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Am J Physiol Renal Physiol (April 27, 2004). doi:10.1152/ajprenal.00427.2003
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Submitted on December 4, 2003
Accepted on April 22, 2004

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

Marisela Varela1, Marcela Herrera2, and Jeffrey L. Garvin2*

1 Division of Nephrology, Henry Ford Hospital, Detroit, MI, USA
2 Divison of Hypertension and Vascular Research, Henry Ford Hospital, Detroit, MI, USA

* To whom correspondence should be addressed. E-mail: jgarvin1{at}hfhs.org.

A high-salt diet enhances 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 hrs, spermine NONOate (SPM; 5 µM) reduced Na/K ATPase activity from 0.44 ± 0.03 to 0.30 ± 0.04 nmol Pi/µg protein/min 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 Pi/µg protein/min). 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 O2- was involved, we measured O2- production. THALs from rats on normal and high salt produced 35.8 ± 0.3 and 23.7 ± 0.8 nmol O2-/min/mg protein, respectively (p < 0.01). Because O2- production differed, we studied the effects of the O2- scavenger Tempol. In the presence of 50 µM Tempol, SPM did not inhibit Na/K ATPase after 120 min (0.50 ± 0.05 vs. 0.52 ± 0.07 nmol Pi/µg protein/min). Propyl gallate, another O2- scavenger, also prevented SPM-induced inhibition of Na/K ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when O2- 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 that this inhibition depends on O2-. NO donors do not inhibit Na/K ATPase in THALs from rats on high salt due to decreased O2- production.




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