Interaction between L-DOPA and 3-O-methyl-L-DOPA for transport in immortalised rat capillary cerebral endothelial cells

The present study aimed to determine the kinetics of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4 and RBE 4B), to define the type of interaction with 3-O-methyl-L-DOPA (3-OM-L-DOPA), sensitivity to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC), N-(methylamino)-isobutyric acid (MeAIB) and sodium. Non-linear analysis of the saturation curves for L-DOPA and 3-OM-L-DOPA revealed in RBE 4 cells K-m values (in mu M) of 72 (53, 91) and 40 (25, 57) and in R BE 4B cells K-m values (in mu M) of 60 (46, 74) and 44 (13, 75), respectively. IC50 values for S-OM-L-DOPA (RBE 4, 642 [542, 759] mu M; RBE 4B, 482 [475, 489] mu M) obtained in the presence of a nearly saturating (250 mu M) concentration of L-DOPA were greater than the corresponding K-i values (RBE 4, 143 [121, 170] mu M; RBE 4B, 93 [92, 95] mu M) obtained in the presence of a nearly saturating (250 mu M) concentration of 3-OM-L-DOPA; this is compatible with a competitive type of interaction between L-DOPA and 3-OM-L-DOPA. Uptake of both L-DOPA and 3-OM-L-DOPA in RBE 4 and RBE 4B cells was sensitive to BHC with similar IC50 values. MeAIB (up to 2.5 mM) was found not to interfere with the uptake of both L-DOPA and 3-OM-L-DOPA. Uptake of (250 mu M) L-DOPA and 3-OM-L-DOPA in the absence of sodium in the incubation medium was similar to that observed in the presence of increasing concentrations of sodium (20-140 mM). Homogenates of both cell lines were endowed with considerable COMT activity. Incubation of RBE 4 and RBE 4B cells with L-DOPA (25 mu M) in the presence of a methyl donor (S-adenosyl-L-methionine) resulted in the formation of 3-OM-L-DOPA; this was abolished by 1 mu M tolcapone. The fractional outflow of intracellular L-DOPA through the luminal and abluminal cell side was not affected by the presence of intracellular 3-OM-L-DOPA. The fractional outflow of exogenous 3-OM-L-DOPA applied from the luminal cell border was similar to that observed for 3-OM-L-DOPA with origin in L-DOPA. It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which L-DOPA and 3-OM-L-DOPA can be taken up, and 3-OM-L-DOPA behaves as a competitive inhibitor for the uptake of L-DOPA. This, however, only occurs for luminal cell inward movement but not for abluminal cell outward movement of the substrates. (C) 1999 Elsevier Science Ltd. All rights reserved.