Regulation of nucleobase transport in LLC-PK1 renal epithelia by protein kinase C

The involvement of protein kinase C (PKC) in the regulation of Na+-dependent and -independent hypoxanthine transport was investigated by exposing confluent monolayers of LLC-PK1 renal epithelia cells to the PKC activator, phorbol 12-myristate 13-acetate (PMA). Chronic exposure (> 2 h) of LLC-PK1 monolayers to 16 nM PMA resulted in approximate to 75% inhibition of Na+-dependent hypoxanthine influx occurring maximally at 8 h and persisting for 72 h. in contrast, PMA had little effect on Na+-independent hypoxanthine influx at 8 h, but longer exposure resulted in stimulation of influx (approximate to 3-fold) that peaked at 24 h and thereafter declined to control levels at 72 h. The effects of PMA were dose-dependent and were associated with changes in V-max of transport (2-4-fold) with no significant change in apparent K-m. 4 alpha-Phorbol, a phorbol ester that does not activate PKC, had no effect on hypoxanthine transport by LLC-PK1 cells. The diacylglycerol kinase inhibitor, R59022 (10 mu M), partially inhibited (28%) Na+-dependent hypoxanthine influx. In addition, the PMA-induced effects on hypoxanthine transport were reversed by Re-31-8220 (1 and 5 mu M) and calphostin C (50 nM), potent and selective inhibitors of PKC. The increase in Na+-independent hypoxanthine influx following exposure to PMA was blocked by the protein synthesis inhibitor, cycloheximide (20 mu M), and correlated with an increase in LLC-PK1 cell proliferation. The PMA-induced decrease in Na+-dependent hypoxanthine transport was independent of PMA effects on cell proliferation and not dependent on protein synthesis. These results are consistent with the proposal that the PMA-induced effects on hypoxanthine transport are due to PKC activation.