Stimulation of P(i) transport in opossum kidney cells by hyposmotic media

Exposure of various cells to hyposmotic media (Hypo) results in a rapid inhibition of both receptor-mediated and fluid-phase endocytosis. We used this maneuver to investigate the role of endocytosis in regulation of Pi transport in opossum kidney (OK) cells. Following exposure to Hypo, Na(+)-dependent Pi uptake increased rapidly, reaching a maximum within 5 min, and remained elevated up to 30 min. This was associated with a simultaneous reduction of horseradish peroxidase uptake. Kinetic studies showed increased apparent Vmax for Pi (9.38 +/- 0.93 vs. 13.08 +/- 1.04 nmol.mg-1.5 min-1 for control and Hypo, respectively; P < 0.05, n = 6) with no change in apparent Km. The effect was specific for Pi with no change in the Na(+)-dependent or -independent uptake of L-proline, L-glutamine, or methyl-alpha-D-glucopyranoside. Stimulation of Pi transport persisted when control and Hypo had identical ionic compositions. Stimulation of Pi transport was rapidly reversed when cells were returned to an isosmotic medium. Preincubation with Hypo at 4 degrees C had no effect on Pi transport. Addition of cycloheximide or actinomycin D did not prevent the increased Pi uptake after exposure to Hypo. The effect also persisted after protein kinase C downregulation. Stimulation of Pi transport by Hypo is consistent with reduced endocytic retrieval of Na(+)-Pi cotransporters from brush-border membrane (BBM), resulting in an increase in their number on the BBM.