Moatter T, Gerritsen M E
Department of Physiology, New York Medical College, Valhalla, New York, USA.
Microcirculation. 1994 Apr;1(1):79-88. doi: 10.3109/10739689409148264.
To determine if the novel cyclooxygenase, COX II, was induced by acidic fibroblast growth factor (aFGF) in rabbit cardiac muscle microvessel (RCME) endothelial cells and to determine the role of protein kinase C (PKC) activation in the mediation of the aFGF induction of COX activity.
Cultured RCME cells were treated with aFGF (200 ng/ml). Induction of COX II activity was assessed by determination of COX activity (PGE2 production), by immunoprecipitation of metabolically labeled COX II, and by Northern analyses. The role of PKC was assessed using phorbol myristate acetate and PKC inhibitors and by determination of PKC activity in cytosol and membrane fractions of RCME cells treated with aFGF and phorbol myristate acetate.
aFGF selectively induced COX II protein and mRNA. Protein kinase C activation was implicated in the transduction of the effects of aFGF for the following reasons: (1) phorbol myristate acetate (PMA), a direct activator of protein kinase C, was a potent inducer of COX II mRNA, COX activity and synthesis of COX II protein. (2) H-7, an inhibitor of PKC, but not the inactive control, HA-1004, blocked aFGF induction of COX II mRNA, COX II protein synthesis, and COX activity. Two additional inhibitors of PKC, calphostin C and staurosporine, also inhibited aFGF induction of COX activity. (3) Downregulation of PKC by overnight incubation with 1 microM PMA blocked subsequent induction of COX II protein synthesis by aFGF. (4) aFGF treatment of RCME cells resulted in the translocation of PKC activity from the cytosol to the membrane fraction. However, aFGF, at concentrations that elicited COX II, neither induced Ca2+ mobilization from intracellular stores nor increased the accumulation of inositol phosphates.
aFGF induces COX II in RCME and this response in mediated, at least in part, by protein kinase C activation. However, aFGF mediated activation of PKC activation must stimulate this kinase through a pathway of signal transduction distinct from inositol phospholipid accumulation or elevation of intracellular Ca2+.
确定新型环氧化酶COX II是否由酸性成纤维细胞生长因子（aFGF）在兔心肌微血管（RCME）内皮细胞中诱导产生，并确定蛋白激酶C（PKC）激活在介导aFGF诱导COX活性中的作用。
用aFGF（200 ng/ml）处理培养的RCME细胞。通过测定COX活性（前列腺素E2生成量）、对代谢标记的COX II进行免疫沉淀以及Northern分析来评估COX II活性的诱导情况。使用佛波酯肉豆蔻酸酯和PKC抑制剂，并通过测定用aFGF和佛波酯肉豆蔻酸酯处理的RCME细胞胞质溶胶和膜组分中的PKC活性来评估PKC的作用。
aFGF选择性地诱导COX II蛋白和mRNA。PKC激活与aFGF效应的转导有关，原因如下：（1）佛波酯肉豆蔻酸酯（PMA），一种PKC的直接激活剂，是COX II mRNA、COX活性和COX II蛋白合成的有效诱导剂。（2）PKC抑制剂H-7，但非无活性对照HA-1004，阻断了aFGF对COX II mRNA、COX II蛋白合成和COX活性的诱导。另外两种PKC抑制剂，钙泊三醇C和星形孢菌素，也抑制了aFGF对COX活性的诱导。（3）用1 microM PMA过夜孵育使PKC下调，阻断了随后aFGF对COX II蛋白合成的诱导。（4）aFGF处理RCME细胞导致PKC活性从胞质溶胶转位到膜组分。然而，能诱导COX II的aFGF浓度既不诱导细胞内储存的Ca2+动员，也不增加肌醇磷酸的积累。
aFGF在RCME中诱导COX II，且这种反应至少部分是由PKC激活介导的。然而，aFGF介导的PKC激活必须通过一条不同于肌醇磷脂积累或细胞内Ca2+升高的信号转导途径来刺激该激酶。
