Acute effects of adenosine triphosphates, cyclic 3',5'-adenosine monophosphates, and follicle-stimulating hormone on cytosolic calcium level in cultured immature rat Ssertoli cells.

The ability of ATP and FSH to induce intracellular calcium [Ca(2+)](i) changes in Sertoli cells is imperfectly understood and reports are conflicting. We have applied the single-cell microfluorometry technique with the calcium probe indo-1 to investigate [Ca(2+)](i) in individual cultured Sertoli cells. When cells were exposed to ATP, cAMP, and FSH, a fast and biphasic increase in [Ca(2+)](i) was obtained in 100%, 70%, and 56% of cells, respectively. Caffeine did not activate Ca(2+) mobilization, while thapsigargin suppressed the peak response. External calcium free-EGTA buffer suppressed the plateau phase, while blockers of voltage-operated Ca(2+) channels did not abolish the response to cAMP and ATP. We conclude that the three messengers mobilized Ca(2+) from intracellular thapsigargin-sensitive stores, which induced a subsequent Ca(2+) influx from the extracellular medium by a voltage-independent Ca(2+) entry. The well-documented mechanisms by which these messengers act on cells support the idea that they release Ca(2+) from smooth endoplasmic reticulum by two different pathways, or that FSH and cAMP first release ATP, which then acts on cells. Among the cells, 77% and 80% responded, respectively, to FSH and cAMP by a delayed long-lasting decrease in [Ca(2+)](i) that was never recorded in the presence of ATP. This suggests that FSH and cAMP also promote a slow redistribution of [Ca(2+)](i) from the exchangeable pool to the bound nonexchangeable pools. Involvement of voltage-operated and voltage-independent calcium channels in the response of Sertoli cells to ATP, FSH, and cAMP is discussed.     

Mobilization of intracellular calcium by peroxynitrite in arteriolar smooth muscle cells from rats

The present study was designed to investigate the possible effects of peroxynitrite (ONOO(-)) on the intracellular calcium concentration ([Ca(2+)](i)) of mesenteric arteriolar smooth muscle cells (ASMCs), and to reveal the underlying mechanisms by using fluorescence imaging analysis. The results showed that ONOO(-) could exert a concentration- and time-dependent but also a dual effect on [Ca(2+)](i). Bolus administration with a low concentration of ONOO(-) (25 microM) decreased [Ca(2+)](i), whereas higher concentrations (50 or 100 microM) increased [Ca(2+)](i) persistently. Further experiments demonstrated that pretreatment of ASMCs with calcium-free medium completely abolished [Ca(2+)](i) increase by 100 microM ONOO(-). Additionally, nifedipine, an antagonist of selective L-type voltage-gated calcium channels (VGCCs), delayed the [Ca(2+)](i) response to ONOO(-), and ryanodine, an inhibitor of intracellular calcium release from the sarcoplasmic reticulum, effectively antagonized [Ca(2+)](i) increase during the late stage of ONOO(-) exposure. Furthermore, [Ca(2+)](i) alteration by ONOO(-) appeared to be intimately associated with the subsequent membrane potential changes. Although the mechanisms by which ONOO(-) alters [Ca(2+)](i) are complex, we conclude that a series of variables such as external calcium influx, activation of VGCCs, intracellular calcium release, and membrane potential changes are involved. The decrease of [Ca(2+)](i) in ASMCs by a low concentration of ONOO(-) may participate in the pathogenesis of low vasoreactivity in shock, and the increase of [Ca(2+)](i) by high concentrations of ONOO(-) may lead to calcium overload with cellular injury.     

EGF enhances Ca(2+) mobilization and capacitative Ca(2+) entry in mouse mammary epithelial cells

Effects of epidermal growth factor (EGF) on the intracellular Ca(2+) ([Ca(2+)](i)) responses to nucleotides, Ca(2+) release from thapsigargin-sensitive stores and capacitative Ca(2+) entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti-PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S-phase of cell cycle. EGF treatment apparently increased the number of PCNA-stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca(2+)](i) response. EGF treatment for 1-2 days in culture, however, enhanced [Ca(2+)](i) responses including [Ca(2+)](i) increase by ATP, UTP and other nucelotides, Ca(2+) release from thapsigargin-sensitive stores, as well as capacitative Ca(2+) entry. Genistein, a tyrosine kinase inhibitor, prevented EGF-induced cell proliferation and the [Ca(2+) ](i) responses in a dose-dependent manner. These results indicate that EGF treatment enhances Ca(2+) mobilization and capacitative Ca(2+) entry, well correlated with cellular proliferation in mammary epithelial cells.     

