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Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca 2+ 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 2.5 s 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 Ca 2+ channels such as Orai1, hence it is required for conducting SOCE activation.

In case of nervous damages, like nervous system trauma or various neurodegenerative diseases such as dementia or Parkinson, several treatments are available to restore neurological function. In spite of these treatments, results are often insufficient or not satisfactory in many neurologic diseases, especially for central nervous system (CNS) lesions. To minimize neurological dysfunction, it is critical to reduce neuronal death, avoiding loss of the synaptic connections, and securing viable neurons to extend axons. Unfortunately, there are no effective strategies to fulfill these basic needs except for some cases of peripheral neural damage up to now. Rescue of damaged neurons, stimulation of neurogenesis and transplantation of nervous tissue are strategies proposed to prevent neurodegenerative disorders. A number of studies have recently reported successful axon regeneration and neurological recovery by using immunosuppressants, such as FK506. Immunosuppressants act as excellent agents for enhancing the rate and extent of axon regeneration and neurological recovery. FK506 and other neuroimmunophilin ligands (NILs) might reverse neuronal degeneration. In several animal models mimicking Parkinson's disease, dementia and surgical damage, NILs induces resprouting, by acting as neurotrophic agents and preventing nerve damage, although more studies are necessary to identify new NILs with neuroprotective action, but lacking the side immunological effects observed in the ligands analyzed to date. This review explores the new clinical role of immunosuppressants in the treatment of nerve surgery of autologous, allografts or xenografts. Results of studies regarding immunosuppressant treatment of nervous system trauma and neurodegenerative diseases, like neurogenic erectile dysfunction, will be here considered.

The immunophilin family includes a large group of proteins with peptidyl prolyl-isomerase activity (PPI-ase). Immunophilins chaperone activity has been documented to be crucial for the correct folding and activation of many proteins. Thus, they have been subjected of intense investigation since they were firstly described in the last decades of the past century. Many of these studies have been focused on leukocyte constitutively expressed immunophilins, due to their relevance in the correct folding, and subsequently, sensitization and activation of the glycoprotein receptor (RGBs) of lymphocyte T CD4+ and Treg, hence regulating immunological responses against pathogen insults. Several clinical trials have been completed in the last decade reporting that administration of immunophilin-binding drugs, derived from macrolide lactones, like cyclosporine A (CsA) and tacrolimus (FK506), induced successful results in preventing organ rejection. By contrast, the expression of immunophilins and their physiological function remain poorly investigated in others cell types, such as platelets, where a reduced number of studies presenting evidences of immunophilins expression and their physiological contribution have been published, despite a number of clinical trials have noticed side effects of these drugs in thrombosis and platelet count, thus suggesting a possible regulatory function of immunophilins in human platelets, which is reviewed here.

Changes in cytosolic free Ca(2+) concentration play a pivotal role in the regulation of platelet functions, from secretion of autocrine and procoagulant factors to reversible or irreversible aggregation. It has long been recognized that platelet agonists release Ca(2+) accumulated into the dense tubular system, the analogue of the endoplasmic reticulum. However, current evidence indicates that Ca(2+) can also be stored and released from a number of acidic organelles, including lysosomes and lysosome-related organelles. Ca(2+) release from the dense tubular system is mediated through phospholipase C-dependent synthesis of inositol 1,4,5-trisphosphate, whereas Ca(2+) efflux from the acidic stores seems to be associated to the second messenger nicotinic acid adenine dinucleotide phosphate. The biochemical and biophysical properties of both Ca(2+) stores in platelets have been reported to show significant differences. Selective discharge of one or both stores depends on the platelet agonist and the concentration used, which further supports the complexity of the Ca(2+) signals that regulate platelet function. In this paper, we summarize the current knowledge on the role of acidic organelles in agonist-evoked Ca(2+) mobilization and highlight recent progress in understanding the functional aspects of the acidic Ca(2+) stores in Ca(2+) signalling and platelet physiology.

Background A novel family of intracellular Ca 2+ -release channels termed two-pore channels (TPCs) has been presented as the receptors of NAADP (nicotinic acid adenine dinucleotide phosphate), the most potent Ca 2+ mobilizing intracellular messenger. TPCs have been shown to be exclusively localized to the endolysosomal system mediating NAADP-evoked Ca 2+ release from the acidic compartments. Objectives The present study is aimed to investigate NAADP-mediated Ca 2+ release from intracellular stores in the megakaryoblastic cell line MEG01. Methods Changes in cytosolic and intraluminal free Ca 2+ concentrations were registered by fluorimetry using fura-2 and fura-ff, respectively; TPC expression was detected by PCR. Results Treatment of MEG01 cells with the H + /K + ionophore nigericin or the V-type H + -ATPase selective inhibitor bafilomycin A1 revealed the presence of acidic Ca 2+ stores in these cells, sensitive to the SERCA inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). NAADP releases Ca 2+ from acidic lysosomal-like Ca 2+ stores in MEG01 cells probably mediated by the activation of TPC1 and TPC2 as demonstrated by TPC1 and TPC2 expression silencing and overexpression. Ca 2+ efflux from the acidic lysosomal-like Ca 2+ stores or the endoplasmic reticulum (ER) results in ryanodine-sensitive activation of Ca 2+ -induced Ca 2+ release (CICR) from the complementary Ca 2+ compartment. Conclusion Our results show for the first time NAADP-evoked Ca 2+ release from acidic compartments through the activation of TPC1 and TPC2, and CICR, in a megakaryoblastic cell line.

