Requirement of the TRPC1 cation channel in the generation of transient Ca2+ oscillations by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is an allosteric protein that responds to extracellular Ca(2+) ([Ca(2+)](o)) and aromatic amino acids with the production of different patterns of oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). An increase in [Ca(2+)](o) stimulates phospholipase C-mediated production of inositol 1,4,5-trisphosphate and causes sinusoidal oscillations in [Ca(2+)](i). Conversely, aromatic amino acid-induced CaR activation does not stimulate phospholipase C but engages an unidentified signaling mechanism that promotes transient oscillations in [Ca(2+)](i). We show here that the [Ca(2+)](i) oscillations stimulated by aromatic amino acids were selectively abolished by TRPC1 down-regulation using either a pool of small inhibitory RNAs (siRNAs) or two different individual siRNAs that targeted different coding regions of TRPC1. Furthermore, [Ca(2+)](i) oscillations stimulated by aromatic amino acids were also abolished by inhibition of TRPC1 function with an antibody that binds the pore region of the channel. We also show that aromatic amino acid-stimulated [Ca(2+)](i) oscillations can be prevented by protein kinase C (PKC) inhibitors or siRNA-mediated PKCalpha down-regulation and impaired by either calmodulin antagonists or by the expression of a dominant-negative calmodulin mutant. We propose a model for the generation of CaR-mediated transient [Ca(2+)](i) oscillations that integrates its stimulation by aromatic amino acids with TRPC1 regulation by PKC and calmodulin.     

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.     

Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.     

P2 receptor-mediated Ca2+ transients in rat cerebral artery smooth muscle cells

Significant Ca(2+) release was previously noted with the activation of L-type Ca(2+) current in rat superior cerebral artery smooth muscle cells. Here we examined whether the P(2X) current that is partly carried by Ca(2+) also triggers Ca(2+) release in this preparation. Application of P(2X) agonists evoked membrane currents and concomitant Ca(2+) transients in whole cell voltage-clamped single cells. The expected increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was calculated from the time-integrated P(2X) current by assuming Ca(2+) is the only charge carrier. The measured increase in [Ca(2+)](i) was plotted as a function of the expected increase in [Ca(2+)](i), and Ca(2+)-buffering power was obtained as a reciprocal of the linear fit to this relationship. Both ryanodine, a Ca(2+)-induced Ca(2+)-release inhibitor, and cADP ribose, a putative activator of Ca(2+)-induced Ca(2+) release, had no significant effects on Ca(2+)-buffering power. These results suggest that Ca(2+) influx through P(2X) receptors does not trigger significant Ca(2+) release. We then examined whether P(2X) responses influence the subsequent P(2Y) response. P(2Y) responses were characterized by measuring the rate of [Ca(2+)](i) increase obtained as the slope of the linear regression to the rising phase of the Ca(2+) transient. During simultaneous application of the P(2X) and P(2Y) agonist, the rate of [Ca(2+)](i) increase was facilitated or suppressed depending on the size of the P(2X) receptor-mediated [Ca(2+)](i) increase. Membrane depolarization close to the Ca(2+) equilibrium potential significantly promoted the rate of [Ca(2+)](i) increase. Our results suggest that the [Ca(2+)](i) increase and membrane depolarization caused by the P(2X) current may regulate the subsequent P(2Y) response.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.     

Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

We examined the role of protein kinase C (PKC) in the mechanism and regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations elicited by an increase in the extracellular concentration of Ca(2+) ([Ca(2+)](e)) in human embryonic kidney 293 cells expressing the Ca(2+)-sensing receptor (CaR). Exposure to the PKC inhibitors bisindolylmaleimide I (GF I) or Ro-31-8220 converted oscillatory responses to transient, non-oscillatory responses, significantly reducing the percentage of cells that showed [Ca(2+)](i) oscillations but without decreasing the overall response to increase in [Ca(2+)](e). Exposure to 100 nm phorbol 12,13-dibutyrate, a direct activator of PKC, eliminated [Ca(2+)](i) oscillations. Addition of phorbol 12,13-dibutyrate at lower concentrations (3 and 10 nm) did not eliminate the oscillations but greatly reduced their frequency in a dose-dependent manner. Co-expression of CaR with constitutively active mutants of PKC (either epsilon or beta(1) isoforms) also reduced [Ca(2+)](i) oscillation frequency. Expression of a mutant CaR in which the major PKC phosphorylation site is altered by substitution of alanine for threonine (T888A) eliminated oscillatory behavior, producing [Ca(2+)](i) responses almost identical to those produced by the wild type CaR exposed to PKC inhibitors. These results support a model in which phosphorylation of the CaR at the inhibitory threonine 888 by PKC provides the negative feedback needed to cause [Ca(2+)](i) oscillations mediated by this receptor.     

Evidence for chloroplast control of external Ca2+-induced cytosolic Ca2+ transients and stomatal closure

The role of guard cell chloroplasts in stomatal function is controversial. It is usually assumed that stomatal closure is preceded by a transient increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in the guard cells. Here, we provide the evidence that chloroplasts play a critical role in the generation of extracellular Ca(2+) ([Ca(2+)](ext))-induced [Ca(2+)](cyt) transients and stomatal closure in Arabidopsis. CAS (Ca(2+) sensing receptor) is a plant-specific putative Ca(2+)-binding protein that was originally proposed to be a plasma membrane-localized external Ca(2+) sensor. In the present study, we characterized the intracellular localization of CAS in Arabidopsis with a combination of techniques, including (i) in vivo localization of green fluorescent protein (GFP) fused gene expression, (ii) subcellular fractionation and fractional analysis of CAS with Western blots, and (iii) database analysis of thylakoid membrane proteomes. Each technique produced consistent results. CAS was localized mainly to chloroplasts. It is an integral thylakoid membrane protein, and the N-terminus acidic Ca(2+)-binding region is likely exposed to the stromal side of the membrane. The phenotype of T-DNA insertion CAS knockout mutants and cDNA mutant-complemented plants revealed that CAS is essential for stomatal closure induced by external Ca(2+). In contrast, overexpression of CAS promoted stomatal closure in the absence of externally applied Ca(2+). Furthermore, using the transgenic aequorin system, we showed that [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients were significantly reduced in CAS knockout mutants. Our results suggest that thylakoid membrane-localized CAS is essential for [Ca(2+)](ext)-induced [Ca(2+)](cyt) transients and stomatal closure.     

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.     

17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3

We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca(2+) ([Ca(2+)](i)) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca(2+)](i), however exposure of cells to E2 in the presence of the microsomal Ca(2+) ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca(2+)](i) compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca(2+)](i). The E2-induced increase in [Ca(2+)](i) was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100muM). The estrogen receptor antagonist ICI 182,780 (1muM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca(2+)](i) from ryanodine-receptor-gated stores. The E2-induced effect on [Ca(2+)](i) could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10muM), the protein kinase A (PKA) inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate (200muM) and the MEK inhibitor PD98059 (10muM). We established E2 rapidly activates the novel PKC isoform PKCvarepsilon, PKA and Erk 1/2 MAPK in a PKCdelta and estrogen-receptor-dependent manner. The E2-induced effect was specific to 17beta-estradiol, as other steroids had no effect on [Ca(2+)](i). We have demonstrated a novel mechanism by which E2 rapidly modulates [Ca(2+)](i) release from ryanodine-receptor-gated intracellular Ca(2+) stores. The signal transduction pathway involves the estrogen receptor coupled to a PKC-PKA-Erk 1/2 signalling pathway.     

Lipoxin A4 stimulates a cytosolic Ca2+ increase in human bronchial epithelium

Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A(4) (LXA(4)) on intracellular [Ca(2+)] ([Ca(2+)](i)) of human bronchial epithelial cell. Exposure of the cells to LXA(4) produced a dose-dependent increase in [Ca(2+)](i) followed by a recovery to basal values in primary culture and in 16HBE14o(-) cells. The LXA(4)-induced [Ca(2+)](i) increase was completely abolished after pre-treatment of the 16HBE14o(-) cells with pertussis toxin (G-protein inhibitor). The [Ca(2+)](i) response was not affected by the removal of external [Ca(2+)] but completely inhibited by thapsigargin (Ca(2+)-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (R(p))-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca(2+) response to LXA(4). However, the response was not affected by chelerytrine chloride (protein kinase C inhibitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA(4) receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o(-) cells. The functional consequences of the effect of LXA(4) on intracellular [Ca(2+)](i) have been investigated on Cl(-) secretion, measured using the short-circuit techniques on 16HBE14o(-) monolayers grown on permeable filters. LXA(4) produced a sustained stimulation of the Cl(-) secretion by 16HBE14o(-) monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca(2+)](i) increase by LXA(4) through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca(2+)-dependent Cl(-) secretion by human airway epithelial cells.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. 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 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

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).     

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, G?6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.     

Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

KB-R7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE.     

Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia

Hypoxic pulmonary vasoconstriction (HPV) requires Ca(2+) influx through store-operated Ca(2+) channels (SOCC) in pulmonary arterial smooth muscle cells (PASMC) and is greater in distal than proximal pulmonary arteries (PA). SOCC may be composed of canonical transient receptor potential (TRPC) proteins and activated by stromal interacting molecule 1 (STIM1). To assess the possibility that HPV is greater in distal PA because store-operated Ca(2+) entry (SOCE) is greater in distal PASMC, we measured intracellular Ca(2+) concentration ([Ca(2+)](i)) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca(2+)-sensitive dye fura 2. Both hypoxia (4% O(2)) and KCl (60 mM) increased [Ca(2+)](i). Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca(2+)-free solutions containing cyclopiazonic acid to deplete Ca(2+) stores in sarcoplasmic reticulum and nifedipine to prevent Ca(2+) entry through L-type voltage-operated Ca(2+) channels. Under these conditions, the increase in [Ca(2+)](i) caused by restoration of extracellular Ca(2+) and the decrease in fura 2 fluorescence caused by Mn(2+) were greater in distal PASMC, indicating greater SOCE. Moreover, the increase in SOCE caused by hypoxia was also greater in distal cells. Real-time quantitative polymerase chain reaction analysis of PASMC and freshly isolated deendothelialized PA tissue demonstrated expression of STIM1 and five of seven known TRPC isoforms (TRPC1 > TRPC6 > TRPC4 > TRPC3 approximately TRPC5). For both protein, as measured by Western blotting, and mRNA, expression of STIM1, TRPC1, TRPC6, and TRPC4 was greater in distal than proximal PASMC and PA. These results provide further support for the importance of SOCE in HPV and suggest that HPV is greater in distal than proximal PA because greater numbers and activation of SOCC in distal PASMC generate bigger increases in [Ca(2+)](i).     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) results from Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through Ca(2+)-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca(2+) entry (CCE) induced by Ca(2+) store depletion represents a major Ca(2+) influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca(2+)-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca(2+) currents and CCE generated by Ca(2+) influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca(2+) channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca(2+)](cyt).     

Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.