Activation of muscarinic acetylcholine receptors induces Ca(2+) mobilization in FRT cells

The effect of the muscarinic receptors agonist carbachol (Cch) on intracellular calcium concentration ([Ca(2+)](i)) and cAMP level was studied in polarized Fischer rat thyroid (FRT) epithelial cells. Cch provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Thapsigargin, a specific microsomal Ca(2+)-ATPase inhibitor, caused a rapid rise in [Ca(2+)](i) and subsequent addition of Cch was without effect. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Ryanodine, an agent that depletes intracellular Ca(2+) stores through stimulation of ryanodine receptors (RyRs), had no effect on [Ca(2+)](i). However, the transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with U73122, a specific inhibitor of phospholipase C (PLC). These data suggest that the Cch-stimulated increment of [Ca(2+)](i) required IP(3) formation and binding to its specific receptors in Ca(2+) stores. Further studies were performed to investigate whether the effect of Cch on Ca(2+) entry into FRT cells was via L-type voltage-dependent Ca(2+) channels (L-VDCCs). Nicardipine, a nonspecific L-type Ca(2+) channel blocker, decreased Cch-induced increase on [Ca(2+)](i), while Bay K-8644, an L-type Ca(2+) channel agonist, slightly increased [Ca(2+)](i) in FRT cells. These data indicate that Ca(2+) entry into these nondifferentiated thyroid cells occurs through an L-VDCC, and probably through another mechanism such as a capacitative pathway. Cch did not affect the intracellular cAMP levels, but its effects on [Ca(2+)](i) were significantly reduced when cells were pretreated with forskolin, suggesting the existence of an intracellular cross-talk between PLC and cAMP mechanisms in the regulation of intracellular Ca(2+) mobilization in neoplastic FRT cells.     

Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor

Growth of preimplantation embryos is influenced by autocrine trophic factors that need to act by the 2-cell stage, but their mode of action is not yet described. This report shows that late zygote and 2-cell stage mouse embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.     

Effects of linoleic acid on intracellular calcium concentration in primarily cultured rat pancreatic β-cells and underlying mechanism

In this study, we investigated the mechanism of linoleic acid-stimulated increase in intracellular calcium concentration ([Ca(2+)](i)) in pancreatic islet β-cells. Pancreatic islet cells were primarily isolated from rats and cultured for the experiments. The cells were loaded with Fluo-3/AM, the indicator of [Ca(2+)](i), and the intensity of Fluo-3 was measured using confocal microscope. The islet β-cells were identified by immunocytochemical staining with insulin antibody after recording. The drugs were given by perfusion system. The results showed that linoleic acid (20 μmol/L) stimulated [Ca(2+)](i) increase with the first peak increase and the following plateau increase. Linoleic acid-stimulated [Ca(2+)](i) increase was partly inhibited by removal of extracellular calcium and by transient receptor potential (TRP) channel blocker, La(3+), and it was totally blocked by exhaustion of intracellular calcium stores and inhibition of phospholipase C. It is concluded that linoleic acid stimulates [Ca(2+)](i) increase in islet β-cells through both extracellular calcium influx via TRP channels and calcium release from intracellular calcium stores.     

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).     

ATP increases intracellular calcium in supraoptic neurons by activation of both P2X and P2Y purinergic receptors

ATP increases intracellular calcium concentration ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system explants loaded with the Ca(2+)-sensitive dye, fura 2-AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin release from hypothalamo-neurohypophyseal system explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca(2+)](i) in dissociated SON neurons. However, the ATP-induced increase in [Ca(2+)](i) persisted after removal of Ca(2+) from the perifusate ([Ca(2+)](o)). This suggested involvement of P2Y purinergic receptors (P2YR), because P2YRs induce Ca(2+) release from intracellular stores, whereas P2XRs are Ca(2+)-permeable ion channels. Depletion of [Ca(2+)](i) stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca(2+)](i) in zero, but not in 2 mM [Ca(2+)](o), indicating that both Ca(2+) influx and release of intracellular Ca(2+) contribute to the ATP response. Ca(2+) influx was partially blocked by cadmium, indicating a contribution of voltage-gated Ca(2+) channels. PPADS (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid), and iso-PPADS, P2XR antagonists, attenuated, but did not abolish, the ATP-induced increase in [Ca(2+)](i). Combined treatment with PPADS or iso-PPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-methylthio-ADP increased [Ca(2+)](i) (with or without tetrodotoxin) that was markedly attenuated by TG. 2-Methylthio-ADP alone induced consistent and larger increases in [Ca(2+)](i) than UTP or UDP. MRS2179, a specific P2Y(1)R antagonist, eliminated the response to ATP in zero [Ca(2+)](o). Thus, both P2XR and P2YR participate in the ATP-induced increase in [Ca(2+)](i), and the P2Y(1)R subtype is more prominent than P2Y(2)R, P2Y(4)R, or P2Y(6)R in SON.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line

