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 μM in the renal cortex and 1.9 ± 0.4 μM in the renal medulla) and by Sch 28080 (Ki of 1.8 ± 0.5 μM and 2.5 ± 0.9 μM 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.
We investigated the effect of the cyclic AMP-protein kinase A (PKA) signalling pathway on renal Na+,K+-ATPase and ouabain-sensitive H+,K+-ATPase. Male Wistar rats were anaesthetized and catheter was inserted through the femoral artery into the abdominal aorta proximally to the renal arteries for infusion of the investigated substances. Na+,K+-ATPase activity was measured in the presence of Sch 28080 to block ouabain-sensitive H+,K+-ATPase and improve specificity of the assay. Dibutyryl-cyclic AMP (db-cAMP) administered at a dose of 10-17 mol/kg per min and 10-6 mol/kg per min increased Na+,K+-ATPase activity in the renal cortex by 34% and 42%, respectively, and decreased it in the renal medulla by 30% and 44%, respectively. db-cAMP infused at 10-6 mol/kg per min increased the activity of cortical ouabain-sensitive H+,K+-ATPase by 33%, and medullary ouabain-sensitive H+,K+-ATPase by 30%. All the effects of db-cAMP were abolished by a specific inhibitor of protein kinase A, KT 5720. The stimulatory effect on ouabain-sensitive H+,K+-ATPase and on cortical Na+,K+-ATPase was also abolished by brefeldin A which inhibits the insertion of proteins into the plasma membranes, whereas the inhibitory effect on medullary Na+,K+-ATPase was partially attenuated by 17-octadecynoic acid, an inhibitor of cytochrome P450-dependent arachidonate metabolism. We conclude that the cAMP-PKA pathway stimulates Na+,K+-ATPase in the renal cortex as well as ouabain-sensitive H+,K+-ATPase in the cortex and medulla by a mechanism requiring insertion of proteins into the plasma membrane. In contrast, medullary Na+,K+-ATPase is inhibited by cAMP through a mechanism involving cytochrome P450-dependent arachidonate metabolites.
Apart from Na+,K+-ATPase, a second sodium pump, Na+-stimulated, K+-independent ATPase (Na+-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na+-ATPase assay based on the method previously used by us to measure Na+,K+-ATPase activity (Acta Biochim Polon.; 2002, 49: 515-27). The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na+-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na+-ATPase activity was detected in the renal cortex (3.5 ± 0.2 μmol phosphate/h per mg protein), but not in the renal medulla. Na+-ATPase was not inhibited by ouabain or an H+,K+-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na+,K+-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na+-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na+,K+-ATPase, medullary Na+,K+-ATPase and cortical Na+-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na+,K+- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na+-ATPase was not different from control. These data indicate that both renal Na+-dependent ATPases are separately regulated and that up-regulation of Na+-ATPase may contribute to Na+ retention and arterial hypertension induced by chronic hyperleptinemia.
Paraoxonase 1 (PON1), contained in plasma high-density lipoproteins, plays an important role in the protection of plasma lipoproteins and cell membranes from oxidative damage. Previous studies indicate that human PON1 is stimulated by high NaCl concentrations. The aim of this study was to characterize in more detail the effect of salts on serum PON1. Paraoxon-hydrolyzing activity of human serum was stimulated by 81.6% following the addition of 1 M NaCl. The effect of NaCl was dose-dependent between 0.5 and 2 M. PON1 activity toward phenyl acetate was reduced by 1 M NaCl by 55.2%. Both the paraoxon- and phenyl acetate-hydrolysing activity was slightly lower in heparinized plasma than in serum, but NaCl had similar stimulatory and inhibitory effects on these activities, respectively. In rat, rabbit, and mouse, NaCl reduced PON1 activity. KCl had a similar effect on human PON1 as NaCl. Sodium nitrite also stimulated human PON1 but much less effectively than chloride salts. In contrast, sucrose, sodium acetate and sodium lactate had no significant effect. NaBr was a less effective PON1 activator than NaCl, whereas the effect of NaJ was non-significant. The activity of human PON1 toward homogentisic acid lactone and γ-decanolactone was unaltered by NaCl. These data indicate that: 1) high concentrations of chlorides stimulate human PON1 activity toward paraoxon but not other substrates, 2) PON1 is inhibited by Cl- in other mammalian species, 3) the potency of human PON1 activation by halogene salts increases with decreasing atomic mass of the halide anion.
We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10-11 mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10-9 and 10-8 mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K0.5 for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.
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