2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.     

P2Y receptor subtypes evoke different Ca2+ signals in cultured aortic smooth muscle cells

Adenine and uridine nucleotides evoke Ca(2+) signals via four subtypes of P2Y receptor in cultured aortic smooth muscle cells, but the mechanisms underlying the different patterns of these Ca(2+) signals are unresolved. Cytosolic Ca(2+) signals were recorded from single cells and populations of cultured rat aortic smooth muscle cells, loaded with a fluorescent Ca(2+) indicator and stimulated with agonists that allow subtype-selective activation of P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Activation of P2Y1, P2Y2, and P2Y6 receptors caused homologous desensitisation, while activation of P2Y2 receptors also caused heterologous desensitisation of the other subtypes. The Ca(2+) signals evoked by each P2Y receptor subtype required activation of phospholipase C and release of Ca(2+) from intracellular stores via inositol 1,4,5-trisphosphate (IP(3)) receptors, but they were unaffected by inhibition of ryanodine or nicotinic acid adenine dinucleotide phosphate (NAADP) receptors. Sustained Ca(2+) signals were independent of the Na(+)/Ca(2+) exchanger and were probably mediated by store-operated Ca(2+) entry. Analyses of single cells established that most cells express P2Y2 receptors and at least two other P2Y receptor subtypes. We conclude that four P2Y receptor subtypes evoke Ca(2+) signals in cultured aortic smooth muscle cells using the same intracellular (IP(3) receptors) and Ca(2+) entry pathways (store-operated Ca(2+) entry). Different rates of homologous desensitisation and different levels of receptor expression account for the different patterns of Ca(2+) signal evoked by each P2Y receptor subtype.     

R-Type Ca<Superscript>2+</Superscript>-channel Activity Is Associated with Chromogranin A Secretion in Human Neuroendocrine Tumor BON Cells

This electrophysiological study was undertaken to investigate the role of voltage-operated Ca(2+) channels (VOCCs) in cultivated human neuroendocrine tumor (NET) cells. Patch-clamp techniques, measurements of intracellular Ca(2+) ([Ca(2+)](i)), and secretion analysis were performed using cultured human NET BON cells. Ba(2+) inward currents through R-type channels (Ca(V)2.3) were measured and identified by SNX-482 (10 n M), a novel voltage-sensitive R-type Ca(2+) channel antagonist. In the presence of nifedipine (5 micro M), omega-Conotoxin GVIA (100 n M) and omega-Agatoxin IVA (20 n M), R-type channel currents were also detectable. Release of Ca(2+) from intracellular Ca(2+) stores by intracellular application of inositol-1,4,5-trisphosphate (InsP(3); 10 micro M) via the patch pipette during whole-cell configuration as well as induction of capacitative Ca(2+) entry (CCE), a passive maneuver to release Ca(2+) from intracellular Ca(2+) stores, led to an increase in [Ca(2+)](i). This effect could be reduced by SNX-482 (20 n M). In addition, SNX-482 (25 n M) also decreased chromogranin A (CgA) secretion, whereas omega-Conotoxin GVIA (500 n M) and nifedipine (5 micro M) failed to reduce CgA secretion. We conclude that these data reveal neuronal R-type channel activity (Ca(V)2.3), for the first time associated with CgA secretion in BON cells. Influx of Ca(2+) by activation of R-type channels may lead to an increase of intracellular Ca(2+), which stimulates CgA secretion. Thus, R-type channels could play an important role in certain clinical characteristics of NETs, such as the hypersecretion syndrome.     

Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.

The mechanisms of agonist-induced Ca(2+) spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP(3)) and a low-affinity Ca(2+) indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP(3) was able to reproduce acetylcholine (ACh)-induced three forms of Ca(2+) spikes: local Ca(2+) spikes and submicromolar (<1 microM) and micromolar (1-15 microM) global Ca(2+) spikes (Ca(2+) waves). These observations indicate that subcellular gradients of IP(3) sensitivity underlie all forms of ACh-induced Ca(2+) spikes, and that the amplitude and extent of Ca(2+) spikes are determined by the concentration of IP(3). IP(3)-induced local Ca(2+) spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca(2+)-induced Ca(2+) release in local Ca(2+) spikes. In contrast, IP(3)- induced global Ca(2+) spikes were consistently faster than those evoked with ACh at all concentrations of IP(3) and ACh, suggesting that production of IP(3) via phospholipase C was slow and limited the spread of the Ca(2+) spikes. Indeed, gradual photolysis of caged IP(3) reproduced ACh-induced slow Ca(2+) spikes. Thus, local and global Ca(2+) spikes involve distinct mechanisms, and the kinetics of global Ca(2+) spikes depends on that of IP(3) production particularly in those cells such as acinar cells where heterogeneity in IP(3) sensitivity plays critical role.     

Stimulation of arachidonic acid release by vasopressin in A7r5 vascular smooth muscle cells mediated by Ca2+-stimulated phospholipase A2

Arachidonic acid (AA) regulates many aspects of vascular smooth muscle behaviour, but the mechanisms linking receptors to AA release are unclear. In A7r5 vascular smooth muscle cells pre-labelled with (3)H-AA, vasopressin caused a concentration-dependent stimulation of 3H-AA release that required phospholipase C and an increase in cytosolic [Ca2+]. Ca2+ release from intracellular stores and Ca2+ entry via L-type channels or the capacitative Ca2+ entry pathway were each effective to varying degrees. Selective inhibitors of PLA2 inhibited the 3H-AA release evoked by vasopressin, though not the underlying Ca2+ signals, and established that cPLA2 mediates the release of AA. We conclude that in A7r5 cells vasopressin stimulates AA release via a Ca2+-dependent activation of cPLA2.     

Polycystin-2 accelerates Ca2+ release from intracellular stores in Caenorhabditis elegans

Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that polycystin-2 is an intracellular Ca(2+) release channel that is required for the normal pattern of Ca(2+) responses involving IP(3) and ryanodine receptor-mediated Ca(2+) release from intracellular stores. Activity of polycystin-2 creates brief cytosolic Ca(2+) transients with increased amplitude and decreased duration. Polycystin-2, along with the IP(3) and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.     

Interactions between inositol 1,4,5-trisphosphate receptors and ryanodine receptors in smooth muscle: one store or two?

This short review proposes a system of simplified functional models describing possible interactions between Ca(2+)-release channels associated with IP(3)Rs and RyRs in smooth muscle, and considers each of these models in the light of the available experimental evidence. Complete separation of IP(3)R- and RyR-gated stores seems to be unusual. Where both receptors release Ca(2+) from a common pool, simple interactions can occur since changes in the activation of one receptor type affects the availability of Ca(2+) for release through the other. Alterations in [Ca(2+)] within the sarcoplasmic reticulum can also affect the open probability of the release channels, and not just the Ca(2+)-flux through the channels when open, e.g., Ca(2+)-release through tonically active IP(3)Rs appears to limit SR Ca(2+)-content in some myocytes, and this modulates RyR activity, as indicated by changes in Ca(2+)-spark frequency. There is also evidence that intracellular release channels may co-operate, leading to positive feedback during activation. In particular, agonist-dependent activation of IP(3)Rs can promote activation of RyRs, amplifying and shaping the resulting Ca(2+)-signal. While there is little direct evidence as to the mechanism responsible for this interaction, some form of Ca(2+)-induced Ca(2+)-release in response to local increases in [Ca(2+)](c) seems likely.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.     

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.     

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Xestospongin B, a competitive inhibitor of IP3-mediated Ca2+ signalling in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells

Xestospongin B, a macrocyclic bis-1-oxaquinolizidine alkaloid extracted from the marine sponge Xestospongia exigua, was highly purified and tested for its ability to block inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release. In a concentration-dependent manner xestospongin B displaced [(3)H]IP(3) from both rat cerebellar membranes and rat skeletal myotube homogenates with an EC(50) of 44.6 +/- 1.1 microM and 27.4 +/- 1.1 microM, respectively. Xestospongin B, depending on the dose, suppressed bradykinin-induced Ca(2+) signals in neuroblastoma (NG108-15) cells, and also selectively blocked the slow intracellular Ca(2+) signal induced by membrane depolarization with high external K(+) (47 mM) in rat skeletal myotubes. This slow Ca(2+) signal is unrelated to muscle contraction, and involves IP(3) receptors. In highly purified isolated nuclei from rat skeletal myotubes, Xestospongin B reduced, or suppressed IP(3)-induced Ca(2+) oscillations with an EC(50) = 18.9 +/- 1.35 microM. In rat myotubes exposed to a Ca(2+)-free medium, Xestospongin B neither depleted sarcoplasmic reticulum Ca(2+) stores, nor modified thapsigargin action and did not affect capacitative Ca(2+) entry after thapsigargin-induced depletion of Ca(2+) stores. Ca(2+)-ATPase activity measured in skeletal myotube homogenates remained unaffected by Xestospongin B. It is concluded that xestospongin B is an effective cell-permeant, competitive inhibitor of IP(3) receptors in cultured rat myotubes, isolated myonuclei, and neuroblastoma (NG108-15) cells.     

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca(2+) in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca(2+) concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca(2+) transients induced by photolysis of caged S1P were caused by Ca(2+) mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.     

RYR2 proteins contribute to the formation of Ca(2+) sparks in smooth muscle

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca(2+) release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca(2+) release events (Ca(2+) sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca(2+) release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca(2+) sparks, Ca(2+)-induced Ca(2+) release, and Ca(2+) waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6(-/-), and RYR3(-/-) mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca(2+) sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca(2+) sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca(2+) -induced Ca(2+) release was similarly augmented in FKBP12.6(-/-), but not in RYR3 null cells relative to wild-type. Finally, Ca(2+) wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca(2+) wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca(2+) sparks in smooth muscle, and the finding of normal Ca(2+) sparks and CICR in RYR3 null mice, indicate that Ca(2+) release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca(2+) sparks, and associated STOCs, in smooth muscle.     

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.     

Role of Ca2+ signaling in initiation of stretch-induced apoptosis in neonatal heart cells

Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca(2+) signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca(2+) ([Ca(2+)](i)). Buffering [Ca(2+)](i) by EGTA-AM, suppressing ryanodine-sensitive Ca(2+) release, and blocking L-type Ca(2+) channels all prevented the stretch-induced apoptosis as assessed by phosphatidylserine exposure and nuclear fragmentation. Notably, Ca(2+) suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca(2+) signaling is the upstream of these events. Since [Ca(2+)](i) did not change without activating mechanosensitive Ca(2+) entry, we conclude that stretch-induced Ca(2+) entry, via the Ca(2+)-induced Ca(2+) release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress.