Juan A. Rosado

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.

Melatonin induces mitochondrial‐mediated apoptosis in human myeloid HL‐60 cells

Abstract:  The role of melatonin in the mediation of apoptotic events has recently gained attention, especially after recent studies have reported that melatonin exerts antiapoptotic actions in normal cells but may activate proapoptotic pathways in some tumor cells. Here, we have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL‐60. Melatonin treatment (1 mm) induced a significant increase in caspase‐3 and ‐9 activities. The effect of melatonin on the activation of caspases was time dependent, reaching a maximum after 12 hr of stimulation, and then decreasing to a minimum after 72 hr. Treatment with melatonin also evoked mitochondrial membrane depolarization and permeability transition pore induction, which caused loss of mitochondrial staining by calcein, and increased cell death by apoptosis/necrosis as demonstrated by propidium iodide positive‐staining of cells after 72 hr of stimulation. In addition, the exposure of cells to melatonin resulted in an activation and association of the proapoptotic proteins Bax and Bid, as well as promoting detectable increases in the expression of both proteins. We conclude that melatonin has proapoptotic and/or oncostatic effects in the human myeloid cell line HL‐60.

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.

Melatonin Reduces Apoptosis Induced by Calcium Signaling in Human Leukocytes: Evidence for the Involvement of Mitochondria and Bax Activation

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.

NATRIURETIC PEPTIDES IN VASCULAR PHYSIOLOGY AND PATHOLOGY

Four major natriuretic peptides have been isolated: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Dendroaspis-type natriuretic peptide (DNP). Natriuretic peptides play an important role in the regulation of cardiovascular homeostasis maintaining blood pressure and extracellular fluid volume. The classical endocrine effects of natriuretic peptides to modulate fluid and electrolyte balance and vascular smooth muscle tone are complemented by autocrine and paracrine actions that include regulation of coronary blood flow and, therefore, myocardial perfusion; modulation of proliferative responses during myocardial and vascular remodeling; and cytoprotective anti-ischemic effects. The actions of natriuretic peptides are mediated by the specific binding of these peptides to three cell surface receptors: type A natriuretic peptide receptor (NPR-A), type B natriuretic peptide receptor (NPR-B), and type C natriuretic peptide receptor (NPR-C). NPR-A and NPR-B are guanylyl cyclase receptors that increase intracellular cGMP concentration and activate cGMP-dependent protein kinases. NPR-C has been presented as a clearance receptor and its activation also results in inhibition of adenylyl cyclase activity. The wide range of effects of natriuretic peptides might be the base for the development of new therapeutic strategies of great benefit in patients with cardiovascular problems including coronary artery disease or heart failure. This review summarizes current literature concerning natriuretic peptides, their receptors and their effects on fluid/electrolyte balance, and vascular and cardiac physiology and pathology, including primary hypertension and myocardial infarction. In addition, we will attempt to provide an update on important issues regarding natriuretic peptides in congestive heart failure.

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.