Modulation of Pig Kidney Na + /K + -ATPase Activity by Cholesterol: Role of Hydration † (original) (raw)
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Biochemistry, 2006
Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na + /K +-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme.
FEBS Letters, 1990
Proximal tubular cells from kidneys of male rats chronically fed with an isotonic NaC1 solution, show a volume increase which is dependent on the length of the treatment with NaC1, when compared with control rats. Parallel to the cell volume increase, there is an increase of the ouabaininsensitive Na-ATPase activity, whereas the ouabain-sensitive Na,K-ATPase activity remains unchanged. These results establish a clear relationship between a chronic Na-diet, kidney cell volume and Na-ATPase activity.
European Journal of Nutrition, 2012
Purpose It has been demonstrated that reabsorption of Na ? in the thick ascending limb is reduced and the ability to concentrate urine can be compromised in undernourished individuals. Alterations in phospholipid and cholesterol content in renal membranes, leading to Na ? loss and the inability to concentrate urine, were investigated in undernourished rats. Methods Sixty-day-old male Wistar rats were utilized to evaluate (1) phospholipid and cholesterol content in the membrane fraction of whole kidneys, (2) cholesterol content and the levels of active Na ? transporters, (Na ? ? K ?) ATPase and Na ?-ATPase, in basolateral membranes of kidney proximal tubules, and (3) functional indicators of medullary urine concentration. Results Body weight in the undernourished group was 73 % lower than in control. Undernourishment did not affect the levels of cholesterol in serum or in renal homogenates. However, membranes of whole kidneys revealed 56 and 66 % reduction in the levels of total phospholipids and cholesterol, respectively. Furthermore, cholesterol and (Na ? ? K ?)ATPase activity in proximal tubule membranes were reduced by 55 and 68 %, respectively. Oxidative stress remained unaltered in the kidneys of undernourished rats. In contrast, Na ?-ATPase activity, an enzyme with all regulatory components in membrane, was increased in the proximal tubules of undernourished rats. Free water clearance and fractional Na ? excretion were increased by 86 and 24 %, respectively, and urinary osmolal concentration was 21 % lower in undernourished rats than controls. Conclusion Lifelong undernutrition reduces the levels of total phospholipids and cholesterol in membranes of renal tubular cells. This alteration in membrane integrity could diminish (Na ? ? K ?)ATPase activity resulting in reduced Na ? reabsorption and urinary concentrating ability.
Biochemical Pharmacology, 1987
Abstraet-The ouabain-insensitive, Na+-stimulated ATPase activity of kidney proximal tubular cells from rats fed a high Na+ diet for 4 months was increased approximately 70% when compared with control (normal diet) rats. The higher ATPase activity was not due to a change in the affinity of the system toward ATP, Mg2+ or Na+. This increase in Na+-ATPase activity may be due to either a higher number of pumps or to a higher turnover rate of the enzyme or both. The oubain-sensitive, Na+,K+-stimulated ATPase activity, on the other hand, did not change with the high sodium diet. These results can be taken as evidence that the Na+,K+-ATPase and the Na+-ATPase of basolateral plasma membranes of proximal tubular cells from rat kidney are two different entities.
Expression, Distribution, and Activity of Na+,K+-ATPase in Normal and Cholestatic Rat Liver
Journal of Histochemistry & Cytochemistry, 1998
Hepatocellular Na+,K+-ATPase is an important driving force for bile secretion and has been localized to the basolateral plasma membrane domain. Cholestasis or impaired bile flow is known to modulate the expression, domain specificity, and activity of various transport systems involved in bile secretion. This study examined Na+,K+-ATPase after ethinylestradiol (EE) treatment and after bile duct ligation (BDL), two rat models of cholestasis. It applied quantitative immunoblotting, biochemical and cytochemical determination of enzyme activity, and immunocytochemistry to the same livers. The data showed a good correlation among the results of the different methods. Neither EE nor BDL induced alterations in the subcellular distribution of Na+,K+-ATPase, which was found in the basolateral but not in the canalicular (apical) plasma membrane domain. Protein expression and enzyme activity showed a small (~10%) decrease after EE treatment and a similar increase after BDL. These modest changes...