P-glycoprotein phosphorylation/dephosphorylation and cellular accumulationof L-DOPA in LLC-GA5 Col300 cells

1 The present work was aimed to study the effect of PKC activation and prot ein-serine/threonine phosphatase (PP1/PP2 A) inhibition on P-glycoprotein ( P-gp) mediated transport of L-DOPA in LLC-GA5 Col300 cells, a renal cell li ne expressing the human P-glycoprotein in the apical membrane. 2 L-DOPA accumulation was a time-and concentration-dependent process with t he following kinetic characteristics: k(in), 57.3 +/- 1.2 pmol mg protein m in(-1) min(-1); k(out), 3.3 +/- 0.1 pmol mg(-1) protein min(-1); A(max), 10 .6 +/- 0.8; K-m, 198 +/- 64 mu M; V-max, 5.2 +/- 0.7 nmol mg protein(-1). 3 Verapamil (25 mu M), a P-glycoprotein inhibitor, markedly increased (appr oximate to 40% increase) the accumulation of a non-saturating concentration of L-DOPA (2.5 mu M) at both initial rate of uptake (IRU, 6 min incubation ) and at steady-state (SS, 30 min incubation). 4 PKC activation with phorbol 12,13-dibutyrate (PDBu, 1, 3 and 10 nM) produ ced a concentration-dependent decrease in L-DOPA accumulation at SS, but no t at IRU. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (10 0 nM), produced no change in L-DOPA accumulation. The effect of PDBu was co mpletely reverted by staurosporine (100 nM). The phosphatase inhibitor okad aic acid (100 nM) reduced by 20% the accumulation of L-DOPA at IRU, but not at SS. 5 It is suggested that P-glycoprotein plays a role in regulation of intrace llular availability of L-DOPA in renal epithelial cells, and phosphorylatio n/dephosphorylation of P-glycoprotein may be involved in the regulation of the transporter.