Ginés M. Salido

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

STIM1 (stromal interaction molecule 1) has recently been proposed to communicate the intracellular Ca2+ stores with the plasma membrane to mediate store-operated Ca2+ entry. Here we describe for the first time that Ca2+ store depletion stimulates rapid STIM1 surface expression and association with endogenously expressed human canonical TRP1 (hTRPC1) independently of rises in cytosolic free Ca2+ 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 Ca2+ stores and hTRPC1 in the plasma membrane, which has been suggested to underlie the activation of store-operated Ca2+ entry in these cells. Electrotransjection of cells with anti-STIM1 antibody, directed toward the N-terminal sequence that includes the Ca2+-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 Ca2+ entry. These findings provide evidence for a role of STIM1 in the activation of store-operated Ca2+ entry probably acting as a Ca2+ sensor.

Orai1 Mediates the Interaction between STIM1 and hTRPC1 and Regulates the Mode of Activation of hTRPC1-forming Ca2+ Channels

Orai1 and hTRPC1 have been presented as essential components of store-operated channels mediating highly Ca2+ selective ICRAC and relatively Ca2+ selective ISOC, respectively. STIM1 has been proposed to communicate the Ca2+ content of the intracellular Ca2+ stores to the plasma membrane store-operated Ca2+ channels. Here we present evidence for the dynamic interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 regulated by depletion of the intracellular Ca2+ stores, using the pharmacological tools thapsigargin plus ionomycin, or by the physiological agonist thrombin, independently of extracellular Ca2+. In addition we report that Orai1 mediates the communication between STIM1 and hTRPC1, which is essential for the mode of activation of hTRPC1-forming Ca2+ 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 Ca2+ entry. These findings support the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of store-operated Ca2+ entry.

Hydrogen Peroxide Generation Induces pp60src Activation in Human Platelets EVIDENCE FOR THE INVOLVEMENT OF THIS PATHWAY IN STORE-MEDIATED CALCIUM ENTRY

Reactive oxygen species, such as H2O2, have been recognized as intracellular messengers involved in several cell functions. Here we report the activation of the tyrosine kinase pp60src by H2O2, a mechanism required for the activation of store-mediated Ca2+ entry (SMCE) in human platelets. Treatment of platelets with H2O2 resulted in a time- and concentration-dependent activation of pp60src. Incubation with GF 109203X, a protein kinase C (PKC) inhibitor, prevented H2O2-induced pp60src activation. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60src by H2O2 is independent of increases in [Ca2+]i. Cytochalasin D, an inhibitor of actin polymerization, significantly reduced H2O2-induced pp60src activation. We found that platelet stimulation with thapsigargin (TG) plus ionomycin (Iono) or thrombin induced rapid H2O2 production, a mechanism independent of elevations in [Ca2+]i. Treatment of platelets with catalase attenuated TG plus Iono- and thrombin-induced activation of pp60src. In addition, catalase as well as the pp60src inhibitor, PP1, reduced both the activation of SMCE and the coupling between the hTrp1 and the IP3R type II without having any effect on the maintenance of SMCE. Consistent with the role of PKC in the activation of pp60src by H2O2, 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 H2O2 production, which acts as a second messenger by stimulating pp60src by a PKC-dependent pathway and is involved in the activation of SMCE in these cells.

Release of calcium from mitochondrial and nonmitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide.

Abstract. In the present study we have studied how [Ca2+]i is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H2O2 caused a slow sustained [Ca2+]i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca2+]i. In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+]i induced by H2O2 was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.

