NO decreases thick ascending limb chloride absorption by reducing Na+-K+-2Cl−cotransporter activity

First published August 9, 2001; 10.1152/ajprenal.0075.2001.—We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption ( JCl−). NaCl transport in the THAL depends on apical Na+-K+-2Cl−cotransporters, apical K+channels, and basolateral Na+-K+-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL JCl−by inhibiting the Na+-K+-2Cl−cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na+]i) and chloride concentrations ([Cl−]i) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na+]ifrom 13.5 ± 1.2 to 9.6 ± 1.6 mM ( P < 0.05) and also decreased [Cl−]i( P < 0.01). We next tested whether NO decreases Na+-K+-2Cl−cotransporter activity by measuring the initial rate of Na+transport. In the presence of SPM in the bath, initial rates of Na+entry were 49.6 ± 6.0% slower compared with control rates ( P < 0.05). To determine whether NO inhibits apical K+channel activity, we measured the change in membrane potential caused by an increase in luminal K+from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K+concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K+permeability could affect NO-induced inhibition of THAL JCl−. In the presence of luminal valinomycin, which increases K+permeability, addition of SPM decreased THAL JCl−by 41.2 ± 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na+-K+-ATPase by measuring oxygen consumption rate (Qo2) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased Qo2to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2· mg protein−1· min−1in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased Qo2by 104 ± 7 and 94 ± 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL JCl−by inhibiting Na+-K+-2Cl−cotransport rather than inhibiting apical K+channels or the sodium pump.