title: NO decreases thick ascending limb chloride absorption by reducing Na(+)-K(+)-2Cl(-) cotransporter activity.
abstract: 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.
authors.affiliation: Division of Hypertension and Vascular Research, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan 48202, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Ortiz
authors.forename: N J
authors.initials: NJ
authors.lastname: Hong
authors.forename: J L
authors.initials: JL
authors.lastname: Garvin
mesh_terms: D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D004553:Electric Conductivity; D005665:Furosemide / Q000494:pharmacology; D008138:Loop of Henle / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009589:Nitrogen Oxides; D009761:Nystatin / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D012965:Sodium Chloride / Q000008:administration & dosage; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D002712:Chlorides; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D026902:Potassium Channel Blockers; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; C091861:spermine nitric oxide complex; D009761:Nystatin; D013096:Spermine; D009569:Nitric Oxide; D012965:Sodium Chloride; D005665:Furosemide; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium