Characterization of ryanodine receptors in oligodendrocytes,type 2 astrocytes,and O-2A progenitors

In this study we have investigated the expression of ryanodine receptors (RyRs), and the ability of caffeine to evoke RyR-mediated elevation of intracellular Ca²⁺ levels ([Ca²⁺]_i) in glial cells of the oligodendrocyte/type 2 astrocyte lineage. Immunocytochemistry with specific antibodies identified ryanodine receptors in cultured oligodendrocytes, type 2 astrocytes, and O-2A progenitor cells, at high levels in the perinuclear region and in a variegated pattern along processes. Glia acutely isolated from rat brain and in aldehyde-fixed sections of cortex were similarly found to express RyRs. Caffeine (5–50 mM) caused an increase in [Ca²⁺]_i in most cultured type 2 astrocytes and in 50% of oligodendrocytes. Responses elicited by caffeine were inhibited by pretreatment with ryanodine (10 μM) or thapsigargin (1 μM), and the peak response was unaffected by removal of [Ca²⁺]_o. O-2A progenitor cells, in contrast, were largely unresponsive to caffeine treatment. Pretreatment with kainate (200 μM) to activate Ca²⁺ entry increased the magnitude of caffeine-evoked [Ca²⁺]_i elevations in type 2 astrocytes and oligodendrocytes, and caused caffeine to activate responses in a significant proportion of previously non-responding O-2A progenitors. In both type 2 astrocytes and oligodendrocytes, caffeine evoked Ca²⁺ changes which propagated as wavefronts from several initiation sites. These wave amplification sites were characterized by significantly higher local Ca²⁺ release kinetics. Our results indicate that several glial cell types express RyRs, and that their functionality differs within different cell types of the oligodendrocyte lineage. In addition, ionotropic glutamate receptor activation fills the caffeine-sensitive Ca²⁺ stores in these cells. J. Neurosci. Res. 52:468–482, 1998. © 1998 Wiley-Liss, Inc.     

Calcium transient activity in cultured murine neural crest cells is regulated at the IP3 receptor

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca²⁺ modulates calcium transient activity. Lanthanum (La³⁺), a general calcium channel antagonist and zero extracellular Ca²⁺, reduces the percentage of cells exhibiting calcium transients (26.2 and 40.5%, respectively) and decreases calcium spiking frequency (4.5 to 1.0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 μM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4,5-triphosphate receptor (IP₃R)-dependent calcium release, with elevated extracellular Mg²⁺ (20 mM), abolished calcium transient activity. Mg²⁺ did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg²⁺ also suppressed thimerosal-induced calcium oscillations (IP₃R-dependent). Small increases in the intracellular calcium concentration ([Ca²⁺]_i), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca²⁺]_i block the transients. Reducing intracellular IP₃ levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP₃R-dependent calcium excitability of the ER.     

Differential inhibition of catecholamine secretion by amitriptyline through blockage of nicotinic receptors,sodium channels,and calcium channels in bovine adrenal chromaffin cells

We investigated the effects of amitriptyline, a tricyclic antidepressant, on [³H]norepinephrine ([³H]NE) secretion and ion flux in bovine adrenal chromaffin cells. Amitriptyline inhibited [³H]NE secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and 70 mM K⁺. The half maximal inhibitory concentration (IC₅₀) was 2 μM and 9 μM, respectively. Amitriptyline also inhibited the elevation of cytosolic calcium ([Ca²⁺]_i) induced by DMPP and 70 mM K⁺ with IC₅₀ values of 1.1 μM and 35 μM, respectively. The rises in cytosolic sodium ([Na⁺]_i) and [Ca²⁺]_i induced by the Na⁺ channel activator veratridine were also inhibited by amitriptyline with IC₅₀ values of 7 μM and 30 μM, respectively. These results suggest that amitriptyline at micromolar concentrations inhibits both voltage-sensitive calcium (VSCCs) and sodium channels (VSSCs). Furthermore, submicromolar concentrations of amitriptyline significantly inhibited DMPP-induced [³H]NE secretion and [Ca²⁺]_i rise, but not veratridine- or 70 mM K⁺-induced responses, suggesting that nicotinic acetylcholine receptors (nAChR) as well as VSCCs and VSSCs can be targeted by amitriptyline. DMPP-induced [Na⁺]_i rise was much more sensitive to amitriptyline than the veratridine-induced rise, suggesting that the influx of Na⁺ and Ca²⁺ through the nAChR itself is blocked by amitriptyline. Receptor binding competition analysis showed that binding of [³H]nicotine to chromaffin cells was significantly affected by amitriptyline at submicromolar concentrations. The data suggest that amitriptyline inhibits catecholamine secretion by blocking nAChR, VSSC, and VSCC. Synapse 29:248–256, 1998. © 1998 Wiley-Liss, Inc.     

