Moore J J, Moore R M, Vander Kooy D
Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109.
J Clin Endocrinol Metab. 1991 May;72(5):1073-80. doi: 10.1210/jcem-72-5-1073.
In previous work we reported that oxytocin activates phospholipase-C (PLC) and increases prostaglandin E2 (PGE2) release in amnion. Whether either of the consequences of activation of PLC by oxytocin, activation of protein kinase-C (PKC) or increases in intracellular calcium, directly results in the production of PGE2 is unknown. Phorbol esters (PMA) and epidermal growth factor (EGF) are also known to increase PGE2 release from amnion. In some tissues these agents are capable of activating the PLC postreceptor cascade system. This study was undertaken primarily to explore the mechanism of oxytocin-induced PGE2 production in amnion and secondarily to determine whether common aspects of PGE2 production by oxytocin, PMA, and EGF include activation of PLC or subsequent steps in this cascade followed by new mRNA/protein production. Involvement of PLC was assessed by inositol phosphate (IP1) turnover. IP1 turnover was increased by oxytocin (2.99 +/- 0.31-fold; P less than 0.01), but not by EGF or PMA. PMA inhibited oxytocin-provoked IP1 turnover (P less than 0.05). PKC involvement was initially evaluated with two PKC inhibitors, H7 and staurosporine. Each inhibited PGE2 production by oxytocin as well as that by PMA and EGF in a dose-dependent fashion. With H7, the IC50 for all agents was 5 microM; the IC50 for staurosporine was 2 nM for PMA and oxytocin and 5 nM for EGF. Agonist-induced PGE2 production was also assessed in cells in which PKC activity had been tachyphylaxed with a high concentration of PMA (400 ng/mL for 48 h). In such cells oxytocin and PMA no longer stimulated (P less than 0.001) PGE2 production, but EGF-stimulated PGE2 production was only slightly reduced. PKC involvement is, thus, implicated for oxytocin and PMA. Other enzymes that are inhibited by H7 and staurosporine are implicated in the production of PGE2 caused by EGF. Although tachyphylaxed cells produced no PGE2 with oxytocin, oxytocin increased intracellular calcium to levels higher than those seen in control cells (435 +/- 102 vs. 286 +/- 1.2) Actinomycin-D (P less than 0.001) and cycloheximide (P less than 0.05) inhibited PGE2 production caused by oxytocin, PMA, and EGF. PGE2 production by oxytocin in human amnion cells proceeds by activation of PKC, followed by new protein and mRNA production. Further, in cells without PKC, oxytocin-induced calcium transients do not increase PGE2. The ability of EGF to stimulate PGE2 in cells with no PKC activity also establishes that PKC activation is not a common intracellular step in the induction of PGE2 production by all agents.
在之前的研究中，我们报道了催产素可激活羊膜中的磷脂酶-C（PLC）并增加前列腺素E2（PGE2）的释放。目前尚不清楚催产素激活PLC的两种后果，即蛋白激酶-C（PKC）的激活或细胞内钙的增加，是否直接导致PGE2的产生。佛波酯（PMA）和表皮生长因子（EGF）也可增加羊膜中PGE2的释放。在某些组织中，这些物质能够激活PLC受体后级联系统。本研究主要探讨催产素诱导羊膜中PGE2产生的机制，其次是确定催产素、PMA和EGF产生PGE2的共同环节是否包括PLC的激活或该级联反应的后续步骤，以及随后新的mRNA/蛋白质的产生。通过肌醇磷酸（IP1）周转率评估PLC的参与情况。催产素可增加IP1周转率（2.99±0.31倍；P<0.01），但EGF或PMA则无此作用。PMA可抑制催产素引起的IP1周转率（P<0.05）。最初使用两种PKC抑制剂H7和星形孢菌素评估PKC的参与情况。二者均以剂量依赖方式抑制催产素以及PMA和EGF诱导的PGE2产生。使用H7时，所有物质的半数抑制浓度（IC50）均为5μM；星形孢菌素对PMA和催产素的IC50为2nM，对EGF的IC50为5nM。还在PKC活性已被高浓度PMA（400ng/mL，作用48小时）快速脱敏的细胞中评估了激动剂诱导的PGE2产生。在这类细胞中，催产素和PMA不再刺激（P<0.001）PGE2产生，但EGF刺激的PGE2产生仅略有减少。因此，PKC参与了催产素和PMA诱导PGE2产生的过程。H7和星形孢菌素抑制的其他酶参与了EGF诱导的PGE2产生。虽然快速脱敏的细胞在接触催产素后不产生PGE2，但催产素可使细胞内钙增加至高于对照细胞的水平（435±102对286±1.2）。放线菌素-D（P<0.001）和放线菌酮（P<0.05）可抑制催产素、PMA和EGF诱导的PGE2产生。人羊膜细胞中催产素诱导PGE2产生是通过激活PKC，随后产生新的蛋白质和mRNA。此外，在没有PKC的细胞中，催产素诱导的钙瞬变不会增加PGE2。EGF在没有PKC活性的细胞中刺激PGE2产生的能力也表明，PKC激活并非所有物质诱导PGE2产生的共同细胞内步骤。
