Vanillic and isovanillic acid were separated well with RP-HPLC using coulometric detection and no interfering peaks were seen. Due our excellent detection capabilities, both reaction products could be seen at low substrate concentrations (Fig. 4). Reproducibility of the analysis was tested for the reaction products. A summary of the characteristics is presented in Table 2. Compared to earlier method utilizing amperometric detection (Nissinen and Männistö 1984), the limit of detection was 10 times lower with only half of the injection volume. In the studied concentration range, the reaction product analysis was linear with less than 10 % variation in precision and accuracy. The precision of the analysis decreased when the same sample was analyzed on subsequent days and additionally when the reaction was made from the same homogenate pool and finally the lowest precision (RSD 37.8 %) was seen when different tissue samples were analyzed. The meta/para ratio calculated for the striatal homogenates was 6.3 (I) and 8.6 (II) suggesting preferential metabolism through S-COMT rather than MB-COMT since at the same reaction conditions the meta/para ratio was closer to that obtained with recombinant S-COMT than that of recombinant MB-COMT (II). In the WKY rats used in the kidney experiments (V) the specific
Figure 4. Chromatograms of A) 0.1 pmol calibration sample (10 ml injection), reaction products obtained from B) recombinant MB-COMT (5 ml injection) and C) recombinant MB-COMT assayed with 1000 mM concentration of ethanol (10 ml injection). The substrate (DHBAc) concentration was 12.5 mM. Peaks: 1=vanillic acid and 2=isovanillic acid. The bar at y-axis denotes 10 nA.
Table 2. Summary of the validation of COMT activity analysis by reversed phase high-performance liquid chromatography with coulometric detection (I). The results are mean ± SD.
COMT activity in the whole brains was 8.52 ± 0.15 pmol/min/mg, which is about one fifth of that in striatal homogenates of the Wistar rats used in other studies (I-III). The brain and kidney specific COMT activities were lower than those of isolated S-COMT but higher than MB-COMT. For example, the specific activites of 86.6 pmol/min/mg protein and 16.5 pmol/min/mg protein for rat brain S-COMT and MB-COMT have been reported (Nissinen 1985). This was apparently due to the use of the lower substrate concentration and unpurified the COMT enzyme preparation. Meta/para ratios were about 2.5.
Kinetics. Kinetic values for the formation of vanillic acid were determined for the recombinant MB-COMT and S-COMT enzymes (II). Apparent Km values were 27.2 ± 1.4 mM and 136 ± 11 mM for recombinant MB-COMT and S-COMT, respectively. The corresponding Vmax values, expressed as mM product formed in 30 min, were 1.8 ± 0.2 and 4.6 ± 1.4. These values agree well with the fact that recombinant MB-COMT has a higher affinity but lower methylation capacity than recombinant S-COMT. The meta/para ratios decreased non-significantly with recombinant MB-COMT from 19 to 13 with increasing substrate concentrations (12.5-300 mM of DHBAc concentration) and remained the same with recombinant S-COMT (from 5.2 to 5.5 with 25-500 mM of DHBAc concentration).
Cell cultures. The analysis of the COMT reaction products from cell culture studies (IV) was performed in a similar way. Artificial CSF with glucose supplement was used since the cell culture media produced background in the chromatograms. The COMT inhibitors did not interfere with the detection system. The reaction with increasing concentrations (12.5-400 uM) of DHBAc was in most cases linear with glial and cocultures (data not shown). Generally, the production of isovanillic acid was below the detection limit and could not be analyzed. A few meta/para ratios suggested a high value (more than 20) which could indicate that most of the metabolism was carried out by MB-COMT compared to S-COMT.
