Ouabain reduces net acid secretion and increases pHi by inhibiting NH4+ uptake on rat tIMCD Na(+)-K(+)-ATPase.
In the rat terminal inner medullary collecting duct (tIMCD), Na+ pump inhibition reduces transepithelial net acid secretion (JtAMM) [JH = total CO2 absorption (JtCO2)+ total ammonia secretion] and increases resting intracellular pH (pHi). The increase in pHi and reduction in JH that follow ouabain addition do not occur in the absence of NH4+ nor when NH4+ is substituted with another weak base. The purpose of this study was to explore the mechanism of the NH4(+)-dependent reduction in JtCO2 and increase in pHi that follow ouabain addition. We hypothesized that NH4+ enters the tIMCD cell through the Na(+)-K(+)-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH4Cl. Since K+ and NH4+ compete for a common binding site on the Na+ pump, increasing extracellular K+ should limit NH4+ (and hence net H+) uptake by the Na+ pump. Upon increasing extracellular K+ concentration from 3 to 12 mM, the NH4(+)-dependent, ouabain-induced increase in pHi and reduction in JtCO2 were attenuated. In the presence but not in the absence of NH4+, reducing Na+ pump activity by limiting Na+ entry reduced JtCO2 and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of HCO3-/CO2 and was not reproduced with BaCl2 or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na(+)-K(+)-2Cl- cotransporter or a HCO3- transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell, NH4+ uptake is mediated by the Na(+)-K(+)-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K+ loading.
Wall
S M
SM
Division of Renal Diseases and Hypertension, University of Texas Medical School at Houston 77030, USA.
D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000493:pharmacokinetics*; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002245:Carbon Dioxide / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007679:Kidney Medulla / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008297:Male; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D015221:Potassium Channels; D000643:Ammonium Chloride; D002034:Bumetanide; D002245:Carbon Dioxide; D001464:Barium; D010042:Ouabain; D000584:Amiloride; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase