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

We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption (J(Cl-)). 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 J(Cl-) 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+](i) from 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 J(Cl-). In the presence of luminal valinomycin, which increases K+ permeability, addition of SPM decreased THAL J(Cl-) 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 (QO(2)) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased QO(2) to 119.1 +/- 19.2 and 125.6 +/- 23.4 nmol O-2. mg protein(-1). min(-1) in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased QO(2) by 104 +/- 7 and 94 +/- 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL J(Cl-) by inhibiting Na+-K+-2Cl(-) cotransport rather than inhibiting apical K+ channels or the sodium pump.