A highly purified suspension of rabbit proximal tubules was cultured in a hormone-supplemented serum-free medium. This primary culture yielded a homogeneous population of cells that demonstrated functional and morphological polarity in mono-layers. The characteristics of the Na-dependent glucose transporter in the luminal membrane were studied by measuring the uptake of alpha-methylglucoside (AMG). The kinetics of Na-dependent AMG uptake were consistent with a single saturable system with an apparent Km of 0.8 mM and Jmax of 0.14 nmol X mg-1 X min-1. AMG permeability was 0.10 microliter X mg-1 X min-1. Uptake was inhibited 95% by 0.1 mM phlorizin and by removal of sodium. The stoichiometry of Na/glucose interaction with the carrier was 2:1. These characteristics are typical of the characteristics described for the late proximal tubule. To examine whether the glucose that enters the cell across the luminal membrane is incorporated into the metabolic pool of the cell, we studied the oxidation of [14C]glucose to 14CO2 in the absence and presence of phlorizin. Significant decarboxylation of [1-14C]glucose and [6-14C]glucose was observed, consistent with the existence of aerobic metabolism and a hexose monophosphate shunt. In the presence of 0.1 mM phlorizin, uptake and oxidation of D-glucose were inhibited to an identical degree, suggesting that luminal uptake is a rate-limiting step in the oxidation of glucose by these proximal tubular cells. These studies indicate that proximal tubular cells in primary culture utilize glucose as an energy source and that the glucose derived from transport across the luminal membrane is incorporated into the metabolic pool of the cell.
We investigated the quantitative relationship between cellular ATP concentration and Na+-K+-ATPase activity as measured by ouabain-sensitive 86Rb influx in rabbit proximal renal cells. Cellular ATP was reduced in a stepwise manner by rotenone (10(-7) to 10(-5) M) and was increased by 10 mM adenosine. During these maneuvers, ouabain-sensitive 86Rb influx was linearly related to cellular ATP and did not saturate up to 9.9 mM ATP. In contrast, Na+-K+-ATPase activity in membranes prepared from these cells saturated at 2.0 mM ATP at various sodium (10-100 mM) and potassium (4-100 mM) concentrations. Sodium-dependent phosphate uptake and alpha-methylglucoside (alpha-MG) uptake were both inhibited to a similar degree when cellular ATP was reduced. We conclude that 1) the ATP requirement for saturation of Na+-K+-ATPase is higher in intact renal cells than in the membranes, and 2) the uptake of phosphate and alpha-MG are similarly influenced by reduction in ATP. This effect of ATP on phosphate and AMG uptake is most likely an indirect one and is secondary to changes in the sodium gradient across the cell.
We examined the effects of acute changes in extracellular and intracellular calcium on transport processes in primary culture of proximal rabbit renal cells. A change in extracellular calcium from 0 to 3 mM inhibited amiloride-sensitive sodium uptake by 30%, and this effect was maximal at 1 mM calcium. Other polyvalent cations (Mn2+, Mg2+, La3+, and Ba2+) produced quantitatively similar inhibition of amiloride-sensitive sodium uptake compared with calcium. An increase in cytosolic calcium produced by calcium loading (20 mM) or by A23187 (20 microM) resulted in an inhibition of 25-40% of amiloride-sensitive sodium uptake. Moreover, quinidine (10(-4)M) and ruthenium red (3 microM), agents presumed to increase cytosolic calcium, inhibited amiloride-sensitive sodium uptake by 20-60%. Both these agents also inhibited sodium-dependent phosphate uptake by 20% but had no effect on ouabain-sensitive 86Rb+ uptake or on sodium-dependent alpha-methylglucoside uptake. Our data indicate that increases in extracellular calcium inhibit amiloride-sensitive sodium uptake and increases in cytosolic calcium inhibit sodium-dependent phosphate and amiloride-sensitive sodium uptakes. The effect of extracellular calcium may be due to charge screening and/or binding to the negatively charged plasma membrane or due to alterations in membrane fluidity.
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