Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca²⁺]_i) that was communicated to surrounding cells. Following propagation of the Ca²⁺ wave, many cells showed asynchronous oscillations in [Ca²⁺]_i. Dantrolene sodium (10 μM) inhibited the increase in [Ca²⁺]_i associated with this Ca²⁺ wave by 60-80%, and prevented subsequent Ca²⁺ oscillations. Despite the markedly decreased magnitude of the increase in [Ca²⁺]_i, the rate of propagation and the extent of communication of the Ca²⁺ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca²⁺]_i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca²⁺]_i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca²⁺ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca²⁺]_i in most cells that was followed by sustained oscillations in [Ca²⁺]_i in some cells. Dantrolene (10 μM) inhibited this initial [Ca²⁺]_i increase caused by glutamate by 65-90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca²⁺ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca²⁺ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP₃) mediates the propagation of Ca²⁺ waves whereas Ca²⁺ -induced Ca²⁺ release amplifies Ca²⁺ waves and generates subsequent Ca²⁺ oscillations.     

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

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.     

An Interaction of Oxytocin Receptors with Metabotropic Glutamate Receptors in Hypothalamic Astrocytes

Hypothalamic astrocytes play a critical role in the regulation and support of many different neuroendocrine events, and are affected by oestradiol. Both nuclear and membrane oestrogen receptors (ERs) are expressed in astrocytes. Upon oestradiol activation, membrane‐associated ER signals through the type 1a metabotropic glutamate receptor (mGluR1a) to induce an increase of free cytoplasmic calcium concentration ([Ca²⁺]_i). Because the expression of oxytocin receptors (OTRs) is modulated by oestradiol, we tested whether oestradiol also influences oxytocin signalling. Oxytocin at 1, 10, and 100 nm induced a [Ca²⁺]_i flux measured as a change in relative fluorescence [ΔF Ca²⁺ = 330 ± 17 relative fluorescent units (RFU), ΔF Ca²⁺ = 331 ± 22 RFU, and ΔF Ca²⁺ = 347 ± 13 RFU, respectively] in primary cultures of female post‐pubertal hypothalamic astrocytes. Interestingly, OTRs interacted with mGluRs. The mGluR1a antagonist, LY 367385 (20 nm ), blocked the oxytocin (1 nm )‐induced [Ca²⁺]_i flux (ΔF Ca²⁺ = 344 ± 19 versus 127 ± 11 RFU, P < 0.001). Conversely, the mGluR1a receptor agonist, (RS)‐3,5‐dihydroxyphenyl‐glycine (100 nm ), increased the oxytocin (1 nm )‐induced [Ca²⁺]_i response (ΔF Ca²⁺ = 670 ± 31 RFU) compared to either compound alone (P < 0.001). Because both oxytocin and oestradiol rapidly signal through the mGluR1a, we treated hypothalamic astrocytes sequentially with oxytocin and oestradiol to determine whether stimulation with one hormone affected the subsequent [Ca²⁺]_i response to the second hormone. Oestradiol treatment did not change the subsequent [Ca²⁺]_i flux to oxytocin (P > 0.05) and previous oxytocin exposure did not affect the [Ca²⁺]_i response to oestradiol (P > 0.05). Furthermore, simultaneous oestradiol and oxytocin stimulation failed to yield a synergistic [Ca²⁺]_i response. These results suggest that the OTR signals through the mGluR1a to release Ca²⁺ from intracellular stores and rapid, nongenomic oestradiol stimulation does not influence OTR signalling in astrocytes.     

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.     

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.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

Effects of hypoxia induced by Na2S2O4 on intracellular calcium and resting potential of mouse glomus cells

