Vasopressin Triggers Phosphorylation of Na‐K‐2Cl Cotransporter (NKCC1) in Rat Inner Medullary Collecting Duct (IMCD)

Abstract

Vasopressin‐induced cell swelling in rat IMCD is dependent on the cotransporter NKCC1. NKCC1 is regulated by phosphorylation at Thr203 and Thr208 in other cell types. Here we used R5, a previously characterized antibody recognizing the pThr203/pThr208 double‐phospho‐form of NKCC1, to study vasopressin's effects in rat IMCD suspensions. The V2R‐selective vasopressin analog dDAVP (1 nM) significantly increased the abundance of phosphorylated NKCC1 (168±15%, n=4), an effect mimicked by cAMP analog CPT‐cAMP and inhibited by protein kinase A (PKA) inhibitor H‐89. Immunoblotting using the R5 antibody cross‐preadsorbed with synthetic mono‐phosphopeptides (pThr203 or pThr208) demonstrated that dDAVP increases phosphorylation at Thr203 but not Thr208. Inhibitors of calmodulin (W7) or myosin light chain kinase (ML‐7) did not prevent NKCC1 phosphorylation. Incubating a nonphosphorylated synthetic peptide spanning Thr203 and Thr208 with purified PKA catalytic subunit failed to phosphorylate the peptide, despite phosphorylation of positive‐control peptide. These results show that dDAVP triggers phosphorylation of NKCC1 at Thr203 in native rat IMCD cells and suggest that the effect is indirectly dependent on PKA.