Dodge K L, Sanborn B M
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, 77030, USA.
Endocrinology. 1998 May;139(5):2265-71. doi: 10.1210/endo.139.5.5963.
The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1-41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1-41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT-cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1-41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since G betagamma activation of phospholipase Cbeta2 (PLCbeta2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCbeta2 via G alpha(i)G betagamma activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLCbeta1 and PLCbeta3, but not PLCbeta2. In PHM1-41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/G alpha(q)-coupled stimulation of PLCbeta1 or PLCbeta3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCbeta2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.
在人子宫肌层细胞系（PHM1-41）和过表达催产素受体的COS-M6细胞中，研究了环磷酸腺苷（cAMP）对催产素刺激的磷脂酰肌醇周转率增加的影响以及可能涉及的途径。用氯苯硫基-cAMP（CPT-cAMP）、福斯可林或松弛素预孵育可抑制PHM1-41细胞中催产素刺激的磷脂酰肌醇周转率，而相对特异性的蛋白激酶A抑制剂H-89可逆转这种抑制作用。CPT-cAMP和瞬时表达的蛋白激酶A催化亚基分别抑制了表达催产素或毒蕈碱M1受体的COS-M6细胞中催产素和卡巴胆碱对[3H]肌醇1,3,4-三磷酸形成的刺激。CPT-cAMP还抑制了PHM1-41细胞中内皮素-1刺激的磷脂酰肌醇周转率，进一步证明了cAMP抑制机制的普遍性。由于磷脂酶Cβ2（PLCβ2）的Gβγ激活是蛋白激酶A的一个假定靶点，因此探讨了催产素受体是否通过Gα(i)Gβγ激活与PLCβ2偶联的可能性。对PHM1-41细胞和COS-M6细胞的蛋白质印迹分析检测到了PLCβ1和PLCβ3，但未检测到PLCβ2。在PHM1-41细胞中，百日咳毒素使催产素刺激的[3H]肌醇1,3,4-三磷酸增加减少了53%，而H-89可完全逆转这种情况。因此，百日咳毒素的抑制作用可能是由cAMP升高的间接作用引起的。这些数据表明，受体/Gα(q)偶联的PLCβ1或PLCβ3刺激可被cAMP通过涉及蛋白激酶A的磷酸化机制抑制，该机制不涉及PLCβ2。在平滑肌中，这种机制可能构成调节收缩和舒张途径之间潜在重要的相互作用。