Isolated and cultured glomus cells, obtained from mouse carotid bodies, were superfused with Ham's F-12 equilibrated with air (mean PO₂, 119 Torr; altitude 1350 m). [Ca²⁺]_o was 3.0 mM. In one experimental series, dual cell penetrations with microelectrodes measured intracellular calcium ([Ca²⁺]_i) and the resting potential (E_m). In another series, [Ca²⁺]_i was measured with Indo-1/AM, dissolved in DMSO. Normoxic cells had a mean E_m of −42.4 mV and [Ca²⁺]_i was about 80 nM (measured with both methods). The calculated calcium equilibrium potential (E_Ca) was 137±0.74 mV. Hypoxia, induced by Na₂S₂O₄ 1 mM, reduced pO₂ to 10–14 Torr. This effect was accompanied by cell depolarization to −19.1 mV. Hypoxia increased [Ca²⁺]_i to 231 nM when detected with Ca-sensitive microelectrodes, but only to 130.2 nM when measured with Indo-1/AM. Calcium increases were preceded by decreases in [Ca²⁺]_i, which also were more pronounced with microelectrode measurements. CoCl₂ 1 mM blocked the hypoxic [Ca²⁺]_i increase and exaggerated the decreases in [Ca²⁺]_i. Correlations between ΔE_m and Δ[Ca²⁺]_i during hypoxia were significant (p<0.05) in 19% of the cells. But, in 29% of them significance was at the p<0.1 level. In the rest (52%), there was no correlation between these parameters. Thus, voltage-gated calcium channels are rare in mouse glomus cells. Their activation by depolarization cannot explain the two to threefold increase in [Ca²⁺]_i seen during hypoxia. More likely, [Ca²⁺]_i increase may be due to hypoxic inactivation of a Ca–Mg ATPase transport system across the cell membrane. The blunting of hypoxic [Ca²⁺]_i increase, seen in Indo-1/AM experiments, is probably due to its solvent (DMSO), which also depresses hypoxic cell depolarization.     

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.     

Calcium dependence of the priming,activation and inactivation of ryanodine receptors in frog motor nerve terminals

We studied the effects of varying extracellular Ca²⁺ ([Ca²⁺]_o) and Ca²⁺ channel density and intracellular loading of Ca²⁺ chelators on stimulation‐induced rises in intracellular Ca²⁺ ([Ca²⁺]_i) in frog motor nerve terminals with Ca²⁺ imaging. The slowly waxing and waning components of rises in [Ca²⁺]_i induced by repetitive tetani were suppressed by blockers of Ca²⁺ pumps of the endoplasmic reticulum (thapsigargin and cyclopiazonic acid) and a blocker of ryanodine receptors [8‐(N,N‐diethylamino)octyl 3,4,5‐trimethoxybenzoate hydrochloride] without affecting the initial quickly‐rising component, thus reflecting the priming (and then subsequent rapid activation) and inactivation phases of Ca²⁺‐induced Ca²⁺ release (CICR) from the endoplasmic reticulum. A short tetanus‐induced rise in [Ca²⁺]_i was proportional to [Ca²⁺]_o, whereas the component of CICR was non‐linearly related to [Ca²⁺]_o with saturation at 0.9 mm . The progressive blockade of Ca²⁺ channels by ω‐conotoxin GVIA caused proportional decreases in CICR and short tetanus‐induced [Ca²⁺]_i rises. Intracellular loading of BAPTA and EGTA reduced the magnitude of CICR as well as short tetanus‐induced rises in [Ca²⁺]_i with a greater effect of BAPTA than EGTA on CICR. The time to peak and the half decay time of CICR were prolonged by a low [Ca²⁺]_o or Ca²⁺ channel blocker or [Ca²⁺]_i chelators. These results suggest that ryanodine receptors sense the high [Ca²⁺]_i transient following single action potentials for triggering CICR, whereas the priming and inactivation processes of CICR sense a slower, persisting rise in [Ca²⁺]_i during and after action potential trains. A model is presented that includes CICR activation in elementary units.     

Different characteristics of cell volume and intracellular calcium ion concentration dynamics between the hippocampal CA1 and lateral cerebral cortex of male mouse brain slices during exposure to hypotonic stress

The mechanism of brain edema is complex and still remains unclear. Our aim was to investigate the regional differences of cell volume and intracellular Ca²⁺ concentration ([Ca²⁺]_i) dynamics during hypotonic stress in male mouse hemi‐brain slices. Brain slices were loaded with the fluorescence Ca²⁺ indicator fura‐2, and cell volume and [Ca²⁺]_i in the lateral cerebral cortex (LCC) and hippocampal CA1 (CA1) region were measured simultaneously during exposure to hypotonic stress using Ca²⁺ insensitive (F360) and Ca²⁺ sensitive fluorescence (F380), respectively. Brain cell swelling induced by hypotonic stress was followed by a regulatory volume change that coincided with an increase in [Ca²⁺]_i. The degrees of change in cell volume and [Ca²⁺]_i were significantly different between the LCC and CA1. The increase in cell volume and [Ca²⁺]_i in the LCC, but not in the CA1, was decreased by the transient receptor potential channel blockers LaCl₃ and GdCl₃. The increase in [Ca²⁺]_i in both the LCC and CA1, was significantly decreased by the intracellular Ca²⁺ modulators thapsigargin and xestospongin C. The K⁺ channel activator isoflurane and Cl^‐ channel blocker NPPB significantly decreased [Ca²⁺]_i in the LCC. This study demonstrated that, between cells located in the LCC and in the CA1, the characteristics of brain edema induced by hypotonic stress are different. This can be ascribed to the different contribution of volume sensitive G‐protein coupled receptor and stretch sensitive Ca²⁺ channels.     