Exposure of HTC rat hepatoma cells to a 33% decrease in extracellular osmolality caused the cytosolic Ca(2+) concentration ([Ca(2+)](i)) to increase transiently by approximately 90 nm. This rise in [Ca(2+)](i) was inhibited strongly by apyrase, grade VII (which has a low ATP/ADPase ratio) but not by apyrase grade VI (which has a high ATP/ADPase ratio) or hexokinase, indicating that extracellular ADP and/or ATP play a role in the [Ca(2+)](i) increase. The hypotonically induced rise in [Ca(2+)](i) was prevented by the prior discharge of the intracellular Ca(2+) store of the cells by thapsigargin. Removal of extracellular Ca(2+) or inhibition of Ca(2+) influx by 1-10 microm Gd(3+) depleted the thapsigargin-sensitive Ca(2+) stores and thereby diminished the rise in [Ca(2+)](i). The hypotonically induced rise in [Ca(2+)](i) was prevented by adenosine 2'-phosphate-5'-phosphate (A2P5P) and pyridoxyl-5'-phosphate-6-azophenyl-2',4'-disulfonate, inhibitors of purinergic P2Y(1) receptors for which ADP is a major agonist. Both inhibitors also blocked the rise in [Ca(2+)](i) elicited by addition of ADP to cells in isotonic medium, whereas A2P5P had no effect on the rise in [Ca(2+)](i) elicited by the addition of the P2Y(2) and P2Y(4) receptor agonist, UTP. HTC cells were shown to express mRNA encoding for rat P2Y(1), P2Y(2), and P2Y(6) receptors. Inhibition of the hypotonically induced rise in [Ca(2+)](i) blocked hypotonically induced K(+) ((86)Rb(+)) efflux, modulated the hypotonically induced efflux of taurine, but had no significant effect on Cl(-) ((125)I-) efflux. The interaction of extracellular ATP and/or ADP with P2Y(1) purinergic receptors therefore plays a role in the response of HTC cells to osmotic swelling but does not account for activation of all the efflux pathways involved in the volume-regulatory response.     

Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea.

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca(2+)](i)) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca(2+)](i) in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca(2+)](i). ATP (1 mM) doubled the [Ca(2+)](i) after a few seconds and further induced a sustained increase of the [Ca(2+)](i). Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropic purinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca(2+)](i). Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT-PCR analysis revealed that two metabotropic receptors (P2Y(1) and P2Y(2)) and two ionotropic receptors (P2X(4) and P2X(7)) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP(3)-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X(7) receptors, which could be deleterious to these cells.     

Modulation of Ca2+ signals by phosphatidylinositol-linked novel D1 dopamine receptor in hippocampal neurons

Recent evidence indicates the existence of a putative novel phosphatidylinositol-linked D1 dopamine receptor in brain that mediates phosphatidylinositol hydrolysis via activation of phospholipase Cbeta. The present work was designed to characterize the Ca(2+) signals regulated by this phosphatidylinositol-linked D(1) dopamine receptor in primary cultures of hippocampal neurons. The results indicated that stimulation of phosphatidylinositol-linked D1 dopamine receptor by its newly identified selective agonist SKF83959 induced a long-lasting increase in basal [Ca(2+)](i) in a time- and dose-dependent manner. Stimulation was observable at 0.1 microm and reached the maximal effect at 30 microm. The [Ca(2+)](i) increase induced by 1 microm SKF83959 reached a plateau in 5 +/- 2.13 min, an average 96 +/- 5.6% increase over control. The sustained elevation of [Ca(2+)](i) was due to both intracellular calcium release and calcium influx. The initial component of Ca(2+) increase through release from intracellular stores was necessary for triggering the late component of Ca(2+) rise through influx. We further demonstrated that activation of phospholipase Cbeta/inositol triphosphate was responsible for SKF83959-induced Ca(2+) release from intracellular stores. Moreover, inhibition of voltage-operated calcium channel or NMDA receptor-gated calcium channel strongly attenuated SKF83959-induced Ca(2+) influx, indicating that both voltage-operated calcium channel and NMDA receptor contribute to phosphatidylinositol-linked D(1) receptor regulation of [Ca(2+)](i).     

