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We recently reported that store-operated [Ca.sub.2+] entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between [Ca.sub.2+] channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as 'secretion-like coupling.' As lot secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced [Ca.sub.2+] entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced [Ca.sub.2+] influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon [Ca.sub.2+] store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskcleton; calcium entry

Use of the patch clamp method facilitates analysis of the influence of various vasoconstrictors on endothelial membrane potential of intact rat aorta. Vasoconstrictor functions on aortic smooth muscle receptors initiate a response that induces oscillations and depolarization in the membrane potential of endothelium. Variations in the input resistance of the endothelium and vasoconstrictor-induced oscillations occur together.

Acetylcholine (ACh)-evoked electrical responses of the endothelium, which does not require vascular smooth muscle activity, produce oscillations in membrane potential. ACh acts on the membrane potential of endothelium of intact rat aorta through the stimulation of phospholipase c, the efflux of Ca2+ from intracellular stores and the raise of intracellular Ca2+ by a mechanism similar to inositol(1,4,5)-triphosphate-mediated agonist responses.

Tumor necrosis factor-[Alpha] (TNF-[Alpha]) is an important component of the early signaling pathways leading to liver regeneration and proliferation, but it is also responsible for several hepatotoxic effects. We have investigated the effect of TNF-[Alpha] on thapsigargin (TG)-induced store-mediated [Ca.sup.2+] entry (SMCE) in the human hepatocellular carcinoma cell line HepG2. In these cells, short-term (10 min) exposure to TNF-[Alpha] slightly increased SMCE. In contrast, long-term (12 h) exposure to TNF-[Alpha] significantly reduced SMCE. This effect was reversed by coincubation with atrial natriuretic peptide (ANP), which itself had no effect on SMCE. Cytochalasin D and latrunculin A, inhibitors of actin polymerization, abolished SMCE. Long-term exposure of HepG2 cells to TNF-[Alpha] abolished TG-induced actin polymerization and membrane association of Ras proteins. When TNF-[Alpha] was added in combination with ANP, these effects were reduced. These findings suggest that in HepG2 cells, TNF-[Alpha] inhibits SMCE by affecting reorganization of the actin cytoskeleton, probably by interfering with the activation of Ras proteins, and that ANP protects against these inhibitory effects of TNF-[Alpha]. calcium influx; actin cytoskeleton; Ras proteins; atrial natriuretic peptide

Rises in cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca 2+ ] cyt , but these only monitor the net effect of manipulations on the processes controlling [Ca 2+ ] cyt (Ca 2+ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca 2+ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca 2+ signalling, we here monitor Ca 2+ flux around the platelet by measuring net Ca 2+ fluxes to or from the extracellular space and the intracellular Ca 2+ stores, which act as the major sources and sinks for Ca 2+ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na + concentration ([Na + ] cyt ), which influences platelet Ca 2+ fluxes via Na + /Ca 2+ exchange. The intracellular store Ca 2+ concentration ([Ca 2+ ] st ) was monitored using Fluo-5N, the extracellular Ca 2+ concentration ([Ca 2+ ] ext ) was monitored using Fluo-4 whilst [Ca 2+ ] cyt and [Na + ] cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca 2+ ] cyt in the absence of extracellular Ca 2+ . PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca 2+ release and Ca 2+ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca 2+ ] cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na + ] cyt which would be expected to reduce Ca 2+ removal via the Na + /Ca 2+ exchanger (NCX). Thrombin-evoked rises in [Na + ] cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn 2+ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca 2+ ] cyt following SERCA inhibition and either removal of extracellular Na + or inhibition of Na + /K + -ATPase activity by removal of extracellular K + or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca 2+ ] cyt by acceleration of SERCA activity, whilst rises in [Ca 2+ ] cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na + /K + -ATPase activity, with the latter limiting the effect of thrombin on rises in [Na + ] cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca 2+ signalling. [ABSTRACT FROM AUTHOR]

Platelet activation is critically regulated by an increase in intracellular calcium concentration ([Ca2+]i). Although Ca2+ release from intracellular Ca2+ stores and subsequent store-operated Ca2+ entry are often thought to be the major contributors to increases in [Ca2+]i evoked by most agonists, high concentrations of thrombin activate a Ca2+ entry pathway that is independent of Ca2+ store depletion (known as ‘non-capacitative cation entry’–NCCE). The channel that conducts NCCE has not previously been clearly identified, and the mechanisms that regulate its activation are also unknown. Here we have investigated NCCE using fura-2-loaded human platelets. To investigate NCCE independently of other Ca2+ signaling pathways, the intracellular Ca2+ stores were first rapidly depleted in the absence of extracellular Ca2+. Sr2+ was then added to monitor maximal store-operated cation influx. Thrombin was then added to stimulate NCCE. Flufenamic acid, which inhibits Ca2+ entry through most TRPC isoforms, but potentiates entry through TRPC6, was found to block store-operated cation entry. In contrast, thrombin-induced NCCE was increased, suggesting the possible involvement of TRPC6. Since TRPC6 is regulated by Src family tyrosine kinases in some cells, we investigated the possible role of this kinase family in NCCE. PP2, a Src family tyrosine kinase inhibitor, completely abolished thrombin-induced NCCE. Furthermore, NCCE was enhanced by phenylarsine oxide and could be directly induced by vanadyl hydroperoxide, both tyrosine phosphatase inhibitors. These data indicate that Src family tyrosine kinase activation is a required step in NCCE activation. In conclusion NCCE may be an important regulator of platelet activation when local thrombin concentrations are high. [ABSTRACT FROM AUTHOR]

Abstract: Recent work has demonstrated a role for Na+/Ca2+ exchange in potentiation of the Ca2+ entry elicited through the human platelet store-operated channel by controlling a Mn2+-impermeable Ca2+ entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na+/Ca2+ exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn2+-impermeable Ca2+ entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca2+ signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca2+ signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion. [Copyright &y& Elsevier]

Abstract: Store-operated calcium entry (SOCE) is a major mechanism for Ca2+ entry in excitable and non-excitable cells. The best-characterised store-operated current is ICRAC, but other currents activated by Ca2+ store depletion have also been reported. The recent identification of the proteins stromal interaction molecule 1 (STIM1) and Orai1 has shed new light on the nature and regulation of SOC channels. STIM1 has been presented as the endoplasmic reticulum (ER) Ca2+ sensor that communicates the content of the Ca2+ stores to the store-operated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and co-clustering with the Ca2+ channel subunit, Orai1. Interestingly, TRPC1, which has long been proposed as a SOC channel candidate, associates with Orai1 and STIM1 in a ternary complex that appears to increase the variability of SOC currents available to modulate cell function. [Copyright &y& Elsevier]

Abstract: Store-operated calcium entry (SOCE) is a major mechanism for Ca2+ influx. Since SOCE was first proposed two decades ago many techniques have been used in attempting to identify the nature of store-operated Ca2+ (SOC) channels. The first identified and best-characterised store-operated current is I CRAC, but a number of other currents activated by Ca2+ store depletion have also been described. TRPC proteins have long been proposed as SOC channel candidates; however, whether any of the TRPCs function as SOC channels remains controversial. This review attempts to provide an overview of the arguments in favour and against the role of TRPC proteins in the store-operated mechanisms of agonist-activated Ca2+ entry. [Copyright &y& Elsevier]

