Päivi Tuomainen

Different in vivo properties of three new inhibitors of catechol O‐methyltransferase in the rat

1 We compared three new catechol O‐methyltransferase (COMT) inhibitors (OR‐611, Ro 40–7592 and CGP 28014; 10 and 30 mg kg−1, i.p.) in male rats given levodopa (l‐DOPA, 50 mg kg−1, i.p.) and carbidopa ((−)‐l‐α‐methyl dopa, 50 mg kg−1, i.p.). In some studies pretreatment with pargyline (80 mg kg−1, i.p.) was used to block the function of monoamine oxidase (MAO). 2 Decreases of hypothalamic and striatal 3‐O‐methyl‐dopa (3‐OMD) levels were used as measures of the inhibition of peripheral COMT. The inhibition of brain COMT activity was estimated by decreases of hypothalamic and striatal homovanillic acid (HVA) and 3‐methoxytyramine (3‐MT; after pargyline) levels. 3 The three COMT inhibitors studied had different individual characteristics. OR‐611 was primarily a peripherally acting COMT inhibitor, decreasing 3‐OMD levels in the striatum (to 31–52%) and in the hypothalamus (to 16–27%) both in the control and pargyline‐treated animals at 1 and 3 h. It did not have any effect on brain HVA and 3‐MT. 3 Ro 40–7592 was a broad spectrum COMT inhibitor decreasing striatal and hypothalamic 3‐OMD (always to < 30%), HVA (to < 50%) and 3‐MT levels (to < 23%) significantly both at 1 and 3 h. It was more potent than OR‐611. 4 CGP 28014 functioned as a weak COMT inhibitor in the periphery inhibiting 3‐OMD formation only at 3 h. In contrast, it was fairly potent in decreasing the brain HVA and 3‐MT levels at 1 h (to 37–22% and 42–35% in the striatum, and to 57–33% and 64–35% in the hypothalamus, respectively) but not at 3 h. Since CGP 28014, unlike OR‐611 and Ro 40–7592, did not generally increase the brain DOPA, dopamine or DOPAC levels, it was not a typical COMT inhibitor.

Comprehensive multidetector HPSEC study on solution properties of cereal arabinoxylans in aqueous and DMSO solutions.

The water-soluble arabinoxylans from wheat flour (high, medium, and low viscosity samples) and rye flour (high viscosity sample) were characterized by (1)H NMR spectroscopy and HPSEC with refractive index, light scattering, and viscometric detectors. These cereal arabinoxylans have recently been used as model arabinoxylans in various studies, but their solution properties have not been previously investigated. In this study, two HPSEC eluent systems were used: the water-based system and DMSO-based system. DMSO seemed to be a better solvent than water, especially for arabinoxylans containing a low amount of arabinose substituents. (1)H NMR spectroscopy indicated the structural differences between the analyzed arabinoxylan samples that also affected the hydrodynamic parameters obtained with HPSEC. Influence of arabinose side groups on the solution conformation of arabinoxylans could not be excluded based on our data, despite the role of arabinose substituents being questioned in previous investigations concerning arabinoxylan conformation in solution.

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.

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.

Beneficial effects of a potassium- and magnesium-enriched salt alternative.

The effects on blood pressure and the development of cardiac hypertrophy of sodium chloride (regular salt) and a novel potassium-, magnesium-, and l-lysine-enriched salt alternative, which in a previous study prolonged the life span of hypertensive rats nearly threefold as compared with the animals receiving regular salt, were compared both in spontaneously hypertensive rats and their hypertension-resistant genetic controls. In particular, the possible protective effect of increased intakes of potassium, magnesium, and l-lysine during a high intake of sodium chloride was examined. Therefore, the salt alternative was added at 1.75 times higher levels to produce the same dietary levels of sodium chloride in the regular salt and the salt alternative groups. Regular salt produced a remarkable left ventricular hypertrophy in both rat strains, but as compared with the respective control groups, it induced an increase of blood pressure only in the spontaneously hypertensive rats. The salt alternative did not induce a rise in blood pressure in either of the rat strains, nor did it produce left ventricular hypertrophy in the hypertension-resistant rats and, in the spontaneously hypertensive animals, significantly less hypertrophy than regular salt. The salt alternative appeared to prevent the sodium chloride-induced volume load since plasma levels of atrial natriuretic peptide were increased in the regular salt groups but remained normal in the salt alternative groups. Therefore, potassium, magnesium, and/or l-lysine of the salt alternative produced a powerful protection against the harmful effects of sodium chloride.

Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches.

Shearzyme (GH10 endo-1,4-beta-D-xylanase) and two different alpha-L-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with alpha-L-Araf (1-->2)-monosubstituted beta-D-Xylp residues or alpha-L-Araf (1-->2)- and (1-->3) doubly substituted beta-D-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: alpha-L-Araf-(1-->2)-[alpha-L-Araf-(1-->3)]-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->4)-D-Xylp, d-II: alpha-L-Araf-(1-->2)-[alpha-L-Araf-(1-->3)]-beta-D-Xylp-(1-->4)-D-Xylp, m-I: alpha-L-Araf-(1-->2)-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->4)-D-Xylp and m-II: alpha-L-Araf-(1-->2)-beta-D-Xylp-(1-->4)-D-Xylp. To our knowledge, this is the first report on structural (1)H and (13)C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly alpha-L-Araf (1-->2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.

Overflow of noradrenaline and dopamine in frontal cortex after [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] (DSP-4) treatment: in vivo microdialysis study in anaesthetized rats

Abstract The changes in the extracellular concentration of endogenous noradrenaline and dopamine in the frontal cortex following pretreatment with noradrenergic neurotoxin DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] were studied by in vivo microdialysis in rats anaesthetized with chloral hydrate.Noradrenaline and dopamine levels in frontal cortex were detected only when the uptake inhibitor, nomifensine (10μM) was present in dialysis fluid. Under those conditions, the Na+ channel agonist veratridine and a depolarising concentration of potassium chloride (60mM), applied locally through the microdialysis probe, increased the overflow of noradrenaline. Tetrodotoxin had an opposite effect. These results indicate that most of the noradrenaline probably arose from exocytotic release. Noradrenaline efflux in the frontal cortex of DSP-4 pretreated rats (52±6.1fmol/sample) did not differ significantly from that of the control animals (69±4.9fmol/sample). Dopamine efflux was not changed either (64±9.6 and 62±3.9fmol/sample, respectively). The α2-adrenoceptor antagonist, atipamezole (3mg/kg i.p.), increased the overflow of noradrenaline in the frontal cortex of saline-treated rats by 100%, whereas in DSP-4 treated rats the increase was only around 30%. The overflow of dopamine was not changed under the conditions described. The effect of atipamezole in DSP-4 treated rats may be of smaller magnitude due to the diminished pool of releasable noradrenaline or due to a downregulation of presynaptic α2-adrenoceptors in the frontal cortex. The perfusion of 60mM KCl at the end of the experiment unexpectedly produced equivalent increases in noradrenaline and dopamine content in dialysates of both vehicle and DSP-4 treated rats. We conclude that the uptake inhibitor, nomifensine, and atipamezole, which had a stronger effect in vehicle-treated animals, reduced the effect of KCl-stimulation and masked the true difference in changes of noradrenaline efflux. Post-mortem tissue concentrations of noradrenaline 7 days after DSP-4 administration (50mg/kg) were significantly reduced in the frontal cortex (54%), hippocampus (62.5%) and to lesser extent in the hypothalamus (27%) as compared to vehicle-treated rats. Dopamine and 3,4-dihydroxyphenylacetic acid concentrations were not changed confirming the efficacy and selectivity of the DSP-4 lesion.These results demonstrate that one week after DSP-4 treatment the extracellular levels of noradrenaline and dopamine as assessed by in vivo microdialysis are not changed in the frontal cortex, but atipamezole-stimulated release of noradrenaline is decreased in DSP-4 treated rats.

