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.
Goligorsky
M S
MS
Department of Medicine, State University of New York, Stony Brook 11794.
Tsukahara
H
H
Magazine
H
H
Andersen
T T
TT
Malik
A B
AB
Bahou
W F
WF
D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D016232:Endothelins / Q000378:metabolism / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D036341:Intercellular Signaling Peptides and Proteins; D008854:Microscopy, Electron; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D009599:Nitroprusside / Q000494:pharmacology; D010455:Peptides; D017466:Receptors, Endothelin / Q000032:analysis / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D014862:Wasp Venoms / Q000494:pharmacology
D000959:Antihypertensive Agents; D016232:Endothelins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D017466:Receptors, Endothelin; D014862:Wasp Venoms; D009599:Nitroprusside; D009569:Nitric Oxide; C002385:linsidomine; C037500:mastoparan; D004247:DNA; D008981:Molsidomine; D002118:Calcium