Miyuki Ota

N-methylation of dopamine-derived 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, (R)-salsolinol, in rat brains : in vivo microdialysis study

Abstract: N‐Methylation of (R)‐1‐methyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline [(R)‐salsolinol] derived from dopamine was proved by in vivo microdialysis study in the rat brain. The striatum was perfused with (R)‐salsolinol and N‐methylated compound was identified in the dialysate using HPLC and electrochemical detection with multichanneled electrodes. N‐Methylation of (R)‐salsolinol was confirmed in three other regions of the brain, the substantia nigra, hypothalamus, and hippocampus. In the substantia nigra, the amount of N‐methylated (R)‐salsolinol was significantly larger than in the other three regions. These results indicate that around dopaminergic neurons, particularly in the substantia nigra, (R)‐salsolinol was methylated into N‐methyl‐(R)‐salsolinol, which has a chemical structure similar to that of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine, the selective dopaminergic neurotoxin. N‐Methylation of tetrahydroisoquinolines and β‐carbolines have already been proven to increase their toxicity to dopaminergic neurons and N‐methylation might be an essential step for these alkaloids to increase their toxicity. On the other hand, after perfusion of (R)‐salsolinol, release of dopamine and 5‐hydroxytryptamine was observed and inhibition of monoamine oxidase was indicated. (R)‐Salsolinol and its derivatives may be candidates for being dopaminergic neurotoxins.

Dopamine-derived endogenous 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-(R)-salsolinol, induced parkinsonism in rat: biochemical, pathological and behavioral studies

Dopamine-derived 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol, Sal) and related compounds were examined for their selective neurotoxicity to dopamine neurons by injection into the rat striatum. Among salsolinol analogs examined, only N-methyl-(R)- salsolinol (NM(R)Sal) induced behavioral changes very similar to those in Parkinsons disease: hypokinesia, stiff tail, limb twitching at rest and postural abnormality. Biochemical analysis showed that after NM(R)Sal injection, NM(R)Sal itself and its oxidation product, 1-2-dimethyl-6,7-dihydroxyisoquinolinium ion (DMDHIQ+) accumulated in the striatum, and also in the substantia nigra definite amount of DMDHIQ+ was detected. Dopamine and noradrenaline were reduced in the striatum and more markedly in the substantia nigra, whereas serotonin and its metabolite were not affected. Morphological analysis revealed selective reduction of tyrosine hydroxylase (TH)-containing neurons in the substantia nigra after continuous NM(R)Sal administration in the striatum. These results demonstrate the selective cytotoxicity of NM(R)Sal to the dopamine neurons in the substantia nigra, and the possible involvement of this 6,7-dihydroxy-isoquinoline in the pathogenesis of Parkinsons disease is discussed.

Peripheral injection of risperidone, an atypical antipsychotic, alters the body weight gains of rats.

