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 approximately 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 (approximately 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 Vmax 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 microM), partially inhibited (28%) Na(+)-dependent hypoxanthine influx. In addition, the PMA-induced effects on hypoxanthine transport were reversed by Ro-31-8220 (1 and 5 microM) 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 microM), 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.
Griffith
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Research School of Biosciences, University of Kent, Canterbury, UK. s.m.jarvis@ukc.ac.uk
Jarvis
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D000818:Animals; D001692:Biological Transport; D002455:Cell Division / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D019271:Hypoxanthine / Q000378:metabolism*; D007700:Kinetics; D018374:LLC-PK1 Cells; D011493:Protein Kinase C / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism
D019271:Hypoxanthine; D003513:Cycloheximide; D012964:Sodium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D013936:Thymidine