HYPEROSMOLALITY REGULATES ENDOTHELIN RELEASE BY MADIN-DARBY CANINE KIDNEY-CELLS

ET release by the renal epithelial Madin-Darby canine kidney (MDCK) ce ll line was investigated under isosmotic (300 mosmol/kg H2O; pH 7.4) a nd hyperosmotic (400, 500, or 600 mosmol/kg H2O) culture and assay con ditions by the use of a specific and sensitive RIA. During isosmotic i ncubation, MDCK cells, which may be of collecting duct origin, secrete d by far more ET into the cell culture supernatant (495.7 +/- 25.5 fmo l.mg of protein-1. 24 h-1) than did the proximol tubule-derived LLC-PK 1 (2.42 +/- 0.20 fmol.mg of protein-1.24 h-1) and opossum kidney (3.12 +/- 0.47 fmol.mg of protein-1.24 h-1) cells. ET secretion by MDCK mon olayers increased progressively within 24 h and then only slightly dec lined up to 48 h. Phosphoramidon (100 mumol/L) inhibited the constitut ive ET synthesis in MDCK cells by 60%, indicating the participation of a phosphoramidon-sensitive ET-converting enzyme in the processing of bigET to ET in these cells. MDCK epithelia grown on filter inserts sho wed a clear polarity in their ET release. The baseline secretion of ET was 2.5 times higher to the basolateral than to the apical side, whic h might be in support of a predominantly basolateral action of the pep tide. Short-term incubation of MDCK cells in hyperosmotic NaCl media f or 24 h dose dependently decreased ET production. When urea was used a s the solute to generate hyperosmolality, ET release by MDCK cells sig nificantly increased. In contrast, when raffinose was added to increas e osmolality to 500 mosmol/kg H2O, a decrease of ET production in a ra nge similar to the effect of NaCl was seen. Furthermore, MDCK cells, w hich were long term adapted to NaCl-induced hyperosmolality (600 mosmo l/kg H2O for 7 months = 14 passages), revealed a markedly lower ET pro duction rate (94.6 +/- 7.7 fmol . mg of protein . 24 h-1; N = 4) than isosmotic controls (445.6 +/- 14. 0 fmol . mg of protein-1 .24 h-1; N = 6; P < 0.001). These findings are consistent with a regulatory effec t of hyperosmolality on ET synthesis along distal nephron portions.