Store-operated Ca Channels Research Articles

Role of symmetric dimethylarginine in vascular damage by increasing ROS via store-operated calcium influx in monocytes

The guanidines asymmetric dimethylarginine (ADMA), a marker of endothelial dysfunction, and its counterpart symmetric dimethylarginine (SDMA), considered inert, are accumulated in chronic kidney disease (CKD). The present study evaluates their effect on monocyte function, since previous data demonstrated leukocyte activation by other guanidino compounds. The effect of ADMA and SDMA on reactive oxygen species (ROS) production in human whole blood at baseline and after N-formyl-methionine-leucine-phenylalanine (fMLP) stimulation was evaluated. By using the fluorescent probe Fluo3-AM, the role of changes in monocytic cytoplasmic calcium ([Ca2+]i) was studied. Thapsigargin, and removal followed by addition of extracellular Ca2+ (Ca2+(ex)), was used to investigate the contribution of store-operated Ca2+-channels (SOCs). SKF96365 was used as a selective inhibitor of the SOCs. A pharmacologic intervention with captopril, known to affect Ca2+ influx, was tested. SDMA enhanced ROS production in fMLP-stimulated monocytes using heparinized blood, and this effect was abolished in EDTA-anticoagulated blood. In the presence of SDMA, an increased Ca2+ entry from the extracellular milieu resulted in an elevated amplitude of the peak [Ca2+]i change triggered by fMLP. None of these effects were seen with ADMA. Depletion of the intracellular stores with thapsigargin in the absence of Ca2+(ex), followed by re-addition of Ca2+(ex) triggered a significantly larger Ca2+ entry after SDMA treatment versus saline. This effect was prevented with SKF96365, as was the SDMA-enhanced oxidative burst after fMLP. Pre-incubation with captopril also reduced the increased ROS production seen with SDMA. SDMA, a uraemic retention solute considered inert, stimulates ROS production of monocytes by acting on Ca2+ entry via SOCs. This pro-inflammatory effect may trigger vascular pathology and may be involved in altering the prevalence of cardiovascular disease in CKD.

An essential role for stromal interaction molecule 1 in neointima formation following arterial injury

There is evidence to suggest that stromal interaction molecule 1 (STIM1) functions as a Ca2+ sensor on the endoplasmic reticulum, leading to transduction of signals to the plasma membrane and opening of store-operated Ca2+ channels (SOC). SOC have been detected in vascular smooth muscle cells (VSMCs) and are thought to have an essential role in the regulation of contraction and cell proliferation. We hypothesized that knockdown of STIM1 inhibits VSMC proliferation and suppresses neointimal hyperplasia. We examined the effect of the knockdown of STIM1 using a rat balloon injury model and cultured rat aortic VSMCs. Interestingly, knockdown of rat STIM1 by adenovirus delivery of small interfering RNA (siRNA) significantly suppressed neointimal hyperplasia in a rat carotid artery balloon injury model at 14 days after injury. The re-expression of human STIM1 to smooth muscle reversed the effect of STIM1 knockdown on neointimal formation. Rat aortic VSMCs were used for the in vitro assays. Knockdown of endogenous STIM1 significantly inhibited proliferation and migration of VSMCs. Moreover, STIM1 knockdown induced cell-cycle arrest in G0/G1 and resulted in a marked decrease in SOC. Replenishment with recombinant human STIM1 reversed the effect of siRNA knockdown. These results suggest STIM1 has a critical role in neointimal formation in a rat model of vascular injury. STIM1 may represent a novel therapeutic target in the prevention of restenosis after vascular interventions.

Ciglitazone induces apoptosis via activation of p38 MAPK and AIF nuclear translocation mediated by reactive oxygen species and Ca 2+ in opossum kidney cells

