Saxena S P, Robertson C, Becker A B, Gerrard J M
Manitoba Institute of Cell Biology, Winnipeg, Canada.
Biochem J. 1991 Jan 15;273(Pt 2)(Pt 2):405-8. doi: 10.1042/bj2730405.
In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.
在先前的报告中，我们已提供证据表明新形成的组胺是人类血小板中的一种细胞内信使。我们研究了蛋白激酶C（PKC）和细胞内钙（Ca2+i）在组胺合成中的作用。用佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）、胶原蛋白和Ca2+离子载体A23187刺激人类血小板，同时使用或不使用PKC抑制剂星形孢菌素。监测血小板聚集、组胺合成以及血小板-2肌动蛋白（47 kDa；P47）和肌球蛋白轻链（20 kDa；P20）蛋白的磷酸化情况。星形孢菌素以剂量依赖的方式抑制PMA和胶原蛋白诱导的聚集、组胺合成以及47 kDa和20 kDa蛋白的磷酸化。对于PMA，星形孢菌素抑制聚集、组胺合成和磷酸化的半数抑制浓度（IC50值）相似，这表明该激动剂诱导的组胺合成可能是PKC激活的结果。相反，胶原蛋白刺激的组胺合成被星形孢菌素抑制时的浓度显著高于抑制聚集（P小于0.005）或血小板-2肌动蛋白磷酸化（P小于0.01）所需的浓度，这表明非PKC机制可能参与了该激动剂诱导的组胺合成。A23187未能诱导血小板中细胞内组胺的合成，而星形孢菌素在显著高于抑制PKC所需的浓度下阻断了A23187诱导的聚集和20 kDa蛋白的磷酸化。当用A23187和PMA联合刺激血小板时，血小板组胺的增加低于单独使用PMA时。这些结果提供了证据，表明血小板中细胞内组胺的合成是PKC激活的结果，并且在[Ca2+]i大幅升高的情况下可能会下调。