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.

STIM1 is a transmembrane protein essential for the activation of store-operated Ca2+ entry (SOCE), a major Ca2+ influx mechanism. STIM1 is either located in the endoplasmic reticulum, communicating the Ca2+ concentration in the stores to plasma membrane channels or in the plasma membrane, where it might sense the extracellular Ca2+ concentration. Plasma membrane-located STIM1 has been reported to mediate the SOCE sensitivity to extracellular Ca2+ through its interaction with Orai1. Here we show that plasma membrane lipid raft domains are essential for the regulation of SOCE by extracellular Ca2+. Treatment of platelets with the SERCA inhibitor thapsigargin (TG) induced Mn2+ entry, which was inhibited by increasing concentrations of extracellular Ca2+. Platelet treatment with methyl-β-cyclodextrin, which removes cholesterol and disrupts the lipid raft domains, impaired the inactivation of Ca2+ entry induced by extracellular Ca2+. Methyl-β-cyclodextrin also abolished translocation of STIM1 to the plasma membrane stimulated by treatment with TG and prevented TG-evoked co-immunoprecipitation between plasma membrane-located STIM1 and the Ca2+ permeable channel Orai1. These findings suggest that lipid raft domains are essential for the inactivation of SOCE by extracellular Ca2+ mediated by the interaction between plasma membrane-located STIM1 and Orai1.

Blood platelets play a crucial role in the primary hemostasis and vessel wall repair. However; platelet hyperactivation is implicated in the pathogenesis of cardiovascular diseases such as thrombosis, atherosclerosis and stroke. Epidemiological data have suggested that regular consumption of fruits and vegetables, which are rich in flavonoids, is associated to a reduction in cardiovascular events. The cardioprotective effect of flavonoids is partly due to the inhibition of platelet function. However; the mechanisms underlying the anti-platelet effect of these compounds remain unclear. The aim of this review is to discuss the role of platelets in cardiovascular disease and to provide an overview of the potential anti-platelet effect of flavonoids, focusing on the various platelet signaling pathways modulated by flavonoids, including oxidative stress, protein tyrosine phosphorylation, calcium mobilization and nitric oxide pathway. The understanding of these mechanisms will be helpful in the development of new anti-platelet agents based on flavonoids with fewer or no adverse effects.

A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca 2+ mobilization, an intracellular signal that is involved in the regulation of a wide variety of cellular functions. Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca 2+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca 2+ mobilization induced by ROS. Keywords: reactive oxygen species; scavengers; antioxidants; intracellular Ca 2+ homeostasis 1. Calcium Homeostasis In eukaryotic cells, Ca 2+ is the most versatile signal involved in the control of cellular processes and functions [1-3]. This versatility derives from the fact that Ca

Transient receptor potential channels convey signaling information from a number of stimuli to a wide variety of cellular functions, mainly by inducing changes in cytosolic Ca2+ concentration. Different members of the TRPC, TRPM and TRPV subfamilies have been reported to play a role in tumorigenesis. Here we show that the estrogen receptor positive and triple negative breast cancer cell lines, MCF7 and MDA-MB-231, respectively, exhibit enhanced expression of the TRPC6 channel as compared to the non-tumoral MCF10A cell line. In vitro TRPC6 knockdown using shRNA impaired MCF7 and MDA-MB-231 cell proliferation, migration and invasion detected by BrdU incorporation, wound healing and Boyden chamber assays, respectively. Using RNAi-mediated TRPC6 silencing as well as overexpression of the pore-dead dominant-negative TRPC6 mutant we have found that TRPC6 plays a relevant role in the activation of store-operated Ca2+ entry in the breast cancer cell lines but not in non-tumoral breast cells. Finally, we have found that TRPC6 interacts with Orai1 and Orai3 in MCF7 and MDA-MB-231 cells and is required for the translocation of Orai1 and Orai3 to the plasma membrane in MDA-MB-231 and MCF7 cells, respectively, upon Ca2+ store depletion. These findings introduce a novel mechanism for the modulation of Ca2+ influx and the development of different cancer hallmarks in breast cancer cells.

