Marala R B, Duda T, Sharma R K
Unit of Regulatory and Molecular Biology, Pennsylvania College of Optometry, Philadelphia 19141.
Mol Cell Biochem. 1993 Mar 10;120(1):69-80. doi: 10.1007/BF00925986.
The endothelial cell has a unique intrinsic feature: it produces a most potent vasopressor peptide hormone, endothelin (ET-1), yet it also contains a signaling system of an equally potent hypotensive hormone, atrial natriuretic factor (ANF). This raises two related curious questions: does the endothelial cell also contain an ET-1 signaling system? If yes, how do the two systems interact with each other? The present investigation was undertaken to determine such a possibility. Bovine pulmonary artery endothelial (BPAE) cells were chosen as a model system. Identity of the ANF receptor guanylate cyclase was probed with a specific polyclonal antibody to the 180 kDa membrane guanylate cyclase (mGC) ANF receptor. A Western-blot analysis of GTP-affinity-purified endothelial cell membrane proteins recognized a 180 kDa band; the same antibody inhibited the ANF-stimulated guanylate cyclase activity; the ANF-dependent rise of cyclic GMP in the intact cells was dose-dependent. By affinity cross-linking technique, a predominant 55 kDa membrane protein band was specifically labeled with [125I]ET-1. ET-1 treatment of the cells showed a migration of the protein kinase C (PKC) activity from cytosol to the plasma membrane; ET-1 inhibited the ANF-dependent production of cyclic GMP in a dose-dependent fashion with an EC50 of 100 nM. This inhibitory effect was duplicated by phorbol 12-myristate 13-acetate (PMA), a known PKC-activator. The EC50 of PMA was 5 nM. A PKC inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), blocked the PMA-dependent attenuation of ANF-dependent cyclic GMP formation. These results demonstrate that the 180 kDa mGC-coupled ANF and ET-1 signaling systems coexist in endothelial cells and that the ET-1 signal negates the ANF-dependent guanylate cyclase activity and cyclic GMP formation. Furthermore, these results support the paracrine and/or autocrine role of ET-1.
它能产生一种极为强效的血管升压肽激素——内皮素（ET - 1），然而它也含有一个同样强效的降压激素——心房利钠因子（ANF）的信号系统。这引发了两个相关的有趣问题：内皮细胞是否也含有ET - 1信号系统？如果是，这两个系统如何相互作用？本研究旨在确定这种可能性。选用牛肺动脉内皮（BPAE）细胞作为模型系统。用针对180 kDa膜鸟苷酸环化酶（mGC）ANF受体的特异性多克隆抗体检测ANF受体鸟苷酸环化酶的特性。对GTP亲和纯化的内皮细胞膜蛋白进行的蛋白质印迹分析识别出一条180 kDa的条带；相同的抗体抑制ANF刺激的鸟苷酸环化酶活性；完整细胞中ANF依赖的环磷酸鸟苷（cGMP）升高呈剂量依赖性。通过亲和交联技术，一条主要的55 kDa膜蛋白条带被[125I]ET - 1特异性标记。用ET - 1处理细胞显示蛋白激酶C（PKC）活性从胞质溶胶迁移到质膜；ET - 1以剂量依赖性方式抑制ANF依赖的cGMP产生，半数有效浓度（EC50）为100 nM。这种抑制作用被佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）（一种已知的PKC激活剂）重复。PMA的EC50为5 nM。一种PKC抑制剂1 -（5 - 异喹啉磺酰基）- 2 - 甲基哌嗪（H - 7）阻断了PMA依赖的ANF依赖的cGMP形成的减弱。这些结果表明，180 kDa的mGC偶联的ANF和ET - 1信号系统在内皮细胞中共存，并且ET - 1信号消除了ANF依赖的鸟苷酸环化酶活性和cGMP形成。此外，这些结果支持ET - 1的旁分泌和/或自分泌作用。
