Superoxide Increases Surface NKCC2 in the Rat Thick Ascending Limbs Via PKC

The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ( ) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% ( < 0.05). -stimulated surface NKCC2 expression was blocked by the scavenger tempol (50 µM). Scavenging HO with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, < 0.05). Inhibition of endogenous NO production with -nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% ( < 0.05). Scavenging with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked -stimulated surface NKCC2 expression ( < 0.05). did not affect NKCC2 phosphorylation at Thr or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. -stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.