Objective— The role of cyclophilins (chaperones that are widely expressed in different cell types, including human platelets) was explored in sarcoendoplasmic calcium (Ca 2+ ) adenosine triphosphatase (SERCA) activity. Methods and Results— Cyclophilin inhibition by cyclosporin A (CsA) evoked a time- and concentration-dependent reduction of Ca 2+ uptake by SERCA2b. However, other Ca 2+ -adenosine triphosphatases expressed in platelets, such as SERCA3 and plasma membrane Ca 2+ adenosine triphophatase, remained unaltered after CsA treatment. Cypermethrin, a non–CsA-related calcineurin inhibitor, did not alter SERCA2b activity. Furthermore, SERCA2b was affected by other CsA analogues, which do not interfere with calcineurin, such as PKF-211-811-NX5 (NIM811) and sanglifehrin A. Inhibition of the immunophilin family members using FK506 (tacrolimus) did not alter SERCA2b ability to sequester Ca 2+ into the dense tubular system. Coimmunoprecipitation experiments confirmed that cyclophilin A associates with SERCA2b and stromal interaction molecule-1 in resting platelets. This interaction is attenuated by the physiological agonist thrombin but enhanced by treatment with CsA or sanglifehrin A. Conclusion— Cyclophilin A is a regulator of SERCA2b in human platelets.

We have evaluated the effect of melatonin on apoptosis evoked by increases in [Ca2+] c in human leukocytes. Our results show that treatment of neutrophils with the calcium mobilizing agonist FMLP or the specific inhibitor of calcium reuptake thapsigargin induced a transient increase in [Ca2+] c . Our results also show that FMLP and thapsigargin increased caspase-9 and -3 activities and the active forms of both caspases. The effect of FMLP and thapsigargin on caspase activation was time-dependent. Similar results were obtained when lymphocytes were stimulated with thapsigargin. This stimulatory effect was accompanied by induction of mPTP, activation of the proapoptotic protein Bax and release of cytochrome c. However, when leukocytes were pretreated with melatonin, all of the apoptotic features indicated above were significantly reversed. Our results suggest that melatonin reduces caspase-9 and -3 activities induced by increases in [Ca2+] c in human leukocytes, which are produced through the inhibition of both mPTP and Bax activation.

SummaryDiabetes mellitus is a disease characterised by hyperglycaemia and associated with several cardiovascular disorders, including angiopathy and platelet hyperactivity, which are major causes of morbidity and mortality in type 2 diabetes mellitus. In type 2 diabetic patients, homo-cysteine levels are significantly increased compared with healthy subjects. Hyperhomocysteinaemia is an independent risk factor for macro-and microangiopathy and mortality. The present study is aimed to investigate the effect of homocysteine on platelet apoptosis. Changes in cytosolic or intraluminal free Ca2+ concentration were determined by fluorimetry. Caspase activity and phosphorylation of the eukaryotic initiation factor 2α (eIF2α) were explored by Western blot. Our results indicate that homocysteine releases Ca2+ from agonist sensitive stores, enhances eIF2α phosphorylation at Ser51 and activates caspase-3 and -9 independently of extracellular Ca2+. Homocysteine induced activation of caspase-3 and -9 was abolished by salubrinal, an agent that prevents endoplasmic reticulum (ER) stress-induced apoptosis. Homo-cysteine-induced platelet effects were significantly greater in type 2 diabetics than in healthy subjects. These findings demonstrate that homocysteine induces ER stress-mediated apoptosis in human platelets, an event that is enhanced in type 2 diabetic patients, which might be involved in the pathogenesis of cardiovascular complications associated with type 2 diabetes mellitus.

Ca(2+) entry, particularly store-operated Ca(2+) entry (SOCE), has been reported to be crucial for a variety of cellular functions. SOCE is a mechanism regulated by the Ca(2+) content of the stores, where the intraluminal Ca(2+) sensor STromal Interaction Molecule 1 (STIM1) has been reported to communicate the filling state of the intracellular Ca(2+) stores to the store-operated Ca(2+)-permeable channels in the plasma membrane, likely involving Orai1 and TRPC proteins, such as TRPC1. Here we have investigated the role of Orai1, STIM1 and TRPC1 in platelet aggregation, an event that occurs during the process of thrombosis and hemostasis. Electrotransjection of cells with anti-STIM1 (25-139) antibody, directed towards the Ca(2+)-binding motif, significantly reduced thrombin-induced aggregation and prevented ADP-evoked response. Extracellular application of the anti-STIM1 antibody, in order to block the function of plasma membrane-located STIM1, reduced thrombin- and ADP-stimulated platelet aggregation to a lesser extent. Introduction of an anti-Orai1 (288-301) antibody, which binds the STIM1-binding site located in the Orai1 C-terminus, or extracellular application of anti-hTRPC1 (557-571) antibody to impair hTRPC1 channel function, significantly reduced thrombin- and ADP-induced platelet aggregation. These findings suggest a role of STIM1, Orai1 and hTRPC1 in thrombin- and ADP-induced platelet aggregation probably through the regulation of Ca(2+) entry, which might become targets for the development of therapeutic strategies to treat platelet hyperactivity and thrombosis disorders.

