Effect of NPC-14686 (Fmoc-L-homophenylalanine) on intracellular Ca2+ levels in human hepatoma cells

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in HA22/VGH human hepatoma cells was explored by using fura-2 as a fluorescent Ca(2+) indicator. NPC-14686 at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. The Ca(2+) signal was reduced by removing extracellular Ca(2+) or by 10 microM nifedipine and was not changed by verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 200 microM NPC-14686-induced Ca(2+) release; and conversely pretreatment with NPC-14686 abolished thapsigargin-induced Ca(2+) release. The Ca(2+) release induced by 200 microM NPC-14686 was not changed by inhibiting phospholipase C with 2 microM U73122. Together, the results suggest that in human hepatoma cells, NPC-14686 induced a [Ca(2+)](i) increase by causing store Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing nifedipine-sensitive Ca(2+) influx.     

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.     

Effect of progesterone on intracellular Ca2+ homeostasis in human myometrial smooth muscle cells

Although it iswell known that progesterone alters uterine contractility and plays animportant role in maintenance of pregnancy, the biochemical mechanismsby which progesterone alters uterine contractility in human gestationare less clear. In this investigation we sought to identifyprogesterone-induced adaptations in human myometrial smooth musclecells that may alter Ca²⁺signaling in response to contractile agents. Cells were treated withvehicle or the progesterone analog medroxyprogesterone acetate (MPA)for 5 days, and intracellular freeCa²⁺ concentration([Ca²⁺]_i)was quantified after treatment with oxytocin (OX) or endothelin (ET)-1.OX- and ET-1-induced increases in[Ca²⁺]_iwere significantly attenuated in cells pretreated with MPA in adose-dependent manner. Progesterone receptor antagonists prevented theattenuated Ca²⁺ transients inducedby MPA. ET_A andET_B receptor subtypes were expressed in myometrial cells, and treatment with MPA resulted insignificant downregulation of ET_Aand ET_B receptor binding. MPA didnot alter ionomycin-stimulated increases in[Ca²⁺]_iand had no effect on inositol trisphosphate-dependent or -independent release of Ca²⁺ from internalCa²⁺ stores. We conclude thatadaptations of Ca²⁺ homeostasis inmyometrial cells during pregnancy may include progesterone-inducedmodification of receptor-mediated increases in[Ca²⁺]_i.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Capsazepine may be mildly cytotoxic.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.     

Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells

Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

Effect of ethanol on intracellular Ca2+ metabolism

The aim of this review is to summarize current thinking on ethanol effects on the Ca2+ homeostasis in the excitable tissue cells. It has been shown that acute exposure to ethanol decreases cytoplasmic Ca2+ concentration due to Ca2+ channels inhibition and Ca2+ pumps activation. Whereas chronic exposure to ethanol increases the intracellular Ca2+ concentration in cells due to activation of passive Ca2+ transport systems and inhibition of energy-dependent Ca2+ transport systems. The emphasis is place on a possible role of pharmacologic agents that preserve Ca2+ homeostasis in protecting against ethanol-induced diseases.     

Effect of endothelin on Ca2+ influx, intracellular free Ca2+ levels and ligand binding to N and L type Ca2+ channels in rat brain

The actions of endothelin, an endogenous vasoconstrictor compound with potent effects on various parameters of Ca2+ metabolism in peripheral tissue, were studied in several neuronal preparations. Endothelin, by itself, did not alter resting intracellular free Ca2+ levels or Ca2+ influx in either rat or chicken brain preparations; nor did it affect depolarization (K+) induced changes in these parameters. Endothelin also had no effect on the binding of [3H]-nitrendipine or [125I]-omega-conotoxin to "L " or "N" type channels respectively nor did it induce the release of endogenous acetylcholine from brain slices. The results show that, despite the proposed role of endothelin on voltage sensitive Ca2+ channels in peripheral tissue and despite the existence of endothelin binding sites on both smooth muscle and neurons, endothelin has no measurable effects on Ca2+ metabolism in neural tissue of central origin.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.     

Effect of intracellular free Ca2+ on leucine transport in Chang liver cells

Addition of Ca2+ ionophore (A23187) to the medium stimulated the Na+-independent leucine transport in Chang liver cells, increasing the cytoplasmic free Ca2+ concentration, irrespective of the presence or absence of extracellular Ca2+. Anticalmodulin drugs, such as chlorpromazine, trifluoperazine, and W-7, significantly inhibited the leucine transport in the cells. The stimulatory effect of A23187 on leucine transport was completely blocked in the presence of the anticalmodulin drug. Two microtubule disrupting drugs, colchicine and colcemid, significantly stimulated leucine transport. On the other hand, taxol, a microtubule stabilizing agent, decreased the stimulatory effect of colchicine on the leucine transport. These results strongly suggest the involvement of Ca2+ and calmodulin in regulation of Na+-independent leucine transport, possibly through control of assembly and disassembly of the microtubule network.     