Lesion studies. Intrastriatal infusion of fluorocitrate, a glial toxin, at 4 nmol dose started to decrease insignificantly striatal COMT activity after 12 h (Fig. 5A) decreasing further at 24 h and 48 h (19 % and 24 %, respectively) (III). The two nmol dose followed insignificantly the same pattern. Surprisingly, after 72 h COMT activity increased with both 2 and 4 nmol doses of fluorocitrate infusion (62 % and 73 % respectively). The meta/para ratio was changed by +30 %, +4% and -7 % after 24 h, 48 h and 72 h, respectively, at 2 nmol dose of fluorocitrate while at 4 nmol dose of fluorocitrate the meta/para ratio was decreased by 3-8 % at the these timepoints. None of these changes were statistically significant. The control meta/para ratios (mean ± sem) with the 2 nmol dose of fluorocitrate were 9.4 ± 1.8, 11.1 ± 2.9 and 8.4 ± 0.91 for 24 h, 48 h and 72 h, respectively, and the control ratios (mean ± sem) with the 4 nmol dose of fluorocitrate were 8.3 ± 0.6, 7.5 ± 0.46 and 8.4 ± 0.45 for 24 h, 48 h and 72 h, respectively. MAO B activity, a marker for astroglia, remained below control levels more predictably with the 2 nmol dose of fluorocitrate throughout the studied period. Alk-PDE activity, a marker of macrophages/microglia, was increased significantly with the 4 nmol dose of fluorocitrate at 48 h and at 72 h with both doses of fluorocitrate. TH activity, a dopaminergic neuronal marker, gave variable results and was not affected significantly by fluorocitrate during the study (Fig. 5B). The control values for specific TH activity were (mean ± sem) 336.8 ± 78.9, 388.7 ± 28.3 and 382.4 ± 54.7 pmol/min/mg protein at 24 h, 48 h and 72 h, respectively, with the 2 nmol dose of fluorocitrate and with the 4 nmol dose of fluorocitrate 508.6 ± 34.6, 615.3 ± 97.4 and 553.7 ± 47.7 pmol/min/mg protein at 24 h, 48 h and 72 h, respectively (n=5-20).
Figure 5. Time course of striatal enzyme activities after intrastriatal infusion of fluorocitrate. A) COMT activity (modified from Fig. 1, III) and B) tyrosine hydroxylase activity. Mean values and sems are presented. Individual specific activities were compared with control side and calculated with paired t-test, * p<0.05, ** p<0.01, n = 3-28.
Immmunohistochemical analysis of the toxin treated rat striata (III), revealed a distinct staining pattern by TH and GFAP (astroglial marker) antisera in control sides of the striata while COMT staining was low and inconclusive with respect to a definitive cellular localization. No OX-42 (microglial marker) immunoreactivity was observed. Fluorocitrate, especially 72 hours after the infusion, caused a decrease of TH and GFAP immunoreactivities in the injection region and an increase of distinguishable COMT reactivity which colocalized with OX-42 in double staining. Further away from the injection site, TH staining was increased while GFAP staining was comparable to control stainings.
Cell cultures. Primary brain cell cultures (IV) were partially characterized by using immunohistochemistry with antiserums against GFAP, an astroglial marker, and against NSE, a neuronal cell marker. The amount of immunopositive cells in a culture was classified and scored from 0 to 5. The ratios expressed in (IV) were calculated from the means of the results shown in Fig 6. All the glial cultures were immunoreactive with GFAP. In neuronal cultures, 1-day basal forebrain was the most neuron-enriched. The number of GFAP positive cells increased during growth from 1 to 6-7 days indicating glial proliferation. In glial/neuronal cocultures, the immunoreactivity was so intense that no quantification could be done. Approximately half of cells were of glial and half were of neuronal origin.
Figure 6. Immunohistochemical characterization of rat brain primary cultured cells. The number of GFAP or NSE stained cells were scored from 0 (no or low amount of stained cells) to 5 (all or almost all cells stained) and the mean + sem for each culture type was calculated, n = 1-7.