1. Risperidone is an atypical antipsychotic drug that possesses 5-hydroxytryptamine 5-HT2 receptor antagonism combined with milder dopamine D2 receptor antagonism. 2. Excessive bodyweight gain is one of the side-effects of antipsychotics. Risperidone treatment causes a greater increase in the body mass of patients than treatment with conventional antipsychotics, such as haloperidol. Therefore, the present study was undertaken in order to address the aetiology of the risperidone-induced bodyweight change in rats by examining the expression of leptin, an appetite-regulating hormone produced in white adipose tissue (WAT), and uncoupling protein (UCP)-1, a substance promoting energy expenditure in the brown adipose tissues (BAT). 3. Eight-week-old male rats were injected subcutaneously with risperidone (0.005, 0.05 or 0.5 mg/kg) twice daily for 21 days. Both bodyweight and food intake were monitored daily. On day 21, rats were decapitated and their serum leptin and prolactin concentrations were measured. Expression levels of leptin, Ucp1 and beta3-adrenoceptor (beta3-AR) genes in WAT and BAT were quantified using real-time polymerase chain reaction amplification. 4. Injection of 0.005 mg/kg risperidone into rats increased food intake and the rate of bodyweight gain, as well as the augmentation of leptin gene expression in WAT. Injection of 0.05 mg/kg risperidone increased food intake and leptin gene expression in WAT, but the rate of bodyweight gain was not affected. Injection of 0.5 mg/kg risperidone caused a reduction in bodyweight gain, as well as enhanced Ucp1 gene expression in BAT and serum prolactin concentrations. The serum leptin concentration and beta3-AR gene expression in WAT and BAT were not affected by injection of 0.5 mg/kg risperidone. 5. Although the changes in food intake observed in risperidone-injected rats were rationalized neither by serum leptin nor prolactin concentrations, the reduction in the rate of bodyweight gain following injection of 0.5 mg/kg can be explained, in part, by increased energy expenditure, as revealed by the remarkable increase in the UCP-1 mRNA expression level in BAT. The role of leptin in risperidone-induced alterations in bodyweight gain remain to be clarified.Ota et al . 1 recently addressed the important issue of developing an animal (rat) model of weight gain induced by novel ‘atypical’ antipsychotics Their study involved risperidone, which is known to induce weight gain in humans. 2 Most, but not all, novel antipsychotics induce weight gain, 3 although the weight gain induced by risperidone in humans is smaller in magnitude that that induced by olanzapine. 4 Olanzapine-induced weight gain has recently been modelled by us in rats 5 and it would be a considerable advantage for work in this area if risperidone-induced weight gain was also modelled effectively in rodents. Novel antipsychotic-induced weight gain has very serious clinical implications, being associated with enhanced morbidity and mortality, as well as reduced compliance. 6 However, the causes of the weight gain induced by many novel antipsychotics remain enigmatic. Many of the wide range of receptors at which novel antipsychotics act are implicated in the control of food intake (e.g. -adrenoceptors, various serotonergic and dopaminergic receptors and histamine H 1 receptors). However, it has, to date, proved impossible, on the basis of clinical studies, to determine the role of any one such receptor in antipsychotic-induced weight gain. 7 It is well documented that the weight gain induced by novel antipsychotics is associated with abnormalities in various hormonal systems, including leptin, 8 insulin 9 and prolactin. 7 Thus, it seems most likely that the development of animal models of novel antipsychotic-induced weight gain in which measures of food intake and bodyweight are supplemented by metabolic and endocrine measures, exactly as in the study by Ota et al ., 1 will prove a very powerful approach with which to advance this field. Indeed, in a recent major review of this area, Baptista et al . 7 highlighted the need to develop such models as a primary current ‘research perspective’. Thus, the type of empirical research reported by Ota et al . 1 is to be highly commended. It is, however, regrettable that examination of the work reported by Ota et al . 1 suggests that the conclusions they have drawn from their data relating to bodyweight and food intake are not supported by the statistical analysis presented and, thus, attempts to relate these findings to metabolic and endocrinological measures may be invalid. In their study, they administered risperidone for 21 days to four groups of rats treated with either vehicle or one of three doses of risperidone. Bodyweights and food intake were recorded daily. These data are reported as being analysed by repeated-measures

Inhibition of tryptophan hydroxylase by (R)- and (S)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines (salsolinols)

The (R)- and (S)-enantiomers of salsolinol, the dopamine-derived tetrahydroisoquinolines, were found to inhibit the activity of tryptophan hydroxylase (TPH), prepared from serotonin-producing murine mastocytoma P-815 cells. Inhibition of TPH by salsolinols was found to be non-competitive with the substrate L-tryptophan. Tryptophan hydroxylase is composed of two elements with different kinetic properties in terms of cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin and these two elements were inhibited by salsolinols in competitive and uncompetitive ways, respectively. Stereoselectivity of salsolinol was not observed, concerning the potency and the type of inhibition on PTH. These data indicate that salsolinols might be naturally occurring inhibitors of indoleamine metabolism.

Resistance to excessive bodyweight gain in risperidone-injected rats.

1. The present study was carried out to explain the resistance of rats injected subcutaneously with risperidone, the atypical antipsychotic drug, for 21 consecutive days at 0.1 mg/kg per day (a dose equivalent to the one used for patients) to result in an excessive bodyweight despite the increase in diet‐uptake in rats against risperidone‐induced decrease in body temperature.

Inhibition of tryptophan hydroxylase by food-derived carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole

Food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), were found to inhibit the activity of tryptophan hydroxylase (TPH) prepared from serotonin-producing murine mastocytomas, P-815 cells. Inhibition of TPH by Trp-P-2 was found to be competitive with the substrate L-tryptophan and non-competitive with the cofactor (6R)-L-erythro-5,6,7, 8-tetrahydrobiopterin. The inhibition proved to be reversible; by dialyzing the sample incubated with Trp-P-2, the enzyme activity could be fully recovered. Among a series of heterocyclic amines examined, Trp-P-1, Trp-P-2 and some other heterocyclic amines inhibited TPH activity. Trp-P-2 and other heterocyclic amines were the newly discovered naturally occurring inhibitors of the indoleamine metabolism.