• The pH gradient across acidic organelles is regulated upon human platelet activation. • Autocrine 5-HT signalling triggers alkalinisation of platelet acidic organelles. • Cytochalasin reduces ADP-evoked Ca2+ release by increasing acidic organelle H + uptake. Human platelets regulate agonist-evoked Ca2+ signalling through Ca2+ release from and sequestration into acidic organelles. Previous studies have pharmacologically characterised the presence of a Ca2+- H + exchanger in these organelles. This exchanger appears to regulate a secondary plateau phase in agonist-evoked cytosolic Ca2+ signals in fura-2-loaded human platelets. Here we demonstrate that cytochalasin D treatment removes the secondary plateau in ADP-evoked Ca2+ signals elicited in the absence of external Ca2+. This effect was reversed by pretreatment with nigericin, a K +/ H + exchanger that short-circuits the Ca2+- H + exchanger. Using Fluo-5N- or Lysosensor Green-loaded cells, cytochalasin D was found to enhance Ca2+ sequestration into acidic organelles by preventing their alkalinisation. Additional experiments demonstrated that ADP-evoked alkalinisation of acidic organelles and subsequent slowing of acidic organellar Ca2+ sequestration was mediated by autocrine 5-HT signalling. Enhancing this 5-HT signalling using fluoxetine overcame the inhibitory effect of cytochalasin D on ADP-evoked Ca2+ signals, indicating that cytochalasin D interferes with 5-HT autocrine signalling. The ability of Cytochalasin D to interfere with autocrine 5-HT signalling was downstream of the 5-HT 2A receptor as secretion of [3H]-5-HT from ADP-stimulated human platelets was not reduced. These data provide the first evidence that the pH gradient across acidic organelles is dynamically regulated upon human platelet activation, and that this can play a significant role in controlling human platelet function by modulating Ca2+- H + exchange and so [Ca2+] i. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Here, we report a novel role for the cysteine protease calpain in store-operated calcium entry. Several structurally and mechanistically unrelated inhibitors of calpain inhibited Ca2+ entry activated in human platelets by thapsigargin-evoked Ca2+ store depletion or the physiological agonist thrombin, whereas inhibitors of other cysteine proteases were without effect. The use of the cell-permeable fluorogenic calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-l-leucyl-l-methionine amide revealed rapid activation of calpain which was closely temporally correlated with Ca2+ store depletion even in the absence of a rise in cytosolic [Ca2+]. Calpain inhibition prevented the tyrosine phosphorylation of several proteins upon Ca2+ store depletion, suggesting that calpain may lie upstream of protein tyrosine phosphorylation that is known to be required for the activation of store-operated Ca2+ entry in human platelets. Earlier studies using calpain inhibitors may need reinterpretation in the light of this finding that calpain plays a role in the activation of physiological Ca2+ entry pathways. [Copyright &y& Elsevier]

Abstract: Platelet activation by potent, Ca2+-mobilizing agonists results in shedding of microparticles that are active in coagulation. Here we show that platelets under storage produce procoagulant microparticles in the absence of agonist. Microparticle formation by resting platelets results from αIIbβ3 signaling to destabilization of the actin cytoskeleton in the absence of calpain activation. Integrin-mediated spreading of platelets over fibrinogen similarly results in microparticle formation. After transfusion of stored platelet preparations to thrombocytopenic patients, the microparticles contribute to coagulant activity in vivo. [Copyright &y& Elsevier]

The role of actin polymerisation in regulating thrombin-evoked Ca 2+ signalling was investigated in human platelets. We have previously reported that cytochalasin D (Cyt D) inhibits thapsigargin-evoked store-operated Ca 2+ entry (SOCE), which is believed to contribute a major component of thrombin-evoked Ca 2+ entry in platelets. In contrast, Cyt D increased thrombin-evoked Ca 2+ entry to 147.5  ±  9.2% and Sr 2+ entry to 134.2  ±  6.4% of control. Similar results were obtained with latrunculin A. This potentiation was not affected if protein kinase C was inhibited using Ro-31-8220, suggesting that it did not involve PKC-dependent non-capacitative Ca 2+ entry. Ca 2+ entry evoked by the PAR-4 agonist, AYPGKF, was increased to 133.7  ±  12.8% of control by Cyt D, whereas Ca 2+ signalling evoked by the PAR-1 agonist, SFLLRN, was unaffected. The PAR-4 antagonist, tcY-NH 2 , abolished the effect of Cyt D on thrombin-evoked Ca 2+ entry. Biotinylation of cell-surface proteins showed that PAR-4 was internalised after stimulation by thrombin. Cyt D reduced this internalisation. These data suggest that Cyt D prevents the internalisation of PAR-4, which may lead to prolonged signalling from this receptor. This may mask a direct effect of Cyt D on the activation of SOCE after the activation of PAR-4. [ABSTRACT FROM AUTHOR]

