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 (O-2(-)) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges O-2(-) and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that O-2(-) 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 O-2(-) produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 mu M) stimulated surface NKCC2 expression by similar to 18 +/- 5% (P < 0.05). O-2(-)-stimulated surface NKCC2 expression was blocked by the O-2(-) scavenger tempol (50 mu M). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 +/- 8% increase from control. P < 0.05). Inhibition of endogenous NO production with N-omega-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% (P < 0.05). Scavenging O-2(-) with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 +/- 4% to 15 +/- 10%, P < 0.05). Protein kinase C inhibition with Go-6976 (100 nM) blocked O-2(-)-stimulated surface NKCC2 expression (P < 0.05). O-2(-) did not affect NKCC2 phosphorylation at Thr(96/101) or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that O-2(-) increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. O-2(-)-stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dah1 salt-sensitive rats.