Importance of membrane-bound catechol-O-methyltransferase in L-DOPA metabolism: a pharmacokinetic study in two types of Comt gene modified mice

Abstract

Background and purpose: Catechol-O-methyltransferase (COMT) metabolizes compounds containing catechol structures and has two forms: soluble (S-COMT) and membrane-bound (MB-COMT). Here we report the generation of a mouse line that expresses MB-COMT but not S-COMT. We compared the effects of deleting S-COMT only or both COMT forms on the pharmacokinetics of oral L-DOPA.

Experimental approach: L-DOPA (10 mg kg(-1)) and carbidopa (30 mg kg(-1)) were given to mice by gastric tube, and samples were taken at various times. HPLC was used to measure L-DOPA in plasma and tissue samples, and dopamine and its metabolites in brain. Immunohistochemistry and Western blotting were used to characterize the distribution of COMT protein isoforms.

Key results: Lack of S-COMT did not affect the levels of L-DOPA in plasma or peripheral tissues, whereas in the full COMT-knock-out mice, these levels were increased. The levels of 3-O-methyldopa were significantly decreased in the S-COMT-deficient mice. In the brain, L-DOPA levels were not significantly increased, and dopamine was increased only in females. The total COMT activity in the S-COMT-deficient mice was 22-47% of that in the wild-type mice. In peripheral tissues, female mice had lower COMT activity than the males.

Conclusions and implications: In S-COMT-deficient mice, MB-COMT in the liver and the duodenum is able to O-methylate about one-half of exogenous L-DOPA. Sexual dimorphism and activity of the two COMT isoforms seems to be tissue specific and more prominent in peripheral tissues than in the brain.

Figure 1

(A) Constructed ∼17 kb targeting vector containing ∼2.5 kb long 5′ homology arm up to Mutated MET2, from Mut-MET2 to floxTK/NEO cassete ∼1.4 kb central arm, ∼4.7 kb floxTKNeo cassette and ∼4 kb short 3′ homology arm. Positions of the Comt exons, vector and selection cassettes are indicated as well as the restriction sites used to linearize and the LoxP1 sites flanking the Neo and TK cassettes removed after recombination in ES cells. Greater detail of the sequence around Met2 in Exon 2 is shown. The DNA sequence of soluble catechol-O-methyltransferase (S-COMT)-deficient mutant is shown, and the sequence of the corresponding protein product is aligned on top (wild type) and bottom (S-COMT). The bases mutated are shown in bold font. A new AgeI restriction site (ACCGGT) for genotyping was introduced along with the Met44Thr mutation. (B) PCR genotyping of S-COMT-deficient animals showing wild-type (S-COMT +/+), heterozygous (S-COMT +/−) and homozygous (S-COMT −/−) genotypes consisting of wild-type allele, mutated allele or both alleles. (C) Western blot analysis visualizing the long (28 kDa) and short (24 kDa) catechol-O-methyltransferase (COMT) transcripts in the liver. (D) Total COMT activities in the liver, duodenum, striatum and prefrontal cortex (PFC) of wild-type (WT) and S-COMT-deficient (S-COMT −/−) mice. ⁺P < 0.05 and ⁺⁺⁺P < 0.001 differs significantly from corresponding male. ***P < 0.001 differs significantly from corresponding wild-type mice. n (male/female) = 12/12 (WT) and 6/6 (S-COMT −/−).

Figure 2

Light-microscopic photomicrographs presenting the distribution of COMT in the duodenal (A–C) and liver (D–F) tissue of wild-type (WT), soluble catechol-O-methyltransferase (S-COMT)-deficient (S-COMT −/−) and catechol-O-methyltransferase (COMT) knock-out (COMT −/−) mice. COMT was visualized with DAB (brown colour) and nuclei were counterstained with Mayer's haematoxylin (blue colour). The lack of S-COMT clearly reduced the intensity of COMT-immunostaining. Moreover, in the S-COMT-deficient liver cells (E), COMT is seen more in the intracellular membranes than the plasma membrane. Scale bars are 20 µm (A–C) and 30 µm (D–F).

Figure 3

Effect of catechol-O-methyltransferase (COMT) genotype [soluble catechol-O-methyltransferase (S-COMT) deficiency or COMT knock-out] on the time course and AUC_{0–120 min} of the plasma (A), liver (B) and duodenal (C) L-DOPA concentrations after oral administration of L-DOPA and carbidopa (10 mg·kg⁻¹ and 30 mg·kg⁻¹ respectively). The data represent group means ± SEM. ⁺P < 0.05 differs significantly from corresponding male. ^#P < 0.05 and ^##P < 0.01 differs significantly from corresponding S-COMT deficient mice. **P < 0.01 and ***P < 0.001 differs significantly from corresponding wild-type (WT) mice. n (male/female) = 13/13 (WT), 6/7 (S-COMT −/−) and 6/7 (COMT −/−).

Figure 4

The effect of catechol-O-methyltransferase (COMT) genotype [soluble catechol-O-methyltransferase (S-COMT) deficiency or COMT knock-out] on the time course and AUC_{0–120 min} of the plasma (A), liver (B) and duodenal (C) 3-O-methyl DOPA (3-OMD) levels after oral administration of L-DOPA and carbidopa (10 mg·kg⁻¹ and 30 mg·kg⁻¹ respectively). The data represent group means ± SEM. ⁺P < 0.05 and ⁺⁺⁺P < 0.001 differs significantly from corresponding male. ^###P < 0.001 differs significantly from corresponding S-COMT-deficient mice. *P < 0.05 and ***P < 0.001 differ significantly from corresponding wild-type (WT) mice. n (male/female) = 13/13 (WT), 6/7 (S-COMT −/−) and 6/7 (COMT −/−).

Figure 5

The effect of genotype [soluble catechol-O-methyltransferase (S-COMT) deficiency or catechol-O-methyltransferase (COMT) knock-out] on the AUC_{0–120 min} of striatal and prefrontal cortex (PFC) levels of L-DOPA (A), 3-O-methyl DOPA (3-OMD) (B), dopamine (C), dihydroxyphenylacetic acid (DOPAC) (D) and HVA (E) after oral administration of L-DOPA and carbidopa (10 mg·kg⁻¹ and 30 mg·kg⁻¹ respectively). The data represent group means ± SEM. ⁺P < 0.05 and ⁺⁺P < 0.01 differ significantly from corresponding male. ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 differ significantly from corresponding S-COMT-deficient mice. *P < 0.05, **P < 0.01 and ***P < 0.001 differ significantly from corresponding wild-type (WT) mice. n (male/female) = 13/13 (WT), 6/7 (S-COMT −/−) and 6/7 (COMT −/−).