Inhibition of Na-K-ATPase in thick ascending limbs by NO depends on O2−and is diminished by a high-salt diet

A high-salt diet enhances nitric oxide (NO)-induced inhibition of transport in the thick ascending limb (THAL). Long exposures to NO inhibit Na-K-ATPase in cultured cells. We hypothesized that NO inhibits THAL Na-K-ATPase after long exposures and a high-salt diet would augment this effect. Rats drank either tap water or 1% NaCl for 7-10 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 microM) reduced Na-K-ATPase activity from 0.44 +/- 0.03 to 0.30 +/- 0.04 nmol P(i).microg protein(-1).min(-1) in THALs from rats on a normal diet (P < 0.03). Nitroglycerin also reduced Na-K-ATPase activity (P < 0.04). After 20 min, SPM had no effect (change -0.07 +/- 0.05 nmol P(i).microg protein(-1).min(-1)). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONOO(-) formed by NO reacting with O(2)(-) was involved, we measured O(2)(-) production. THALs from rats on normal and high salt produced 35.8 +/- 0.3 and 23.7 +/- 0.8 nmol O(2)(-).min(-1).mg protein(-1), respectively (P < 0.01). Because O(2)(-) production differed, we studied the effects of the O(2)(-) scavenger tempol. In the presence of 50 microM tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 +/- 0.05 vs. 0.52 +/- 0.07 nmol P(i).microg protein(-1).min(-1)). Propyl gallate, another O(2)(-) scavenger, also prevented SPM-induced inhibition of Na-K-ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when O(2)(-) levels were increased with xanthine oxidase and hypoxanthine. We concluded that NO inhibits Na-K-ATPase after long exposures when rats are on a normal diet and this inhibition depends on O(2)(-). NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased O(2)(-) production.