Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca²⁺]_i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca²⁺]_i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl₃). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca²⁺]_i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF^- ₄), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca²⁺ resulted in a transient rise in [Ca²⁺]_i . Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca²⁺]_i . These results suggest that dephostatin can mobilize Ca²⁺ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.     

Differential regulation of intracellular calcium oscillations by mitochondria and gap junctions

Fluctuations of intracellular Ca²⁺ ([Ca²⁺]_i) regulate a variety of cellular functions. The classical Ca²⁺ transport pathways in the endoplasmic reticulum (ER) and plasma membrane are essential to [Ca²⁺]_i oscillations. Although mitochondria have recently been shown to absorb and release Ca²⁺ during G protein-coupled receptor (GPCR) activation, the role of mitochondria in [Ca²⁺]_i oscillations remains to be elucidated. Using fluo-3-loaded human teratocarcinoma NT2 cells, we investigated the regulation of [Ca²⁺]_i oscillations by mitochondria. Both the muscarinic GPCR agonist carbachol and the ER Ca²⁺-adenosine triphosphate inhibitor thapsigargin (Tg) induced [Ca²⁺]_i oscillations in NT2 cells. The [Ca²⁺]_i oscillations induced by carbachol were unsynchronized among individual NT2 cells; in contrast, Tg-induced oscillations were synchronized. Inhibition of mitochondrial functions with either mitochondrial blockers or depletion of mitochondrial DNA eliminated carbachol—but not Tg-induced [Ca²⁺]_i oscillations. Furthermore, carbachol-induced [Ca²⁺]_i oscillations were partially restored to mitochondrial DNA-depleted NT2 cells by introduction of exogenous mitochondria. Treatment of NT2 cells with gap junction blockers prevented Tg-induced but not carbachol-induced [Ca²⁺]_i oscillations. These data suggest that the distinct patterns of [Ca²⁺]_i oscillations induced by GPCR and Tg are differentially modulated by mitochondria and gap junctions.     

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⁺.     

Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

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.     

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.     

NMDA Receptor-Mediated Calcium Influx in Cerebral Cortical Synaptosomes of the Hypoxic Guinea Pig Fetus

Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca²⁺]_i observed under hypoxic conditions is the result of an NMDA-mediated Ca²⁺ influx. Changes of [Ca²⁺]_i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca²⁺]_i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca²⁺]_i was not observed in a Ca²⁺ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca²⁺]_i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca²⁺ via the altered NMDA receptor with subsequent release of Ca²⁺ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.     

Ca as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

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.     

Glucagon receptor mediates calcium signaling by coupling to Gαq/11 and Gαi/o in HEK293 cells

Glucagon induces intracellular Ca²⁺ ([Ca²⁺]_i) elevation by stimulating glucagon receptor (GCGR). Such [Ca²⁺]_i signaling plays important physiological roles, including glycogenolysis and glycolysis in liver cells and the survival of β-cells. Previous studies indicated that phospholipase C (PLC) might be involved in glucagon-mediated [Ca²⁺]_i response. Other studies also debated whether cAMP accumulation mediated by GCGR/Gα_s coupling contributes to [Ca²⁺]_i elevation. But the exact mechanisms remain uncertain. In the present study, we found that glucagon induces [Ca²⁺]_i elevation in HEK293 cells expressing GCGR. Removing extracellular Ca²⁺ did not affect glucagon-stimulated [Ca²⁺]_i response. But depleting the intracellular Ca²⁺ store by thapsigargin completely inhibited glucagon-induced [Ca²⁺]_i response. Experiments with forskolin and adenylyl cyclase inhibtor revealed that cAMP is not the cause of [Ca²⁺]_i response. Further studies with Gα_q/11 RNAi and pertussis toxin (PTX) indicated that both Gα_q/11 and Gα_i/o are involved. Combination of Gα_q/11 RNAi and Gα_i/o inhibition almost completely abolished glucagon-induced [Ca²⁺]_i signaling.     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

Oscillations of cytosolic free calcium in bombesin-stimulated HIT-T15 cells

The mechanism underlying the generation of cytosolic free Ca²⁺ ([Ca²⁺_i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca²⁺]_i oscillations with variable patterns. The bombesin-induced [Ca²⁺]_i oscillations could last more than 1 h and glucose was required for maintaining these [Ca²⁺ fluctuations. Bombesin-evoked [Ca²⁺]_i oscillations were dependent on extracellular Ca²⁺ entry and were attenuated by membrane hype rpolarization or by L-type Ca²⁺ channel blockers. These [Ca²⁺]_i oscillations were apparently not associated with fluctuations in plasma membrane Ca²⁺ permeability as monitored by the Mn²⁺ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca²⁺ stores, respectively, by inhibiting Ca²⁺-ATPase of endoplasmic reticulum and by affecting Ca²⁺-induced Ca²⁺ release, disrupted bombesin-induced [Ca²⁺]_i oscillations. 4-chloro-m-resol raised [Ca²⁺]_i by mobilizing an intracellular Ca²⁺ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca²⁺]_i It raised [Ca²⁺]_i by promoting Ca²⁺ entry while inhibiting bombesin-elicited [Ca²⁺]_i oscillations. Our results suggest that in bombesin-elicited [Ca²⁺]_i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca²⁺ stores; and (ii) the Ca²⁺ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca²⁺]_i oscillations. The interplay between intracellular Ca²⁺ stores and voltage-sensitive and voltage-insensitive extracellular Ca²⁺ entry determines the [Ca²⁺]_i oscillations evoked by bombesin.     