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.     

Increase of intracellular calcium induced by oxytocin in hypothalamic cultured astrocytes

A recent study demonstrated oxytocin (OT) receptors on hypothalamic cultured astrocytes (Di Scala-Guenot and Strosser, 1992). The attempt in the present paper was to determine a possible intracellular calcium mobilization induced by OT receptor activation in these cells. Using the microspectrofluorimetric technique with fura-2 as calcium indicator, brief applications of OT on single astrocytes induced a transient and reversible dose-dependent increase of intracellular calcium concentration ([Ca²⁺]_i) in most of the cells tested. In a few cells, OT application triggered intracellular calcium oscillations. Repetitive applications of OT generally produced a decreasing calcium signal, suggesting a desensitization of the receptor. OT-induced calcium release was prevented by a prior or simultaneous application of an OT antagonist. The origin of the calcium mobilization was assessed during conditions where no extracellular calcium was available. Neither removal of extracellular calcium nor addition of a calcium channel blocker, cadmium 100 μM, in the bathing solution, did affect the calcium response to OT, demonstrating that release of intracellular calcium is solely involved in the OT-induced [Ca²⁺]_i increase. The OT-induced calcium mobilization was abolished after thapsigargin application (100 nM). This indicates that the calcium response to OT application was principally associated with activation of the IP₃-sensitive calcium stores. Taken together these results demonstrate that OT receptors previously detected on hypothalamic cultured astrocytes are functional receptors which transduction pathways involve calcium mobilization from IP₃-sensitive stores. © 1994 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.     

Different stimulatory opioid effects on intracellular Ca in SH-SY5Y cells

Present study revealed the stimulatory effects of δ opioid receptor on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in SH-SY5Y cells. Fura-2 based single cell fluorescence ratio (F345/F380) was used to monitor the fluctuation of [Ca²⁺]_i. Application of the selective delta-opioid receptor agonist alone, [D-Pen^2,5]-enkephalin (DPDPE), hardly had any effects on cells cultivated for 3–10 days. However, after the cells had been pre-stimulated with cholinoceptor agonist, carbachol, variable calcium elevation was found in 59% of the cultures. The response was naltridole-reversible and dose-dependent, and was abolished completely by thapsigargin (TG) treatment but not by administration of CdCl₂ or 0-Ca²⁺ bath solutions. DPDPE-mediated [Ca²⁺]_i elevation was abolished by pertussis toxin (PTX) pretreatment but not cholera toxin (CTX), indicating coupling via G proteins of G_i/G_o subfamily. In 17.5% of the responding cells, biphase response was found which may be due to both the stimulatory and the inhibitory effects of opioid. On the other hand, in acutely dissociated cells, DPPDE alone induced [Ca²⁺]_i increase in 50% of the cultures. The probability and the amplitude of the elevation were decreased considerably by application of nifedipine or 0-Ca²⁺ bath solution and was little affected by application of TG. DPDPE activated [Ca²⁺]_i increase via a PTX-insensitive and CTX-sensitive pathway suggesting coupling through G_s subunit. All these indicated the opioid modulated the intracellular Ca²⁺ regulation system through different pathways. SH-SY5Y cell line might be a suitable model for the investigation of the complex mechanism which underlies opioid function.     

Inhibition of noradrenaline stimulated increase in [Ca]i in cultured astrocytes by chronic treatment with a therapeutically relevant lithium concentration

Chronic treatment of mouse astrocytes in primary cultures with 1 mM lithium chloride for 7–14 days decreased the basal level of free cytosolic calcium concentration ([Ca²⁺]_i) from 50–70 nM to 70% of this value and reduced the increase in [Ca²⁺]_i caused by exposure to 1 μM noradrenaline (normally to 500–700 nM) by almost one half. A similar, but much smaller, response to serotonin was unaffected by chronic treatment with lithium. Acute exposure to lithium (30 min) had no effect on either basal or noradrenaline stimulated [Ca²⁺]_i The dependence on chronic, versus acute treatment suggests that this effect may be related to the therapeutic effect of lithium as a mood-stabilizing drug, which likewise requires chronic treatment. Since good evidence is found that noradrenaline increases [Ca²⁺]_i by activation of the phosphoinositol second messenger system the present findings are also consistent with literature data that lithium acts by interfering with this system.     