Inhibition of human brain aromatic l-amino acid decarboxylase by cooked food-derived 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and other heterocyclic amines

A carcinogenic, food-derived heterocyclic amine, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), was found to inhibit aromatic L-amino acid decarboxylase isolated from human brainstem. Trp-P-2 inhibited the enzyme activity toward L-DOPA more markedly than that toward 5-hydroxytryptophan. The inhibition was competitive to a cofactor of the enzyme, pyridoxal-5-phosphate, and the Ki value of Trp-P-2 was 163 microM. The enzyme activity could be fully recovered after removal of Trp-P-2 by gel filtration, which indicates that the inhibition was reversible. Among a series of heterocyclic amines examined for their effects on the activity toward L-DOPA, Trp-P-2 was the most potent inhibitor, followed by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, then Trp-P-1. Another heterocyclic amine, 2-amino-3-methyl-9H-pyrido[2,3-b]indole also inhibited the enzyme. The inhibition of the decarboxylase activity by these heterocyclic amines may affect the catecholamine metabolism in human brain.

Exon 3 of tyrosine hydroxylase gene: lack of association with Japanese schizophrenic patients.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine (DA) biosynthesis.1,2 Exon 3 of the human TH gene encodes the sequence from Ser31 to Glu104 of type 1 enzyme,3,4 which contains the critical parts for regulation of the catalytic activity. The amino acid residues Gly36-Arg37-Arg38 were identified as a key sequence for DA to exert its inhibitory effect on catalytic activity.5–7 Therefore, we screened the nucleotide sequences of exon 3 from 201 Japanese patients with schizophrenia to explain the elevation in the synaptic or presynaptic DA concentrations in the schizophrenic brain,8–11 based on the hypothesis that any mutation changing the amino acid sequence Gly36-Arg37-Arg38 would result in the elevation of DA synthesis, due to a reduced inhibitory effect of DA on the catalytic activity.5–7 However, no mutated sequences of exon 3 and both exon–intron boundaries were detected in any of the patients examined. Polymorphisms generating Val81 and Met81 were compared of the distributions of genotype and allele between the patients and 175 Japanese healthy controls, which did not suggest an association between the polymorphism and schizophrenia. These results indicate that exon 3 of the human TH gene lacks association with schizophrenia in Japanese patients.

Structural studies of condensation products of biogenic amines as inhibitors of trytophan hydroxylase

The effects of condensation products of dopamine and indoleamines on the activity of tryptophan hydroxylase (TPH) were evaluated to determine the structures associated with modulation of this enzyme activity. The compounds having a catechol structure, such as 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, markedly inhibited the activity of the enzyme prepared from the rat brain. The inhibition was non-competitive in terms of both the biopterin cofactor and the substrate L-tryptophan. Substitution on the one or two positions of catechol isoquinolines did not affect the inhibitory activity towards TPH. Among these compounds, a charged substance, 1,2[N]-dimethyl-6,7-dihydroxy-isoquinolinium ion, was an extremely potent inhibitor; the Ki values were 0.88 +/- 0.17 and 0.64 +/- 0.08 microM (mean +/- S.D.) in terms of the substrate and cofactor, respectively. By contrast, the condensation products of tryptophan and tryptamine with acetaldehyde scarcely affected TPH activities. 1-Methyl-1,2,3,4-tetrahydroisoquinoline, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) were almost inactive. These results indicated that the catechol structure recognized and combined with TPH at a binding site different from that of the substrate or cofactor and the positive charge on the dopamine-derived substance enhanced the affinity to TPH. The selective inhibition of TPH by dopamine-derived catechol isoquinolines was discussed in relationship to the interactions between catecholamines, indoleamines and their metabolites in the brain under physiological and pathological conditions.

Risperidone reduces mRNA expression levels of Sulfonylurea Receptor 1 and TASK1 in PC12 cells

Electrophysiological and immunohistochemical studies have demonstrated that glucose-sensing neurons in the hypothalamus contain both ATP-sensitive K(+) (K(ATP)) and tandem-pore K(+) (TASK1 and TASK3) channels and that glucose-induced depolarization or hyperpolarization of these neurons function as an important link between glucose-excited or glucose-inhibited neurons and feeding behavior. Medication with atypical antipsychotics increases the appetite of schizophrenic patients and thus causes increases in body weight. Therefore, the present study investigates mRNA expression levels of the genes encoding the components of these K(+) channel subsets in PC12 cells cultured with risperidone (an atypical antipsychotic) and in the hypothalami of rats subcutaneously injected for 21 consecutive days with 0.1 or 0.01 mg/kg/day of risperidone. The mRNA expression levels of various genes were not obviously altered in rat hypothalami. However, the mRNA expression levels for sulfonylurea receptor 1, a component affording nucleotide-binding folds to K(ATP) channels, and TASK1 were down-regulated in PC12 cells cultured with 50 microM risperidone for 24h, but the amount of intracellular ATP in these cells was not affected by the drug. Collectively, these results indicate that the amplitude of the current through these K(+) channels in PC12 cells might be modulated as a pharmacological effect of risperidone.