COULD CYTOPLASMIC CONCENTRATION GRADIENTS FOR SODIUM AND ATP EXIST IN INTACT RENAL-CELLS

In renal cells, the Na+ pump maintains a transmembrane concentration gradient for sodium ensuring the net reabsorption of sodium with or without cotransported species. This process requires a significant fraction of the ATP turnover of proximal tubules and thick ascending limbs. To understand the potential regulatory influences of Na+ and ATP on the activity of the Na+ pump in these nephron segments, the apparent kinetics of the membrane-bound Na+ -K+ ATPase and of the cellular Na+ pump were studied in different preparations of dog proximal tubules and thick ascending limbs (tubular suspensions, tissue homogenates, and basolateral membrane vesicles) obtained from dog kidney cortex and red medulla. Two determinant kinetic parameters, i.e., the apparent Michaelis constant (K-m) and the saturating concentrations for sodium and ATP, were compared with the intracellular concentrations of Na+ and ATP measured under physiological conditions. In both types of tubules, the apparent K-m value for Na+ (5-15 mM) is set well below the measured mean intracellular concentration of sodium (50-60 mM), suggesting that the Na+ pump should be saturated by sodium ions under normal conditions. Nevertheless, a modest increment of the Na concentration in the vicinity of the pump, obtained by equilibrating the intra- and extra-cellular sodium concentrations at various extracellular [Na+] with nystatin, increases the activity of the Na+ pump in intact cortical tubules and thick ascending limbs, even when the extracellular [Na+] is set at the estimated intracellular [Na+], demonstrating that the pump is not saturated by sodium in situ. Similarly, the kinetics of the renal Na+ pump as a function of the ATP concentration suggested that the pump should be saturated by ATP in physiological conditions, since in both tissues the cellular ATP level (3-6 mM) is higher than the concentration required to achieve saturation of this activity (<2.5 mM). However, in renal cortical tubules, the steady-state intracellular [Na+] is affected by modest changes of ATP concentration, suggesting that the Na+ pump is not functionally saturated by ATP. Our data suggest that concentration gradients for Na+ and ATP may exist in the cytosol of renal cells. These gradients would be related to the polarity of sodium transport and of the ATP-consuming and ATP-regenerating processes in intact cells. According to this scheme, the intracellular concentration of both Na+ and ATP would be lower in the vicinity of the Na+ pump, the site of active extrusion of sodium and ATP utilization, than the mean cellular concentrations measured using classical techniques. Such an organization would amplify the signals given to the cell membranes by changes in ATP concentration.