Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity.

Abstract

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.

• Authors: Jerzy Bełtowski, Anna Jamroz-Wiśniewska, Jarosław Nazar, Grazyna Wójcicka
• Journal: Acta biochimica Polonica (Acta Biochim Pol) Vol. 51 Issue 4 Pg. 1003-14 ( 2004) ISSN: 0001-527X [Print] Poland
• PMID: 15625572 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Leptin
• Ouabain
• Sodium
• Sodium-Potassium-Exchanging ATPase
• Potassium

Topics

• Animals
• Diet
• Drug Resistance
• Kidney (drug effects, enzymology)
• Leptin (pharmacology)
• Male
• Models, Animal
• Obesity (enzymology, etiology)
• Ouabain (pharmacology)
• Potassium (pharmacology)
• Rats
• Sodium (pharmacology)
• Sodium-Potassium-Exchanging ATPase (analysis, antagonists & inhibitors, metabolism)
• Spectrophotometry (methods)