Antagonistic actions of renal dopamine and 5‐hydroxytryptamine: effects of amine precursors on the cell inward transfer and decarboxylation

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Abstract
1 The present work was designed to examine the interference of L‐3,4‐dihydroxyphenylalanine (L‐DOPA) on the cell inward transport of L‐5‐hydroxytryptophan (L‐5‐HTP) and on its decarboxylation by aromatic L‐amino acid decarboxylase (AAAD) in rat isolated renal tubules. 2 The accumulation of both L‐5‐HTP and L‐DOPA in renal tubules was found to occur through non‐saturable and saturable mechanisms. The kinetics of the saturable component L‐5‐HTP and L‐DOPA uptake in renal tubules were as follows: L‐5‐HTP, Vmax = 24.9 ± 4.5 nmol mg−1 protein h−1 and Km = 121 (95% confidence limits: 75, 193) μm (n = 5); L‐DOPA, Vmax = 58.0 ± 4.3 nmol mg−1 protein h−1 and Km = 135 (97, 188) μm (n = 5). When the saturation curve of L‐5‐HTP tubular uptake was performed in the presence of L‐DOPA (250 μm), the maximal rate of accumulation of L‐5‐HTP in renal tubules was found to be markedly (P < 0.01) reduced (Vmax = 10.5 ± 1.7 nmol mg−1 protein h−1, n = 4); this was accompanied by a significant (P < 0.05) increase in Km values (325 [199, 531] μm, n = 4). 3 L‐DOPA (50 to 2000 μm) was found to produce a concentration‐dependent decrease (38% to 91% reduction) in the tubular uptake of 5‐HTP; the Ki value (in μm) of L‐DOPA for inhibition of L‐5‐HTP uptake was found to be 29.1 (13.8, 61.5) (n = 6). 4 At the highest concentration tested the organic anion inhibitor, probenecid (10 μm) produced no significant (P = 0.09) changes in L‐5‐HTP and L‐DOPA uptake (18% and 22% reduction, respectively). The organic cation inhibitor, cyanine 863 (1‐ethyl‐2‐[1,4‐dimethyl‐2‐phenyl‐6‐pyrimidinylidene)methyl]‐quinolinium) produced a potent inhibitory effect on the tubular uptake of L‐5‐HTP (Ki = 212 [35, 1289] nM, n = 8), being slightly less effective against L‐DOPA uptake (Ki = 903 [584, 1396] nM, n = 5). The cyanine derivatives 1,1‐diethyl‐2,4‐cyanine (decynium 24) and 1,1‐diethyl‐2,2‐cyanine (decynium 22) potently inhibited the tubular uptake of both L‐5‐HTP (Ki = 100 [49, 204] and 120 [26, 561] nM, n = 4–6, respectively) and L‐DOPA (Ki = 100 [40, 290] and 415 [157, 1094] nM, n = 5, respectively). 5 The Vmax and Km values for AAAD using L‐DOPA as the substrate (Vmax = 479.9 ± 74.0 nmol mg−1 protein h−1; Km = 2380 [1630, 3476] μm; n = 4) were both found to be significantly (P < 0.01) higher than those observed when using L‐5‐HTP (Vmax = 81.4 ± 5.2 nmol mg−1 protein h−1, Km = 97 [87, 107] μm, n = 10). The addition of 5 mM L‐DOPA to the incubation medium reduced by 30% (P < 0.02) the maximal rate of decarboxylation of L‐5‐HTP (Vmax = 56.7 ± 3.1 nmol mg−1 protein h−1, n = 10) and resulted in a significant (P < 0.05) increase in Km values (249 [228, 270] μm, n = 10). 6 The results presented suggest that L‐5‐HTP and L‐DOPA are using the same transporter (most probably, the organic cation transporter) in order to be taken up into renal tubular cells; L‐DOPA exerts a competitive type of inhibition upon the tubular uptake and decarboxylation of L‐5‐HTP. The decrease in the formation of 5‐HT as induced by L‐DOPA may also depend on a decrease in the rate of its decarboxylation by AAAD.