title: Intracellular ramification of endothelin signal.
abstract: Effects of porcine 1-21 endothelin (ET-1) on [Ca2+]i, [Na+]i, and [Cl-]i and on membrane potential were studied in individual mesangial (MC) and vascular smooth muscle (VSMC) cells using microspectrofluorimetry of fura-2, SBFI, SPQ, and bis-oxonol, respectively. ET-1 increased [Ca2+]i by fivefold, showing an immediate and a sustained phase of response. Ca(2+)-free medium and nifedipine pretreatment significantly curtailed the sustained phase of response to ET-1. These findings were confirmed in studies of vascular ring preparations, demonstrating that Ca2+ influx may account for at least 50% of contraction. ET-1 caused immediate and sustained depolarization of MC and VSMC. This could not be attributed to Na+ influx, since fluorescence of SBFI was not affected by ET-1 and Na(+)-free medium did not abolish the ET-1-induced membrane depolarization. Studies of SPQ fluorescence changes induced by ET-1 revealed an increase in fluorescence intensity consistent with the decrease in [Cl-]i. A Cl- channel blocker, IAA-94, abolished changes in SPQ fluorescence and curtailed sustained phases of membrane depolarization and [Ca2+]i elevation in response to ET-1, but did not affect KCl-induced [Ca2+]i transients. IAA-94 also attenuated the ET-1-induced contraction of aortic rings. Microinjection of either calcium gluconate or inositol 1,4,5-trisphosphate (IP3) in SPQ-loaded cells resulted in an increase in fluorescence mimicking the effect of ET-1. These changes were blunted by pretreatment of cells with BAPTA and incubation in Ca(2+)-free medium. When IP3 was microinjected into fura-2-loaded MC, this resulted in immediate and sustained elevation of [Ca2+]i. In conclusion, generation of IP3 results in mobilization of intracellular Ca2+ stores and activation of Cl- channels. Ensuing Cl- efflux causes membrane depolarization and, in turn, activation of voltage-dependent Ca2+ channels, resulting in sustained elevation of [Ca2+]i which is indispensable for the full-scale contraction produced by ET-1.
authors.affiliation: Department of Medicine, State University of New York, Stony Brook 11794-8152.
authors.forename: K
authors.initials: K
authors.lastname: Iijima
authors.forename: L
authors.initials: L
authors.lastname: Lin
authors.forename: A
authors.initials: A
authors.lastname: Nasjletti
authors.forename: M S
authors.initials: MS
authors.lastname: Goligorsky
mesh_terms: D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D004232:Diuretics / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology*; D005456:Fluorescent Dyes; D005920:Glomerular Mesangium / Q000187:drug effects / Q000502:physiology*; D006016:Glycolates / Q000494:pharmacology; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009543:Nifedipine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D004232:Diuretics; D016232:Endothelins; D005456:Fluorescent Dyes; D006016:Glycolates; C015765:MK 473; D009543:Nifedipine; D002118:Calcium