TRPC (canonical transient receptor potential) channel subunits have been shown to assemble into homo- or hetero-meric channel complexes, including different Ca 2+ -handling proteins, required for the activation of CCE (capacitative Ca 2+ entry) or NCCE (non-CCE) pathways. In the present study we found evidence for the dynamic interaction between endogenously expressed hTRPC6 (human TRPC6) with either both Orai1 and STIM1 (stromal interaction molecule 1) or hTRPC3 to participate in CCE or NCCE. Electrotransjection of cells with an anti-hTRPC6 antibody, directed towards the C-terminal region, reduces CCE induced by TPEN [ N , N , N ′, N ′-tetrakis-(2-pyridylmethyl)-ethylenediamine], which reduces the intraluminal free Ca 2+ concentration. Cell stimulation with thrombin or extensive Ca 2+ -store depletion by TG (thapsigargin)+ionomycin enhanced the interaction between hTRPC6 and the CCE proteins Orai1 and STIM1. In contrast, stimulation with the diacylglycerol analogue OAG (1-oleoyl-2-acetyl- sn -glycerol) displaces hTRPC6 from Orai1 and STIM1 and enhances the association between hTRPC6 and hTRPC3. The interaction between hTRPC6 and hTRPC3 was abolished by dimethyl-BAPTA [1,2-bis-( o -aminophenoxy)ethane- N , N , N ′, N ′-tetra-acetic acid] loading, which indicates that this phenomenon is Ca 2+ -dependent. These findings support the hypothesis that hTRPC6 participates both in CCE and NCCE through its interaction with the Orai1–STIM1 complex or hTRPC3 respectively.

Background/Aims The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca 2+ homeostasis, we aimed to investigate the effect of these proteins on Ca 2+ mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment. Methods Ca 2+ mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry. Results Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca 2+ concentration, and reduced Ca 2+ concentration into the stores. Phenylephrine-evoked Ca 2+ release, Ca 2+ entry and extrusion by the plasma membrane Ca 2+ -ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca 2+ mobilization and increased calpain activation were observed in replicon-containing cells. Conclusions NS5A and core proteins induce oxidative stress-mediated Ca 2+ homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.

Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca2+ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca2+ release and entry and further increased Ca2+ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.

Store-operated calcium entry (SOCE) is a major mechanism for Ca(2+) entry in excitable and non-excitable cells. The best-characterised store-operated current is I(CRAC), but other currents activated by Ca(2+) 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) Ca(2+) sensor that communicates the content of the Ca(2+) stores to the store-operated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and co-clustering with the Ca(2+) 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.

Store-operated calcium entry (SOCE) is a major mechanism for Ca(2+) influx. Since SOCE was first proposed two decades ago many techniques have been used in attempting to identify the nature of store-operated Ca(2+) (SOC) channels. The first identified and best-characterised store-operated current is I(CRAC), but a number of other currents activated by Ca(2+) 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 Ca(2+) entry.

Summary. Background: Thrombin is a physiological platelet agonist that activates apoptotic events, including cytochrome c release and phosphatidylserine exposure; however, the mechanisms underlying these events remain unclear. Objectives: The present study is aimed to investigate whether thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak. Methods: Changes in the mitochondrial membrane potential were registered using the dye JC-1; Bid, Bax and Bak translocation to the mitochondria was detected by immunoprecipitation and Western blotting in samples from mitochondrial and cytosolic fractions. Results: Treatment of platelets with thrombin or ADP induces activation and mitochondrial association of active Bid, Bax and Bak. Translocation of Bid and Bax to the mitochondria was reduced by cytochalasin D, latrunculin A or jasplakinolide. Platelet exposure to exogenous H2O2 (10 μm) results in activation of Bid and Bax, which was found to be similar to the effect of thrombin. Thrombin evokes mitochondrial membrane depolarization, which is attenuated by catalase. Conclusion: Our results indicate that thrombin induces activation and mitochondrial translocation of Bid, Bax and Bak, which is likely to be one of the apoptotic events in human platelets.

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.

Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.

