OSMOLAR REGULATION OF ENDOTHELIN SIGNALING IN RAT RENAL MEDULLARY INTERSTITIAL-CELLS

We tested the hypothesis that endothelin (ET) responsiveness in the re nal medulla is modulated by ambient osmolarity. Cultured renal medulla ry interstitial cells (RMICs) were incubated from 3 to 24 h in isosmol ar culture medium (300 mOsm/kg H2O) or media rendered hyperosmolar (60 0 mOsm/kg H2O) by the addition of urea. Under hyperosmolar conditions, the peak of ET-evoked Ca2+ transient was blunted by 45-58% (P < 0.02) and PGE(2) accumulation decreased from 16- to 2-fold above basal valu es (P < 0.001). To explore whether hyperosmolar conditions blunt intra cellular signaling via modulation of receptor number or expression, ki netics of ET binding and Northern blot analysis of ET(A) receptor mRNA was performed. Under hyperosmolar conditions, ET(A) receptor density was reduced by 84% versus isosmolar conditions (238+/-12 vs, 14501+/-1 84 fmol/ mg) (P < 0.01). In contrast to the ligand binding studies, ET (A) receptor mRNA was increased by 58% (P < 0.05) in cells grown under hyperosmolar versus isosmolar media. These observations indicate that in the hyperosmolar setting, ET-evoked intracellular signaling is blu nted in RMICs due to ET receptor downregulation. Since ET(A) receptor mRNA is increased under hyperosmolar conditions, we conclude that ET r eceptor downregulation is the consequence of either decreased translat ion of message, increased degradation of receptor peptide, or increase d internalization of specific receptor sites.