuropeptide secretion from HNS terminals.     

Flow cytometric study of mitochondrial dysfunction after AMPA receptor activation

The effect of AMPA-receptor stimulation on MMP and on the concentration of intracellular calcium ([Ca²⁺]_i) was studied in dissociated CGC from rat pups, by flow cytometry. In the presence of cyclothiazide, AMPA induced a sodium-independent decrease in MMP up to 30.7 ± 2.5%. This effect was antagonized by CNQX and NBQX. Mepacrine and dibucaine reversed the effect of AMPA on MMP, suggesting that it is mediated by a release of arachidonic acid. AMPA alone induced a slight (about 7%) increase in [Ca²⁺]_i. In the presence of cyclothiazide, AMPA induced a concentration-dependent [Ca²⁺]_i increase up to 29.10 ± 2.10% that was not reversed by flunarizine. This increase was similar to that observed in a Na⁺-free medium, and was antagonized by CNQX and NBQX, but not by MK-801. Mitochondria play a key role in the modulation of [Ca²⁺]_i since a significant [Ca²⁺]_i increase was found in the presence of FCCP. On the other hand, the dantrolene-sensitive calcium pools do not participate in the [Ca²⁺]_i increase induced by stimulation of AMPA receptors. It is concluded that when AMPA-receptor desensitization is blocked, a decrease in MMP and an increase in [Ca²⁺]_i occurs, which could be additional events to potentiate neuronal cell death induced by glutamate. J. Neurosci. Res. 52:684–690, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of [Ca2+]i Transients in Rat Adrenal Chromaffin Cells by Neuropeptide Y Role for a cGMP-dependent Protein Kinase-activated K+ Conductance

The effects of neuropeptide Y on the intracellular level of Ca²⁺ ([Ca²⁺]_i) were studied in cultured rat adrenal chromaffin cells loaded with fura-2. A proportion (16%) of cells exhibited spontaneous rhythmic [Ca²⁺]_i oscillations. In silent cells, oscillations could be induced by forskolin and 1,9–dideoxyforskolin. This action of forskolin was not modified by H-89, an inhibitor of protein kinase A. Spontaneous [Ca²⁺_i fluctuations and [Ca²⁺]_i fluctuations induced by forskolin- and 1,9-dideoxyforskolin were inhibited by neuropeptide Y. Increases in [Ca²⁺]_i induced by 10 and 20 mM KCI but not by 50 mM KCI were diminished by neuropeptide Y. However, neuropeptide Y had no effect on [Ca²⁺]_i increases evoked by (-)BAY K8644 and the inhibitory effect of neuropeptide Y on responses induced by 20 mM KCI was not modified by o-conotoxin GVIA, consistent with neither L- nor N-type voltage-sensitive Ca²⁺ channels being affected by neuropeptide Y. Rises in [Ca²⁺]_i provoked by 10 mM tetraethylammonium were not decreased by neuropeptide Y, suggesting that K⁺ channel blockade reduces the effect of neuropeptide Y. However, [Ca²⁺]_i transients induced by 1 mM tetraethylammonium and charybdotoxin were still inhibited by neuropeptide Y, as were those to 20 mM KCI in the presence of apamin. The actions of neuropeptide Y on [Ca²⁺]_i transients provoked by 20 and 50 mM KCI, 1 mM tetraethylammonium, (-)BAY K8644 and charybdotoxin were mimicked by 8–bromo-cGMP. In contrast, 8–bromo-CAMP did not modify responses to 20 mM KCI or 1 mM tetraethylammonium. The inhibitory effects of neuropeptide Y and 8–bromo-cGMP on increases in [Ca²⁺]_i induced by 1 mM tetraethylammonium were abolished by the Rp-8–pCPT-cGMPS, an inhibitor of protein kinase G, but not by H-89. A rapid, transient increase in cGMP level was found in rat adrenal medullary tissues stimulated with 1 μM neuropeptide Y. Rises in [Ca²⁺]_i produced by DMPP, a nicotinic agonist, but not by muscarine, were decreased by neuropeptide Y. Our data suggest that neuropeptide Y activates a K⁺ conductance via a protein kinase G-dependent pathway, thereby opposing the depolarizing action of K⁺ channel blocking agents and the associated rise in [Ca²⁺]_i.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Intracellular calcium signalling in peripheral cells of patients with bipolar affective disorder

Summary Consistent with previous studies, elevated free intracellular calcium ion concentrations ([Ca²⁺]_i) were found in blood platelets and lymphocytes of patients with mania and bipolar depression. Incubation with an ultrafiltrate of plasma from patients with bipolar illness had no effect on intracellular calcium ion concentration in platelets from normal subjects, suggesting that elevated [Ca²⁺]_i is not due to a circulating factor. As was true in an earlier study of the effect of lithium on platelets, incubation with therapeutic levels of carbamazepine lowered [Ca²⁺]_i in lymhocytes from affectively ill patients but not controls. Increased [Ca²⁺]_i in peripheral cells may reflect a diffuse change in cellular homeostasis and may contribute to mixtures as well as rapid alternations of activity of affective, behavioral and physiologic systems in bipolar illness. Correction of the abnormality may at least be a marker of a relevant therapeutic action if it is not the action itself.     

Amyotrophic Lateral Sclerosis Immunoglobulins Increase Intracellular Calcium in a Motoneuron Cell Line

A hybrid motoneuron cell line (VSC4.1) was used as a model system to study the relationship between alterations in intracellular calcium and subsequent cell death induced by immunoglobulin fractions purified from sera of patients with ALS. Using fluo-3 fluorescence imaging, immunoglobulins from 8 of 10 patients with ALS were found to induce transient increases in intracellular calcium ([Ca²⁺]_i) in differentiated VSC4.1 cells. These transient [Ca²⁺]_iincreases required extracellular calcium entry through voltage-gated calcium channels sensitive to synthetic FTX and to high concentrations (>1 μM) of ω-agatoxin IVa. The incidence of transient [Ca²⁺]_iincreases induced by ALS immunoglobulins correlated with the extent of cytotoxicity induced by the same ALS immunoglobulins in parallel cultures of VSC4.1 cells. Furthermore, manipulations which blocked transient [Ca²⁺]_iincreases (addition of synthetic FTX or ω-agatoxin IVa) also inhibited the cytotoxic effects of ALS immunoglobulins. No transient calcium increases were observed in VSC4.1 cells following addition of immunoglobulins from 7 neurologic disease control patients. However, transient [Ca²⁺]_iincreases were observed following addition of immunoglobulins from 4 of 5 patients with myasthenia gravis (MG). The [Ca²⁺]_ichanges induced by MG immunoglobulins were not blocked by s-FTX, suggesting that they result from a different mechanism than those induced by ALS immunoglobulins. These results suggest that immunoglobulins from patients with ALS can induce transient increases in intracellular calcium in a motoneuron cell line, which may represent early events in the cascade of processes leading to injury and death of susceptible cells.     

Evidence That Actin Depolymerization Protects Hippocampal Neurons against Excitotoxicity by Stabilizing [Ca]i

Calcium influx through glutamate receptors and voltage-dependent channels mediates an array of functional and structural responses in neurons. However, unrestrained Ca²⁺ influx can injure and kill neurons; a mechanism implicated in both acute and chronic neurodegenerative disorders. Data reported here indicate that depolymerization of actin filaments can stabilize intracellular free calcium levels ([Ca²⁺]_i) and protect hippocampal neurons against excitotoxic injury. Studies with fluorescein-labeled phalloidin showed that cytochalasin D and glutamate each induced actin filament depolymerization. The microfilament-disrupting agent cytochalasin D protected cultured rat hippocampal neurons against glutamate toxicity, whereas the actin filament-stabilizing agent jasplakinolide potentiated glutamate toxicity. The microtubule-disrupting agent colchicine was ineffective in protecting neurons against glutamate toxicity. Cytochalasin D did not protect neurons against calcium ionophore toxicity or iron toxicity, indicating that its actions were not due to nonspecific effects on Ca²⁺ or free radical metabolism. Cytochalasin D markedly attenuated kainate-induced damage to hippocampus of adult rats, suggesting an excitoprotective role for actin depolymerization in vivo. Elevations of [Ca²⁺]_i induced by glutamate were attenuated in cultured hippocampal neurons pretreated with cytochalasin D and potentiated in neurons pretreated with jasplakinolide. The [Ca²⁺]_i response to a Ca²⁺ ionophore was unaffected by cytochalasin D, suggesting that actin depolymerization reduced Ca²⁺ influx through membrane channels. Taken together with previous patch clamp data, our findings suggest that depolymerization of actin in response to Ca²⁺ influx may serve as a feedback mechanism to attenuate potentially toxic levels of Ca²⁺ influx.