The hyperactivation of platelets is involved in the cardiovascular complications associated with type 2 diabetes mellitus. Altered platelet behavior contributes to the angiopathies associated with diabetes. A number of mechanisms involved in platelet activation are altered in diabetes. Platelets from type 2 diabetic patients show an enhanced endogenous reactive oxygen species production and a reduced antioxidant capability, which increase the activity of several tyrosine kinases, such as the Bruton's tyrosine kinase, MAP kinases or proteins of the SRC family. Oxidative stress is also involved in the abnormal intracellular calcium homeostasis observed in platelets from type 2 diabetics, including an enhanced resting cytosolic calcium concentration and calcium release and entry in response to agonists. Moreover, diabetes alters the bioavailability of nitric oxide in platelets. Basal nitric oxide synthase activity is reduced in homogenates of platelets obtained from patients with type 2 diabetes mellitus. The study of these abnormalities might be helpful in the development of new pharmacological strategies to reduce platelet activation in type 2 diabetes mellitus.

Ca+release-activated Ca2+(CRAC) channels are activated when free Ca2+concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+chelator that prevented TG-evoked cytosolic free Ca2+concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+entry.

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.

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 μM 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.

Two separate Ca2+ stores have been reported in human platelets: the dense tubular system (DTS) and lysosome-like acidic organelles. Recent work has reported that Ca2+ release from the DTS is mediated by the generation of inositol 1,4,5-trisphosphate, whereas Ca2+ efflux from the acidic stores is mostly linked to nicotinic acid adenine dinucleotide phosphate. Platelet agonists release Ca2+ selectively from one or both stores, which provides additional insight into the complexity of Ca2+ signaling and the cellular functions activated. Here, we review the role of multiple Ca2+ mobilizing messengers and Ca2+ stores in the activation of specific functions in platelets in response to different physiologic agonists.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 μM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.

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.

Platelet hyperaggregability is involved in the pathogenesis of type 2 diabetes mellitus. Thrombin-evoked platelet aggregation includes the activation of several intracellular pathways, including endogenous generation of reactive oxygen species (ROS), Ca2+ mobilization and protein tyrosine phosphorylation. Here we show that crude aqueous extract from Urtica dioica reduces thrombin-evoked aggregation in platelets from healthy donors and diabetics, in a concentration-dependent manner. U. dioica extract showed a potent antioxidant activity and prevented thrombin-evoked ROS generation in platelets from healthy and diabetic donors. Treatment with U. dioica extract reduced Ca2+ entry induced by thrombin or selective depletion of the two Ca2+ stores in platelets (without altering Ca2+ release), reduced protein tyrosine phosphorylation and reversed the abnormal Ca2+ mobilization and tyrosine phosphorylation in platelets from diabetics. We conclude that extract from U. dioica shows antiaggregant actions and might be used for the treatment and/or prevention of cardiovascular complications associated with type 2 diabetes mellitus.

Summary. Background: Thrombin is a major physiological platelet agonist that activates a number of cell functions including aggregation. Platelet stimulation with thrombin has been shown to result in the development of apoptotic events, including activation of caspases-3 and -9, cytochrome c release and phosphatidylserine (PS) exposure; however, the mechanism underlying the activation of apoptosis remains unclear. Objectives: In the present study, we aim to investigate whether endogenously generated reactive oxygen species upon thrombin stimulation is required for the activation of apoptosis in human platelets. Methods: Changes in the mitochondrial membrane potential were registered using the dye JC-1; caspase-3 and -9 activity was determined from the cleavage of their respective specific fluorogenic substrates; PS externalization was estimated using annexin V-fluorescein isothicyanate and cytochrome c release was detected by Western blotting in samples from the mitochondrial and cytosolic fractions. Results: Treatment of platelets with thrombin stimulates mitochondrial membrane potential depolarization and endogenous generation of H2O2. Platelet exposure to exogenous H2O2 results in cytochrome c release and activation of caspases-9. In addition, H2O2 induces the activation of caspase-3 and PS exposure by a mechanism dependent on cytochrome c release and caspase-9 activation. Finally, thrombin-evoked development of apoptotic events was impaired by treatment with catalase. Conclusion: Our results indicate that thrombin-induced apoptosis is likely mediated by endogenous generation of H2O2 in human platelets.

