Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca²⁺]_i) followed by a sustained rise. In Ca²⁺-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca²⁺]_i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca²⁺]_i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca²⁺]_i and inhibited the following [Ca²⁺]_i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca²⁺]_i. Muscarine-induced sustained [Ca²⁺]_i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca²⁺]_i rise and Mn²⁺ influx in response to muscarine were suppressed by a voltage-dependent Ca²⁺ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca²⁺ entry, but also Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca²⁺ channels may function as one of the Ca²⁺ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

Bcl-2 Protects Against Apoptosis in Neuronal Cell Line Caused by Thapsigargin-Induced Depletion of Intracellular Calcium Stores

Abstract: The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca²⁺-ATPase, was investigated in GT1-7 cells, a murine hypothalamic cell line. Treatment of these cells with 50 or 100 nM thapsigargin greatly reduced cell viability at 24 and 48 h. These doses of thapsigargin induced a rapid rise in free cytosolic Ca²⁺ ([Ca²⁺]_i), followed by a sustained increase. Addition of EGTA to chelate extracellular Ca²⁺ diminished somewhat the size of the initial increase of [Ca²⁺]_i caused by thapsigargin, and abolished the sustained increase. The sustained increase could also be abolished by addition of La³⁺ and by SKF 96365, a drug selective for receptor-mediated calcium entry, but not by verapamil or flunarizine. Pretreatment with 50 µM BAPTA/AM, a cytosolic Ca²⁺ chelator, inhibited the peak [Ca²⁺]_i caused by thapsigargin but did not inhibit the sustained elevation of [Ca²⁺]_i. Neither EGTA nor BAPTA/AM inhibited the cell death induced by thapsigargin. The cell death was characterized by DNA fragmentation (“laddering”), nuclear condensation and fragmentation, and was inhibited by protein synthesis inhibitor cycloheximide, all characteristic of apoptotic cell death. Overexpression of the proto-oncogene bcl-2 in GT1-7 cells inhibited significantly DNA fragmentation, nuclear condensation and fragmentation, and cell death induced by thapsigargin. However, Bcl-2 did not alter either basal [Ca²⁺]_i or the elevation of [Ca²⁺]_i induced by thapsigargin. Our results suggest that abnormal Ca²⁺ release from endoplasmic reticulum caused by thapsigargin induces GT1-7 death by apoptosis and that this effect does not depend on Ca²⁺ influx from the extracellular space. Bcl-2 inhibited apoptosis induced by thapsigargin, but the mechanism is unlikely to be inhibition of endoplasmic reticular Ca²⁺ release in GT1-7 neuronal cells.     

Insulin restores expression of adenosine kinase in streptozotocin-induced diabetes mellitus rats

The effects of extracellular Mg²⁺ on both dynamic changes of [Ca²⁺]_i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca²⁺ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca²⁺]_i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca²⁺]_i (150 nM) for several hours. The initial [Ca²⁺]_i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca²⁺]_i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg²⁺ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg²⁺, caused an increase in basal [Mg²⁺]_i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg²⁺, 1 M thapsigargin induces, in 10 mM Mg²⁺, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca²⁺]_i and a lower Ca²⁺ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca²⁺]_i which was inhibited by high extracellular and intracellular Mg²⁺.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca²⁺]_i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Thapsigargin at concentrations between 10?nM and 10 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺ indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺]_i rise. This Ca²⁺ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²⁺ channels or by modulation of protein kinase C activity. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²⁺ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²⁺]_i rise, suggesting that thapsigargin released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²⁺]_i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²⁺ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca²⁺]_i rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2-sensitive Ca²⁺ channels. Thapsigargin also induced cell death via Ca²⁺-dependent pathways and Ca²⁺-independent apoptotic pathways.     

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca²⁺ stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca²⁺ imaging, we found that the depletion of ER/SR Ca²⁺ stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca²⁺ ([Ca²⁺]_i), followed by sustained increase depending on extracellular Ca²⁺. But, TRP channels inhibitor (SKF96365), not L-type channels or the Na⁺/Ca²⁺ exchanger inhibitors, inhibited [Ca²⁺]_i relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl₃) or by an increased extracellular Ca²⁺([Ca²⁺]_o) increased the concentration of intracelluar Ca²⁺, whereas, the sustained elevation of [Ca²⁺]_i was reduced in the presence of SKF96365. Similarly, the duration of [Ca²⁺]_i increase was also shortened in the absence of extracellular Ca²⁺. Western blot analysis showed that GdCl₃ increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl₃. Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca²⁺-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.     

