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 oxide serves as a physiological brake of ET-1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ETA 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-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca2+]i recovery, and counteracted the development of homologous desensitization by a cyclic GMP-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 lys9-biotinylated ET-1 (ET-1 [BtK9]), 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-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET-1 from ETA receptor (EC50 = 75 microM and 6 microM, 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 [BtK9] from ETA 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+]i mobilization, hence inhibiting end-responses to ET-1.