TERMINATION OF ENDOTHELIN SIGNALING - ROLE OF NITRIC-OXIDE

Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxi de 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) tran sients 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, acceler ated the rate of [Ca2+], recovery, and counteracted the development of homologous desensitization by a cyclic CMP-independent mechanism, sug gesting an alternative mode for NO modulation of ET-1 responses. Stimu lation of CHO-ET cells with mastoparan, a wasp Venom acting directly o n G proteins (bypassing receptor activation), was inhibited by NO, rev ealing a postreceptoral target for NO-induced modulation of [Ca2+]i mo bilization. Using a lys(9)-biotinylated ET-1 (ET-1 [BtK(9)]), binding sites were ''mapped'' in CHO-ET cells. Receptor-ligand complexes did n ot exhibit spontaneous dissociation during 60-min observations. Quanti tative fluorescence microscopy revealed that SNP or SIN-I caused a rap id, concentration-dependent, and reversible dissociation of biotinylat ed 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 mechanis ms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptor al pathway for [Ca2+], mobilization, hence inhibiting end-responses to ET-1. (C) 1994 Wiley-Liss, Inc.