Stimulation of Ca2+ Entry in Lactotrophs and Somatotrophs from Immature Rat Pituitary by N-Terminal Fragments of Proopiomelanocortin

We have previously shown that 10–12 kDa N-terminal fragments of rat proopiomelanocortin (POMC) and human POMC_1–76 stimulate mitosis and/or differentiation in lactotrophs of early postnatal rat pituitary. A truncated form, POMC_1–26, mimics the differentiation-inducing but not the mitogenic action of the former peptides. To further characterize these two biological responses, the present study compared changes in the intracellular free calcium concentration ([Ca²⁺]_i) in response to POMC_1–76 and POMC_1–26 in isolated pituitary cells from 14-day-old female rats. Calcium (Ca²⁺) responses were also used as a guide to determine whether the responsive cells belong to the lactosomatotroph lineage. Application of POMC_1–76 or POMC_1–26 induced a maintained oscillating [Ca²⁺]_i increase in a small population of cells. Increasing doses of the peptides did not affect the magnitude and the frequency of [Ca²⁺]_i oscillations but clearly augmented the number of responding cells. Approximately 2% of the cells responded at 0.1 nM POMC_1–76 or 5 nM POMC_1–26, and 11–13% of the cells responded at 10 nM and 500 nM of the respective peptides. About one-third of the cells responsive to these peptides also showed a [Ca²⁺]_i increase in response to growth hormone-releasing peptide-6 (GHRP-6) while, in a small number of responsive cells, [Ca²⁺]_i was depressed by dopamine, suggesting that the former cells are somatotrophs and the latter lactotrophs. This interpretation was confirmed by immunocytochemical identification of prolactin and growth hormone (GH) in the cells. Of the cells showing Ca²⁺ response to POMC_1–76, approximately one-third contained GH and another third prolactin. The remainder contained neither GH nor prolactin. Comparable results were obtained with POMC_1–26. The rise of [Ca²⁺]_i induced by the N-terminal POMC peptides persisted after depletion of intracellular Ca²⁺ stores by thapsigargin. Removal of Ca²⁺ from the extracellular medium or addition of cadmium completely abolished both the POMC_1–76- and POMC_1–26-induced [Ca²⁺]_i increase. Nifedipine inhibited the Ca²⁺ response to both peptides, although only in 55% of the responsive cells. Depletion of some isoforms of protein kinase C by preincubation with the phorbol ester PMA for 24 h did not modify the Ca²⁺ responses. In contrast, blockade of the protein kinase A pathway with Rp-cAMPs partially inhibited the POMC_1–76- or POMC_1–26-induced [Ca²⁺]_i increase. The present data show that, in immature pituitary cells, POMC_1–76 induces an increase in [Ca²⁺]_i through extracellular Ca²⁺ influx, possibly mediated in part by protein kinase A activation. The active domain of POMC_1–76 seems to comprise its N-terminal moiety. The data support the hypothesis that POMC_1–76 exerts a specific function in the development of different members of the lactosomatotroph lineage and that the peptide mobilizes different subsets of cells within this lineage, by a mechanism determined by its concentration.     

P2U nucleotide receptor activation in rat glial cell line induces [Ca2+]i oscillations which depend on cytosolic pH

In single rat glioma cells, the signal transduction process activated by the UTP sensitive purinergic nucleotide receptor was studied by determining [Ca²⁺]_i by Fura-2 fluorescence and measuring pH by BCECF fluorescence to elucidate the control of [Ca²⁺]_i oscillations by intracellular pH. Addition of UTP for long time periods (some min) causes a [Ca²⁺]_i response composed of i) an initial large peak and a following sustained increase (160 s duration), and ii) subsequent regular [Ca²⁺]_i oscillations (amplitude 107 nM, frequency 1.5 oscillations per min). The maintenance of the [Ca²⁺]_i oscillations depends on the continued presence of agonist. The oscillations are abolished by reducing extracellular Ca²⁺ concentration. The interaction of UTP receptors and bradykinin receptors during the [Ca²⁺]_i oscillations was investigated because previous studies have already shown that the peptide causes comparable [Ca²⁺]_i oscillations. During [Ca²⁺]_i oscillations induced by UTP or bradykinin, long-term admission of both hormones (400–500 s) causes a large initial response superimposed on regular [Ca²⁺]_i oscillations. Short pulses (12 s) of the second agonist given in any phase of the oscillations induce large [Ca²⁺]_i peaks. In both cases, the following oscillations are not disturbed. The influence of cytosolic pH was studied by alkalinizing pH_i by application of NH₄Cl. [Ca²⁺]_i oscillations stop after addition of NH₄Cl. Recovery of NH₄Cl-induced alkalinization is reduced by furosemide. To the same degree, the interruption of [Ca²⁺]_i oscillations is significantly prolonged in the presence of furosemide. Thus cytosolic alkalinization suppresses hormone-induced [Ca²⁺]_i oscillations in rat glioma cells. The understanding of the molecular mechanism of this interference of pH should provide an important contribution for unravelling the function of cytosolic pH in cellular signal transduction. © 1996 Wiley-Liss, Inc.     

