1. The present work has examined the effects of short- (30 min) and long-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 microM), L-DOPA was rapidly accumulated in LLC-PK1 cells (cultured in collagen-treated plastic) attaining equilibrium at 30 min of incubation. Non-linear analysis of the saturation curves revealed a Km of 113+/-16 microM and a Vmax of 5581+/-297 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 500 microM) for 6 min accumulate newly-formed dopamine by a saturable process with apparent Km and Vmax values of 31+/-6 microM and 1793+/-91 pmol mg(-1) protein 6 min(-1), respectively. The fractional outflow of newly-formed dopamine was found to be 20%. Up to 200 microM of intracellular newly-formed dopamine, the outward transfer of the amine was found to be a non-saturable process. 4. Short-term exposure to CsA (0.3, 1.0 and 3.0 microg ml(-1)) was found not to change the intracellular concentrations of newly-formed dopamine, but increased the levels of dopamine in the incubation medium (143% to 224% increase) and the total amount of dopamine formed (31% to 59% increase). Long-term exposure to CsA (0.03 to 3.0 microg 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 medium. Both short- and long-term exposure to CsA resulted in a concentration-dependent increase in the fractional outflow of newly-formed dopamine. 5. Short-term exposure to CsA (3.0 microg 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 availability (15% reduction; P<0.05). 6. Detection of P-glycoprotein activity was carried out by measuring verapamil- or UIC2-sensitive rhodamine 123 accumulation. Both UIC2 (3 microg ml(-1)) and verapamil (25 microM) significantly increased the accumulation of rhodamine 123 in LLC-PK1 cells. A 30 min exposure to CsA was found not to affect the accumulation of rhodamine 123 in the presence of verapamil (25 microM), 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-glycoprotein. The effects of CsA on the fractional outflow of newly-formed dopamine appear to be mediated by a different mechanism.

The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70-80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na(+) in LLC-PK1 cells.

P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport.

1. Human multidrug resistance protein 1 (MRP1) is a 190 kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. We recently demonstrated that glutathione (GSH) is required for the labelling of the C-terminal half of MRP1 with a photoanalog of agosterol A (azido AG-A). In this study, we further characterized the GSH-dependent photolabelling site of azido AG-A on MRP1. 2. An epitope-inserted MRP1, MRP1 1222HA, which has two hemagglutinin A (HA) epitopes in the extracellular loop between transmembrane segment (TM) 16 and TM17 of the transporter, could bind azido AG-A in a GSH-dependent manner. 3. Protease digestion of the photolabelled MRP1 1222HA, followed by immunoprecipitation with an anti-HA antibody suggested that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. 4. Arg(1210) in human MRP2 that corresponds to Arg(1202) in human MRP1 has an important role in the transporting activity of MRP2. Therefore, we replaced the Arg residue at position 1202 of MRP1 with Gly. Whereas photolabelling of the mutant MRP1 R1202G was greatly reduced, it retained leukotriene C(4) (LTC(4)) transport activity and conferred Vincristine resistance in LLC-PK1 cells. 5. In summary, this study demonstrated that the GSH-dependent azido AG-A photolabelling site on MRP1 resides in the region within TM14-17 and the cytoplasmic region proximate to the C-terminus of TM17. The charged amino acid Arg(1202) proximate to TM helix 16 is of critical importance for the GSH-dependent photolabelling of MRP1 with azido AG-A. Arg(1202) itself or the region nearby Arg(1202) may be involved in azido AG-A photolabelling.