INWARD TRANSPORT OF H-3 MPP(- EVIDENCE FOR INTERACTION WITH CATECHOLAMINES() IN FRESHLY ISOLATED RAT HEPATOCYTES )

1-Methyl-4-phenylpyridinium (MPP(+)), the neurotoxic metabolite of 1-m ethyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mec hanism(s) involved in the hepatic uptake of MPP(+) remains partially u nknown. The aim of the present study was to further characterize the h epatic uptake of H-3-MPP(+), namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocy tes) with MPP(+) transport. The accumulation of H-3-MPP(+) in isolated rat hepatocytes occurred through saturable and non-saturable mechanis ms. The kinetics of the saturable component of H-3-MPP(+) uptake was a s follows: V-max = 181.3 +/- 11.1 pmol mg protein(-1) min(-1) and K-m = 47.1 mu M (27.9, 66.3) (n = 5). The diffusion constant (in ml mg pro tein(-1) min(-1)) for the non-saturable uptake of H-3-MPP(+) was 0.000 68 (0.00052, 0.00083) (n = 5). From the analysis of the time course of H-3-MPP(+) accumulation at a substrate concentration of 100 nM H-3-MP P(+), it was found that the rate constant of inward transport of H-3-M PP(+) into hepatocytes (k(in)) was 15.7 +/- 3.8 mu l mg protein(-1) mi n(-1) the rate constant of outward transport of H-3-MPP(+) from hepato cytes (k(out)) was 0.077 +/- 0.023 min(-1) and the equilibrium accumul ation (A(max)) of H-3-MPP(+) was 20.2 +/- 2.0 pmol mg protein(-1) (n = 36). Decynium22 (1,1'-diethyl-2,2'-cyanide; 1 mu M) significantly red uced k(in) to 6.1 +/- 1.8 mu l mg protein(-1) min(-1) (P < 0.05) and t he equilibrium accumulation (A(max)) of H-3-MPP(+) to 9.6 +/- 1.3 pmol mg protein(-1) (P < 0.005) (n = 36). H-3-MPP(+) accumulation (in cell s incubated with 200 nM H-3-MPP(+)) was sensitive to (-)-adrenaline, ( -)-isoprenaline, (-)-dopamine, (+/-)-adrenaline and (-)-noradrenaline. The most potent catecholamine in inhibiting H-3-MPP(+) uptake was (-) -adrenaline, with an IC50 of 99 (22, 449) mu M (n = 6). (-)-Adrenaline competitively inhibited H-3-MPP(+) uptake, as it significantly increa sed the K-m value of H-3-MPP(+) uptake (to 125.4 mu M (63.6; 187.1); P < 0.02; n = 3) but did not change the V-max value. The cyanine-deriva tives decynium22 and cyanine863 thyl-2-([1,4-dimethyl-2-phenyl-6-pyrim idinylidene] methyl)quinolinium), which inhibit uptake(2) as well as t he apical type of the renal transporter for organic cations, potently inhibited H-3-MPP(+) uptake with IC50's of 1.4 (0.4-5.3) (n = 6) and 6 .5 (2.6-16) (n = 4) mu M, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition (wit h either pargyline (500 mu M) + Ro 01-2812 (3,5-dinitropyrocatechol; 2 mu M) or pargyline (500 mu M) + U-0521 (3,4-dihidroxy-2-methyl-propio phenone; 12 mu M)), (-)-adrenaline (up to 1 mM) had no inhibitory effe ct on the uptake of H-3-MPP(+). Moreover, the uptake of H-3-MPP(+) in the presence of pargyline + Ro 01-2812 was significantly lower (66.9 /- 30.4%; P < 0.05; n = 4) than in the absence of these compounds. The refore, the effect of these MAO and COMT inhibitors on H-3-MPP(+) upta ke was examined. Interestingly enough, pargyline, Ro 01-2812 and U-052 1 were found to inhibit the uptake of H-3-MPP(+) (in cells incubated w ith 200 nM H-3-MPP(+)): 500 mu M pargyline, 2 mu M Ro 01-2812 and 100 mu M U-0521 decreased the accumulation of H-3-MPP(+) to 38.1 +/- 6.8 ( n = 5), 60.5 +/- 10.1 (n = 7) and 71.3 +/- 14.5 (n = 7) % of control, respectively. It is concluded that H-3-MPP(+) is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catech olamines, decynium22 and cyanine863, and to the enzyme inhibitors parg yline, Ro 01-2812 and U-0521.