Complex estrogenic regulation of catechol-O-methyltransferase (COMT) in rats.

Catechol-O-methyltransferase (COMT) activity depends on gender, age and physiological status suggesting that estrogen may regulate COMT activity. In fact, estrogens down-regulate the function of COMT promoters in cell cultures. On the other hand, COMT may play an important role in estrogen-induced cancers due to its ability to inactivate estrogen metabolites and thereby lowering the levels of these potential carcinogens. In this study, we explored the effect of estrogen on COMT activity in vivo in rats. Male and female Wistar rats received 14-day treatments with either estradiol (100 µg/kg/day; s.c.) or tamoxifen (500 µg/kg/day; s.c.), respectively; in addition ovariectomized rats were studied. COMT activity and COMT protein expression were measured from various brain- and peripheral tissues. Although we found a regulatory function of estrogen, its effects were sex and tissue dependent. Antagonizing the effects of estrogen via tamoxifen increased COMT protein expression in several central and peripheral tissues. However, amounts of COMT protein and COMT activities did not always match. Generally, COMT activities were quite resistant to the effects of tamoxifen and estradiol. Estradiol, unexpectedly, doubled the amount of COMT protein in the prostate but exhibited down-regulatory function in the prefrontal cortex and kidneys. Ovariectomy by itself, however, had only minor effects on COMT activity and expression. It is noteworthy that the estrogen down-regulation and tamoxifen up-regulation of COMT were best substantiated in the prefrontal cortex and kidneys where COMT is physiologically important for dopamine metabolism.

MPTP and MPP+ target specific aminergic cell populations in larval zebrafish.

Larval zebrafish offers a good model to approach brain disease mechanisms, as structural abnormalities of their small brains can be correlated to quantifiable behavior. In this study, the structural alterations in one diencephalic dopaminergic nucleus induced by 1-methyl-4-phenylpyridinium (MPP+), a toxin inducing Parkinson's disease in humans, and those found in several neuronal groups after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the pretoxin, were associated with decreased swimming speed. Detailed cell counts of dopaminergic groups indicated a transient decline of tyrosine hydroxylase expressing neurons up to about 50% after MPTP. The MPTP effect was partly sensitive to monoamine oxidase inhibitor deprenyl. Detailed analysis of the developing catecholaminergic cell groups suggests that the cell groups emerged at their final positions and no obvious significant migration from the original positions was seen. One 5-HT neuron group was also affected by MPTP treatment, whereas other groups remained intact, suggesting that the effect is selective. New nomenclature for developing catecholaminergic cell groups corresponding to adult groups is introduced. The diencephalic cell population consisting of groups 5,6 and 11 was sensitive to both MPTP and MPP+ and in this respect resembles mammalian substantia nigra. The results suggest that MPTP and MPP+ induce a transient functional deficit and motility disorder in larval zebrafish.

The COMT inhibitor, entacapone, reduces levodopa-induced elevations in plasma homocysteine in healthy adult rats.

Levodopa treatment has been shown to increase plasma homocysteine levels in Parkinson's disease (PD) patients and this may lead to an increased risk for coronary arterial diseases. Levodopa is metabolised via O-methylation by catechol-O-methyltransferase (COMT) using S-adenosyl-L-methionine (SAM) as the methyl donor, this leading to the subsequent formation of homocysteine. In this study, the effects of the COMT inhibitor, entacapone, on levodopa-induced hyperhomocysteinaemia were studied in rats. Using a single dose acute treatment paradigm, entacapone (10 or 30 mg/kg) prevented the levodopa (30 or 100 mg/kg) induced rise in plasma homocysteine levels in a dose-dependent manner. Five-day sub-chronic treatment with levodopa (3 x 100 mg/kg per day) resulted in a marked rise in plasma homocysteine levels when measured 2 hours post-treatment on Day 5. These levels fell but remained greater than baseline at 8 hours post-treatment on Day 5. Consistent with findings in the acute treatment test paradigm, the co-administration of entacapone (30 mg/kg) significantly (p<0.001) reduced levodopa-induced hyperhomocysteinaemia for up to 2 hours post-treatment on Day 5 of the sub-chronic study. These results suggest that entacapone may reduce levodopa-induced hyperhomocysteinaemia in PD patients.

