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Protein kinase C activates non-capacitative calcium entry in human platelets

Juan A. Rosado and Stewart O. Sage

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. 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.

6. 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.

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