Generation of repetitive Ca transients by bombesin requires intracellular release and influx of Ca through voltage-dependent and voltage independent channels in single HIT cells

In the present study, the bombesin-induced changes in cytosolic free Ca²⁺ ([Ca²⁺]_i) were investigated in single Fura-2 loaded SV-40 transformed hamster β-cells (HIT). Bombesin (50–500 pM) caused frequency-modulated repetitive Ca²⁺ transients. The average frequency of the Ca²⁺ transients induced by bombesin (200 pM) was 0.58 ± 0.02 min⁻¹ (n = 121 cells). High concentrations of bombesin (≥ 2 nM) triggered a large initial Ca²⁺ transient followed by a sustained plateau or by a decrease to basal levels. In Ca²⁺- free medium, bombesin caused only one or two Ca²⁺ transients and withdrawal of extracellular Ca²⁺ abolished the Ca²⁺ transients. The voltage-dependent Ca²⁺ channel (VDCC) blockers, verapamil (50 μM) and nifedipine (10 μM), reduced amplitude and frequency of the Ca²⁺ transients and stopped the Ca²⁺ transients in some cells. Thapsigargin caused a sustained rise in [Ca²⁺]_i) in the presence of extracellular Ca²⁺ while in its absence the rise in [Ca²⁺]_i) was transient. Verapamil (50 μM) inhibited the thapsigargin-induced increase in [Ca²⁺], by about 50%. Depletion of intracellular Ca²⁺ stores by repetitive stimulation with increasing concentrations of bombesin or thapsigargin in Ca²⁺-free medium caused an agonist-independent increase in [Ca²⁺]_i) when extracellular Ca²⁺ was restored, which was larger than in control cells that had been incubated in Ca²⁺-free medium for the same period of time. This rise in [Ca²⁺]_i and the thapsigargin-induced increase in [Ca²⁺]_i) were only partly inhibited by VDCC-blockers. Thus, depletion of the agonist-sensitive Ca²⁺ pool enhances Ca²⁺ influx through VDCC and voltage-independent Ca²⁺ channels (VICC). In conclusion, the bombesin-induced Ca²⁺ response in single HIT cells is periodic in nature with frequency-modulated repetitive Ca²⁺ transients. Intracellular Ca²⁺ is mobilized during each Ca²⁺ transient, but Ca²⁺ influx through VDCC and VICC is required for maintaining the sustained nature of the Ca²⁺ response. Ca²⁺ influx in whole or part is activated by a capacitative Ca²⁺ entry mechanism.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia

Astrocytes play pivotal roles in both the physiology and the pathophysiology of the brain. They communicate with each other via extracellular messengers as well as through gap junctions, which may exacerbate or protect against pathological processes in the brain. However, their roles during the acute phase of ischemia and the underlying cellular mechanisms remain largely unknown. To address this issue, we imaged changes in the intracellular calcium concentration ([Ca²⁺]_i) in astrocytes in mouse cortical slices under oxygen/glucose deprivation (OGD) condition using two-photon microscopy. Under OGD, astrocytes showed [Ca²⁺]_i oscillations followed by larger and sustained [Ca²⁺]_i increases. While the pharmacological blockades of astrocytic receptors for glutamate and ATP had no effect, the inhibitions of gap junctional intercellular coupling between astrocytes significantly advanced the onset of the sustained [Ca²⁺]_i increase after OGD exposure. Interestingly, the simultaneous recording of the neuronal membrane potential revealed that the onset of the sustained [Ca²⁺]_i increase in astrocytes was synchronized with the appearance of neuronal anoxic depolarization. Furthermore, the blockade of gap junctional coupling resulted in a concurrent faster appearance of neuronal depolarizations, which remain synchronized with the sustained [Ca²⁺]_i increase in astrocytes. These results indicate that astrocytes delay the appearance of the pathological responses of astrocytes and neurons through their gap junction-mediated intercellular network under OGD. Thus, astrocytic gap junctional networks provide protection against tissue damage during the acute phase of ischemia.     

Altered calcium homeostasis and cell injury in silica-exposed alveolar macrophages

There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca²⁺]_i. However, the mechanism of silica- induced cytotoxicity relative to [Ca²⁺]_i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 μm in diameter) and the fluctuation in their [Ca²⁺]_i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca²⁺]_i in a dose-dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca²⁺]_o markedly inhibited the [Ca²⁺]_i rise (≈90%), suggesting that Ca²⁺ influx from extracellular source is a major mechanism for silica-induced [Ca²⁺]_i rise. When used at low doses but sufficient to cause a transient [Ca²⁺]_i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca²⁺]_o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca²⁺ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca²⁺ influx is inhibited by the Ca²⁺ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca²⁺ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca²⁺]_i rise was not due to the impairment of Ca²⁺-pumps, which restricts Ca²⁺ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca²⁺]_i rise. Taken together, these results suggest that the elevation of [Ca²⁺]_i caused by silica is due mainly to Ca²⁺ influx through plasma membrane Ca²⁺ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca²⁺ leakage during phagocytosis was attributed to the silica-induced [Ca²⁺]_i rise. © 1993 Wiley-Liss, Inc.     

Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes

Abstract: Rilmenidine, a ligand for imidazoline and α₂-adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca²⁺]_i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca²⁺]_i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca²⁺-free medium, indicating that rilmenidine evokes release of Ca²⁺ from intracellular stores. However, the sustained elevation of Ca²⁺ was completely dependent on extracellular Ca²⁺, consistent with rilmenidine activating Ca²⁺ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca²⁺ influx pathway. The α₂-adrenergic antagonist rauwolscine attenuated the increase in [Ca²⁺]_i induced by clonidine (a selective α₂ agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca²⁺ release from intracellular stores and Ca²⁺ influx by a mechanism independent of α₂-adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca²⁺ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.     

Separate analysis of nuclear and cytosolic Ca2+ concentrations in human umbilical vein endothelial cells

Ca²⁺ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca²⁺ concentration in cytosol ([Ca²⁺]_i) and nuclei ([Ca²⁺]_n). After calibration, [Ca²⁺]_n was constantly higher than [Ca²⁺]_i before and after the chelation of extracellular Ca²⁺ suggesting an active Ca²⁺ accumulation system on nuclear membrane. [Ca²⁺]_n was also constantly higher than [Ca²⁺]_i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca²⁺]_n and [Ca²⁺]_i was identical, and [Ca²⁺]_i gradient towards the nucleus and peripheral or central [Ca²⁺]_n rise was observed after these stimulations. From these results, [Ca²⁺]_n is not only regulated by the active Ca²⁺ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca²⁺]_n or [Ca²⁺]_i rise from cell to cell; single spike or oscillatory change of [Ca²⁺]_n and [Ca²⁺]_i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca²⁺, suggesting that FCS stimulated [Ca²⁺]_n and [Ca²⁺]_i rise solely depending on Ca²⁺ influx from extracellular medium. The higher concentration of [Ca²⁺]_n and heterogeneous [Ca²⁺]_n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc.     

Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca²⁺]_i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca²⁺]_i in a dose-dependent manner. This elevation of [Ca²⁺]_i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca²⁺ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca²⁺]_i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca²⁺-free condition, orexin A failed to induce any changes of Ca²⁺ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca²⁺]_i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca²⁺ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca²⁺]_i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca²⁺]_i in neurons from deep layers of PFC, which depends on extracellular Ca²⁺ influx via L-type Ca²⁺ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.     

Ca2+ release and Ca2+ influx in Chinese hamster ovary cells expressing the cloned mouse B2 bradykinin receptor: tyrosine kinase inhibitor-sensitive and -insensitive processes

A cDNA encoding a mouse B₂ bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B₂ BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). The initial elevation in [Ca²⁺]_i was abolished by thapsigargin pretreatment in Ca²⁺-free medium. The second phase was dependent on external Ca²⁺. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca²⁺ stores required extracellular Ca²⁺ for refilling. Ca²⁺ influx induced by BK and thapsigargin was confirmed by Mn²⁺ entry through Ca²⁺ influx pathways producing Mn²⁺ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca²⁺]_i increase during the sustained phase and the rate of Mn²⁺ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B₂ BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca²⁺]_i increases, by mobilization of intracellular Ca²⁺ and store-depletion-mediated Ca²⁺ influx, the latter of which is tyrosine phosphorylation-dependent.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

Calcium Sequestering Ability of Mitochondria Modulates Influx of Calcium through Glutamate Receptor Channel

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca²⁺ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca²⁺ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca²⁺]_i followed by a slow and persistently rising phase. The rapid increase in [Ca²⁺]_i was mainly due to influx of Ca²⁺ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca²⁺ in the extracellular medium elicited a small transient rise in [Ca²⁺]_i which can be attributed to the mobilization of Ca²⁺ from IP₃ sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca²⁺ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca²⁺ uniporter. These results imply that mitochondria sequester the Ca²⁺ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca²⁺ caused permeability transition and released the sequestered Ca²⁺ which is manifested as slow rise in [Ca²⁺]_i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca²⁺]_i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca²⁺]_i response. Combined treatment with oligomycin and rotenone further diminished the [Ca²⁺]_i response and also abolished the CCCP mediated rise in [Ca²⁺]_i. However, rotenone alone had no effect on glutamate induced [Ca²⁺]_i response. These changes in glutamate-induced [Ca²⁺]_i response could not be explained on the basis of deficient mitochondrial Ca²⁺ sequestration or ATP dependent Ca²⁺ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca²⁺ buffering processes leading to elevation of [Ca²⁺]_i. In contrast our results showed repression of Ca²⁺ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca²⁺ sequestering ability of mitochondria, the glutamate induced Ca²⁺ influx could be impeded. To validate this, influx of Mn²⁺ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca²⁺ sequestration may desensitize the NMDA receptor operated channel.     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.