title: Protein kinase C activates non-capacitative calcium entry in human platelets.
abstract: 1. In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. 2. Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. 3. Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. 4. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. 5. Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. 6. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non-capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores.
authors.affiliation: Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Rosado
authors.forename: S O
authors.initials: SO
authors.lastname: Sage
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000097:blood; D015220:Calcium Channels / Q000097:blood* / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D047630:Depsipeptides; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D047028:Macrocyclic Compounds; D018613:Microscopy, Confocal; D010080:Oxazoles / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism; D058469:Receptors, Purinergic P2X; D013324:Strontium / Q000097:blood; D013917:Thrombin / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D002413:Cations, Divalent; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D007211:Indoles; D047028:Macrocyclic Compounds; D010080:Oxazoles; D010456:Peptides, Cyclic; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; C108644:xestospongin A; C057531:jasplakinolide; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium; C064758:Ro 31-8220; D013324:Strontium