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 inhibits transepithelial HCO₃/^- absorption (JHCO₃/^-) by inhibiting apical membrane Na⁺/H⁺ exchange. To examine signaling mechanisms involved in this regulatory response, MTALs were isolated and perfused in vitro with 25 mH HCO₃/^- solutions (290 mosmol/kg H₂O). Osmolality was increased in lumen and bath solutions by addition of 300 mM mannitol or 75 mM NaCl. Addition of mannitol reduced JHCO₃/^- by 60% and addition of NaCl reduced JHCO₃/^- by 50%. With the protein tyrosine kinase (PTK) inhibitor genistein (7 μM) or herbimycin A (1 μM) in the bath, addition of mannitol reduced JHCO₃/^- only by 11% and addition of NaCl reduced JHCO₃/^- only by 15%. Staurosporine (10^-7 M) or forskolin (10^-6 M) in the bath had no effect on inhibition of JHCO₃/^- by hypertonic NaCl. Genistein had no effect on inhibition of JHCO₃/^- by vasopressin (a cyclic AMP-dependent process) or stimulation of JHCO₃/^- by prostaglandin E₂ (a protein kinase C-dependent process). Under isosmotic conditions, addition of genistein or herbimycin A to the bath increased JHCO₃/^- by 30% through stimulation of apical membrane Na⁺/H⁺ exchange. Addition of the tyrosine phosphatase inhibitor molybdate (50 μM) to the bath reproduced the inhibition of JHCO₃/^- observed 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 pathway that functions independent of regulation by cyclic AMP and protein kinase C, and 2) a constitutive PTK activity inhibits apical membrane Na⁺/H⁺ exchange and HCO₃/^absorption under isosmotic conditions. Our results suggest that tyrosine phosphorylation is a critical step in inhibition of the apical Na⁺/H⁺ exchanger isoform NHE-3 by hyperosmolality.