Receptor binding profile and anxiolytic-type activity of deramciclane (EGIS-3886) in animal models

The present series of experiments was done to characterize the properties of deramciclane, a new antiserotonergic drug, in both receptor binding studies in vitro and in a number of anxiolytic, antidepressant, and antidopaminergic tests in rodents. A striking property of deramciclane was its high affinity to both 5‐HT2A and 5‐HT2C receptors (Ki = 8.7–27 nM/l). Deramciclane had also a moderate affinity to dopamine D2 and sigma receptors but did not interact with any of the adrenergic receptors.

Presence of 1->3-linked 2-O-β-D-xylopyranosyl-α-L-arabinofuranosyl side chains in cereal arabinoxylans.

The presence of a fairly uncommon side chain 2-O-beta-D-xylopyranosyl-alpha-L-arabinofuranosyl in arabinoxylans (AX) from eight different cereal by-products was investigated, using (1)H NMR spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after Shearzyme (GH10 endo-1,4-beta-D-xylanase) hydrolysis. This disaccharide side group was present in significant amounts in AX extracted from corn cobs and barley husks. For the first time, it was also detected in AX from oat spelts and rice husks, and in lesser amounts in wheat straw AX. Arabinoxylo-oligosaccharide (AXOS) containing the 2-O-beta-D-Xylp-alpha-L-Araf side chain was purified from the oat spelt AX hydrolysate and the structure was fully analyzed using 1D and 2D NMR spectroscopy. The AXOS was identified as beta-D-Xylp-(1-->2)-alpha-L-Araf-(1-->3)-beta-D-Xylp-(1-->4)-D-Xyl. To our knowledge, such a structure with 2-O-beta-D-Xylp-alpha-L-Araf attached to the O-3 of the nonreducing end of xylobiose has not been described previously. New information on substitution of AX from various cereal by-products was obtained by combining NMR and enzyme-assisted HPAEC-PAD analysis.

Comparison of two new inhibitors of catechol O‐methylation on striatal dopamine metabolism: a microdialysis study in rats

1 Effects of two new inhibitors of catechol O‐methylation (CGP 28014 and entacapone; 30 mg kg−1, i.p.) were compared by means of brain microdialysis in rats treated with l‐3,4‐dihydroxyphenylalanine (l‐dopa)/carbidopa (50/50 mg kg−1, i.p., respectively) or saline. 2 In saline‐treated rats, CGP 28014 maximally (max) increased striatal dopamine and 3,4‐dihydroxyphenylacetic acid (DOPAC) effluxes by 41% and 49%, respectively, whereas homovanillic acid (HVA) levels were decreased by 71%. 3 In the presence of l‐dopa/carbidopa, a peripherally active inhibitor of catechol O‐methyltransferase (COMT) entacapone had a short‐lasting increasing effect on l‐dopa efflux. Compared to the effects of l‐dopa/carbidopa alone 3‐O‐methyldopa (3‐OMD) levels were effectively reduced (max 79%) by entacapone, but not by CGP 28014. 4 Entacapone, in contrast to CGP 28014, increased striatal dopamine efflux (max 492% of that after l‐dopa/carbidopa alone). Also DOPAC levels were increased by entacapone (255% at 180 min), but not significantly by CGP 28014 (159% at 180 min). 5 Both compounds initially decreased HVA efflux. The effect of CGP 18014 was longer‐lasting. By the end of the measurement, entacapone even increased HVA levels (max 259%). 6 Our results demonstrate that entacapone is a peripheral COMT inhibitor and support the view that CGP 18014 is mainly a centrally acting inhibitor of O‐methylation.