The basal COMT activities were similar as found in other studies with striatal tissues (I-III). Glial cells, prepared from various parts of the rat brain, displayed similar COMT activity indicating about equal distribution between different parts of the brain (Fig. 7). Cerebellar glial cultures, which had the highest COMT activity, differed from both 1-day neuron-enriched cultures and from both glial/neuronal cocultures. In other glial cultures, a partial glial dominance of COMT activity over neurons was also found compared to basal forebrain neuron-enriched cell cultures. COMT activity in striatal and hypothalamic glial cultures, which did not differ from each other, was higher than in 1-day basal forebrain neuron-enriched culture.
Figure 7. Basal specific COMT activities in primary cultures of the rat brain cells. Values are mean + sem, statistics: one-way ANOVA followed by Tukey's test, *: p<0.001, **: p<0.01, ***: p<0.05, n = 6-26.
Kidney. In rat kidney tissue homogenates (V), the specific COMT activity was highest in cortical sections (399 ± 104 pmol/min/mg) being about ten times higher than in striatal homogenates. In the outer medulla homogenates, the COMT activity (210 ± 48 pmol/min/mg) was approximately half of the cortical activity and nearly twice as high as in papillar tissues (123 ± 24 pmol/min/mg), which had the lowest activity. The meta/para ratios were approximately 5-9. These values resemble those of recombinant S-COMT (II) suggesting the primary response being attributable to S-COMT rather than MB-COMT.
Recombinant COMT. To test the effect of ethanol on COMT activity in vitro (II), the most pure enzyme preparations i.e. recombinant forms of COMT, were used. Ethanol did not affect the coulometric detection system (Fig. 4C). As the ethanol concentration increased, the formation of both reaction products, i.e. vanillic acid and isovanillic acid, by recombinant MB-COMT tended to decrease and this fall reached statistical significance at 1000 mM ethanol concentration (51 % and 57 % decrease in the formation of vanillic acid and isovanillic acid, respectively). With recombinant S-COMT, only the formation of vanillic acid was affected by 1000 mM ethanol concentration. With both recombinant enzymes the meta/para ratio was increased at 1000 mM ethanol concentration (from 13.2 to 19.0 and from 5.3 to 7.7 with recombinant MB-COMT and recombinant S-COMT, respectively). Due to the opposite effect of ethanol on COMT activities, ethanol was not anticipated to interfere with the COMT assay. With striatal homogenate, 1000 mM concentration of ethanol decreased the formation of vanillic acid (10 %) and isovanillic acid (30 %, p<0.001) (Fig. 8).
Figure 8. Effect of ethanol on striatal COMT activity in vitro. Values (mean ± sem) were obtained from three independent experiments. Statistics: one-way ANOVA followed by Tukey's test, *: p<0.001, n = 15-16.
The effect of 1000 mM concentration of ethanol to apparent kinetic values was tested with recombinant COMT enzymes. Ethanol decreased both Km and Vmax values of MB-COMT indicating a mixed type of inhibition mechanism (Table 3). With S-COMT, the Vmax was increased. With increasing substrate concentrations, the meta/para ratio of recombinant MB-COMT was decreased (from 28 to 15) by 1000 mM ethanol concentration, which differed from the corresponding control value at the lowest concentration of DHBAc (p<0.01). With increasing concentrations of the substrate, 1000 mM concentration of ethanol did not affect the meta/para ratios (7.3-7.7) of recombinant S-COMT, while all meta/para values differed from corresponding control values (p<0.01). In striatal tissue, only a decrease in the formation of isovanillic was observed.
Table 3. Effect of 1000 mM ethanol on the apparent kinetics of vanillic acid with recombinant COMT enzymes. Values are means ± sem, n = 3.
DHBAc concentrations were 12.5-300 mM and 25-500 mM for the MB-COMT and the S-COMT, respectively, with 2-5 replicate samples. The values were obtained from the double reciprocal plots without or with 1000 mM ethanol in the assay. Vmax is expressed as mmol/l product formed in 30 min. (10 ml of the sample analyzed with HPLC). ap<0.05, bp< 0.01 vs corresponding control (t-test).
