TERMINATION OF ENDOTHELIN SIGNALING - ROLE OF NITRIC-OXIDE
TERMINATION OF ENDOTHELIN SIGNALING - ROLE OF NITRIC-OXIDE
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DOI：
10.1002/jcp.1041580313
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发表时间：
1994-03-01
影响因子：
5.6
通讯作者：
BAHOU, WF
中科院分区：
文献类型：
--
作者：
GOLIGORSKY, MS;TSUKAHARA, H;BAHOU, WF
负责终止ET-1信号的细胞机制知之甚少。为了检验一氧化氮作为ET-1信号传导的生理制动的假设，研究了用ET（A）受体cDNA（CHO-ET）稳定转染的中国仓鼠卵巢（CHO）细胞。CHO-ET对ET-1的反应具有强烈的[Ca 2 +]（i）瞬变，并产生了持久的同源脱敏。供体一氧化氮（NO），3-吗啉代-sydnonimine盐酸盐（SIN-I），或硝普钠（SNP）降低这些反应的幅度，加快[Ca 2 +]，恢复率，并抵消了发展的同源脱敏的循环CMP-独立的机制，这表明一种替代模式NO调制ET-1的反应。刺激CHO-ET细胞与mastoparan，黄蜂毒液直接作用于G蛋白（绕过受体激活），被NO抑制，揭示了一个postreceptoral目标NO诱导的调制[Ca 2 +]i动员。使用lys（9）-生物素化的ET-1（ET-1 [BtK（9）]），在CHO-ET细胞中“定位”结合位点。受体-配体复合物在60分钟的观察过程中没有表现出自发解离。定量荧光显微镜显示，SNP或SIN-1引起生物素化的ET-1从ET（A）受体上快速、浓度依赖性和可逆的解离（EC（50）分别为75 μ M和6 μ M），这种作用不能被8-溴-环GMP模拟。内皮细胞与CHO-ET的“三明治”共培养显示，内皮细胞激活NO产生类似地导致ET-1 [BtK（9）]从ET（A）受体上解离。我们假设NO通过双重机制在ET-1信号传导的生理终止中起作用：（1）从其受体中置换结合的ET-1，从而防止同源脱敏，和（2）干扰[Ca ~（2+）]动员的受体后途径，从而抑制对ET-1的末端反应。(C)1994 Wiley-Liss，Inc.
Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET-1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ET(A) receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca2+](i) transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl (SIN-I), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca2+], recovery, and counteracted the development of homologous desensitization by a cyclic CMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp Venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca2+]i mobilization. Using a lys(9)-biotinylated ET-1 (ET-1 [BtK(9)]), binding sites were ''mapped'' in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60-min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-I caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET-1 from ET(A) receptor (EC(50) = 75 mu M and 6 mu M, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. ''Sandwich'' co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK(9)] from ET(A) receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca2+], mobilization, hence inhibiting end-responses to ET-1. (C) 1994 Wiley-Liss, Inc.