We have previously demonstrated that the synthetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist ciglitazone induces apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) and nuclear translocation of apoptosis inducing factor (AIF) in opossum kidney (OK) renal epithelial cells. However, the precise mechanism by which ciglitazone induces activation of p38 MAPK and the role of AIF in the induction of the apoptosis are not defined. This study was therefore undertaken to determine whether the roles of reactive oxygen species (ROS) generation and intracellular Ca 2+ in the ciglitazone-induced activation of p38 MAPK and whether AIF nuclear translocation is responsible for the ciglitazone-induced apoptosis in OK renal epithelial cells. Ciglitazone caused generation of ROS and an increase in intracellular Ca 2+. Ciglitazone-induced cell death was reduced by the antioxidant Trolox, the Ca 2+ chelator EGTA, and the store-operated Ca 2+ channels (SOCC) blocker lanthanum chloride (La 3+), indicating involvement of ROS and Ca 2+ in the ciglitazone-induced cell death. Ciglitazone-induced intracellular Ca 2+ increase was decreased by Trolox, while ROS generation was not affected by EGTA and La 3+, suggesting that ROS generation promote the increase of intracellular Ca 2+. Transfection of small interfering RNA (siRNA) of p38 MAPK or vector expressing microRNA (miRNA) of AIF prevented the ciglitazone-induced cell death. Activation of p38 MAPK, mitochondrial membrane depolarization, and AIF nuclear translocation induced by ciglitazone were inhibited by Trolox, EGTA and La 3+. Taken together, these results suggest that ROS-dependent intracellular Ca 2+ increase is responsible for activation of p38 MAPK and nuclear translocation of AIF by ciglitazone.

Mitochondrial inhibitors activate influx of external Ca 2+ in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca 2+ concentration ([Ca 2+] i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na +/Ca 2+ exchange inhibitor) increases [Ca 2+] i with at least two differing profiles. These increases depend on the presence of extracellular Ca 2+, which indicates they involve Ca 2+ uptake and not only mitochondrial Ca 2+ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn 2+. Store-operated Ca 2+ channel (SOC) blockers (Ni 2+, SKF96365 and Gd 2+) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca 2+ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca 2+ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca 2+] i, apparently oligomycin also has an extra mitochondrial target.

New insights into the activation mechanism of store-operated calcium channels: roles of STIM and Orai

The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past two decades. Store-operated Ca2+-release-activated Ca2+ (CRAC) channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement. In a set of breakthrough studies over the past two years, stromal interaction molecule 1 (STIM1, the ER Ca2+ sensor) and Orai1 (a pore-forming subunit of the CRAC channel) have been identified. Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels (SOCCs).

Protease-activated Receptor-2 Increases Exocytosis via Multiple Signal Transduction Pathways in Pancreatic Duct Epithelial Cells

Protease-activated receptor-2 (PAR-2) is activated when trypsin cleaves its NH(2) terminus to expose a tethered ligand. We previously demonstrated that PAR-2 activates ion channels in pancreatic duct epithelial cells (PDEC). Using real-time optical fluorescent probes, cyan fluorescence protein-Epac1-yellow fluorescence protein for cAMP, PH(PLC-delta1)-enhanced green fluorescent protein for phosphatidylinositol 4,5-bisphosphate, and protein kinase Cgamma (PKCgamma)-C1-yellow fluorescence protein for diacylglycerol, we now define the signaling pathways mediating PAR-2 effect in dog PDEC. Although PAR-2 activation does not stimulate a cAMP increase, it induces phospholipase C to hydrolyze phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Intracellular Ca(2+) mobilization from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores and a subsequent Ca(2+) influx through store-operated Ca(2+) channels cause a biphasic increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), measured with Indo-1 dye. Single-cell amperometry demonstrated that this increase in [Ca(2+)](i) in turn causes a biphasic increase in exocytosis. A protein kinase assay revealed that trypsin also activates PKC isozymes to stimulate additional exocytosis. Paralleling the increased exocytosis, mucin secretion from PDEC was also induced by trypsin or the PAR-2 activating peptide. Consistent with the serosal localization of PAR-2, 1 microm luminal trypsin did not induce exocytosis in polarized PDEC monolayers; on the other hand, 10 microm trypsin at 37 degrees C damaged the epithelial barrier sufficiently so that it could reach and activate the serosal PAR-2 to stimulate exocytosis. Thus, in PDEC, PAR-2 activation increases [Ca(2+)](i) and activates PKC to stimulate exocytosis and mucin secretion. These functions may mediate the reported protective role of PAR-2 in different models of pancreatitis.

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).