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.     

GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: Their role in catecholamine secretion

The activation of GABA_B receptors of adrenal chomaffin cells produces an increase of [Ca²⁺]_i measured by fura-2 AM techniques. GABA_B agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca²⁺, values 332 ± 60.9 and 306 ± 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca²⁺ buffer. The GABA_B-induced increase of [Ca²⁺]_i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca²⁺ channels as the dihydropiridine nifedipine 50 μM was able to decrease it more than 60%, while ω-conotoxin, which blocks N-type channels, did not produce any change in the GABA_B-evoked Ca²⁺ increment. The second component was due to a release of Ca²⁺ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca²⁺]_i. GABA_B receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca²⁺ store. In a low Ca²⁺ buffer after treatment with 2 μM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca²⁺]_i, whereas 4 mM caffeine had no effect on GABA_B response. This intracellular Ca²⁺ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABA_B receptors. In fact, the specific agonists after 10 minutes incubation produced a dosedependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 μM baclofen and 3-APPA, and it was 3.63 ± 0.75 and 3.2 ± 1.5 times the basal levels (7.3 ± 0.3 pmol/10⁶ cells), respectively. In the absence of extracellular Ca²⁺, GABA_B-evoked catecholamine secretion and cyclic AMP formation were reduced more than 70%, suggesting an important role of extracellular Ca²⁺ in GABA_B mechanisms in adrenal chromaffin cells. © 1995 Wiley-Liss, Inc.     

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.     

Spatiotemporal Distribution of Ca2+ Following Axotomy and Throughout the Recovery Process of Cultured Aplysia Neurons

This study investigates the alterations in the spatiotemporal distribution pattern of the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) during axotomy and throughout the recovery process of cultured Aplysia neurons, and correlates these alterations with changes in the neurons input resistance and trans-membrane potential. For the experiments, the axons were transected while imaging the changes in [Ca²⁺]_i with fura-2, and monitoring the neurons’resting potential and input resistance (R_i) with an intracellular microelectrode inserted into the cell body. The alterations in the spatiotemporal distribution pattern of [Ca²⁺]_i were essentially the same in the proximal and the distal segments, and occurred in two distinct steps: concomitantly with the rupturing of the axolemma, as evidenced by membrane depolarization and a decrease in the input resistance, [Ca²⁺]_i increased from resting levels of 0.05 – 0.1 μM to 1 – 1.5 μM along the entire axon. This is followed by a slower process in which a [Ca²⁺]_i front propagates at a rate of 11 – 16 μm/s from the point of transection towards the intact ends, elevating [Ca²⁺]_i to 3 – 18 μM. Following the resealing of the cut end 0.5 – 2 min post-axotomy, [Ca²⁺]_i recovers in a typical pattern of a retreating front, travelling from the intact ends towards the cut regions. The [Ca²⁺]_i recovers to the control level 7 – 10 min post-axotomy. In Ca²⁺-free artificial sea water (2.5 mM EGTA) axotomy does not lead to increased [Ca²⁺]_i and a membrane seal is not formed over the cut end. Upon reperfusion with normal artificial sea water, [Ca²⁺]_i is elevated at the tip of the cut axon and a membrane seal is formed. This experiment, together with the observations that injections of Ca²⁺, Mg²⁺ and Na⁺ into intact axons do not induce the release of Ca²⁺ from intracellular stores, indicates that Ca²⁺ influx through voltage gated Ca²⁺ channels and through the cut end are the primary sources of [Ca²⁺]_i following axotomy. However, examination of the spatiotemporal distribution pattern of [Ca²⁺]_i following axotomy and during the recovery process indicates that diffusion is not the dominating process in shaping the [Ca²⁺]_i gradients. Other Ca²⁺ regulatory mechanisms seem to be very effective in limiting these gradients, thus enabling the neuron to survive the injury.     

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.     

Simultaneous Monitoring of Cytosolic Free Calcium and Exocytosis at the Single Cell Level

Quinacrine, a fluorescent basic molecule, accumulates in secretory granules of pituitary cells, as was revealed by its colocalization with immunoreactive prolactin. Thus quinacrine fluorescence may be used to monitor secretory activity at the single cell level. Rat pituitary cells in primary culture were loaded with quinacrine and stimulated with physiological secretagogues, such as thyrotrophin-releasing hormone or bradykinin, which induced a multiphasic lowering of fluorescence, corresponding to the loss of quinacrine contained in exocytosed granules. Quinacrine was further used in combination with the fluorescent calcium probe fura-2, in order to monitor simultaneously exocytosis and variations in the cytosolic free calcium concentration, [Ca²⁺]_i. With an appropriate selection of the excitation wavelengths, in dual excitation microfluorimetry experiments, it was possible to distinguish between fluorescence changes due to altered [Ca²⁺]_i versus quinacrine exocytosis. Transient elevations of [Ca²⁺]_i were provoked in individual pituitary cells by enhancing calcium influx through voltage gated channels. In part of the cells an initial increase in [Ca²⁺]_i coincided with stimulated quinacrine release. The approach was also applied to cells of the neuroblastoma line NCB20, where stimulation with bradykinin caused a transient rise in [Ca²⁺]_i, concomitantly with enhanced exocytosis. No increase in exocytosis was ever detected without an elevation of [Ca²⁺]_i, suggesting that in both cellular systems, an increase in [Ca²⁺]_i, is absolutely necessary, but not sufficient to induce secretion.     

Ryanodine receptor-mediated [Ca]i release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

The hypothesis that intracellular calcium ([Ca²⁺]_i) release in glomus cells via ryanodine receptor (RyR) activation by caffeine may be independent of natural stimuli and chemosensory discharge was tested in the rat carotid body (CB). CB type I cells were isolated, plated and preloaded with calcium-sensitive fluorescent probe, Indo-1AM. With the increase of caffeine dose (0–50 mM) cytosolic calcium ([Ca²⁺]_c) increased from 85±15 nM to 1933±190 nM (n=6) at normoxia (P ₂=125–130 Torr, P ₂=25–30 Torr, pH 7.30–7.35). Hypoxia (P ₂=10–15 Torr) increased and hypocapnia (P ₂=7–9 Torr) decreased the cytoplasmic calcium [Ca²⁺]_c levels, independent of caffeine. Caffeine-related [Ca²⁺]_c increase was the same in the presence and the absence of extracellular calcium ([Ca²⁺]_o), indicating the source of Ca²⁺ ions is the cellular store. Permeabilization of the cell membrane with saponin (25 μg/ml) retained the caffeine response. Additional treatment of the cells with 50 μM ryanodine (an inhibitor of the caffeine-activated RyR site) abolished caffeine-stimulated response. In vitro CB chemosensory (carotid sinus nerve, CSN) responses to hypoxia (P ₂=35–40 Torr) were not altered by caffeine. These results suggest that [Ca²⁺]_i stores in CB cells, mobilized by RyR activation, do not participate in the CSN responses to natural stimuli.     

Effect of tacrine on intracellular calcium in cholinergic SN56 neuronal cells

We have found earlier that the depolarization-induced release of acetylcholine from the brain could be inhibited by tacrine (tetrahydroaminoacridine) but the mechanism of this action of tacrine was not clarified (S. Tu?ek, V. Dole?al, J. Neurochem. 56 (1991) 1216). We have now investigated whether tacrine has an effect on the changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) induced by depolarization. Experiments were performed on the cholinergic SN56 neuronal cell line with Fura-2 fluorescence technique of calcium imaging. The depolarization by 71 mmol/l K⁺ evoked minimum increases of [Ca²⁺]_i up to day 5 in culture. Then the response gradually increased and reached a plateau after 7 days in culture. A similar time course was observed for acetylcholinesterase activity. The effect of K⁺ ions was concentration-dependent and the concentration of 71 mmol/l K⁺ evoked maximum [Ca²⁺]_i responses. The increases of [Ca²⁺]_i did not occur in the absence of extracellular calcium. They were mediated by high voltage-activated calcium channels of the L-type and the N-type. Nifedipine (2 μmol/l; L-type calcium channel blocker) and ω-conotoxin GVIA (100 nmol/l; N-type calcium channel blocker) diminished the response to 71 mmol/l K⁺ by 53% and 39%, respectively, and their effects were additive (decrease to 8% of controls). Non-selective inorganic blocker of voltage-activated calcium channels LaCl₃ (0.1 mmol/l) decreased the response by 83%. Tacrine attenuated the [Ca²⁺]_i response in a concentration-dependent manner. At a concentration of 10 μmol/l it inhibited the [Ca²⁺]_i response by 55% and its inhibitory effect was additive with that of ω-conotoxin GVIA but not with that of nifedipine. An equimolar concentration of paraoxon, an irreversible inhibitor of cholinesterases, had no influence on [Ca²⁺]_i response. Tacrine exhibited the same inhibitory effect when paraoxon was present. In conclusion, our data indicate that high-voltage-activated calcium channels of the L-type and the N-type are both present in the SN56 cells but that they are fully expressed only after 6–7 days in culture. Tacrine attenuates the influx of calcium by inhibiting the L-type calcium channels. This inhibitory effect is not a consequence of the anticholinesterase activity of tacrine. The finding that low micromolar concentrations of tacrine may interfere with calcium-dependent events is likely to be of importance for the evaluation of the therapeutic potential of the drug.     

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.     

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.     

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.     

Cerebrospinal fluid from amyotrophic lateral sclerosis has no effect on intracellular free calcium in cultured cortical neurons

The data from the literature regarding the presence of a neurotoxic factor in amyotrophic lateral sclerosis (ALS) plasma or cerebrospinal fluid (CSF) remain controversial. As a new approach to this question, we have studied the effect of CSF from ALS patients on the temporal dynamics of the intracellular free calcium concentration ([Ca²⁺]_i) of murine cortical neurons in cultures using Fura-2 fluorescence videomicroscopy and single-cell imaging. CSF from seven ALS patients and controls was added at dilutions up to 20% to cortical neuronal cultures. The in vitro inhibition of CSF on [³H]kainic acid binding showed that the CSF did not contain any substances other than glutamate itself in larger amounts. At the concentrations used, the CSF did not have any effect on [Ca²⁺]_i or on the neuronal responsiveness as defined by the ability of the cells to respond with a transient increase in [Ca²⁺]_i to depolarization induced by KCl. The disturbance of the intracellular calcium homeostasis is one of the key mechanisms of action of excitotoxic compounds mediating delayed neuronal cell death by stimulation of glutamate receptor subtypes. In this study, CSF from ALS patients did not induce immediate rises in [Ca²⁺]_i or disturbances of the intracellular calcium homeostasis when measured over a period of 2 h.     

Pharmacological characterization of the specific binding of [H]ryanodine to rat brain microsomal membranes

High-affinity binding of [³H]ryanodine has been characterized in rat brain microsomal fractions. Membrane fractions from 4 brain regions (cerebral cortex, cerebellum, hippocampus and brainstem) have been isolated using sucrose density gradient purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the presence of a high-molecular weight protein (M_r 320kDa), similar to that of ryanodine receptor from muscle sarcoplasmic reticulum (SR). In the presence of high salt (1 M KCl), [³H]ryanodine binds to low density (0.8 M sucrose) cortical microsomal fraction with high affinity (K_d 1.5nM), and with the highest capacity (B_max 330fmol/mg protein). Kinetic analysis of the binding suggests multiple available binding sites for ryanodine. Binding of ryanodine is Ca²⁺ dependent (ED₅₀ 1 μM) and inhibited by Mg²⁺ and Ruthenium red. Adenine nucleotides have a biphasic effect on the binding of [³H]ryanodine. At low Ca²⁺ concentration caffeine and daunorubicin enhance the binding of [³H]ryanodine. The inositol 1,4,5-trisphosphate (IP₃) binding inhibitor, heparin, has no effect on ryanodine binding, and ryanodine and caffeine do not influence the binding of [³H]IP₃, which is enriched in the cerebellar fractions. These data demonstrate significant quantitative differences in the pharmacology of brain and muscle receptors and raise the question as to the physiological role of ryanodine binding proteins in the central nervous system and whether it is coupled to an endoplasmatic reticulum (ER) Ca²⁺ release channel.     

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage‐sensitive calcium channels

The existence of voltage-sensitive Ca²⁺ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca²⁺ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca²⁺ concentration ([Ca²⁺]_i) variations induced by K⁺ depolarization and modified by specific Ca²⁺ channel agonists and antagonists. At rest, [Ca²⁺]_i was 90 ± 20 nm in both cell types. Microperifusion of high-K⁺ solution (50 mm ) for 1 s increased [Ca²⁺]_i to 290 ± 50 nm in type I (n = 20) and to 440 ± 50 nm in type II cells (n = 10). In Ca²⁺-free medium, K⁺ did not alter [Ca²⁺]_i. The specific L-type Ca²⁺ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K⁺-induced [Ca²⁺]_i increase in both cell types with maximum effect at 2 μm and 400 nm , respectively. Ni²⁺, a T-type Ca²⁺ channel blocker, reduced K⁺-evoked Ca²⁺ responses in a dose-dependent manner. For elevated Ni²⁺ concentrations, the response was differently affected by Ni²⁺ alone, or combined to nitrendipine (500 nm ). In optimal conditions, nitrendipine and Ni²⁺ strongly depressed by 95% the [Ca²⁺]_i increases. By contrast, neither ω-agatoxin IVA (1 μm ), a specific P- and Q-type blocker, nor ω-conotoxin GVIA (1 μm ), a specific N-type blocker, affected K⁺-evoked Ca²⁺_i responses. These results provide the first direct evidence that L- and probably T-type channels control the K⁺-induced Ca²⁺ influx in both types of sensory cells.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

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.