Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity.
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. 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 mumol 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.
Bełtowski
Jerzy
J
Department of Pathophysiology, Medical University, Lublin, Poland. patfiz@asklepios.am.lublin.pl
Jamroz-Wiśniewska
Anna
A
Nazar
Jarosław
J
Wójcicka
Grazyna
G
D000818:Animals; D004032:Diet; D004351:Drug Resistance; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D020738:Leptin / Q000494:pharmacology*; D008297:Male; D023421:Models, Animal; D009765:Obesity / Q000201:enzymology* / Q000209:etiology; D010042:Ouabain / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*
D020738:Leptin; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium