Inward transport of 3H-MPP+ in freshly isolated rat hepatocytes: evidence for interaction with catecholamines.
1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mechanism(s) involved in the hepatic uptake of MPP+ remains partially unknown. The aim of the present study was to further characterize the hepatic uptake of 3H-MPP+, namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocytes) with MPP+ transport. The accumulation of 3H-MPP+ in isolated rat hepatocytes occurred through saturable and non-saturable mechanisms. The kinetics of the saturable component of 3H-MPP+ uptake was as follows: Vmax = 181.3 +/- 11.1 pmol mg protein-1 min-1 and Km = 47.1 microM (27.9, 66.3) (n = 5). The diffusion constant (in ml mg protein-1 min-1) for the non-saturable uptake of 3H-MPP+ was 0.00068 (0.00052, 0.00083) (n = 5). From the analysis of the time course of 3H-MPP+ accumulation at a substrate concentration of 100 nM 3H-MPP+, it was found that the rate constant of inward transport of 3H-MPP+ into hepatocytes (k(in)) was 15.7 +/- 3.8 microliters mg protein-1 min-1, the rate constant of outward transport of 3H-MPP+ from hepatocytes (kout) was 0.077 +/- 0.023 min-1 and the equilibrium accumulation (Amax) of 3H-MPP+ was 20.2 +/- 2.0 pmol mg protein-1 (n = 36). Decynium22 (1,1'-diethyl-2,2'-cyanide; 1 microM) significantly reduced kin to 6.1 +/- 1.8 microliters mg protein-1 min-1 (P < 0.05) and the equilibrium accumulation (Amax) of 3H-MPP+ to 9.6 +/- 1.3 pmol mg protein-1 (P < 0.005) (n = 36). 3H-MPP+ accumulation (in cells incubated with 200 nM 3H-MPP+) was sensitive to (-)-adrenaline, (-)-isoprenaline, (-)-dopamine, (+/-)-adrenaline and (-)-noradrenaline. The most potent catecholamine in inhibiting 3H-MPP+ uptake was (-)-adrenaline, with an IC50 of 99 (22, 449) microM (n = 6). (-)-Adrenaline competitively inhibited 3H-MPP+ uptake, as it significantly increased the Km value of 3H-MPP+ uptake (to 125.4 microM (63.6; 187.1); P < 0.02; n = 3) but did not change the Vmax value. The cyanine-derivatives decynium22 and cyanine863 (1-ethyl-2-([1,4-dimethyl-2-phenyl-6-pyrimidinylidene] methyl)quinolinium), which inhibit uptake2 as well as the apical type of the renal transporter for organic cations, potently inhibited 3H-MPP+ uptake with IC50's of 1.4 (0.4-5.3) (n = 6) and 6.5 (2.6-16) (n = 4) microM, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition (with either pargyline (500 microM) + Ro01-2812 (3,5-dinitropyrocatechol; 2 microM) or pargyline (500 microM) + U-0521 (3,4-dihidroxy-2-methyl-propiophenone; 12 microM)), (-)-adrenaline (up to 1 mM) had no inhibitory effect on the uptake of 3H-MPP+. Moreover, the uptake of 3H-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. Therefore, the effect of these MAO and COMT inhibitors on 3H-MPP+ uptake was examined. Interestingly enough, pargyline, Ro 01-2812 and U-0521 were found to inhibit the uptake of 3H-MPP+ (in cells incubated with 200 nM 3H-MPP+): 500 microM pargyline, 2 microM Ro 01-2812 and 100 microM U-0521 decreased the accumulation of 3H-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 3H-MPP+ is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catecholamines, decynium22 and cyanane863, and to the enzyme inhibitors pargyline, Ro 01-2812 and U-0521.
Martel
F
F
Department of Biochemistry, Faculty of Medicine, Porto, Portugal.
Martins
M J
MJ
Azevedo
I
I
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000502:physiology*; D002395:Catecholamines / Q000378:metabolism*; D015259:Dopamine Agents / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008995:Monoamine Oxidase / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000037:antagonists & inhibitors; D011726:Pyridinium Compounds / Q000493:pharmacokinetics*; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D002352:Carrier Proteins; D002395:Catecholamines; D015259:Dopamine Agents; D018377:Neurotransmitter Agents; D011726:Pyridinium Compounds; D011804:Quinolines; C100254:pseudoisocyanine; C026560:1-(4-methoxyphenyl)pyridinium; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D008995:Monoamine Oxidase