Catecholamine metabolism in the brain by membrane-bound and soluble catechol-o-methyltransferase (COMT) estimated by enzyme kinetic values.

A kinetic model was constructed to reevaluate the catecholamine metabolism in hypothetical brain homogenates. Earlier published kinetic values of recombinant membrane-bound (MB-) COMT and soluble (S-) COMT were combined with data suggesting that MB-COMT represents 70% and 30% of total COMT protein in human and rat brain, respectively. In the rat brain model L-DOPA and 3,4-dihydroxybenzoic acid were O-methylated mainly via S-COMT, while dopamine and noradrenaline, at low concentrations, were O-methylated slightly more by MB-COMT. In the human brain model dopamine and noradrenaline were metabolized primarily by MB-COMT. The ratio of meta (3-methoxy) over para (4-methoxy) product formation from 3,4-dihydroxybenzoic acid was higher for MB-COMT than S-COMT. It is suggested that MB-COMT clearly predominates the O-methylation of dopamine and noradrenaline also in vivo. Additionally, meta/para ratios could support the enrichment of either isoform of COMT in a homogenate sample.

The role of dopamine-metabolizing enzymes in the regulation of renal sodium excretion in the rat.

The intrarenal natriuretic hormone dopamine (DA) is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). We have previously shown that inhibition of COMT by entacapone results in a potent D1-like receptor-mediated natriuretic response. The present study was performed using anaesthetized rats to compare the importance of MAO and COMT in DA-mediated natriuresis by use of the MAO inhibitor phenelzine. Urinary sodium and DA excretion remained unchanged after MAO inhibition, while excretion of the main metabolite dihydroxyphenylacetic acid (DOPAC) decreased by 55%. The response was unaltered if 5-hydroxytryptamine receptors (5-HT1A) were blocked during MAO inhibition. We also investigated the specific renal activities of MAO and COMT in rat renal cortex during DA-influenced natriuresis. Specific COMT activity in the renal cortex was reduced by 13% after isotonic sodium loading (5% of body mass) whereas renal MAO-A and MAO-B activities remained unaltered. Furthermore, preliminary data obtained from spontaneously hypertensive rats, whose basal urinary DA excretion is higher than that of normotensive Wistar-Kyoto rats, show a tendency for renal COMT activity to be lower. It is concluded that MAOinhibition by phenelzine does not alter sodium excretion. Furthermore, specific renal cortical COMT activity is reduced during partly D1-like receptor-mediated natriuresis, whereas MAO activity remains unchanged. The results suggest that MAO is less important than COMT in regulating DA-mediated natriuresis in the rat kidney.

Effect of intracerebral 6-nitronoradrenaline, an endogenous catechol-O-methyltransferase (COMT) inhibitor, on striatal dopamine metabolism in anaesthetised rats.

6-Nitronoradrenaline, a bioactive compound recently identified in the brain, is known to inhibit catechol-O-methyltransferase. To study its effect on dopamine metabolism, it was administered into rat striatum via a microdialysis probe. Other nitrated catechols (6-nitrodopamine, 6-nitro-DOPAC and 5-nitro-HVA) were studied for comparison. Tolcapone, a selective catechol-O-methyltransferase inhibitor, was used as a positive reference compound. Both 6-nitronoradrenaline and tolcapone increased striatal extracellular dopamine levels during the perfusion (at 100 microM concentration but not at 10 microM) and decreased the efflux of homovanillic acid. Tolcapone, but not other nitrated catechols, increased 3,4-dihydroxyphenylacetic acid efflux. None of the compounds inhibited MAO-B activity at 100 microM or lower. At 1 mM, 6-nitrodopamine inhibited MAO-B by 60%. Compared to tolcapone, other nitrated catechols were very weak COMT inhibitors in vitro. Neither tolcapone nor 6-nitronoradrenaline modified the metabolism of L-dopa which was given peripherally. In binding studies, both 6-nitronoradrenaline and other nitrocatechols failed to affect the dopamine transporter even at high micromolar concentrations. In conclusion, exogenous 6-nitronoradrenaline can act as a COMT inhibitor in the striatum and elevate striatal dopamine levels without inhibiting dopamine reuptake. Whether endogenous 6-nitronoradrenaline can be formed also in vivo in the striatum and act as a regulator of dopaminergic tone remains to be determined.

