EVIDENCE FOR THE INVOLVEMENT OF P-GLYCOPROTEIN ON THE EXTRUSION OF TAKEN UP L-DOPA IN CYCLOSPORINE-A TREATED LLC-PK1 CELLS

1 The present work has examined the effects of short- (30 min) and lon g-term (14 h) exposure to cyclosporine A (CsA) on the uptake of L-DOPA , its decarboxylation to dopamine and the cellular extrusion of taken up L-DOPA and of newly-formed amine in monolayers of LLC-PK1, cells. 2 In the presence of benserazide (50 mu M), L-DOPA was rapidly accumula ted in LLC-PK1 cells (cultured in collagen-treated plastic) attaining equilibrium at 30 min of incubation. Non-linear analysis of the satura tion curves revealed a K-m, of 113+/-16 mu M and a V-max,, of 5581+/-2 97 pmol mg(-1) protein 6 min(-1). 3 In the absence of benserazide, LLC -PK1 cells incubated with increasing concentrations of L-DOPA (10 to 5 00 mu M) for 6 min accumulate newly-formed dopamine by a saturable pro cess with apparent K-m, and V-max,, values of 31+/-6 mu M and 1793+/-9 1 pmol mg(-1) protein 6 min(-1), respectively. The fractional outflow of newly-formed dopamine was found to be 20%. Up to 200 mu M of intrac ellular newly-formed dopamine, the outward transfer of the amine was f ound to be a non-saturable process. 4 Short-term exposure to CsA (0.3, 1.0 and 3.0 mu g ml(-1)) was found not to change the intracellular co ncentrations of newly-formed dopamine, but increased the levels of dop amine in the incubation medium (143% to 224% increase) and the total a mount of dopamine formed (31% to 59% increase). Long-term exposure to CsA (0.03 to 3.0 mu g ml(-1)) reduced the total amount of dopamine (15 % to 39% reduction) and the intracellular levels of the amine (11% to 56% reduction), without changing dopamine levels in the incubation med ium. Both short-and long-term exposure to CsA resulted in a concentrat ion-dependent increase in the fractional outflow of newly-formed dopam ine. 5 Short-term exposure to CsA (3.0 mu g ml(-1)) reduced the apical extrusion of intracellular L-DOPA by 15% (P<0.05), whereas long-term exposure to CsA reverted this effect and decreased its intracellular a vailability (15% reduction; P<0.05). 6 Detection of P-glycoprotein act ivity was carried out by measuring verapamil-or UIC2-sensitive rhodami ne 123 accumulation. Both UIC2 (3 mu g ml(-1)) and verapamil (25 mu M) significantly increased the accumulation of rhodamine 123 in LLC-PK1 cells. A 30 min exposure to CsA was found not to affect the accumulati on of rhodamine 123 in the presence of verapamil (25 mu M), whereas a 14 h exposure to CsA was found to reduce the accumulation of rhodamine 123. 7 In conclusion, the increase and the reduction in the formation of dopamine after short-and long-term exposure to CsA, respectively, correlate with the effects of the immunosuppressant on the apical cell extrusion of taken up L-DOPA, suggesting the involvement of P-glycopr otein. The effects of CsA on the fractional outflow of newly-formed do pamine appear to be mediated by a different mechanism.