| First Author | Nakamura S | Year | 2006 |
| Journal | J Lab Clin Med | Volume | 147 |
| Issue | 1 | Pages | 45-51 |
| PubMed ID | 16443004 | Mgi Jnum | J:106404 |
| Mgi Id | MGI:3618449 | Doi | 10.1016/j.lab.2005.08.013 |
| Citation | Nakamura S (2006) H+-ATPase activity in selective disruption of H+-K+-ATPase alpha 1 gene of mice under normal and K-depleted conditions. J Lab Clin Med 147(1):45-51 |
| abstractText | 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(+)-ATPase plays an important role in compensatory proton secretion in the HKAalpha1 KO mice under normal conditions. |
