Protein kinase C alpha enhances sodium-calcium exchange during store-operated calcium entry in mouse platelets

ArticleinCell calcium 48(6):333-40 · December 2010with15 Reads
DOI: 10.1016/j.ceca.2010.10.006 · Source: PubMed
A rise in intracellular calcium concentration ([Ca(2+)](i)) is necessary for platelet activation. A major component of the [Ca(2+)](i) elevation occurs through store-operated Ca(2+) entry (SOCE). The aim of this study was to understand the contribution of the classical PKC isoform, PKCα to platelet SOCE, using platelets from PKCα-deficient mice. SOCE was reduced by approximately 50% in PKCα(-/-) platelets, or following treatment with bisindolylmaleimide I, a PKC inhibitor. However, TG-induced Mn(2+) entry was unaffected, which suggests that divalent cation entry through store-operated channels is not directly regulated. Blocking the autocrine action of secreted ADP or 5-HT on its receptors did not reproduce the effect of PKCα deficiency. In contrast, SN-6, a Na(+)/Ca(2+) exchanger inhibitor, did reduce SOCE to the same extent as loss of PKCα, as did replacing extracellular Na(+) with NMDG(+). These treatments had no further effect in PKCα(-/-) platelets. These data suggest that PKCα enhances the extent of SOCE in mouse platelets by regulating Ca(2+) entry through the Na(+)/Ca(2+) exchanger.
    • "The role of individual isoforms in platelet activation has been investigated using isoform-specific inhibitors and mice deficient in single isoforms [4–7,9–19]. This has led to the conclusion that the classical isoforms play positive roles in platelet activation, with PKCα playing the predominant role, supported by PKCβ, while the novel isoforms play minor or inhibitory roles [10]. "
    [Show abstract] [Hide abstract] ABSTRACT: In comparison to the classical isoforms of protein kinase C (PKC), the novel isoforms are thought to play minor or inhibitory roles in the regulation of platelet activation and thrombosis. To measure the levels of PKCθ and PKCε and to investigate the phenotype of mice deficient in both novel PKC isoforms. Tail bleeding and platelet activation assays were monitored in mice and platelets from mice deficient in both PKCθ and PKCε. PKCε plays a minor role in supporting aggregation and secretion following stimulation of the collagen receptor GPVI in mouse platelets but has no apparent role in spreading on fibrinogen. PKCθ, in contrast, plays a minor role in supporting adhesion and filopodial generation on fibrinogen but has no apparent role in aggregation and secretion induced by GPVI despite being expressed at over 10 times the level of PKCε. Platelets deficient in both novel isoforms have a similar pattern of aggregation downstream of GPVI and spreading on fibrinogen as the single null mutants. Strikingly, a marked reduction in aggregation on collagen under arteriolar shear conditions is observed in blood from the double but not single-deficient mice along with a significant increase in tail bleeding. These results reveal a greater than additive role for PKCθ and PKCε in supporting platelet activation under shear conditions and demonstrate that, in combination, the two novel PKCs support platelet activation.
    Full-text · Article · Jul 2012
    • "Platelet SOCE may also involve TRPC channels, since TRPCs have been found to associate with Orai1 [27,28]. In addition, TG induces extensive Na + entry in human and mouse platelets [29,30], whereas Orai1 is reported to have a very low Na + permeability in the presence of extracellular Ca 2+ [31,32] , suggesting that there may be components of the SOC in addition to Orai1. Consistent with possible roles for Orai1 and/or TRPCs, we found that BTP-2 was an effective blocker of TG-induced SOCE. "
    [Show abstract] [Hide abstract] ABSTRACT: Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca2+ signalling. Two distinct Ca2+ entry pathways amplify and sustain platelet Ca2+ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca2+ entry (SOCE) and non-capacitative Ca2+ entry (NCCE) in thrombin-induced Ca2+ signalling and PS exposure by using two Ca2+ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca2+ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca2+ signalling at higher thrombin concentrations. However, despite being involved in Ca2+ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca2+ entry is an important regulator of thrombin-induced PS exposure in platelets.
    Full-text · Article · Jul 2011
  • [Show abstract] [Hide abstract] ABSTRACT: The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺](i)) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺](i) levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺](i) in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺](i) rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺](i) release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺](i) rise. Carvedilol at 5-50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²⁺](i) rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.
    Article · Jun 2011
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