Pedro C. Redondo

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.

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.

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.

Evidence for secretion-like coupling involving pp60src in the activation and maintenance of store-mediated Ca2+ entry in mouse pancreatic acinar cells

Store-mediated Ca2+ entry (SMCE) is one of the main pathways for Ca2+ influx in non-excitable cells. Recent studies favour a secretion-like coupling mechanism to explain SMCE, where Ca2+ entry is mediated by an interaction of the endoplasmic reticulum (ER) with the plasma membrane (PM) and is modulated by the actin cytoskeleton. To explore this possibility further we have now investigated the role of the actin cytoskeleton in the activation and maintenance of SMCE in pancreatic acinar cells, a more specialized secretory cell type which might be an ideal cellular model to investigate further the properties of the secretion-like coupling model. In these cells, the cytoskeletal disrupters cytochalasin D and latrunculin A inhibited both the activation and maintenance of SMCE. In addition, stabilization of a cortical actin barrier by jasplakinolide prevented the activation, but not the maintenance, of SMCE, suggesting that, as for secretion, the actin cytoskeleton plays a double role in SMCE as a negative modulator of the interaction between the ER and PM, but is also required for this mechanism, since the cytoskeleton disrupters impaired Ca2+ entry. Finally, depletion of the intracellular Ca2+ stores induces cytoskeletal association and activation of pp60(src), which is independent on Ca2+ entry. pp60(src) activation requires the integrity of the actin cytoskeleton and participates in the initial phase of the activation of SMCE in pancreatic acinar cells.

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

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

Store-operated Ca2+ entry: Vesicle fusion or reversible trafficking and de novo conformational coupling?

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

A role for SNAP‐25 but not VAMPs in store‐mediated Ca2+ entry in human platelets

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

Store-Operated Ca 2+ Entry

Store-operated Ca(2+) entry (SOCE) is an ubiquitous and major mechanism for Ca(2+) influx in mammalian cells with important physiological relevance. Since the discovery of SOCE in 1986 both, the mechanism that communicates the amount of Ca(2+) accumulated in the intracellular Ca(2+) stores to the plasma membrane channels and the nature of the capacitative channels, have been a matter of intense investigation. During the last decade, two of the major elements of SOCE, STIM1, the Ca(2+) sensor of the intracellular Ca(2+) compartments, and Orai1, the protein forming the channel that conducts the capacitative Ca(2+) release-activated current I (CRAC), were identified. Together with these proteins, different homologues, including STIM2, Orai2 and Orai3, were identified, although their relevance in SOCE has not been fully characterized yet. Before the identification of STIM1 and Orai1, TRPC proteins were found to be involved in SOCE in different cell types, more likely conducting the non-selective capacitative current described as I (SOC). Current evidence indicates that STIM1, Orai1 and TRPC proteins dynamically interact forming a ternary complex that mediates SOCE in a number of cellular models. The dynamic interaction of STIM1 with Orai1, TRPCs or both might provide an explanation to the distinct capacitative currents described in different cell types.

A role for 5,6‐epoxyeicosatrienoic acid in calcium entry by de novo conformational coupling in human platelets

A major pathway for Ca2+ entry in non‐excitable cells is activated following depletion of intracellular Ca2+ stores. A de novo conformational coupling between elements in the plasma membrane (PM) and Ca2+ stores has been proposed as the most likely mechanism to activate this capacitative Ca2+ entry (CCE) in several cell types, including platelets. Here we report that a cytochrome P450 metabolite, 5,6‐EET, might be a component of the de novo conformational coupling in human platelets. In these cells, 5,6‐EET induces divalent cation entry without having any detectable effect on Ca2+ store depletion. 5,6‐EET‐induced Ca2+ entry was sensitive to the CCE blockers 2‐APB, lanthanum, SKF‐96365 and nickel and impaired by incubation with anti‐hTRPC1 antibody. Ca2+ entry stimulated by low concentrations of thapsigargin, which selectively depletes the dense tubular system and induces EET production, was impaired by the cytochrome P450 inhibitor 17‐ODYA, which has no effect on CCE mediated by depletion of the acidic stores using 2,5‐di‐(tert‐butyl)‐1,4‐hydroquinone. We have found that 5,6‐EET‐induced Ca2+ entry requires basal levels of H2O2, which might maintain a redox state favourable for this event. Finally, our results indicate that 5,6‐EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca2+ store towards the PM to facilitate de novo coupling between IP3R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6‐EET in TG‐induced coupling between IP3R type II and hTRPC1 and subsequently CCE is compatible with the de novo conformational coupling in human platelets.