Cell cultures. The effect of COMT inhibitors (IV) on COMT activity was tested in primary glial, neuronal and glial/neuronal co-cultures of brain cells. The inhibitors with a nitrocatechol structure, entacapone and tolcapone, decreased COMT activity in all glial, neuron-enriched and glial/neuronal cocultures (Fig. 9). In contrast, a pyridine derivative, CGP 28014, did not affect COMT activity in any of these cultures. Since the effect of nitrocatechol-type inhibitors was usually higher than 50 % inhibition of COMT activity, exact IC50 values were not obtained. The approximated concentrations of the nitrocatechol drugs inhibiting COMT activity in glial cell cultures by 50 % were 10-20 nM for tolcapone and 45-150 nM for entacapone. In glial/neuronal co-cultures the estimated 50 % inhibitory concentrations were 15 nM in both cultures for tolcapone and 45 nM in basal forebrain coculture and 100 nM in midbrain coculture for entacapone. This corresponds to about 3-7 times greater potency of tolcapone than entacapone. In neuron-enriched cultures, the efficacy was slightly better in 1- day cultures than in 6-7 day cultures suggesting modest sensitivity of neuronal COMT to nitrocatechol drugs. The approximated 50 % inhibitory concentrations of the drugs in neuron-enriched cultures were 15-60 nM for tolcapone and 20-75 nM for entacapone, suggesting equal potency of both drugs. In all glial and neuronal/glial cocultures at 30 nM concentration tolcapone was more efficient than entacapone (p<0.05). This was true also at 150 nM concentration in striatal glial culture and 1-day basal forebrain cultures (p<0.05) in addition to glial/midbrain neuronal co-cultures (p<0.05) at 300 nM concentration of the drugs.
Figure 9. The efficacy of COMT inhibitors on specific COMT activity in primary cultures of rat brain cells. For the sake of clarity, only the results for 30 nM and 300 nM concentration of the drugs are shown. A) Glia. The cell cultures were grown for 35-41 days. B) Neurons. The cultures were grown for 1 day (1 d) or 6-7 days (7 d). C) Neuronal/glial coculture. The cocultures were grown for 7 days after plating of neurons on top of 18-36 day-old glia. With the exception of 30 nM concentration of entacapone in midbrain glial culture, the drugs decreased significantly COMT activity (p<0.05 at least).
Kidney COMT activity. Peripheral actions of entacapone on COMT activity (V) were studied by analyzing COMT activity ex vivo in different regions of rat kidney. COMT activity decreased similarly in all sections of the kidney, i.e. cortex, outer medulla and papilla. Two and three hours after entacapone treatment (30 mg/kg, i.p.) the activity was decreased by nearly 100 %. To evaluate a possible correlation of central nervous system COMT activity to that of kidney, the whole brain COMT activity was analyzed after the same entacapone treament. The whole brain COMT activity was reduced by about 40 % one hour after entacapone treatment and had returned to its basal level three hours after entacapone administration.
Kidney function. The effect of certain compounds on rat kidney function (V) were also studied. Entacapone (30 mg/kg, i.p.) increased natriuresis by more than five-fold. The entacapone induced natriuretic effect was suppressed by dopamine receptor type 1 (D1) antagonism with SCH23390 (30 mg/kg/h) by about 60 % whereas D2 antagonism with sulpiride was without any effect on natriuresis. COMT inhibition with entacapone caused a trend towards transient increase of dopamine excretion while DOPAC excretion was increased by more than three-fold with or without antagonist treatments. Entacapone alone or with dopamine receptor antagonists did not affect the kidney hemodynamic responses (GFR, RPF or MAP). L-DOPA infusion (60 mg/kg/h) for one hour increased natriuresis by two-fold, and this could be blocked with D1 antagonist treatment. Dopamine excretion increased by more than 17-fold and DOPAC excretion was elevated by two-fold.