L-alanine accumulation in isolated tubuli from rat kidney cortex.

Tubuli were prepared from rat renal cortex by modification of the method adopted from HOWARD and PESCH (J.Biol. Chem. 243, 3105, 1968). At 37 degrees C tubuli showed constant pyruvate consumption and glucose production for more than one hour. Intracellular electrolyte and amino acid concentrations were measured after separating the tubuli from the incubation medium by a layer of silicon oil in 5 seconds. During incubation at 37 degrees C L-alanine was accumulated 2.6 fold in the tubuli, while intracellular Na+ concentration dropped to about 50 mEq/l. At 27 degrees C, when pyruvate consumption and glucose production was reduced to less than half compared with 37 degrees C incubation, the respective gradients were higher. L-alanine accumulation was reduced by 2,4-dinitrophenol, antimycin A, ouabain and p-hydroxymercuribenzoate and was completely abolished by the reversal of the Na+ concentration gradient between extracellular and intracellular spaces. At 4 degrees C, when erergy production in tubuli ceased, L-alanine could be concentrated in the case of a Na+o/Na+i ratio greater than unity but not at a lower ratio. It is concluded that the accumulation of L-alanine is a direct function of the energy provided by the Na+ concentration gradient and is mediated by a postulated carrier, which can be injured by sulfhydryl group reagents like p-hydroxymercuribenzoate.