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 (tropolone), non-competitive (S-adenosyl-l-homocysteine; SAHC) and tight-binding (tolcapone and 3,5-dinitrocatechol) inhibitors. COMT activity was evaluated by the ability to methylate adrenaline (0.1 to 2000 microM) 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 micrograms/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 and 3 nM, respectively), whereas in liver the IC50 values for tolcapone against liver MB- and S-COMT (IC50's of 123 and 795 nM, respectively) were markedly higher than those observed in the brain. By contrast, when inhibition studies were performed with a fixed concentration of COMT (15 nM), as determined by the Ackermann-Potter equation, tolcapone was found to be endowed with 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 potent 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 used. Tropolone, a competitive inhibitor, was much less potent than tolcapone and 3,5-dinitrocatechol in inhibiting S- and MB-COMT from both brain and liver 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 amount of enzyme was used, SAHC was found to be endowed with the same potency against S- and MB-COMT from brain and liver. In the final series of experiments the inhibitory effect of tolcapone was examined under in vitro ex vivo 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 tolcapone 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 activity was reduced by 38% only. In conclusion, the results reported here show that tolcapone is particularly potent in inhibiting MB-COMT from liver and brain under in vivo experimental conditions, though it does not discriminate between MB- and S-COMT under in vitro experimental conditions when using the same amount of enzyme in the assay. Copyright 1999 Elsevier Science B.V.