Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2+ channels

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca(2+) selective I(CRAC) and relatively Ca(2+) selective I(SOC), respectively. STIM1 has been proposed to communicate the Ca(2+) content of the intracellular Ca(2+) stores to the plasma membrane store-operated Ca(2+) channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca(2+) stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca(2+). In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca(2+) permeable channels. Electrotransjection of cells with anti-Orai1 antibody, directed toward the C-terminal region that mediates the interaction with STIM1, and stabilization of an actin cortical barrier with jasplakinolide prevented the interaction between STIM1 and hTRPC1. Under these conditions hTRPC1 was no longer involved in store-operated calcium entry but in diacylglycerol-activated non-capacitative Ca(2+) entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca(2+) entry.     

A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: evidence against the de novo conformational coupling hypothesis

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.     

SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets

Two agonist-releasable Ca(2+)stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca(2+)entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca(2+)-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca(2+)content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca(2+)in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 microM TBHQ, individually or simultaneously, accelerated Ca(2+) store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca(2+) influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca(2+) extrusion by PMCA or the Na(+)/Ca(2+) exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.     

Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 microM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 microM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.     

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca(2+) entry (SOCE) regulated by two independent Ca(2+) stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP(3) receptor (IP(3)RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca(2+) entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca(2+)-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca(2+) stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca(2+) stores and the DTS, the major releasable Ca(2+) store in platelets. These findings were confirmed by using Sr(2+) as a surrogate for Ca(2+) entry. Colchicine attenuated the coupling between IP(3)RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP(3)RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca(2+). These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca(2+) stores.     

Functional relevance of the de novo coupling between hTRPC1 and type II IP3 receptor in store-operated Ca2+ entry in human platelets

Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is regulated by the filling state of the intracellular Ca2+ stores. We have previously reported that a de novo conformational coupling between the type II IP3 receptor (IP3RII) and hTRPC1 channel occurs after depletion of the intracellular Ca2+ stores in human platelets, which might be involved in the activation of SOCE in these cells. Here we present for the first time direct evidence for the functional relevance of the coupling between hTRPC1 and IP3RII in SOCE in human platelets. Our data suggest that at least two pathways may contribute to SOCE in these cells. An early component, insensitive to cytochalasin D (Cyt D), is followed by a late component which is sensitive to Cyt D. Introduction of a peptide corresponding to IP3RII(317-334) (IP3BD-peptide(317-334)) in the cells by electrotransjection impairs the coupling between hTRPC1 and IP3RII but not the interaction between hTRPC1 and STIM1 induced by store depletion. Coimmunoprecipitation experiments indicated that endogenously expressed hTRPC1 interacts with the IP3BD-peptide(317-334). Electrotransjection of cells with IP3BD-peptide(317-334), significantly attenuated the late stage of Ca2+ and Mn2+ entry induced by 10 nM thapsigargin (TG) or 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ), providing evidence for a functional role of the de novo coupling between hTRPC1 and IP3RII in the activation of SOCE in human platelets.     

A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: Evidence against the de novo conformational coupling hypothesis

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP₃ receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca²⁺ but not Mn²⁺ or Na⁺ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca²⁺ but not Mn²⁺ or Na⁺ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na⁺/Ca²⁺ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca²⁺ but not Mn²⁺ or Na⁺ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca²⁺ permeable pathways activated following Ca²⁺ store depletion in human platelets: A Ca²⁺-permeable, hTRPC1-containing SOC and reverse Na⁺/Ca²⁺ exchange, which is activated following Na⁺ entry through the SOC and requires a functional actin cytoskeleton.     

Interaction of STIM1 with endogenously expressed human canonical TRP1 upon depletion of intracellular Ca2+ stores

STIM1 (stromal interaction molecule 1) has recently been proposed to communicate the intracellular Ca(2+) stores with the plasma membrane to mediate store-operated Ca(2+) entry. Here we describe for the first time that Ca(2+) store depletion stimulates rapid STIM1 surface expression and association with endogenously expressed human canonical TRP1 (hTRPC1) independently of rises in cytosolic free Ca(2+) concentration. These events require the support of the actin cytoskeleton in human platelets, as reported for the coupling between type II inositol 1,4,5-trisphosphate receptor in the Ca(2+) stores and hTRPC1 in the plasma membrane, which has been suggested to underlie the activation of store-operated Ca(2+) entry in these cells. Electrotransjection of cells with anti-STIM1 antibody, directed toward the N-terminal sequence that includes the Ca(2+)-binding region, prevented the migration of STIM1 toward the plasma membrane, the interaction between STIM1 and hTRPC1, the coupling between hTRPC1 and type II inositol 1,4,5-trisphosphate receptor, and reduced store-operated Ca(2+) entry. These findings provide evidence for a role of STIM1 in the activation of store-operated Ca(2+) entry probably acting as a Ca(2+) sensor.     

STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.     

A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets

We have previously suggested that store-mediated Ca2+ entry (SMCE) in human platelets may be activated by a secretion-like coupling model, involving de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP(3)RII) to the putative Ca2+ entry channel, hTRPC1. In other cells, hTRPC1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane. Here we have shown that hTRPC1 is largely associated with detergent-resistant platelet membranes, from which it is partially released when the cells are depleted of cholesterol by treatment with methyl-beta-cyclodextrin (MBCD). MBCD treatment inhibited thapsigargin (TG)-evoked SMCE in a concentration-dependent manner, reducing it to 38.1+/-4.1% at a concentration of 10mM. Similarly, the Ca2+ entry evoked by thrombin (1unit/ml) was reduced to 48.2+/-4.5% of control following MBCD (10mM) treatment. Thrombin- and TG-evoked coupling between IP(3)RII and hTRPC1 was also reduced following cholesterol depletion. These results suggest that hTRPC1 is associated with LRDs in human platelets and that these domains are important for its participation in SMCE.     

Two distinct Ca2+ compartments show differential sensitivity to thrombin, ADP and vasopressin in human platelets

Recent studies propose the existence of two distinct Ca2+ compartments in human platelets based on the expression of different SERCA isoforms with distinct sensitivity to thapsigargin and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Using fura-2-loaded human platelets we have found that depletion of the TBHQ sensitive store reduces thrombin--but not ADP--or vasopressin (AVP)-induced Ca2+ release. Redistribution of cytosolic Ca2+ after thrombin stimulation resulted in overloading of the TBHQ-sensitive store. This phenomenon was not observed with ADP or AVP. We found that NAADP decreases the Ca2+ concentration into the stores in permeabilized platelets, which is prevented by depletion of the TBHQ-sensitive store. Nimodipine, an inhibitor of the NAADP receptor, reduced thrombin-induced Ca2+ release from the TBHQ-sensitive stores, without having any effect on the responses elicited by ADP or AVP. Finally, the phospholipase C inhibitor, U-73122, abolished ADP- and AVP-induced Ca2+ release, suggesting that their responses are entirely dependent on IP3 generation. In contrast, treatment with both U-73122 and nimodipine was required to abolish thrombin-induced Ca2+ release. We suggest that thrombin evokes Ca2+ release from TBHQ-sensitive and insensitive stores, which requires both NAADP and IP3, respectively, while ADP and AVP exert an IP3-dependent release of Ca2+ from the TBHQ-insensitive compartment in human platelets.     

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells

It is generally thought that receptor-operated Ca2+ entry is related to store-operated or capacitative Ca2+ entry mechanism. Recent evidence suggests that non-capacitative Ca2+ entry pathways are also involved in receptor activated Ca2+ influx in many different kinds of cells. In this study, we studied whether alpha1-adrenoreceptor (alpha1-AR)-activated Ca2+ entry is coupled to both capacitative and non-capacitative pathways in A10 vascular smooth muscle cells by fura-2 fluorescence probe and conventional whole-cell patch clamp techniques. We found that both thapsigargin (TG) and phenylephrine (Phe) induced transient increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in Ca2+-free medium, and subsequent addition of Ca2+ evoked a sustained [Ca2+]i rise. When the membrane potential was held at -60 mV, both TG and Phe activated inward currents, which were inhibited by GdCl3(Gd3+), 0Na+/0Ca2+ solution and 1-{beta[3-(4-mehtoxyphenyl)propoxy]-4-methoxypheneth-yl}-1H- imidazole hydro-chloride (SK&F96365), but not by nifedipine. When Ca2+ store was depleted by TG in Ca2+-free solution, Phe failed to further evoke [Ca2+]i rise. However, when capacitative Ca2+ entry was activated by TG in the medium containing Ca2+, 10 microM Phe further increased [Ca2+]i. At the same concentration, TG activated an inward cation current, subsequent addition of Phe also further induced an inward cation current. Furthermore, the amplitudes of [Ca2+]i increase and current density induced by Phe in the presence of TG were less than that induced by Phe alone. Our results suggest that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca2+ influx induced by activation of alpha1-AR in A10 vascular smooth muscle cells.     

Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition

Capacitative calcium entry or store-operated calcium entry in nonexcitable cells is a process whereby the activation of calcium influx across the plasma membrane is signaled by depletion of intracellular calcium stores. Transient receptor potential (TRP) proteins have been proposed as candidates for store-operated calcium channels. Human TRPC3 (hTRPC3), an extensively studied member of the TRP family, is activated through a phospholipase C-dependent mechanism, not by store depletion, when expressed in HEK293 cells. However, store depletion by thapsigargin is sufficient to activate hTRPC3 channels when expressed in DT40 avian B-lymphocytes. To gain further insights into the differences between hTRPC3 channels generated in these two expression systems and further understand the role of hTRPC3 in capacitative calcium entry, we examined the effect of two well characterized inhibitors of capacitative calcium entry, Gd3+ and 2-aminoethoxydiphenyl borane (2APB). We confirmed that in both DT40 cells and HEK293 cells, 1 microm Gd3+ or 30 microm 2APB completely blocked calcium entry due to receptor activation or store depletion. In HEK293 cells, 1 microm Gd3+ did not block receptor-activated hTRPC3-mediated cation entry, whereas 2APB had a partial (approximately 60%) inhibitory effect. Interestingly, store-operated hTRPC3-mediated cation entry in DT40 cells was also partially inhibited by 2APB, whereas 1 microm Gd3+ completely blocked store-operated hTRPC3 activity in these cells. Furthermore, the sensitivity of store-operated hTRPC3 channels to Gd3+ in DT40 cells was similar to the endogenous store-operated channels, with essentially 100% block of activity at concentrations as low as 0.1 microm. Finally, Gd3+ has a rapid inhibitory effect when added to fully developed hTRPC3-mediated calcium entry, suggesting a direct action of Gd3+ on hTRPC3 channels. The distinct action of these inhibitors on hTRPC3-mediated cation entry in these two cell types may result from their different modes of activation and may also reflect differences in basic channel structure.     

Mechanisms of phospholipase C-regulated calcium entry

In a variety of cell types, activation of phospholipase C-linked receptors results in the generation of intracellular Ca2+ signals comprised of components of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. This entry of Ca2+ occurs by either of two general mechanisms: the release of stored Ca2+ can activate, by an unknown mechanism, store-operated channels in the plasma membrane, a process known as capacitative calcium entry. Alternatively, second messengers generated at the plasma membrane can activate Ca2+ channels more directly, a non-capacitative calcium entry process. This review summarizes current knowledge of the underlying signaling mechanisms and the nature of the channel molecules responsible for these two general categories of regulated Ca2+ entry.     

Role of the actin cytoskeleton in store-mediated calcium entry in glioma C6 cells

The effects of actin cytoskeleton disruption by cytochalasin D and latrunculin A on Ca2+ signals evoked by ADP, UTP or thapsigargin were investigated in glioma C6 cells. Despite the profound alterations of the actin cytoskeleton architecture and cell morphology, ADP and UTP still produced cytosolic calcium elevation in this cell line. However, calcium mobilization from internal stores and Ca2+ influx through store-operated Ca2+ channels induced by ADP and UTP were strongly reduced. Cytochalasin D and latrunculin A also diminished extracellular Ca2+ influx in unstimulated glioma C6 cells previously incubated in Ca2+ free buffer. In contrast, the disruption of the actin cytoskeleton had no effect on thapsigargin-induced Ca2+ influx in this cell line. Both agonist- and thapsigargin-generated Ca2+ entry was significantly decreased by the blocker of store-operated Ca2+ channels, 2-aminoethoxydiphenylborate. The data reveal that two agonists and thapsigargin activate store-operated Ca2+ channels but the mechanism of activation seems to be different. While the agonists evoke a store-mediated Ca2+ entry that is dependent on the actin cytoskeleton, thapsigargin apparently activates an additional mechanism, which is independent of the disruption of the cytoskeleton.     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Characterization of the calcium signaling system in the submandibular cell line SMG-C6

Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, alpha1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Consistently, 1, 4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.     

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca²⁺ signalling. Two distinct Ca²⁺ entry pathways amplify and sustain platelet Ca²⁺ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca²⁺ entry (SOCE) and non-capacitative Ca²⁺ entry (NCCE) in thrombin-induced Ca²⁺ signalling and PS exposure by using two Ca²⁺ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca²⁺ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca²⁺ signalling at higher thrombin concentrations. However, despite being involved in Ca²⁺ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca²⁺ entry is an important regulator of thrombin-induced PS exposure in platelets.