bafilomycin-a1 and Potassium-Deficiency

The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by two luminal proton ATPases, H(+)-ATPase and H(+)-K(+)-ATPase (HKA), both of which are in the intercalated cells (ICs) of OMCD. We showed previously that HKAalpha1 (gastric H(+)-K(+)-ATPase) activity is the essential H(+)-K(+)-ATPase activity under normal conditions, and that HKAalpha2 (colonic H(+)-K(+)-ATPase) is induced and mediates increased proton-secretion under K-depleted conditions. To better understand the role of H(+)-ATPase (potassium-independent) in acid secretion and the relationship between H(+)-ATPase and a specific HKA isoform, we examined H(+)-ATPase activity in the H(+)-K(+)-ATPasealpha1 knockout (KO) mice under normal and K-depleted conditions. Mice were fed a potassium-free diet and studied after 7 days. Segments of the OMCD were perfused in vitro, and intracellular pH (pH(i)) was measured by ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. The isolated OMCD tubules obtained from mice fed a potassium-free diet were examined by fluorescent immunocytochemistry with an antibody to the 31-kDa subunit of H(+)-ATPase (E-11) and were compared with those obtained from a normal diet. In the absence of Na(+) and K(+), the H(+)-ATPase-mediate pH(i) recovery rates were 6.7 +/- 1.1 x 10(-4) units/s (n = 7 ICs) in wild-type (WT) mice and increased to 8.7 +/- 1.8 x 10(-4) (P < 0.05; n = 6) in HKAalpha1 KO mice. K-independent proton transport activity was significantly inhibited by the H(+)-ATPase inhibitor bafilomycin A(1) (BAF, 10 nM) with luminal applied in both WT and KO mice. Comparison of the results indicated upregulation of BAF-sensitive H(+)-ATPase activity in KO mice. To determine the intracellular localization of H(+)-ATPase in the intercalated cells of OMCD, we dissected the OMCD and performed fluorescent immunocytochemistry with the H(+)-ATPase antibody in the WT and KO mice. In the WT mice, on normal diet, H(+)-ATPase staining distributed diffusely throughout the intercalated cells and was slightly polarized to the apical plasma membrane in the KO mice, consistent with increase in the H(+)-ATPase-mediate pH(i) recovery in the KO mice. One week of a potassium-free diet resulted in a significant increase in the degree of H(+)-ATPase polarization at the apical plasma membrane in both WT and KO mice. Hypokalemia stimulates H(+)-ATPase in the intercalated cells of OMCD of both WT and KO mice. The enhanced activity of H(+

Topics: Animals; Cell Membrane; Enzyme Inhibitors; H(+)-K(+)-Exchanging ATPase; Kidney Tubules, Collecting; Macrolides; Mice; Mice, Knockout; Potassium; Potassium Deficiency; Proton Pump Inhibitors; Proton-Translocating ATPases; Sodium; Up-Regulation

In the present study, we have investigated the effects of dietary potassium depletion on the activity and distribution of the H+-ATPase in the distal nephron of the Sprague-Dawley rat. H+-ATPase activity was assessed from the change in transepithelial potential difference (Vte) in response to bafilomycin A1 during perfusion of the late distal tubule in vivo, with solutions containing inhibitors of known ion channels. Bafilomycin A1 caused a negative deflection in Vte in control animals, an effect that was significantly enhanced during potassium depletion (P < 0.01). The distribution of H+-ATPase within the population of intercalated cells was assessed using a specific monoclonal antibody (E11). Hypokalemia was associated with a highly significant redistribution of the staining pattern (P < 0. 001), with an increase in the percentage of cells displaying immunoreactivity in the apical membrane. These results indicate that dietary potassium depletion increases electrogenic H+-ATPase activity in the rat distal tubule; this may be associated with increased insertion of pumps into the apical membrane.

Topics: Animals; Anti-Bacterial Agents; Electrophysiology; Enzyme Inhibitors; Immunohistochemistry; Kidney; Kidney Tubules, Collecting; Kidney Tubules, Distal; Macrolides; Male; Nephrons; Potassium; Potassium Deficiency; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Reference Values

The cellular mechanism of luminal acidification (bicarbonate reabsorption) was studied in cortical distal tubules of rat kidney. The stopped-flow microperfusion technique was applied to early distal (ED) and late distal (LD) segments, perfused with bicarbonate Ringer solution to which specific inhibitors were added, to measure bicarbonate reabsorption [HCO3 flux (JHCO3)]. pH and transepithelial potential difference (Vt) were recorded by double-barreled H+ exchange resin/reference (1 M KCl) electrodes. Amiloride increased stationary pH and reduced Vt in both early and late segments. Hexamethylene-amiloride (HMA), a specific Na(+)-H+ exchange blocker, reduced JHCO3 in both segments (ED by 43.6 and LD by 40.3%) without affecting Vt. Benzamil, an Na(+)-channel blocker, reduced Vt by 75.9 in ED and 74.9% in LD but had no significant effect on acidification in both segments. The specific inhibitor of H(+)-ATPase, bafilomycin A1, inhibited LD JHCO3 at a concentration of 2 x 10(-7) M by 49%, but ED was inhibited by 24% only at 2 x 10(-6) M. Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase, reduced JHCO3 by 35% in LD of K(+)-depleted rats but not in control rats and had no effect on ED. These data indicate that, in ED, bicarbonate reabsorption is mediated mostly by Na(+)-H+ exchange. In LD, there is evidence for contribution of Na(+)-H+ exchange, vacuolar H(+)-ATPase, and H(+)-K(+)-ATPase (in K(+)-depleted rats) to bicarbonate reabsorption.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Antifungal Agents; Bicarbonates; Epithelium; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Kidney Cortex; Kidney Tubules, Distal; Kinetics; Macrolides; Male; Membrane Potentials; Perfusion; Potassium Deficiency; Proton Pump Inhibitors; Proton-Translocating ATPases; Rats; Rats, Wistar