Effects of tolcapone upon soluble and membrane-bound brain and liver catechol-O-methyltransferase

The present study was aimed to evaluate the sensitivity of soluble (S) and membrane bound (MB) catechol-O-methyltransferase (COMT) from rat brain and liver to inhibitors which interact with the enzyme as competitive (tropolon e), non-competitive (S-adenosyl-L-homocysteine; SAHC) and tight-binding (to lcapone and 3,5-dinitrocatechol) inhibitors. COMT activity was evaluated by the ability to methylate adrenaline (0.1 to 2000 mu M) to metanephrine in the presence of a saturating concentration of the methyl donor (S-adenosyl- L-methionine). When using a fixed amount of total protein (2 mu g/ml), but variable concentrations of COMT, the inhibitory potency of tolcapone upon S - and MB-COMT activity in the brain was in the low nM range (IC50's of 2 an d 3 nM, respectively), whereas in Liver the IC50 values for tolcapone again st liver MB- and S-COMT (IC50's of 123 and 795 nM, respectively) were marke dly higher than those observed in the brain. By contrast, when inhibition s tudies were performed with a fixed concentration of COMT (15 nM), as determ ined by the Ackermann-Potter equation, tolcapone was found to be endowed wi th the same potency (in the low nM range) in inhibiting S- and MB-COMT from both brain and liver. As for tolcapone, 3,5-dinitrocatechol was more poten t against MB- than against S-COMT when a fixed amount of total protein was used, but showed the same potency when a fixed concentration of COMT was us ed. Tropolone, a competitive inhibitor, was much less potent than tolcapone and 3,5-dinitrocatechol in inhibiting S- and MB-COMT from both brain and l iver and its potency was found not to depend on enzyme concentration. SAHC, a non-competitive inhibitor, behaved similarly to tight-binding inhibitors when a fixed amount of total protein was used. By contrast, when a fixed a mount of enzyme was used, SAHC was found to be endowed with the same potenc y against S- and MB-COMT from brain and liver. In the final series of exper iments the inhibitory effect of tolcapone was examined under in vitro ex vi vo conditions, using the same concentration of COMT (15 nM). One hour after its oral administration, tolcapone (0.3 to 30 mg/kg) was found to be much more potent against MB-COMT than against S-COMT. In the liver, 0.3 mg/kg to lcapone resulted in 82% inhibition of MB-COMT and 31% inhibition of S-COMT. In the brain, 3.0 mg/kg tolcapone inhibited 78% MB-COMT, whereas S-COMT ac tivity was reduced by 38% only. In conclusion, the results reported here sh ow that tolcapone is particularly potent in inhibiting MB-COMT from liver a nd brain under in vivo experimental conditions, though it does not discrimi nate between MB- and S-COMT under in vitro experimental conditions when usi ng the same amount of enzyme in the assay. (C) 1999 Elsevier Science B.V. A ll rights reserved.