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

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) microM (n = 5); L-DOPA, Vmax = 58.0 +/- 4.3 nmol mg-1 protein h-1 and Km = 135 (97, 188) microM (n = 5). When the saturation curve of L-5-HTP tubular uptake was performed in the presence of L-DOPA (250 microM), 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] microM, n = 4). 3. L-DOPA (50 to 2000 microM) was found to produce a concentration-dependent decrease (38% to 91% reduction) in the tubular uptake of 5-HTP; the Ki value (in microM) 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 microM) 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]-quino linium) 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] microM; 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] microM, 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] microM, 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.