Pyridoxine uptake by rat renal proximal tubular cells

Uptake of [3H]pyridoxine by freshly isolated rat renal proximal tubular cells was temperature dependent and exhibited saturation kinetics, with estimated Kt and Vmax of 1.3 microM and 14 pmol/(10(6) cells.0.5 min), respectively. Pyridoxamine, 4'-deoxypyridoxine and 5'-deoxypyridoxal were as effective as unlabeled pyridoxine in inhibiting tracer uptake, whereas pyridoxamine-5'-phosphate, pyridoxal and 4'-pyridoxic acid had no effect, and other vitameric forms and structural analogs caused intermediate inhibition of cellular vitamin uptake. After 30 min, 88% of accumulated radioactive vitamin B-6 in cell extracts had been metabolized to phosphorylated forms and pyridoxal, which supports metabolic trapping as a mechanism responsible for the cellular accumulation observed. Initial uptake was significantly decreased in the presence of ethionine and ouabain; however, carbonylcyanide-p-trifluoromethoxyphenylhydrazone had no effect. Partial to complete substitution of NH4Cl and LiCl for NaCl had no effect, while partial substitution with RbCl caused significant dose-related inhibition of initial pyridoxine uptake. Amiloride also significantly decreased initial uptake. These data are consistent with a facilitated uptake process for pyridoxine that has substrate specificity and may be modulated by sodium-hydrogen exchange and/or pH gradient effects. Cellular uptake is followed by intracellular metabolic trapping catalyzed by pyridoxal kinase.