Background/Aims: we have analyzed the mechanisms of calcium entry and release in platelets obtained from bile duct-ligated rats (BDL), two (cholestasis group) and four weeks after surgery. Methods: platelets were washed and loaded with fura-2. Cytosolic calcium concentration ([Ca2+] i}) was determined in cell suspensions by means of fluorescence spectroscopy. Results: Basal [Ca2+] i} was similar in platelets from BDL rats as compared with those from their respective controls, both in the absence and presence of extracellular calcium. Platelet stimulation with thrombin in the absence and presence of extracellular calcium induced a rapid rise in [Ca2+] i} that was of greater magnitude in platelets from BDL rats than in controls. Calcium storage was significantly elevated in platelets from BDL rats, as well as the activity of the sarcoendoplasmic reticulum calcium ATP-ase (SERCA). Capacitative calcium entry, as evaluated by inhibition of SERCA with thapsigargin, is also altered in platelets from BDL rats, showing lower rates of calcium entry. Conclusions: chronic BDL alters intracellular calcium homeostasis in platelets, so that an enhanced calcium release is evoked by thrombin, which may be due to an increased amount of calcium stored in the intracellular organelles and secondary to an enhanced activity of SERCA. These alterations are already evident before cirrhosis is completely developed, during the cholestasis phase.

SummaryWe have investigated the intracellular mechanisms involved in microtubular remodelling by thrombin and its possible involvement in platelet aggregation and secretion. Platelet stimulation with thrombin induces a time- and concentration-dependent regulation of the microtubular content, which was found to be maximally effective at the concentration 0.1 U/ml. Thrombin (0.1 U/ml) evoked an initial decrease in the microtubule content detectable at 5 seconds (sec) and reached a minimum 10 sec after stimulation.The microtubular content then increased, exceeding basal levels again approximately 30 sec after stimulation. Inhibition of tyrosine phosphatases using vanadate abolished thrombin-induced microtubular depolymerisation while inhibition of tyrosine kinases by methyl-2,5-dihydroxycinnamate prevented microtubule polymerisation.Thrombin activates the cytosolic Brutons tyrosine kinase (Btk) and Src proteins. Inhibition of Btk or Src by LFM-A13 or PP1, respectively, abolished thrombin-induced microtubular polymerisation, while maintaining intact its ability to induce initial depolymerisation. Microtubular disruption by colchicine significantly reduced thrombin induced platelet aggregation and ATP secretion. Similar results were observed after inhibition of microtubular disassembly by paclitaxel.These findings indicate that thrombin induces microtubular remodelling by modifying the balance between protein tyrosine phosphorylation and dephosphorylation. The former seems to be required for microtubular polymerisation, while tyrosine dephosphorylation is required for microtubular depolymerisation. Both, initial microtubular disassembly and subsequent polymerisation are required for thrombin-induced platelet aggregation and secretion in human platelets.

Changes in cytosolic Ca(2+) concentration ([Ca(2+)]c) regulate granule secretion in different cell types. Thrombin activates PAR1 and PAR4 receptors and promotes release of Ca(2+) from distinct intracellular stores, which, in turn, activates store-operated Ca(2+) entry (SOCE). A crucial step during platelet function is the release of physiological agonists stored in secretory granules to the extracellular compartment during activation. We aim to study the role of Ca(2+) mobilization from the extracellular compartment or from different intracellular stores in platelet granule secretion. By using flow cytometry, we have found that α- and δ-granules are secreted in thrombin-stimulated platelets in the absence of extracellular Ca(2+), and in a concentration-dependent manner. Our findings show that thrombin-stimulated granule secretion depends on Ca(2+) mobilization from intracellular stores. Analysis of the kinetics of granule secretion reveals that platelet stimulation with thrombin results in rapid release of α-granules which precedes the secretion of δ-granules. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 573-586 of TRPC6, inhibited thrombin-evoked δ-granule exocytosis. Our results indicate that the mechanisms underlying thrombin-induced α- and δ-granule secretion show differences in dependency on Ca(2+) mobilization.

The ion Ca(2+) is a ubiquitous second messenger that mediates a variety of cellular functions. Dysfunction of the mechanisms involved in Ca(2+) homeostasis underlies a number of pathological processes, including cancer. Store-operated Ca(2+) entry (SOCE) is a major mechanism for Ca(2+) entry modulated by the intracellular Ca(2+) stores. The Ca(2+)-selective store-operated current (ICRAC) is mediated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and the store-operated Ca(2+) (SOC) channel Orai1, while other non-selective cation currents (ISOC) involves the participation of members of the canonical transient receptor potential (TRPC) channel family, including TRPC1. Distinct isoforms of the key components of SOCE have been described in mammalian cells, STIM1 and 2, Orai1-3 and TRPC1-7. In cancer cells, SOCE has been reported to play an important role in cell cycle progression and proliferation, migration, metastasis and evasion of apoptosis. Changes in the expression of the key elements of SOCE and Ca(2+) homeostasis remodeling have been account to play important roles in the phenotypic changes observed in transformed cells. Despite there are differences in the expression level of the molecular components of SOCE, as well as in the relevance of the STIM, Orai and TRPC isoforms in SOCE and tumorigenesis among cancer cell types, there is a body of evidence supporting an important role for SOCE underlying the phenotypic modifications of cancer cells that propose STIM and the SOC channels as suitable candidate targets for future prognostic or therapeutic strategies. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.