Regulation of dopamine-induced natriuresisby the dopamine-metabolizing enzyme catechol-O-methyltransferase.

Dopamine (DA) is an intrarenal natriuretic hormone involved in sodium homeostasis. A study was performed to elucidate two possible regulatory pathways of DA-induced natriuresis, i.e., metabolism and precursor delivery. This was done by use of an intraperitoneal injection of a catechol-O-methyltransferase (COMT) inhibitor, entacapone, or intravenous infusion of the DA precursor, L-dopa. Entacapone (30 mg/kg i.p.) induced a more than fivefold increase in renal sodium excretion which occurred without changes in renal haemodynamics. The natriuretic response was highly dependent on DA D(1)-like receptor activation, since the selective D(1)-like receptor antagonist SCH23390 attenuated the natriuretic response by 61%, while the selective D(2)-like receptor antagonist sulpiride was ineffective. The urinary excretion of DA did not increase. Infusion of L-dopa (60 microg/h/kg) only induced a twofold increase in sodium excretion, but the urinary excretion of DA increased more than 17-fold. The L-dopa-induced natriuretic response occurred without increments in glomerular filtration rate and could be blocked with the D(1)-like receptor antagonist SCH23390. It is concluded that the DA-metabolizing enzyme COMT is involved in the regulation of the natriuretic effect of intrarenal DA. It may be speculated that intrarenal DA activity is not primarily determined on the basis of delivered precursor, but on that of the level of DA metabolism.

Increase of catechol-O-methyltransferase activity in rat brain microglia after intrastriatal infusion of fluorocitrate, a glial toxin.

Striatal catechol-O-methyltransferase (COMT), monoamine oxidase B (MAO-B; an astroglial enzyme), alkaline phosphodiesterase I (PDE; a microglia/macrophage marker) and tyrosine hydroxylase (TH; catecholaminergic neuron marker) activities were analyzed biochemically 1-3 days after infusion of fluorocitrate, an astrocyte damaging agent. Astrocytes, microglia and neurons were stained immunohistochemically with specific antibodies (against glial fibrillary acidic protein, OX-42 and TH, respectively) and with COMT antiserum. Three days after fluorocitrate infusion the activity of MAO-B was reduced, whereas COMT and PDE activities were increased. The elevation of COMT immunoreactivity co-localized to microglial cells, but not to astrocytes. In conclusion, this is the first report indicating that microglia contains COMT activity which may be increased in pathological conditions.

Validation of assay of catechol-O-methyltransferase activity in human erythrocytes.

The multistep assay of specific catechol-O-methyltransferase (COMT) activity in human erythrocytes was validated. Enzyme preparations from lysed erythrocytes were incubated with a substrate (3,4-dihydroxybenzoic acid) in the presence of Mg2+ and S-adenosylmethionine. The reaction products (vanillic acid and isovanillic acid) were analyzable by HPLC with electrochemical detection directly in the incubation medium after protein precipitation. The precision was calculated in order to define the random variability associated with the method by intra-assay and inter-assay relative standard deviations (RSDs) for the assays of both reaction products and protein. The intra-assay RDSs for the specific activities were between 4.8 and 11.9% (n = 5-6) at two levels of COMT activity. The inter-assay RSDs were between 6.4 and 14.2% (n = 5-6), respectively. The total variation was mostly caused by the protein assay and the HPLC assay, and contributions from the sample preparation and incubation steps were minor. Some results from the clinical application of the erythrocyte COMT assay are also reported. For both normal volunteers and patients having Parkinson's disease, a single 400 mg dose of entacapone, a peripherally acting COMT inhibitor, decreased the erythrocyte COMT activity. The application demonstrates that the assay was able to detect differences between the subjects and the effect of COMT inhibition in the clinical study.

Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection.

We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day.