Intracellular Ca2+ homeostasis in rat round spermatids

Intracellular calcium, [Ca²⁺]_i, can regulate meiotic progression of mammalian oocytes. However, the role of [Ca²⁺]_i in the regulation of the spermatogenic process and its cellular homeostatic mechanisms in spermatogenic cells has not been elucidated. Using intracellular fluorescent probes for Ca²⁺ and immunodetection of plasma membrane (PM) Ca²⁺-ATPases, we report that: a) rat round spermatids maintain [Ca²⁺]_i levels of 60 ± 5 nM (SEM), as estimated with fluo-3 in single cells or fura-2 in cells in suspension; b) these cells regulate [Ca²⁺]_i by actively extruding it using a PM Ca²⁺-ATPase; c) rat spermatids also actively transport Ca²⁺ by sarco-endoplasmic reticulum type ATPases (SERCA); d) rat spermatids possess non-mitochondrial intracellular Ca²⁺_i stores insensitive to thapsigargin but releasable by ionomycin; and e) rat spermatids do not activate Ca²⁺ entry mechanisms by the release of Ca²⁺ from SERCA-regulated stores. These results demonstrate that rat round spermatids can generate modulated intracellular Ca²⁺ signals upon activation of Ca²⁺ channels or Ca²⁺ release from intracellular stores.     

Bergmann glial cells in situ express endothelinB receptors linked to cytoplasmic calcium signals

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca²⁺]_i in Bergmann glial cells in cerebellar slices acutely isolated from 20–25 day-old mice. The intracellular calcium concentration ([Ca²⁺]_i) was monitored using Fura-2-based ([Ca²⁺]_i) microfluorimetry. The ET-triggered ([Ca²⁺]_i) transients were mimicked by ET, receptor agonist BO-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca²⁺]_i in Ca2+-free extracellular solution and the ET-triggered [Ca²⁺]_i elevation was blocked by 500 nM thapsigargin indicating that the [Ca²⁺]_i was released from InsP3 sensitive intracellular pools. The ET-triggered [Ca²⁺]_i increase in Ca²⁺-free solution was shorter in duration. Restoration of normal extracellular [Ca²⁺] briefly after the ET application induced a second [Ca²⁺]_i increase indicating the presence of a secondary Ca²⁺ influx which prolongs the Ca²⁺ signal. Pre-application of 100 μM ATP or 10 μM noradrenaline blocked the ET response suggesting the involvement of a common Ca²⁺ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca²⁺ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ET_B receptors which induce the generation of intracellular [Ca²⁺]_i signals by activation of Ca²⁺ release from InsP₃-sensitive intracellular stores followed by a secondary Ca²⁺ influx.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Intracellular Ca2+ Homeostasis in Trypomastigotes of Trypanosoma cruzi

ABSTRACT Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺]_i) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]_o (preloaded cells) increased [Ca²⁺]_i < 20 nM whereas total cell Ca²⁺ increased by 1.5 to 2.0 pmole/cell. This amount of Ca²⁺ would be expected to increase [Ca²⁺]_i to > 10 μM suggesting active sequestration of Ca²⁺. We tested the hypothesis that maintenance of [Ca²⁺]_i involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca²⁺]_i through well characterized pathways in higher eukaryotic cells were employed. [Ca²⁺], responses in the presence or absence of [Ca²⁺]o were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺]_i homeostasis. In the presence of 0.5 mM [Ca²⁺]_o, the ability of several agents to increase [Ca²⁺]_i was magnified in the order ionomycin ? nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca²⁺], observed only in response to monensin. Manoalide, an inhibitor of phospholipase A₂, enhanced the accumulation of [Ca²⁺]_i due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca²⁺]_i involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca²⁺ may involve phospholipase A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca²⁺]_i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca²⁺]_i, which began to decrease after 3 min. Moreover, [Ca²⁺]_i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca²⁺]_i efflux. Phe-induced [Ca²⁺]_i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca²⁺-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca²⁺]_i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance ⁴⁵Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca²⁺-ATPase (SERCA), respectively, did not elicit any changes in [Ca²⁺]_i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca²⁺]_i, PMCA activity and ⁴⁵Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and ⁴⁵Ca-uptake capability decreased from day 9, at the same time [Ca²⁺]_i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca²⁺]_i and subsequent long-term increase in [Ca²⁺]_i.     

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Diindolylmethane at concentrations of 20–50 µM induced [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Diindolylmethane-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca²⁺]_i rise in PC3 cells by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A₂-sensitive store-operated Ca²⁺ channels. Diindolylmethane caused cell death in which apoptosis may participate.