Store-operated Ca(2+) entry (SOCE) is an important mechanism for Ca(2+) influx in non-excitable cells, also present in excitable cells. The activation of store-operated channels (SOCs) is finely regulated by the filling state of the intracellular agonist-sensitive Ca(2+) compartments, and both, the mechanism of sensing the Ca(2+) stores and the nature and functional properties of the SOCs, have been a matter of intense investigation and debate. The identification of STIM1 as the endoplasmic reticulum Ca(2+) sensor and both Orai1, as the pore-forming subunit of the channels mediating the Ca(2+)-selective store-operated current, and the members of the TRPC subfamily of proteins, as the channels mediating the cation-permeable SOCs, has shed new light on the underlying events. This review summarizes the initial hypothesis and the current advances on the mechanism of activation of SOCE.

Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.

Store-operated Ca2+ entry (SOCE) is an important mechanism for Ca2+ influx in non-excitable cells, also present in excitable cells. The activation of store-operated channels (SOCs) is finely regulated by the filling state of the intracellular agonist-sensitive Ca2+ compartments, and both, the mechanism of sensing the Ca2+ stores and the nature and functional properties of the SOCs, have been a matter of intense investigation and debate. The identification of STIM1 as the endoplasmic reticulum Ca2+ sensor and both Orai1, as the pore-forming subunit of the channels mediating the Ca2+-selective store-operated current, and the members of the TRPC subfamily of proteins, as the channels mediating the cation-permeable SOCs, has shed new light on the underlying events. This review summarizes the initial hypothesis and the current advances on the mechanism of activation of SOCE.

Physiological agonists increase cytosolic free Ca2+ concentration to regulate a number of cellular processes. The platelet thrombin receptors, PAR (protease-activated receptor) 1 PAR-4 and GPIb-IX-V (glycoprotein Ib-IX-V) have been described as potential contributors of thrombin-induced platelet aggregation. Platelets present two separate Ca2+ stores, the DTS (dense tubular system) and acidic organelles, differentiated by the distinct sensitivity of their respective SERCAs (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPases) to TG (thapsigargin) and TBHQ [2,5-di-(tert-butyl)-1,4-hydroquinone]. However, the involvement of the thrombin receptors in Ca2+ release from each Ca2+ store remains unknown. Depletion of the DTS using ADP, which releases Ca2+ solely from the DTS, in combination with 10 nM TG, to selectively inhibit SERCA2 located on the DTS reduced Ca2+ release evoked by the PAR-1 agonist, SFLLRN, and the PAR-4 agonist, AYPGKF, by 80 and 50% respectively. Desensitization of PAR-1 and PAR-4 or pre-treatment with the PAR-1 and PAR-4 antagonists SCH 79797 and tcY-NH2 reduced Ca2+ mobilization induced by thrombin, and depletion of the DTS after desensitization or blockade of PAR-1 and PAR-4 had no significant effect on Ca2+ release stimulated by thrombin through the GPIb-IX-V receptor. Converse experiments showed that depletion of the acidic stores using TBHQ reduced Ca2+ release evoked by SFLLRN or AYPGKF, by 20 and 50% respectively, and abolished thrombin-stimulated Ca2+ release through the GPIb-IX-V receptor when PAR-1 and PAR-4 had been desensitized or blocked. Our results indicate that thrombin-induced activation of PAR-1 and PAR-4 evokes Ca2+ release from both Ca2+ stores, while activation of GPIb-IX-V by thrombin releases Ca2+ solely from the acidic compartments in human platelets.

Aim: Natriuretic peptide receptor A (NPR-A) is the main physiological receptor for atrial natriuretic peptide (ANP). Maximal activation of NPR-A guanylyl cyclase (GC) requires ANP binding and ATP interaction with a putative cytoplasmic site. This study investigates the regulatory effect of ATP on GC-coupled NPR-A activity in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Methods: Cyclic GMP production and competitive inhibition of [125I]ANP1−28 binding were performed in solubilized glomerular and papillary renal membranes. Results: Here, we report that incubation of renal glomerular and papillary membranes with ATP induced a concentration-dependent increase in basal and ANP1−28-stimulated GC activity that was significantly greater in SHR than in age-matched WKY. ATPγS was more effective than ATP and induced a greater stimulation of cGMP production in SHR than in WKY. In contrast, in solubilized membranes ATP exerted an inhibitory role on basal and ANP1−28-induced GC activity, suggesting that an accessory protein is required for ATP-induced GC activation. ATP increases NPR-A affinity for ANP1−28 and decreased Bmax in crude and solubilized membranes. Kinetic analysis of GC-coupled NPR-A revealed that ATP reduced the Km and increased the Vmax, an effect that was greater in SHR. Conclusion: Our observations indicate that ATP exerts a greater net effect on NPR-A in SHR than in WKY, which might explain the greater rate of cGMP production observed in SHR compared to WKY.