Activation of alpha‐1 adrenergic receptors increases cytosolic calcium in neurones of the paraventricular nucleus of the hypothalamus

Norepinephrine (NE) activates adrenergic receptors (ARs) in the hypothalamic paraventricular nucleus (PVN) to increase excitatory currents, depolarise neurones and, ultimately, augment neuro‐sympathetic and endocrine output. Such cellular events are known to potentiate intracellular calcium ([Ca²⁺]_i); however, the role of NE with respect to modulating [Ca²⁺]_i in PVN neurones and the mechanisms by which this may occur remain unclear. We evaluated the effects of NE on [Ca²⁺]_i of acutely isolated PVN neurones using Fura‐2 imaging. NE induced a slow increase in [Ca²⁺]_i compared to artificial cerebrospinal fluid vehicle. NE‐induced Ca²⁺ elevations were mimicked by the α₁‐AR agonist phenylephrine (PE) but not by α₂‐AR agonist clonidine (CLON). NE and PE but not CLON also increased the overall number of neurones that increase [Ca²⁺]_i (ie, responders). Elimination of extracellular Ca²⁺ or intracellular endoplasmic reticulum Ca²⁺ stores abolished the increase in [Ca²⁺]_i and reduced responders. Blockade of voltage‐dependent Ca²⁺ channels abolished the α₁‐AR induced increase in [Ca²⁺]_i and number of responders, as did inhibition of phospholipase C inhibitor, protein kinase C and inositol triphosphate receptors. Spontaneous phasic Ca²⁺ events, however, were not altered by NE, PE or CLON. Repeated K⁺‐induced membrane depolarisation produced repetitive [Ca²⁺]_i elevations. NE and PE increased baseline Ca²⁺, whereas NE decreased the peak amplitude. CLON also decreased peak amplitude but did not affect baseline [Ca²⁺]_i. Taken together, these data suggest receptor‐specific influence of α₁ and α₂ receptors on the various modes of calcium entry in PVN neurones. They further suggest Ca²⁺ increase via α₁‐ARs is co‐dependent on extracellular Ca²⁺ influx and intracellular Ca²⁺ release, possibly via a phospholipase C inhibitor‐mediated signalling cascade.     

Gonadotropin releasing hormone modulates γ-aminobutyric acid-evoked intracellular calcium increase in immortalized hypothalamic gonadotropin releasing hormone neurons

To examine the functional role of calcium signaling in the interactive modulation of gonadotropin releasing hormone (GnRH) neurons by γ-aminobutyric acid (GABA) and GnRH itself, we analyzed the intracellular calcium level ([Ca²⁺]_i), using fura-2AM fluorescent dye in immortalized hypothalamic GT1-1 cells. GT1-1 cells showed spontaneous [Ca²⁺]_i oscillations, which were dependent on extracellular Ca²⁺ level, L-type Ca²⁺ channel and SK-type K⁺ channel. When GABA or a specific GABA_A type receptor agonist, muscimol was applied to the media, [Ca²⁺]_i rapidly increased through L-type Ca²⁺ channel in a dose-dependent manner, and subsequently decreased below the basal level without any oscillation. However, a specific GABA_B type receptor agonist, baclofen showed no effect. On the other hand, application of GnRH or its potent agonist buserelin, rapidly abolished the spontaneous [Ca²⁺]_i oscillations. Interestingly, a prior treatment with buserelin abolished GABA-evoked increase in [Ca²⁺]_i in a noncompetitive manner. Since buserelin also blocked K⁺-evoked increase in [Ca²⁺]_i, we suggest that GnRH may block spontaneous [Ca²⁺]_i oscillation through modulating the L-type [Ca²⁺]_i channel activity. These results show that GABAergic agents may exert both stimulatory and inhibitory controls over the GnRH neuronal activity, and GnRH can block the stimulatory effect of GABA, implicating the possible existence of an ultrashort feedback circuit.