Anne-Françoise Steulet

Inhibition of catechol-O-methyltransferase (COMT) as well as tyrosine and tryptophan hydroxylase by the orally active iron chelator, 1,2-dimethyl-3-hydroxypyridin-4-one (L1, CP20), in rat brain in vivo

The orally active iron chelator, 1,2-dimethyl-3-hydroxypyridin-4-one (L1, CP20) proposed for reduction of iron overload in hemoglobinopathic patients, was studied in rats with respect to its ability to interfere with dopamine (DA) and serotonin (5-HT) metabolism. At 100 mg/kg i.p., it reduced the levels of DA, 5-HT, 5-hydroxyindoleacetic acid and particularly homovanillic acid in the rat striatum for several hours. These effects were shown to result from concomitant inhibition of catechol-O-methyltransferase (COMT; EC2.1.1.6), tyrosine [tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating) (EC 1.14.16.2)] and tryptophan hydroxylase [tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating) (EC 1.14.16.4)], with similar time-courses. COMT was inhibited with a threshold dose of about 1 mg/kg i.p. and an ED50 of about 10 mg/kg i.p. as determined by the conversion of exogenous L-dihydroxyphenylalanine (L-DOPA) to its O-methylated derivative. Tyrosine and tryptophan hydroxylase activities as measured by the accumulation of DOPA and 5-hydroxytryptophan, respectively, after central decarboxylase inhibition, were inhibited in striatum and cortex, with threshold doses of 3-10 mg/kg and ED50s of about 20-30 mg/kg i.p. or p.o. While COMT inhibition by L1 is probably related to the structural similarity of the latter drug with the normal enzyme substrates, tyrosine and tryptophan hydroxylase inhibition is more likely due to coordination to iron bound to these enzymes. Desferrioxamine at 100 mg/kg i.p. did not show comparable effects. It is not known whether this relates to poor brain and/or cell penetration, or whether multidentate chelators are less suitable as inhibitors of aromatic amino acid hydroxylases.

Effects of selective dopamine D1 and D2 receptor agonists on the rate of GABA synthesis in mouse brain

The effects of dopamine D1 and D2 receptor agonists and antagonists on the rate of GABA synthesis in four regions of mouse brain (corpus striatum, cerebellum, cortex and hippocampus) were examined after irreversible inhibition of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19; GABA-T) by gabaculine. The dopamine D2 receptor agonists PPHT, LY 171555 and RU 24213 exerted a dose-related inhibitory effect on GABA synthesis in these four regions. The decreases in the rate of GABA formation were prevented by the dopamine D2 receptor antagonist S(-)-sulpiride. The dopamine D1 receptor agonists SKF 77434 and SKF 38393 augmented gabaculine-induced GABA accumulation in the corpus striatum only, and this effect was blocked by the dopamine D1 receptor antagonist SCH 23390. However, SKF 81297 and SKF 82958, two other dopamine D1 receptor agonists, did not affect or only marginally altered the rate of GABA synthesis. Stimulation of D2 receptors thus induces a decrease in the rate of GABA formation in the four brain areas examined, whereas stimulation of D1 receptors either increases GABA synthesis in the corpus striatum or does not alter it. This effect appears to be independent of the degree of receptor occupancy.

Determination of rat brain and plasma levels of the orally active GABAB antagonist 3-amino-propyl-n-butyl-phosphinic acid (CGP 36742) by a new GC/MS method

An involvement of GABAergic neurons has been suggested in the process of memory consolidation based on anatomical evidence and increasing physiological and biochemical data. With the advent of orally active GABAB antagonists, such as CGP 36742, the question of their therapeutic value, for example in Alzheimers disease, becomes relevant. Therefore, a new GC/MS method was developed to determine the concentration of CGP 36742 (3-amino-propyl-n-butyl phosphinic acid) in various intra- and extracerebral tissues after different routes of application. The compound was chemically derivatised in a two-step process (acylation of the amino group and esterification of the phosphinic acid). The limit of detection of the method was 0.01 microgram/g tissue and 0.0005 microgram/mL plasma. The time-course after i.p. treatment showed peak levels of CGP 36742 between 30 min and 1 hr after injection. After a dose of 100 mg/kg, the concentration in the brain ranged from 1 to 1.4 microgram/g or 6 to 8 microM, assuming that 1 mg tissue equals 1 microL (i.e., below the IC50 of the interaction with GABAB receptors as measured by [3-3H]-aminopropyl-phosphinic acid binding [35 microM]). These results are discussed in light of the psychopharmacological effects (improvement of cognitive performance of rats) of CGP 36742 observed at very low oral doses.

Effects of CGP 28014 on the in vivo release and metabolism of dopamine in the rat striatum assessed by brain microdialysis

The effects on rat striatal dopamine (DA) metabolism of systemic and local administration of CGP 28014, an inhibitor of catechol-O-methyl-transferase (COMT), were studied by in vivo microdialysis. CGP 28014 (30 mg/kg i.p.) significantly reduced the levels of homovanillic acid (HVA), but did not modify DA and 3,4-dihydroxyphenylacetic acid (DOPAC). The intrastriatal administration (via the microdialysis probe) of 5, 7.5, 10, and 20 mM of CGP 28014 elicited a concentration-dependent, several-fold increase in extracellular DA but did not alter the levels of HVA and DOPAC. Thus, the effects of CGP 28014 observed after i.p. injection (decrease in HVA levels) are different from those measured after intrastriatal administration (increase in DA release). Therefore, the inhibition of COMT is likely to be due to the action of a metabolite of CGP 28014 formed in the periphery and not in the brain.

Reversal by apomorphine of the gabaculine-induced GABA accumulation in mouse cortex

To test the assumption that in the mice cortex the rate of accumulation of gamma-aminobutyric acid (GABA) after irreversible inhibition of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19; GABA-T) represents an index of GABA turnover, we examined whether the reversal of the gabaculine-induced accumulation of GABA elicited by apomorphine was due to a decrease in GABA turnover or to a modulation of the activity of the GABA-T inhibitor. Therefore, we simultaneously measured the action of apomorphine on gabaculine-induced accumulation of GABA and on GABA-T activity. In vitro, apomorphine (3 and 30 microM) did not alter the concentration-dependent inhibition of GABA-T by gabaculine. Ex vivo, apomorphine (2 x 0.5 mg/kg s.c.) markedly decreased (69%) gabaculine-induced (150 mg/kg i.p.) accumulation of GABA. This drug had no direct effect on GABA-T activity, but significantly reduced from 83 to 71% the inhibition of GABA-T by gabaculine. The linear correlation found between GABA levels and GABA-T activity allowed the quantification of the decrease in GABA turnover elicited by apomorphine. The results showed that apomorphine decreased significantly (P less than 0.001) the rate of GABA synthesis from 7.48 to 3.36 micromol GABA/g per h, if the partial reversal of gabaculine-induced inhibition of GABA-T is considered and 2.44 micromol/g per h if not. Apomorphine effect on GABA accumulation is mainly due to a decrease of the rate of GABA synthesis and to a lesser extent to a reversal of the inhibitory activity of gabaculine. Thus, inhibition of GABA-T by gabaculine is a sensitive and reliable method for the estimation of the rate of synthesis.