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.

The mechanisms involved in the effect of ethanol on Ca2+ entry and aggregability have been investigated in human platelets in order to shed new light on the pathogenesis of alcohol consumption. Ethanol (50 mM) induced H2O2 production in platelets by Ca2+-dependent and independent mechanisms. Ca2+ entry induced by ethanol was impaired by catalase. Ethanol reduced SOCE mediated by depletion of the 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive acidic stores but enhances SOCE regulated by the dense tubular system. This effect was abolished by treatment with catalase or the sulphydryl group reducing agent dithiotreitol (DTT). Similarly, the anti-aggregant effect of ethanol was prevented by platelet treatment with catalase or DTT. In conclusion we provide considerable evidence that ethanol alters Ca2+ entry and reduces thrombin-induced aggregation as a result of the generation of H2O2 and the oxidation of sulphydryl groups in human platelets.

Platelets express, among others, initiator caspase 9 and effector caspase 3. Upon activation by physiological agonists, calcium ionophores or under shear stress they might develop apoptotic events. Although it is well known that the cytoskeletal network plays a crucial role in apoptosis, the relationship between caspases 3 and 9 and the cytoskeleton is poorly understood. Here we demonstrate that the physiological agonist thrombin is able to induce activation of caspases 3 and 9 in human platelets and significantly increases the amount in the cytoskeleton of the active forms of both caspases and the procaspases 3 and 9. After stimulation with thrombin the amount of active caspases 3 and 9 in the cytosolic and cytoskeletal fractions were significantly reduced in Ro-31-8220-treated cells, which demonstrates that caspases activation and association with the cytoskeleton needs the contribution of PKC. Inhibition of actin polymerization by cytochalasin D inhibits translocation and activation of both caspases, suggesting that thrombin stimulates caspase 3 and 9 activation and association with the reorganizing actin cytoskeleton. Finally, our results show that inhibition of thrombin-induced caspase activation has no effect on their translocation to the cytoskeleton although impairment of thrombin-evoked caspase translocation has negative effects on caspase activity, suggesting that translocation to the cytoskeleton might be important for caspase activation by thrombin in human platelets.

Apoptosis has been shown to be associated with changes in cytosolic free calcium concentration ([Ca(2+)](c)). Here we show that the agonist thrombin induces activation of caspases 9 and 3 and translocation of the caspase active forms and procaspases to the cytoskeleton in human platelets. Dimethyl-BAPTA loading did not affect thrombin-induced caspase 9 and 3 activation or translocation suggesting that these responses are independent of increases in [Ca(2+)](c). Treatment with thapsigargin plus ionomycin, to induce extensive Ca(2+) store depletion and subsequent increase in [Ca(2+)](c), stimulates caspase activation although it was unable to induce caspase translocation to the cytoskeleton. Similar results were observed in cells loaded with dimethyl-BAPTA, suggesting that activation of caspases 9 and 3 by thapsigargin plus ionomycin does not require rises in [Ca(2+)](c). These findings suggest that thrombin-induced caspase 9 and 3 activation and translocation are independent on rises in [Ca(2+)](c) but might require store depletion in human platelets.

Reactive oxygen species can induce several biological processes by stimulating signal transduction components such as cytosolic free calcium concentration. The physiological significance of the role of biological oxidants in the regulation of calcium signalling pathway as well as the mechanisms of the oxidant-stimulation of signal transduction are discussed in this review.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in platelets and other cells. De novo conformational coupling between elements in the plasma membrane and Ca2+ stores, where the actin cytoskeleton plays an important regulatory role, has been proposed as the most likely mechanism to activate SOCE in platelets. Here we have examined for the first time changes in platelet F-actin levels on a subsecond time scale. Using stopped-flow fluorimetry and a quenched-flow approach, we provide evidence for the involvement of cofilin in actin filament reorganization and SOCE in platelets. Thrombin (0.1 U/mL) evoked an initial decrease in F-actin that commenced within 0.1 second and reached a minimum 0.9 second after stimulation, prior to the activation of SOCE. F-actin then increased, exceeding basal levels approximately 2.5 seconds after stimulation. Thrombin also induced cofilin dephosphorylation and activation, which paralleled the changes observed in F-actin, and rapid Btk activation. Inhibition of cofilin dephosphorylation by LFM-A13 resulted in the loss of net actin depolymerization and an increased delay in SOCE initiation. These results suggest that cofilin is important for the rapid actin remodeling necessary for the activation of SOCE in platelets through de novo conformational coupling.