Stimulation of intracellular Ca2+ oscillations by diacylglycerol in human myometrial cells

Stimulation of G-protein coupled membrane receptors linked to phospholipase C results in production of the second messengers diacylglycerol and inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from the endoplasmic reticulum, which triggers increased Ca2+ influx across the plasma membrane, so-called capacitative calcium entry. DAG can also activate plasma membrane calcium-permeable channels but the mechanism is still not fully understood. In the pregnant human myometrial cell line PHM1 and in primary myometrial cells, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analogue of diacylglycerol, induced variable oscillatory patterns of intracellular free Ca2+. Similar behavior was seen with Sr2+ entry. The Ca2+ oscillations were not blocked by a broad spectrum of protein kinase C inhibitors, including chelerytrine, bisindolylmaleimide I and calphostin C, and were enhanced and prolonged by RHC-80267, an inhibitor of diacylglycerol lipase. The OAG-induced oscillatory response was not dependent on Ca2+ release from the endoplasmic reticulum but required extracellular Ca2+. Our results indicate that diacylglycerol directly activates cation channels in PHM1 and primary myometrial cells and promotes intracellular Ca2+ oscillations by actions independent of intracellular Ca2+ -ATPase activity and protein kinase C involvement.     

Elevated intracellular Ca2+ affects Lii-Nao countertransport in human red blood cells

Changes in cytoplasmic Ca2+ concentration and in Lii-Nao countertransport activity have been shown to be associated with essential hypertension. Elevated intracellular free [Ca2+], as well as abnormalities of Ca2+ binding and transport have been reported in cells from different tissues of hypertensive laboratory animals and essential hypertensive patients. Similarly, enhanced rates of Lii-Nao countertransport and the modified pattern of the temperature dependence of this activity in red blood cells from essential hypertensive patients have been previously demonstrated. The aim of the present study was to investigate possible interaction between changes in intracellular free [Ca2+] and the Lii-Nao exchange in human red blood cells. The ionophore ionomycin was used to allow Ca2+ incorporation into the cells in a dose-dependent manner. The elevation of intracellular [Ca2+], in turn, resulted in enhanced Li+ efflux from the cells. At 3 microM, ionomycin selectively and significantly enhanced the Lii-Nao countertransport but not Li+ leakage from the cells. EGTA totally abolished the effect of ionomycin, indicating that the effect is directly related to Ca2+. As low as 0.4 microM Ca2+ caused a statistically significant effect. The maximal effect of Ca2+ on the Lii-Nao countertransport was achieved around the external pH range of 6.8-7.5. In contrast, the leakage of Li+ was significantly enhanced by Ca2+ at a pH of 7.4 and above. Ca2+ did not affect the Km of the Lii-Nao countertransport for Li+. Amiloride, which inhibits Na+/H+ exchange, inhibited only 10% of the Ca2+-enhanced countertransport. It is concluded that Ca2+ may play a role in the regulation of Lii-Nao countertransport in erythrocytes.     

Uridine uptake inhibition in Novikoff hepatoma cells by perturbants of intracellular Ca2+ and K+ levels

The rate of uptake of uridine into the acid-soluble fraction of Novikoff hepatoma cells is inhibited by low concentrations of the ionophores A23187 and gramicidin and other perturbants of intracellular cation levels. Inhibition of uridine uptake by A23187 is dependent on Ca2+ and is reduced by serum and high levels of Mg2+. The effectiveness of A23187 is dependent on the Ca2+/Mg2+ ratio rather than the absolute concentration of either ion. Inhibition of uridine uptake by gramicidin is not significantly affected by serum or divalent cations. Other effectors of monovalent cation flux such as ouabain and valinomycin also inhibit uridine uptake. These results indicate that net uptake of uridine may be influenced by intracellular levels of certain monovalent and divalent inorganic cations.     

Increased intracellular Ca2+ induces Ca2+ influx in human T lymphocytes.

One current hypothesis for the initiation of Ca2+ entry into nonelectrically excitable cells proposes that Ca2+ entry is linked to the state of filling of intracellular Ca2+ stores. In the human T lymphocyte cell line Jurkat, stimulation of the antigen receptor leads to release of Ca2+ from internal stores and influx of extracellular Ca2+. Similarly, treatment of Jurkat cells with the tumor promoter thapsigargin induced release of Ca2+ from internal stores and also resulted in influx of extracellular Ca2+. Initiation of Ca2+ entry by thapsigargin was blocked by chelation of Ca2+ released from the internal storage pool. The Ca2+ entry pathway also could be initiated by an increase in the intracellular concentration of Ca2+ after photolysis of the Ca(2+)-cage, nitr-5. Thus, three separate treatments that caused an increase in the intracellular concentration of Ca2+ initiated Ca2+ influx in Jurkat cells. In all cases, Ca(2+)-initiated Ca2+ influx was blocked by treatment with any of three phenothiazines or W-7, suggesting that it is mediated by calmodulin. These data suggest that release of Ca2+ from internal stores is not linked capacitatively to Ca2+ entry but that initiation is linked instead by Ca2+ itself, perhaps via calmodulin.     

Effect of desipramine on Ca2+ levels and growth in renal tubular cells

The in vitro effect of desipramine on renal tubular cell is unknown. In Madin-Darby canine kidney (MDCK) cells, the effect of desipramine on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Desipramine (>25 microM) caused a rapid and sustained rise of [Ca2+]i in a concentration-dependent manner (EC50=50 microM). Desipramine-induced [Ca2+]i rise was prevented by 40% by removal of extracellular Ca2+ but was not altered by L-type Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which desipramine failed to release more Ca2+; in addition, pretreatment with desipramine partly decreased thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not change desipramine-induced [Ca2+]i rise. Incubation with 10-100 microM desipramine enhances or inhibits cell proliferation in a concentration- and time-dependent manner. The inhibitory effect of desipramine on proliferation was not extracellular Ca2+-dependent. Apoptosis appears to contribute to desipramine-induced cell death. Together, these findings suggest that desipramine increases baseline [Ca2+]i in renal tubular cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and can cause apoptosis.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).