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2002 | 49 | 2 | 515-527
Article title

Spectrophotometric method for the determination of renal ouabain-sensitive H+,K+ -ATPase activity.

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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.
Physical description
  • Attmane-Elakeb A, Boulanger H, Vernimmen C, Bichara M. (1997) Apical location and inhibition by arginine vasopressin of K+/H+ antiport of the medullary thick ascending limb of rat kidney. J Biol Chem.; 272: 25668-77.
  • Beil W, Staar U, Sewing KF. (1987) SCH 28080 is a more selective inhibitor than SCH 32651 at the K+ site of gastric K+/H+-ATPase. Eur J Pharmacol.; 139: 349-52.
  • Bełtowski J, Górny D, Marciniak A. (1998a) The mechanism of Na+,K+-ATPase inhibition by atrial natriuretic factor in rat renal medulla. J Physiol Pharmacol.; 49: 271-83.
  • Bełtowski J, Górny D, Marciniak A. (1998b) Biphasic effect of protein kinase C on rat renal cortical Na+,K+-ATPase. J Physiol Pharmacol.; 49: 627-37.
  • Bełtowski J, Wójcicka G, Górny D, Marciniak A. (1999) Human leptin administered intraperitoneally decreases renal medullary but not cortical Na+,K+-ATPase in the rat. Physiol Res.; 48 Suppl 1: S56.
  • Buffin-Meyer B, Younes-Ibrahim M, Barlet-Bas C, Cheval L, Marsy S, Doucet A. (1997) K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct. Am J Physiol.; 272, F124-31.
  • Caruso-Neves C, Rangel LB, Vives D, Vieyra A, Coka-Guevara S, Lopes AG. (2000) Ouabain-insensitive Na+-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule. Biochim Biophys Acta.; 1468: 107-14.
  • Chiou S, Vesely DL. (1995) Kaliuretic peptide: the most potent inhibitor of Na+-K+ ATPase of the atrial natriuretic peptides. Endocrinology.; 136: 2033-9.
  • Codina J, Cardwell J, Gitomer JJ, Cui Y, Kone BC, Dubose Jr TD. (2000) Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells. Am J Physiol Cell Physiol.; 279: C1319-26.
  • Doucet A, Marsy S. (1987) Characterization of K-ATPase activity in distal nephron: stimulation by potassium depletion. Am J Physiol.; 253: F418-23.
  • Doucet A. (1997) H+, K+-ATPase in the kidney: localization and function in the nephron. Exp Nephrol.; 5: 271-6.
  • Fejes-Tóth G, Fejes-Tóth AN. (2001) Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells. Am J Physiol.; 281: F318-25.
  • Feraille E, Doucet A. (2001) Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev.; 81: 345-418.
  • Garg LC, Narang N. (1988) Ouabain-insensitive K-adenosine triphosphatase in distal nephron segments of the rabbit. J Clin Invest.; 81: 1204-8.
  • Giebisch G, Wang W. (1996) Potassium transport: from clearance to channels and pumps. Kidney Int.; 49: 1624-31.
  • Giebisch G. (1998) Renal potassium transport: mechanisms and regulation. Am J Physiol.; 274: F817-33.
  • Hurst RO. (1964) The determination of nucleotide phosphorus with a stannous chloride- hydrazine sulphate reagent. Can J Biochem.; 42: 287-92.
  • Jörgensen PL. (1974) Purification and characterisation of (Na+K+)-ATPase. III. Purification from outer medulla of mammalian kidney after selective removal of membrane components with sodium dodecylsulphate. Biochim Biophys Acta.; 356: 36-52.
  • Khundimiri SJ, Lederer, A. (2002) PTH and DA regulate Na-K ATPase through divergent pathways. Am J Physiol Renal Physiol.; 282: F512-22.
  • Kiroytcheva M, Cheval L, Carranza ML, Martin PY, Favre H, Doucet A, Feraille E. (1999) Effect of cAMP on the activity and the phosphorylation of Na+,K+-ATPase in rat thick ascending limb of Henle. Kidney Int.; 55: 1819-31.
  • Laroche-Joubert N, Marsy S, Doucet A. (2000) Cellular origin and hormonal regulation of K+-ATPase activities sensitive to Sch-28080 in rat collecting duct. Am J Physiol Renal Physiol.; 279: F1053-59.
  • Lowry OH, Rosebrough NI, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem.; 193: 265-75.
  • Muto S. (2001) Potassium transport in the mammalian collecting duct. Physiol Rev.; 81: 85-116.
  • Rabinowitz L. (1996) Aldosterone and potassium homeostasis. Kidney Int.; 49: 1738-42.
  • Sabolic I, Brown D, Verbavatz JM, Kleinman J. (1994) H+-ATPases of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole. Am J Physiol.; 266: F868-77.
  • Sangan P, Rajendran VM, Mann AS, Kashgarian M, Binder HJ. (1997) Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion. Am J Physiol.; 272: C685-96.
  • Shyjan AW, Cena V, Klein DC, Levenson R. (1990) Differential expression and enzymatic properties of the Na+,K+-ATPase α3 isoenzyme in rat pineal glands. Proc Natl Acad Sci U S A.; 87: 1178-82.
  • Silver RB, Choe H, Frindt G. (1998) Low-NaCl diet increases H-K-ATPase in intercalated cells from rat cortical collecting duct. Am J Physiol.; 275: F94-F102.
  • Silver RB, Soleimani M. (1999) H+-K+-ATPases: regulation and role in pathophysiological states. Am J Physiol.; 276: F799-F811.
  • Sweeney G, Nu W, Kanani R, Klip A. (2000) Regulation of the Na,K-pump by leptin in 3T3-L1 fibroblasts. Endocrinology.; 141: 1277-80.
  • Wang Z, Rabb H, Craig T, Burnham C, Shull GE, Solemani M. (1997) Ischemic-reperfusion injury in the kidney: overexpression of colonic H+,K+-ATPase and suppression of NHE-3. Kidney Int.; 51: 1106-15.
  • Wesson DE. (1998) Na/H exchange and H-K ATPase increase tubule acidification in chronic alkalosis. Kidney Int.; 53: 945-51.
  • Wingo CS, Smolka AJ. (1995) Function and structure of H-K-ATPase in the kidney. Am J Physiol.; 269: F1-16.
  • Younes-Ibrahim M, Barlet-Bas C, Buffin-Meyer B, Cheval L, Rajerison R, Doucet A. (1995) Ouabain-sensitive and -insensitive K-ATPases in rat nephron: effect of K depletion. Am J Physiol.; 268: F1141-7.
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