S1885 TGFβ Utilizes NF-κB to Mediate PTEN Suppression in a Smad-Independent Manner in Pancreatic Cancer Cells

Background: Transforming growth factor-β (TGFβ) is a key tumor suppressor that is involved in cell differentiation and normal pancreatic cell homeostasis. In pancreatic cancer, the suppressor function is lost in part by events that affect the TGFβ-SMAD signaling pathway, such as bi-allelic loss of SMAD4 and oncogenic K-RAS activation that slows nuclear translocation of receptor-activated SMADs. With loss of TGFβ-SMAD signaling, TGFβ promotes cellular proliferation via SMAD-independent signaling pathways. We have shown that TGFβ downregulates the tumor suppressor PTEN in a SMAD-independent manner via activation of PKCα in pancreatic cancer cells, but the mechanisms leading to PKCα activation by TGFβ are unexplored. We examined if TGFβ could activate a Ca2+-dependent pathway, particularly Ca2+ entry through the plasmamembrane store-operated Ca2+ channel (SOC).We hypothesized that TGFβ increases cytoplasmic free Ca2+ ([Ca]cyt) via SOC after activation of the IP3 receptor (IP3R) on the endoplasmic reticulum membrane. Methods: BxPc-3 human pancreatic cancer cells were treated with 10 ng/ml TGFβ, 100 mM 2-APB (inhibitor for both IP3R and SOC), 30 mM La3+ (a selective SOC inhibitor), and the effect on [Ca]cyt were measured by a digital MetaFluor Imaging System using the fluorescent Ca2+-sensitive dye, fura 2-AM. Separated membrane and cytosolic fraction proteins were probed for PKCα by Western blotting. Results: TGFβ activates PKCα by 15 minutes after treatment, as evidenced by translocation from the cytosolic to the membrane fraction. TGFβ increased [Ca]cyt within 100 seconds after treatment (∆F 0.25 ± 0.02, n=38, P<0.01), while pretreatment with 2-APB inhibited increase in [Ca]cyt (∆F 0.01 ± 0.01, 98% inhibition, n=46, P<0.001). Pre-treatment of the cells with 2-APB prevented TGFβ-induced PKCα activation. Pretreatment with La3+ totally abolished the TGFβ-induced increase in [Ca]cyt (n=34, P<0.01). Conclusion: TGFβ may activate IP3R that triggers release of intracellular Ca2+ and Ca2+ entry via SOC. These intracellular Ca2+ stores are important for TGFβ-induced PKCα activation and subsequent tumor cell proliferation. Our data is the first to demonstrate that TGFβ can mobilize intracellular Ca2+ stores to activate proliferative PKCα signaling in pancreatic cells. Future treatments of pancreatic cancer and other cancers may involve manipulation of intracellular Ca2+ levels by interventions towards Ca2+-dependent pathways.

S1884 TGFβ Activates PKCα Through the Store-Operated CA 2+ Channel (SOC) in Pancreatic Cancer Cells

S1884 TGFβ Activates PKCα Through the Store-Operated CA 2+ Channel (SOC) in Pancreatic Cancer Cells

Knockdown of Stromal Interaction Molecule 1 (STIM‐1) Decreases Store‐operated Calcium Entry (SOCE) and Attenuates Hypoxic Calcium Response in Pulmonary Artery Smooth Muscle Cells (PASMC)

The increased intracellular Ca2+ concentration ([Ca2+]i) caused by acute hypoxia in PASMC requires Ca2+ release from sarcoplasmic reticulum (SR) and influx through store‐operated Ca2+ channels (SOCC). Recent evidence in a variety of cell types, including smooth muscle, suggests that STIM‐1 senses Ca2+ concentration within the SR lumen and transduces activation of SOCC upon SR Ca2+ release and depletion. To determine if STIM‐1 plays a role in the [Ca2+]i response to hypoxia in PASMC, we measured [Ca2+]i with the fluorescent Ca2+‐sensitive dye, Fura‐2, in primary cultures of rat distal PASMC treated with nontargeted small interfering RNA (siRNA) or siRNA targeted to STIM‐1. Compared to nontargeted siRNA, STIM‐1 siRNA caused: 1) a 92% decrease in STIM‐1 mRNA, as measured by real‐time quantitative PCR; 2) an 81% decrease in STIM‐1 protein, as measured by Western blotting; 3) no change in mRNA for STIM‐2, a related protein that may regulate STIM‐1; 4) 74% inhibition of the increase in [Ca2+]i caused by restoration of extracellular Ca2+ in cells perfused with Ca2+‐free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete SR Ca2+ stores and nifedipine to block Ca2+ entry through L‐type voltage‐operated Ca2+ channels; 5) 77% inhibition of increase in [Ca2+]i caused by hypoxia (4% O2) in PASMC perfused with normal KRBS; and 6) no inhibition of the increase in [Ca2+]i caused by equimolar (60 mM) substitution of KCl for NaCl in the normal KRBS perfusate. These results indicate that STIM‐1 is expressed in rat distal PASMC and plays an important role in SOCE and the [Ca2+]i response to acute hypoxia in these cells.

Store-operated Ca 2+ channels and Stromal Interaction Molecule 1 (STIM1) are targets for the actions of bile acids on liver cells

Cholestasis is a significant contributor to liver pathology and can lead to primary sclerosis and liver failure. Cholestatic bile acids induce apoptosis and necrosis in hepatocytes but these effects can be partially alleviated by the pharmacological application of choleretic bile acids. These actions of bile acids on hepatocytes require changes in the release of Ca 2+ from intracellular stores and in Ca 2+ entry. However, the nature of the Ca 2+ entry pathway affected is not known. We show here using whole cell patch clamp experiments with H4-IIE liver cells that taurodeoxycholic acid (TDCA) and other choleretic bile acids reversibly activate an inwardly-rectifying current with characteristics similar to those of store-operated Ca 2+ channels (SOCs), while lithocholic acid (LCA) and other cholestatic bile acids inhibit SOCs. The activation of Ca 2+ entry was observed upon direct addition of the bile acid to the incubation medium, whereas the inhibition of SOCs required a 12 h pre-incubation. In cells loaded with fura-2, choleretic bile acids activated a Gd 3+-inhibitable Ca 2+ entry, while cholestatic bile acids inhibited the release of Ca 2+ from intracellular stores and Ca 2+ entry induced by 2,5-di-( tert-butyl)-1,4-benzohydro-quinone (DBHQ). TDCA and LCA each caused a reversible redistribution of stromal interaction molecule 1 (STIM1, the endoplasmic reticulum Ca 2+ sensor required for the activation of Ca 2+ release-activated Ca 2+ channels and some other SOCs) to puncta, similar to that induced by thapsigargin. Knockdown of Stim1 using siRNA caused substantial inhibition of Ca 2+-entry activated by choleretic bile acids. It is concluded that choleretic and cholestatic bile acids activate and inhibit, respectively, the previously well-characterised Ca 2+-selective hepatocyte SOCs through mechanisms which involve the bile acid-induced redistribution of STIM1.

Orai1–STIM1 formed store-operated Ca 2+ channels (SOCs) as the molecular components needed for Pb 2+ entry in living cells

Heavy metal lead (Pb 2+) is a pollutant and causes severe toxicity when present in human tissues especially the nervous system. Recent reviews have suggested that Pb 2+ can target Ca 2+-related proteins within neurons and that Ca 2+ channels might be a candidate for Pb 2+ entry. This study's main aim was to identify the functional entry pathway of Pb 2+ into living cells. We firstly characterized the endogenous expression of Orai1 and STIM1 mRNA together with the level of thapsigargin (TG) stimulated capacitative Ca 2+ entry in PC12 and HeLa cells; this was done by RT–PCR and time-lapse Ca 2+ imaging microscopy, respectively. Our data supported Orai1 and STIM1 as contributing to store-operated Ca 2+ channel (SOC) basal activity. Secondly, using the indo-1 quenching method with the SOC blocker 2-APB, we observed that Pb 2+ was able to enter cells directly through unactivated SOCs without TG pretreatment. Thirdly, we further demonstrated that co-expression of Orai1 and STIM1 differentially enhanced SOC functional activity (4-fold with PC12 and 5-fold with HeLa cells) and Pb 2+ entry (5- to 7-fold with PC12 and 2-fold with HeLa cells). Furthermore, after a 1 h of Pb 2+ exposure, the depolarization- and histamine-induced Ca 2+ responses were significantly decreased in both PC12 and HeLa cells in a dose-dependent manner. This result indicated that the decreased Ca 2+ responses were, in part, due to Pb 2+ entry. In summary, our results suggest that SOCs are responsible for Pb 2+ permeation and that the Orai1–STIM1 protein complex formed by functional SOCs is one of the molecular components involved in Pb 2+ entry.

Phospholipase C-gamma1 is required for the activation of store-operated Ca2+ channels in liver cells.

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

Some assembly required: Constructing the elementary units of store-operated Ca 2+ entry

The means by which Ca 2+ store depletion evokes the opening of store-operated Ca 2+ channels (SOCs) in the plasma membrane of excitable and non-excitable cells has been a longstanding mystery. Indirect evidence has supported local interactions between the ER and SOCs as well as long-range interactions mediated through a diffusible activator. The recent molecular identification of the ER Ca 2+ sensor (STIM1) and a subunit of the CRAC channel (Orai1), a prototypic SOC, has now made it possible to visualize directly the sequence of events that links store depletion to CRAC channel opening. Following store depletion, STIM1 moves from locations throughout the ER to accumulate in ER subregions positioned within 10–25 nm of the plasma membrane. Simultaneously, Orai1 gathers at discrete sites in the plasma membrane directly opposite STIM1, resulting in local CRAC channel activation. These new studies define the elementary units of store-operated Ca 2+ entry, and reveal an unprecedented mechanism for channel activation in which the stimulus brings a channel and its activator/sensor together for interaction across apposed membrane compartments. We discuss the implications of this choreographic mechanism with regard to Ca 2+ dynamics, specificity of Ca 2+ signaling, and the existence of a specialized ER subset dedicated to the control of the CRAC channel.

Store-Operated Ca 2+ Influx and Expression of TRPC Genes in Mouse Sinoatrial Node

Store-operated Ca(2+) entry was investigated in isolated mouse sinoatrial nodes (SAN) dissected from right atria and loaded with Ca(2+) indicators. Incubation of the SAN in Ca(2+)-free solution caused a substantial decrease in resting intracellular Ca(2+) concentration ([Ca(2+)](i)) and stopped pacemaker activity. Reintroduction of Ca(2+) in the presence of cyclopiazonic acid (CPA), a sarcoplasmic reticulum Ca(2+) pump inhibitor, led to sustained elevation of [Ca(2+)](i), a characteristic of store-operated Ca(2+) channel (SOCC) activity. Two SOCC antagonists, Gd(3+) and SKF-96365, inhibited 72+/-8% and 65+/-8% of this Ca(2+) influx, respectively. SKF-96365 also reduced the spontaneous pacemaker rate to 27+/-4% of control in the presence of CPA. Because members of the transient receptor potential canonical (TRPC) gene family may encode SOCCs, we used RT-PCR to examine mRNA expression of the 7 known mammalian TRPC isoforms. Transcripts for TRPC1, 2, 3, 4, 6, and 7, but not TRPC5, were detected. Immunohistochemistry using anti-TRPC1, 3, 4, and 6 antibodies revealed positive labeling in the SAN region and single pacemaker cells. These results indicate that mouse SAN exhibits store-operated Ca(2+) activity which may be attributable to TRPC expression, and suggest that SOCCs may be involved in regulating pacemaker firing rate.

CRAC channel activity in C. elegans is mediated by Orai1 and STIM1 homologues and is essential for ovulation and fertility.