Store-operated Ca2+ entry (SOCE), a mechanism regulated by the filling state of the intracellular Ca2+ stores, is a major pathway for Ca2+ influx. Hypotheses to explain the communication between the Ca2+ stores and plasma membrane (PM) have considered both the existence of small messenger molecules, such as a Ca2+-influx factor (CIF), and both stable and de novo conformational coupling between proteins in the Ca2+ store and PM. Alternatively, a secretion-like coupling model based on vesicle fusion and channel insertion in the PM has been proposed, which shares some properties with the de novo conformational coupling model, such as the role of the actin cytoskeleton and soluble N-ethylmaleimide (NEM)-sensitive-factor attachment proteins receptor (SNARE) proteins. Here we review recent progress made in the characterization of the de novo conformational coupling and the secretion-like coupling models for SOCE. We pay particular attention into the involvement of SNARE proteins and the actin cytoskeleton in both SOCE models. SNAREs are recognized as proteins involved in exocytosis, participating in vesicle transport, membrane docking, and fusion. As with secretion, a role for the cortical actin network in Ca2+ entry has been demonstrated in a number of cell types. In resting cells, the cytoskeleton may prevent the interaction between the Ca2+ stores and the PM, or preventing fusion of vesicles containing Ca2+ channels with the PM. These are processes in which SNARE proteins might play a crucial role upon cell activation by directing a precise interaction between the membrane of the transported organelle and the PM. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

We recently reported that store-operated Ca2+ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as ‘secretion-like coupling.’ As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. [ABSTRACT FROM AUTHOR]

Store-mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non-excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome-associated protein (SNAP-25) and the vesicle-associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP-25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin- (TG) and agonist-evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP-25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP-25 has a role in SMCE in human platelets. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that tumor necrosis factor (TNF)-alpha inhibits store-mediated calcium entry in the human hepatocellular carcinoma cell line HepG2. Regulation of atrial natriuretic peptide immunoreactivity by TNF-alpha; Stimulation of divalent cation entry by thapsigarin; Influence of TNF-alpha on membrane associations of Ras proteins.

In human platelets and other non-excitable cell types depletion of the intracellular calcium stores promotes calcium entry across the plasma membrane. Although the mechanism of this store-mediated calcium entry remains uncertain, it has been suggested that a tyrosine phosphorylation step could be involved. In support of this hypothesis various tyrosine kinase inhibitors have been shown to reduce store-mediated calcium entry in platelets, although this inhibition is never complete. Here we investigate the properties of store-mediated calcium entry in human platelets during the time course of its activation. Our data suggest that at least two pathways may contribute to store-mediated calcium entry in these cells. An early component, activated soon after the initiation of Ca[sup 2+] store depletion, is insensitive to trivalent cations, SKF 96365 and tyrosine kinase inhibitors. This is followed by a second component which is inhibited by La[sup 3+], SKF 96365 and by tyrosine kinase inhibitors. These results suggest a role for tyrosine kinases in generating only the later stages of store-mediated calcium entry in platelets and may explain the incomplete inhibition of this pathway by inhibitors of tyrosine kinases. [ABSTRACT FROM AUTHOR]

The Ca2+ responses of rat platelets are dominated by the influx of extracellular Ca2+ across the plasma membrane [Heemskerk, J. W. M., Feijge, M. A. H.. Rietman, E, & Hornstra, G. (1991) FEBS Lett. 284, 223], which allows the study of Ca2+ entry into these cells by measuring increases in cytosolic Ca2+ concentration, [Ca2+]. Several pieces of evidence indicated that, as in human platelets [Sage, S. O., Reast, R., & Rink, T. J. (1990) Biochem. J. 265. 675-680; Alonso, M., Alvarez, J., Montero, M., Sanchez, A. & García-Sancho, J. (1991) Biochem. J. 280, 783-789], agonist-stimulated Ca2+ entry was linked to the mobilisation of Ca2+ from intracellular stores: there was good correlation between the potency of receptor agonists in elevating [Ca2+] in the presence or absence of external CaCl2: agonist-induced Ca2+ entry was inhibited to a similar degree as internal mobilisation by activators of cAMP-dependent or cGMP-dependent protein kinase or by the phospholipase C inhibitor, U73122: thapsigargin (an inhibitor of endomembrane Ca2+-ATPases) evoked store depletion and Ca2+ entry, which were both reduced by prior activation of cAMPdependent or cGMP-dependent protein kinase but were not affected by U73122. In platelets with depleted Ca2+ stores, the addition of CaCl2 resulted in a considerable entry of Ca2+ which was insensitive to cAMP-dependent and cGMP-dependent protein kinase activation. In control platelets with full Ca2+ stores, CaCl2 potentiated the thrombin-induced generation of my-inositol phosphates, suggesting that Ca2+ entry potentiated phospholipase C activity. Taken together, these results indicate that Ca2+ entry in rat platelets, (a) is mostly secondary to store depletion, (b) is not directly downregulated by cAMP-dependent and cGMP-dependent protein kinase, but indirectly by inhibition of store depletion. (c) can proceed in the absence of phospholipase C activation, but is stimulated by this activity probably by increased mobilisation of Ca2+ from the stores. These results lead to the concept that a major part of receptor-mediated Ca2+ entry in rat platelets is regulated in an indirect way by factors that stimulate or inhibit the degree of Ca2+ mobilisation from the internal stores. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article related to expression of TRPV1 ion channels in human and mouse blood platelets and published in the previous issues of the magazine.

Abstract: Two mechanisms for store-operated Ca2+ entry (SOCE) regulated by two independent Ca2+ 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 IP3 receptor (IP3RII) 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 Ca2+ entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca2+-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca2+ stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca2+ stores and the DTS, the major releasable Ca2+ store in platelets. These findings were confirmed by using Sr2+ as a surrogate for Ca2+ entry. Colchicine attenuated the coupling between IP3RII 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 IP3RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca2+. 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 Ca2+ stores. [Copyright &y& Elsevier]

Store-operated Ca 2+ entry (SOCE) is a functionally relevant mechanism for Ca 2+ influx present in electrically excitable and non-excitable cells. Regulation of Ca 2+ entry through store-operated channels is essential to maintain an appropriate intracellular Ca 2+ homeostasis and prevent cell damage. Calcium-release activated channels exhibit Ca 2+ -dependent inactivation mediated by two temporally separated mechanisms: fast Ca 2+ -dependent inactivation takes effect in the order of milliseconds and involves the interaction of Ca 2+ with residues in the channel pore while slow Ca 2+ -dependent inactivation (SCDI) develops over tens of seconds, requires a global rise in [Ca 2+ ] cyt and is a mechanism regulated by mitochondria. Recent studies have provided evidence that the protein SARAF (SOCE-associated regulatory factor) is involved in the mechanism underlying SCDI of Orai1. SARAF is an endoplasmic reticulum (ER) membrane protein that associates with STIM1 and translocate to plasma membrane-ER junctions in a STIM1-dependent manner upon store depletion to modulate SOCE. SCDI mediated by SARAF depends on the location of the STIM1-Orai1 complex within a PI(4,5)P 2 -rich microdomain. SARAF also interacts with Orai1 and TRPC1 in cells endogenously expressing STIM1 and cells with a low STIM1 expression and modulates channel function. This review focuses on the modulation by SARAF of SOCE and other forms of Ca 2+ influx mediated by Orai1 and TRPC1 in order to provide spatio-temporally regulated Ca 2+ signals. [ABSTRACT FROM AUTHOR]

Abstract: Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca2+ entry mechanism (SOCE). Recently, regulation of STIM1 by glycosylation and phosphorylation on serine/threonine or proline residues has been described; however other modes of phosphorylation that are important for activating SOCE in platelets, such as tyrosine phosphorylation, have been poorly investigated. Here we investigate the latency of STIM1 phosphorylation on tyrosine residues during the first steps of SOCE activation. Human platelets were stimulated and fixed at desired times using rapid kinetic assays instruments, and immunoprecipitation and western blotting techniques were then used to investigate the pattern of STIM1 tyrosine phosphorylation during the first steps of SOCE activation. We have found that maximal STIM1 tyrosine phosphorylation occurred 2.5s after stimulation of human platelets with thapsigargin (Tg). STIM1 localized in the plasma membrane were also phosphorylated in platelets stimulated with Tg. By using chemical inhibitors that target different members of the Src family of tyrosine kinases (SKFs), two independent signaling pathways involved in STIM1 tyrosine phosphorylation during the first steps of SOCE activation were identified. We finally conclude that STIM1 tyrosine phosphorylation is a key event for the association of STIM1 with plasma membrane Ca2+ channels such as Orai1, hence it is required for conducting SOCE activation. [Copyright &y& Elsevier]

Platelets are activated by adhesion to vascular collagen via the immunoglobulin receptor, glycoprotein VI (GPVI). This causes potent signaling toward activation of phospholipase Cγ2, which bears similarity to the signaling pathway evoked by T- and B-cell receptors. Phosphoinositide 3-kinase (PI3K) plays an important role in collagen-induced platelet activation, because this activity modulates the autocrine effects of secreted ADP. Here, we identified the PI3K isoforms directly downstream of GPVI in human and mouse platelets and determined their role in GPVI-dependent thrombus formation. The targeting of platelet PI3Kα or -13 strongly and selectively suppressed GPVI-induced Ca2+ mobilization and inositol 1,4,5-triphosphate production, thus demonstrating enhancement of phospholipase Cγ2 by PI3Kαβ. That PI3Kα and -β have a non-redundant function in GPVI- induced platelet activation and thrombus formation was concluded from measurements of: (i) serine phosphorylation of Akt, (ii) dense granule secretion, (iii) intracellular Ca2+ increases and surface expression of phosphatidylserine under flow, and (iv) thrombus formation, under conditions where PI3Kα/β was blocked or p85α was deficient. In contrast, GPVI-induced platelet activation was insensitive to inhibition or deficiency of PI3Kδ or -γ. Furthermore, PI3Kα/β, but not PI3Kγ, contributed to GPVI-induced Rap1b activation and, surprisingly, also to Rap1b-independent platelet activation via GPVI. Together, these findings demonstrate that both PI3Kα and -β isoforms are required for full GPVI-dependent platelet Ca2+ signaling and thrombus formation, partly independently of Rap1b. This provides a new mechanistic explanation for the anti-thrombotic effect of PI3K inhibition and makes PI3Kα an interesting new target for anti-platelet therapy. [ABSTRACT FROM AUTHOR]

Abstract: Two agonist-releasable Ca2+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 Ca2+entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca2+-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca2+content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca2+in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 μM TBHQ, individually or simultaneously, accelerated Ca2+ 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 Ca2+ influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca2+ extrusion by PMCA or the Na+/Ca2+ 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. [Copyright &y& Elsevier]

A major pathway for stimulated Ca2+ entry in nonexcitable cells is activated following depletion of intracellular Ca2+ stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca2+ stores has been proposed as the most likely mechanism to activate this store-mediated Ca2+ entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca2+ pools, which are differentially modulated by the actin cytoskeleton. Ca2+ entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca2+ entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca2+ entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca2+ entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM. [ABSTRACT FROM AUTHOR]

Reactive oxygen species, such as H[sub 2]O[sub 2], have been recognized as intracellular messengers involved in several cell functions. Here we report the activation of the tyrosine kinase pp60[sup 8rc] by H[sub 2]O[sub 2], a mechanism required for the activation of store-mediated Ca[sup 2+] entry (SMCE) in human platelets. Treatment of platelets with H[sub 2]O[sub 2] resuited in a time- and concentration-dependent activation of pp60[sup src]. Incubation with GF 109203X, a protein kinase C (PKC) inhibitor, prevented H[sub 2]O[sub 2]-induced pp60[sub src] activation. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60[sup src] by H[sub 2]O[sub 2] is independent of increases in [Ca[sup 2+]][sub i]. Cytochalasin D, an inhibitor of actin polymerization, significantly reduced H[sub 2]O[sub 2]-induced pp60[sup src] activation. We found that platelet stimulation with thapsigargin (TG) plus ionomycin (Iono) or thrombin induced rapid H[sub 2]O[sub 2] production, a mechanism independent of elevations in [Ca[sup 2+]][sub i]. Treatment of platelets with catalase attenuated TG plus Iono- and thrombin-induced activation of pp60[sup src]. In addition, catalase as well as the pp60[sup src] inhibitor, PP1, reduced both the activation of SMCE and the coupling between the hTrp1 and the IP[sub 3]R type II without having any effect on the maintenance of SMCE. Consistent with the role of PKC in the activation of pp60[sup src] by H[sub 2]O[sub 2], the PKC inhibitors GF 109202X and Ro-31-8220 were found to reduced SMCE in platelets. This study suggests that platelet activation with TG plus Iono or thrombin is associated with H[sub 2]O[sub 2] production, which acts as a second messenger by stimulating pp60[sup src] by a PKC-dependent pathway and is involved in the activation of SMCE in these cells. [ABSTRACT FROM AUTHOR]