Biocficmical Pitamracology, Vol. 36, No. 1, pp. 7-11, 1987, Rited in Great Britain. RW-295y87 $3.00 + 0.00 Pergamon Journals Ltd. HIGH SODIUM DIET AND Na+-STIMULATED ATPase ACTIVITIES IN BASOLATERAL PLASMA MEMBRANES FROM RAT KIDNEY PROXIMAL TUBULAR CELLS MARIA ANTONIETA OBANDO, REINALDO MAR~N, TERESA PROVERBIOand FULGENCIQ PROVERBIO* Centro de Bioffsica y Bioqufmica, Instituto Venezolano de Investigaciones Cientfficas, Caracas lOlOA, Venezuela zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP (Received 16 July 1985; zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM accepted 26 Ferrule 1986) 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. Two me~h~isms of cellular Na* extrusion have been demonstrated to occur in kidney cortex slices (rich in proximal tubules) from several mammals, including rats [l-5]: (1) exchange of Na+ for K+, and (2) extrusion of Na+ accompanied by Cl- and water. Two types of Mg2+-dependent, Na+-stimulated ATPase activity have also been shown to be present in basolateral plasma membranes of cells from guinea pig and rat kidney cortex slices [6,7]: (1) the ouabain- sensitive, Na+,K+-ATPase, and (2) the ouabain- insensitive, Na+-ATPase. The Na+,K+-ATPase is considered to generate the energy supply for the extrusion of Na+ exchanged for IV. The Na+- ATPase is considered to generate the energy supply for the extrusion of Na+ ac~ompa~ed by Cl- and water [6-H]. Even though the two ATPase systems have very different characteristics [9,10], the exist- ence of the Na+-ATPase is still controversial. Changes in the Na+ intake may well produce dif- ferential changes in renal Na+ transport mechanisms and, consequently, changes in Na+-ATPase activi- ties. With this in mind, we fed 25day-old rats an isotonic NaCl solution. After 4months, we found that the Na+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from the experimental rats was about twice the activity of control rats. The Na+, K+-ATPase activity, on the other hand, was similar in both groups. STALLS AND ~THODS Sprague-Dawley rats (25 days old) were separated into two groups: a control group was given tap water to drink and an experimental group was given a solution of 0.9% NaCl to drink. Every month, some of the control and the experimental rats were killed, and basolateral plasma membranes from their kidney * To whom correspondence should be addressed. proximal tubular cells were prepared and assayed for ATPase activity. Isolation zyxwvutsrqponmlkjihgfedcbaZYXWVUTS of b~ol~ter~l plasma membranes. The basolateral plasma membrane enriched fractions were prepared as follows. Three-month-old, healthy male Sprague-Dawley rats were anesthetized with ether and immediately decapitated. The kidneys were removed, decapsulated, and collected in a medium containing 250 mM sucrose/20 mM Tris- HCl (pH 7.2)/0.5 mM dithiothreitol/0.2 mM phenylmethylsulfonylfluoride (sucrose/Tris/DlT/ PMSF medium), at 4”. The outermost slices of the kidney cortex (which are rich in proximal tubules [12]) were homogenized and centrifuged at 4”, using a modification of the method of Kinsella et al. (orig- inally developed for dog kidney cortex) [13]. The slices were homogenized at 4”, with eight strokes at 2500 rpm in an Eberbach homogenizer with a tight- fittin Teflon pestle, in 3 voi./g of tissue of sucrose/ Tris DTT/PMSF medium. DTT and PMSF were B used to avoid possible oxidation of the ATPases and to inhibit proteases respectively. These agents were not used originally by Kinsella et al. [13]. The hom- ogenate (usually 240 mg protein) was spun at 1000 g for 10min and the supernatant fraction was saved. The pellet was resuspended with sucrose/Tris/DTT/ PMSF medium and recentrifuged at 1OOOg for 10 min. The resulting supernatant fraction was com- bined with the previous supernatant. This mixture was spun at 9500 g for 10 min. The supernatant and the soft, lighter upper portion of the pellet were combined and spun at 48,000 g for 20 min. The soft, lighter upper portion of the pellet was resuspended in a solution of 25 mM Tris-HCl (pH 7.2)/100 mM mannitol/2 mM CaClr/l mM MgC12/1 mM MnC12 to a final protein concentration of 4-6 mg/ml. This membrane suspension was kept on ice for 90min and then spun at 14OOg for 12 min. The ellet was resuspended with the same solution (Tris P mannitol/