The Ca(2+) release-activated Ca(2+) (CRAC) channel is a plasma membrane Ca(2+) entry pathway activated by endoplasmic reticulum (ER) Ca(2+) store depletion. STIM1 proteins function as ER Ca(2+) sensors and regulate CRAC channel activation. Recent studies have demonstrated that CRAC channels are encoded by the human Orai1 gene and a homologous Drosophila gene. C. elegans intestinal cells express a store-operated Ca(2+) channel (SOCC) regulated by STIM-1. We cloned a full-length C. elegans cDNA that encodes a 293 amino acid protein, ORAI-1, homologous to human and Drosophila Orai1 proteins. ORAI-1 GFP reporters are co-expressed with STIM-1 in the gonad and intestine. Inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signalling regulates C. elegans gonad function, fertility and rhythmic posterior body wall muscle contraction (pBoc) required for defecation. RNA interference (RNAi) silencing of orai-1 expression phenocopies stim-1 knockdown and causes sterility and prevents intestinal cell SOCC activation, but has no effect on pBoc or intestinal Ca(2+) signalling. Orai-1 RNAi suppresses pBoc defects induced by intestinal expression of a STIM-1 Ca(2+)-binding mutant, indicating that the proteins function in a common pathway. Co-expression of stim-1 and orai-1 cDNAs in HEK293 cells induces large inwardly rectifying cation currents activated by ER Ca(2+) depletion. The properties of this current recapitulate those of the native SOCC current. We conclude that C. elegans expresses bona fide CRAC channels that require the function of Orai1- and STIM1-related proteins. CRAC channels thus arose very early in animal evolution. In C. elegans, CRAC channels do not play obligate roles in all IP(3)-dependent signalling processes and ER Ca(2+) homeostasis. Instead, we suggest that CRAC channels carry out highly specialized and cell-specific signalling roles and that they may function as a failsafe mechanism to prevent Ca(2+) store depletion under pathophysiological and stress conditions.

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.

Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities

An increased understanding of the genetic, molecular and cellular mechanisms responsible for the development of polycystic kidney disease has laid out the foundation for the development of rational therapies. Many animal models where these therapies can be tested are currently available. This review summarizes the rationale for these treatments, the results of preclinical trials and the prospects for clinical trials, some already in early phases of implementation.

Initiation Site of Ca2+ Entry Evoked by Endoplasmic Reticulum Ca2+ Depletion in Mouse Parotid and Pancreatic Acinar Cells

PurposeIn non-excitable cells, which include parotid and pancreatic acinar cells, Ca2+ entry is triggered via a mechanism known as capacitative Ca2+ entry, or store-operated Ca2+ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca2+ stores, which acts as an important factor triggering Ca2+ entry. However, both the mechanism of store-mediated Ca2+ entry and the molecular identity of store-operated Ca2+ channel (SOCC) remain uncertain.Materials and MethodsIn the present study we investigated the Ca2+ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.ResultsTreatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca2+-ATPase, in an extracellular Ca2+ free state, and subsequent exposure to a high external calcium state evoked Ca2+ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca2+ channel, completely blocked Tg-induced Ca2+ entry. Microfluorometric imaging showed that Tg-induced Ca2+ entry started at a basal membrane, not a apical membrane.ConclusionThese results suggest that Ca2+ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.

Heregulinβ activates store-operated Ca 2+ channels through c-erbB2 receptor level-dependent pathway in human breast cancer cells

The heregulinβ (HRGβ) is a ligand to activate c-erbB2/c-erbB3 interaction and can subsequently increases cytosolic [Ca 2+] i. In the two human breast cancer cell lines, MCF-7 shows a low c-erbB2 expression level, whereas SK-BR-3 overexpress c-erbB2 receptor. In this article, we have found that in MCF-7, HRGβ induced Ca 2+ release from the endoplasmic reticulums (ER) and subsequently activated Ca 2+ entry via store-operated Ca 2+ channel (SOC). However, in SK-BR-3, HRGβ failed to induce Ca 2+ release and Ca 2+entry. RNA interference to decrease c-erbB2 level in SK-BR-3 resulted in reactivation of HRGβ-evoked Ca 2+ release and Ca 2+ entry via SOC, which was similar to that of MCF-7. In addition, in the absence of HRGβ, a constitutive activation of SOC was observed in SK-BR-3 rather than in MCF-7 and c-erbB2-siRNA treated SK-BR-3. Compared to the cells with low c-erbB2 level, c-erbB2 might tend to interact with c-erbB3 in the resting state in the cells with high c-erbB2 level, which resulted in different [Ca 2+] i responses to HRGβ. In SK-BR-3, the Ca 2+ mobilization in the presence or in the absence of HRGβ was completely blocked by PLC inhibitor U73122. In summary, our results indicate that HRGβ-induced SOC was regulated by c-erbB2 level and dependent on activation of PLC in human breast cancer cells.