The aim of this work was to develop a method for renal H+,K+-ATPase measurement based on the previously used Na+ ,K+ -ATPase assay (Bełtowski et al.: J Physiol Pharmacol.; 1998, 49: 625-37). ATPase activity was assessed by measuring the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Both ouabain-sensitive and ouabain-resistant K+ -stimulated and Na+ -independent ATPase activity was detected in the renal cortex and medulla. These activities were blocked by 0.2 mM imidazolpyridine derivative, Sch 28080. The method for ouabain- sensitive H+ ,K -ATPase assay is characterized by good reproducibility, linearity and recovery. In contrast, the assay for ouabain-resistant H ,K -ATPase was unsatisfactory, probably due to low activity of this enzyme. Ouabain-sensitive H+ ,K+ -ATPase was stimulated by K + with Km of 0.26 ± 0.04 mM and 0.69 ±0.11 mM in cortex and medulla, respectively, and was inhibited by ouabain (Ki of 2.9 ± 0.3 uM in the renal cortex and 1.9 ± 0.4 uM in the renal medulla) and by Sch 28080 (Ki of 1.8 ± 0.5 uM and 2.5 ± 0.9 uM in cortex and medulla, respectively). We found that ouabain-sensitive H+ ,K+ -ATPase accounted for about 12% of total ouabain-sensitive activity in the Na+ ,K+-ATPase assay. Therefore, we suggest to use Sch 28080 during Na+,K+-ATPase measurement to block H+ ,K+ -ATPase and improve the assay specificity. Leptin administered intraperitoneally (1 mg/kg) decreased renal medullary Na+ ,K+ -ATPase activity by 32.1% at 1 h after injection but had no effect on H+ ,K+ -ATPase activity suggesting that the two renal ouabain-sensitive ATPases are separately regulated.
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