HYPEROSMOLALITY STIMULATES NA-K-ATPASE GENE-EXPRESSION IN INNER MEDULLARY COLLECTING DUCT CELLS

Primary cultures of inner medullary collecting duct (IMCD) cells of ra ts were incubated in hyperosmotic media to determine the effects on Na -K-ATPase alpha(1)- and beta(1)-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH(2)O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media suppleme nted with NaCl, mannitol, or raffinose caused two- to fourfold increas es in the alpha(1)-subunit mRNA accumulation and five- to eightfold in creases in the beta(1)-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmo lar urea medium had no effect at any time. When NaCl or mannitol was a dded to the media in amounts ranging from 300 to 600 mosmol/kgH(2)O, t he maximal effects on both alpha(1)- and beta(1)-subunit mRNA accumula tion occurred at 500 mosmol/kgH(2)O. In urea-supplemented medium, howe ver, there was no significant change at any level of osmolality. The u pregulation of alpha(1)- and beta(1)-subunit mRNA induced by hyperosmo tic mannitol- or raffinose-supplemented media was markedly inhibited b y removal of Na from the culture medium. Furthermore, pretreatment wit h a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha(1)- and beta(1)-subunit mRNA in IMCD cells expos ed to hyperosmotic media treated with NaCl or mannitol. When IMCD cell s were incubated with hyperosmotic media (500 mosmol/kgH(2)O) suppleme nted with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium h ad no significant effect on Na-K-ATPase activity. These results demons trate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha(1)- and beta(1)-subunit mRNA accumulatio ns in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha(1) and beta(1)-subunit mRNA leads to an increase in Na-K-ATPase activity; and 3) the above up regulation of alpha(1)- and beta(1)-subunit mRNA in response to hypero smotic media requires, at least in part, the presence of Na in the ext racellular medium and the de novo synthesis of intermediate proteins.