HYPEROSMOLALITY INHIBITS BICARBONATE ABSORPTION IN RAT MEDULLARY THICK ASCENDING LIMB VIA A PROTEIN-TYROSINE KINASE-DEPENDENT PATHWAY

In the rat medullary thick ascending limb (MTAL), hyperosmolality inhi bits transepithelial HCO, absorption (JHCO(3)(-)) by inhibiting apical membrane Na+/H+ exchange, To examine signaling mechanisms involved in this regulatory response, MTALs were isolated and perfused in vitro w ith 25 mM HCO3- solutions (290 mosmol/kg H2O). Osmolality was increase d in lumen and bath solutions by addition of 300 mM mannitol or 75 mM NaCl. Addition of mannitol reduced JHCO(3)(-) by 60% and addition of N aCl reduced JHCO(3)(-) by 50%. With the protein tyrosine kinase (PTK) inhibitor genistein (7 mu M) or herbimycin A (1 mu M) in the bath, add ition of mannitol reduced JHCO(3)(-) only by 11% and addition of NaCl reduced JHCO(3)(-) only by 15%. Staurosporine (10(-7) M) or forskolin (10(-6) M) in the bath had no effect on inhibition of JHCO(3)(-) by hy pertonic NaCl. Genistein had no effect on inhibition of JHCO(3)(-) by vasopressin (a cyclic AMP-dependent process) or stimulation of JHCO(3) (-) by prostaglandin E(2) (a protein kinase C-dependent process). Unde r isosmotic conditions, addition of genistein or herbimycin A to the b ath increased JHCO(3)(-) by 30% through stimulation of apical membrane Na+/H+ exchange, Addition of the tyrosine phosphatase inhibitor molyb date (50 mu M) to the bath reproduced the inhibition of JHCO(3)(-) obs erved with hyperosmolality. These data indicate that 1) the effect of hyperosmolality to inhibit MTAL HCO3- absorption through inhibition of apical membrane Na+/H+ exchange is mediated via a PTK-dependent pathw ay that functions independent of regulation by cyclic AMP and protein kinase C, and 2) a constitutive PTR activity inhibits apical membrane Na+/H+ exchange and HCO3- absorption under isosmotic conditions, Our r esults suggest that tyrosine phosphorylation is a critical step in inh ibition of the apical Na+/H+ exchanger isoform NHE-3 by hyperosmolalit y.