Takuwa N, Kumada M, Yamashita K, Takuwa Y
Department of Physiology, Faculty of Medicine, University of Tokyo, Japan.
J Biol Chem. 1991 Aug 5;266(22):14237-43.
A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.
一种肽类促分裂原蛙皮素可激活磷脂酶C - 蛋白激酶C信号通路，它能使预先标记的瑞士3T3成纤维细胞释放的[3H]花生四烯酸及其代谢产物（[3H]AA）呈剂量依赖性增加，且对米帕林敏感。该效应在时间上由两个阶段组成，即最初的短暂爆发，这一阶段基本不依赖细胞外Ca2+，以及随后的持续阶段，此阶段绝对依赖细胞外Ca2+。最初的短暂[3H]AA释放与蛙皮素诱导的预先标记细胞中45Ca外流同时发生：用Ca2+螯合剂喹碘方加载细胞后，这两种反应均显著减弱。然而，蛙皮素诱导的细胞内Ca2+动员本身不足以作为最初短暂[3H]AA释放的信号，因为A23187能有效刺激45Ca外流，其程度与蛙皮素相当，但在无细胞外Ca2+时无法诱导[3H]AA释放。将细胞外Ca2+降至0.03 mM可消除蛙皮素诱导的[3H]AA释放的第二个持续阶段，尽管相同操作对蛙皮素对磷脂酶C和蛋白激酶C激活的影响几乎没有作用。蛋白激酶C激活剂佛波醇12,13 - 二丁酸酯可诱导细胞外Ca(2+)依赖性、缓慢发展的[3H]AA释放持续增加，并显著增强蛙皮素诱导的[3H]AA释放的两个阶段。细胞蛋白激酶C的下调完全消除了佛波醇二丁酸酯的所有作用，并部分抑制蛙皮素诱导的[3H]AA释放的第二个阶段，但不抑制第一个阶段。这些结果表明，蛙皮素诱导的受体介导的磷脂酶A2激活涉及多种机制，包括第一阶段的细胞内Ca2+动员、第二阶段的蛋白激酶C激活加Ca2+内流，以及两个阶段均存在的独立于细胞内Ca2+动员或蛋白激酶C的未知机制。