Effect of erythromycin on ATP-induced intracellular calcium response in A549 cells

ATP induced a biphasic increase in the intracellular Ca(2+)concentration ([Ca(2+)](i)), an initial spike, and a subsequent plateau in A549 cells. Erythromycin (EM) suppressed the ATP-induced [Ca(2+)](i) spike but only in the presence of extracellular calcium (Ca(2+)(o)). It was ineffective against ATP- and UTP-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation and UTP-induced [Ca(2+)](i) spike, implying that EM perturbs Ca(2+) influx from the extracellular space rather than Ca(2+)release from intracellular Ca(2+) stores via the G protein-phospholipase C-Ins(1,4,5)P(3) pathway. A verapamil-sensitive, KCl-induced increase in [Ca(2+)](i) and the Ca(2+) influx activated by Ca(2+) store depletion were insensitive to EM. 3'-O-(4-benzoylbenzoyl)-ATP evoked an Ca(2+)(o)-dependent [Ca(2+)](i) response even in the presence of verapamil or the absence of extracellular Na(+), and this response was almost completely abolished by EM pretreatment. RT-PCR analyses revealed that P2X(4) as well as P2Y(2), P2Y(4), and P2Y(6) are coexpressed in this cell line. These results suggest that in A549 cells 1) the coexpressed P2X(4) and P2Y(2)/P2Y(4) subtypes contribute to the ATP-induced [Ca(2+)](i) spike and 2) EM selectively inhibits Ca(2+) influx through the P2X channel. This action of EM may underlie its clinical efficacy in the treatment of airway inflammation.     

Transthyretin oligomers induce calcium influx via voltage-gated calcium channels

The deposition of transthyretin (TTR) amyloid in the PNS is a major pathological feature of familial amyloidotic polyneuropathy. The aim of the present study was to examine whether TTR could disrupt cytoplasmic Ca(2+) homeostasis and to determine the role of TTR aggregation in this process. The aggregation of amyloidogenic TTR was examined by solution turbidity, dynamic light scattering and atomic force microscopy. A nucleation-dependent polymerization process was observed in which TTR formed low molecular weight aggregates (oligomers < 100 nm in diameter) before the appearance of mature fibrils. TTR rapidly induced an increase in the concentration of intracellular Ca(2+) ([Ca(2+)](i)) when applied to SH-SY5Y human neuroblastoma cells. The greatest effect on [Ca(2+)](i) was induced by a preparation that contained the highest concentration of TTR oligomers. The TTR-induced increase in [Ca(2+)](i) was due to an influx of extracellular Ca(2+), mainly via L- and N-type voltage-gated calcium channels (VGCCs). These results suggest that increasing [Ca(2+)](i) via VGCCs may be an important early event which contributes to TTR-induced cytotoxicity, and that TTR oligomers, rather than mature fibrils, may be the major cytotoxic form of TTR.     

Increase of [Ca(2+)](i) via activation of ATP receptors in PC-Cl3 rat thyroid cell line.

In PC-Cl3 rat thyroid cell line, ATP and UTP provoked a transient increase in [Ca(2+)](i), followed by a lower sustained phase. Removal of extracellular Ca(2+) reduced the initial transient response and completely abolished the plateau phase. Thapsigargin (TG) caused a rapid rise in [Ca(2+)](i) and subsequent addition of ATP was without effect. The transitory activation of [Ca(2+)](i) was dose-dependently attenuated in cells pretreated with the specific inhibitor of phospholipase C (PLC), U73122. These data suggest that the ATP-stimulated increment of [Ca(2+)](i) required InsP(3) formation and binding to its specific receptors in Ca(2+) stores. Desensitisation was demonstrated with respect to the calcium response to ATP and UTP in Fura 2-loaded cells. Further studies were performed to investigate whether the effect of ATP on Ca(2+) entry into PC-Cl3 cells was via L-type voltage-dependent Ca(2+) channels (L-VDCC) and/or by the capacitative pathway. Nifedipine decreased ATP-induced increase on [Ca(2+)](i). Addition of 2 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment of the cells with TG or with 100 microM ATP in Ca(2+)-free medium. These data indicate that Ca(2+) entry into PC-Cl3 stimulated with ATP occurs through both an L-VDCC and through a capacitative pathway. Using buffers with differing Na(+) concentrations, we found that the effects of ATP were dependent of extracellular Na(+), suggesting that a Na(+)/Ca(2+) exchange mechanism is also operative. These data suggest the existence, in PC-Cl3 cell line, of a P2Y purinergic receptor able to increase the [Ca(2+)](i) via PLC activation, Ca(2+) store depletion, capacitative Ca(2+) entry and L-VDCC activation.     

Altered intracellular calcium homeostasis in cerebellar granule cells of prion protein-deficient mice

Previous studies have indicated that recombinant cellular prion protein (PrP(C)), as well as a synthetic peptide of PrP(C), affects intracellular calcium homeostasis. To analyze whether calcium homeostasis in neurons is also affected by a loss of PrP(C), we performed microfluorometric calcium measurements on cultured cerebellar granule cells derived from prion protein-deficient (Prnp(0/0)) mice. The resting concentration of intracellular free calcium [Ca(2+)](i) was found to be slightly, but significantly, reduced in Prnp(0/0) mouse granule cell neurites. Moreover, we observed a highly significant reduction in the [Ca(2+)](i) increase after high potassium depolarization. Pharmacological studies further revealed that the L-type specific blocker nifedipine, which reduces the depolarization-induced [Ca(2+)](i) increase by 66% in wild-type granule cell somas, has no effect on [Ca(2+)](i) in Prnp(0/0) mouse granule cells. Patch-clamp measurements, however, did not reveal a reduced calcium influx through voltage-gated calcium channels in Prnp(0/0) mice. These data clearly indicate that loss of PrP(C) alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage-gated calcium channels.     

Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

Cholecystokinin-8 induces intracellular calcium signaling in cultured myenteric neurons from neonatal guinea pigs

The responsiveness of cultured myenteric neurons to cholecystokinin (CCK-8) was examined using fura-2-based digital microfluorimetric measurement of intracellular calcium ([Ca(2+)](i)). CCK-8 (10(-10)-10(-6)M) evoked concentration-dependent increases in percentage of neurons responding (8-52%) and delta[Ca(2+)](i) (76-169 nM). Gastrin (1 microM) also induced an increase in [Ca(2+)](i) in 29+/-6% of neurons (delta[Ca(2+)](i): 71+/-3 nM). L-364,718, an antagonist for the CCK-A receptor, blocked [Ca(2+)](i) response to CCK-8. Removal of extracellular calcium eliminated CCK-induced [Ca(2+)](i) increments, as did the addition of the calcium channel inhibitors nickel (1mM) and lanthanum (5mM). Nifedipine (1-50 microM) dose-dependently attenuated CCK-caused [Ca(2+)](i) responses. CCK evokes [Ca(2+)](i) signaling in myenteric neurons by the influx of extracellular calcium, likely through L-type calcium channels.     

Effects of Staphylococcus aureus-hemolysin A on calcium signalling in immortalized human airway epithelial cells

Part of the innate defence of bronchial epithelia against bacterial colonization is secretion of salt and water which generally depends on coordinated actions of receptor-mediated cAMP- and calcium signalling. The hypothesis that Staphylococcus aureus-virulence factors interfere with endogenous signals in host cells was tested by measuring agonist-mediated changes in [Ca(2+)](i) in S9 cells upon pre-incubation with bacterial secretory products. S9 cells responded to mAChR-activation with calcium release from intracellular stores and capacitative calcium influx. Treatment of cells with culture supernatants of S. aureus (COL) or with recombinant alpha-hemolysin (Hla) resulted in time- and concentration-dependent changes in [Ca(2+)](i). High concentrations of Hla (2000 ng/ml) resulted in elevations in [Ca(2+)](i) elicited by accelerated calcium influx. A general Hla-mediated permeabilization of S9 cell membranes to small molecules, however, did not occur. Lower concentrations of Hla (200 ng/ml) induced a reduction in [Ca(2+)](i)-levels during the sustained plateau phase of receptor-mediated calcium signalling which was abolished by pre-incubation of cells with carboxyeosin, an inhibitor of the plasma membrane calcium-ATPase. This indicates that low concentrations of Hla change calcium signalling by accelerating pump-driven extrusion of Ca(2+) ions. In vivo, such a mechanism may result in attenuation of calcium-mediated cellular defence functions and facilitation of bacterial adherence to the bronchial epithelium.