Termination of endothelin signaling: Role of nitric oxide

Abstract

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⁺], 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⁺], 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⁺] 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 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN‐1 caused a rapid, concentration‐dependent, and reversible dissociation of biotinylated ET‐ 1 from ET_A receptor (EC₅₀ = 75 μM and 6 μM, 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 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.