Competitive and non-competitive inhibition of l-3,4-dihydroxyphenylalanine uptake in Opossum kidney cells

Abstract

The present study aimed to determine the kinetics of l-3,4-dihydroxyphenylalanine ( l-DOPA) uptake in Opossum kidney (OK) cells and to define the type of inhibition produced by l-5-hydroxytryptophan ( l-5-HTP), cyanine 863 and 3,3″-diethyloxacarbocyanine (3,3″-DOC). Non-linear analysis of the saturation curves revealed for l-DOPA a K m (in μM) of 129 (114, 145) and a V max (in nmol mg protein −1 6 min −1) of 30.0±0.4. IC 50 values for l-5-HTP (1454 μM) obtained in the presence of a nearly saturating (250 μM) concentration of l-DOPA were almost 4-fold those obtained when non-saturating (25 μM) concentrations of l-DOPA were used (330). IC 50 values for cyanine 863 and 3,3″-DOC (638 and 353 μM) obtained in the presence of a nearly saturating (250 μM) concentration of l-DOPA were similar to those obtained when non-saturating (25 μM) concentrations of l-DOPA were used (654 and 339 μM). V max values (in nmol mg protein −1 6 min −1) for l-DOPA uptake were identical in the absence (36.4±0.7) and the presence of l-5-HTP (39.2±1.3), but K m values ( μM) were significantly greater ( P<0.05) when l-DOPA uptake was studied in the presence of l-5-HTP (121 (100, 142) versus 318 (237, 399)). In contrast, the effect of cyanine 863 and 3,3″-DOC was to cause a significant reduction in V max values without significant changes in K m values. It is concluded that l-5-HTP exerts a competitive type of inhibition of l-DOPA uptake in cultured OK cells, whereas both cyanine 863, an organic cation transport inhibitor and 3,3″-DOC behave as non-competitive inhibitors.