INTRACELLULAR RAMIFICATION OF ENDOTHELIN SIGNAL

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. Ca2+-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 Ca2+-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.