Ca2+ accumulation into acidic organelles mediated by Ca2+- and vacuolar H+-ATPases in human platelets

Most physiological agonists increase cytosolic free [Ca2+]c (cytosolic free Ca2+ concentration) to regulate a variety of cellular processes. How different stimuli evoke distinct spatiotemporal Ca2+ responses remains unclear, and the presence of separate intracellular Ca2+ stores might be of great functional relevance. Ca2+ accumulation into intracellular compartments mainly depends on the activity of Ca2+- and H+-ATPases. Platelets present two separate Ca2+ stores differentiated by the distinct sensitivity to thapsigargin and TBHQ [2,5-di-(t-butyl)-1,4-hydroquinone]. Although one store has long been identified as the dense tubular system, the nature of the TBHQ-sensitive store remains uncertain. Treatment of platelets with GPN (glycylphenylalanine-2-naphthylamide) impaired Ca2+ release by TBHQ and reduced that evoked by thrombin. In contrast, GPN did not modify Ca2+ mobilization stimulated by ADP or AVP ([arginine]vasopressin). Treatment with nigericin, a proton carrier, and bafilomycin A1, an inhibitor of the vacuolar H+-ATPase, to dissipate the proton gradient into acidic organelles induces a transient increase in [Ca2+]c that was abolished by previous treatment with the SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) 3 inhibitor TBHQ. Depleted acidic stores after nigericin or bafilomycin A1 were refilled by SERCA 3. Thrombin, but not ADP or AVP, reduces the rise in [Ca2+]c evoked by nigericin and bafilomycin A1. Our results indicate that the TBHQ-sensitive store in human platelets is an acidic organelle whose Ca2+ accumulation is regulated by both Ca2+- and vacuolar H+-ATPases.

TRPC channels and store-operated Ca2+ entry

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.

Thrombin induces apoptotic events through the generation of reactive oxygen species in human platelets

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.

Ethanol stimulates ROS generation by mitochondria through Ca2+ mobilization and increases GFAP content in rat hippocampal astrocytes.

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca2+]c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H2DCFDA for the determination of changes in [Ca2+]c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca2+ chelator BAPTA (10 microM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 microM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 microM antimycin plus 10 microM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca2+]c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.

Early caspase‐3 activation independent of apoptosis is required for cellular function

A number of pro‐apoptotic stimuli induce the activation of caspase‐9, an initiator protease that activates executioner caspases, such as caspase‐3, leading to the development of programmed cell death. Here we demonstrate that cell (platelets and pancreatic acinar cells) stimulation with agonists induces a bimodal activation of caspase‐3. The early caspase‐3 activation occurs within 1 min of stimulation and is independent on caspase‐9 or mitochondrial cytochrome c release suggesting that is a non‐apoptotic event. The ability of agonists to induce early activation of caspase‐3 is similar to that observed for other physiological processes. Activation of caspase‐3 by physiological concentrations of cellular agonists, including thrombin or CCK‐8, is independent of rises in cytosolic calcium concentration but requires PKC activation, and is necessary for agonist‐induced activation of the tyrosine kinases Btk and pp60src and for several cellular functions, including store‐operated calcium entry, platelet aggregation, or pancreatic secretion. Thus, early activation of caspase‐3 seems to be a non‐apoptotic event required for cellular function. J. Cell. Physiol. 209: 142–152, 2006.

Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1.

Store-operated Ca2+ entry (SOCE) is a mechanism regulated by the filling state of the intracellular Ca2+ stores that requires the participation of the Ca2+ sensor STIM1, which communicates the Ca2+ content of the stores to the plasma membrane Ca2+-permeable channels. We have recently reported that Orai1 mediates the communication between STIM1 and the Ca2+ channel hTRPC1. This event is important to confer hTRPC1 store depletion sensitivity, thus supporting the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of SOCE. Here we have explored the relevance of lipid rafts in the formation of the STIM1-Orai1-hTRPC1 complex and the activation of SOCE. Disturbance of lipid raft domains, using methyl-β-cyclodextrin, reduces the interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 upon depletion of the intracellular Ca2+ stores and attenuates thapsigargin-evoked Ca2+ entry. These findings suggest that TRPC1, Orai1 and STIM1 form a heteromultimer associated with lipid raft domains and regulated by the intracellular Ca2+ stores.