Effects of Difenamizole on Content of Catecholamines and Metabolites in Mouse Brain
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EFFECTS OF DIFENAMIZOLE ON CONTENT OF CATECHOLAMINES AND METABOLITES IN MOUSE BRAIN Toshitaka NABESHIMA, Kazumasa YAMAGUCHI and Tsutomu KAMEYAMA Department of Chemical Pharmacology, Fuculty of Pharmaceutical Sciences, o University, Nagoya 468, Japan Meij Accepted April 13, 1978 In earlier studies in mice, a favorable correlation was found between the analgesic activity and cerebral concentration of 1,3-diphenyl-5-(2-dimethylaminopropionamide) pyrazole[difenamizole, DFZ] (1). In a subsequent study, Nabeshima and Kameyama reported that an analgesic effect was observed after intracerebral injection of DFZ in mice (2). DFZ markedly suppressed conditioned behavior maintained by a fixed ratio schedule of reinforcement, by differential reinforcement of a low rate schedule, and by the shuttle-box method (2, 3). Emotional behavior in rats with bilateral ablations of the olfactory bulb and aggressive behavior induced by isolation in male mice were suppressed by DFZ in anti nociceptive doses (4). These behavioral effects of DFZ were similar to the actions of low doses of chlorpromazine (CPZ) (2-4), in support of the view that DFZ acts via the central nervous system. It has been pointed out that central catecholaminergic and tryptaminergic mechanisms play a role in the analgesic action of morphine (5). We previously studied the effects of drugs which modify catecholaminergic or tryptaminergic mechanisms on the analgesic action of DFZ and morphine, and we suggested that the analgesic action of DFZ may be mediated by dopamine (DA), whereas that of morphine appears to be related to 5-hydroxy tryptamine and norepinephrine (NE) (6, 7). In the present investigation, the effects of analgesic doses of DFZ on cerebral catecholamines and their metabolites were investigated in mice. Male ddY mice, weighing 20-22 g, were used throughout the study. DFZ was given orally as a suspension in 5`/ gum arabic solution. Controls were given the vehicle only. DFZ concentrations were based on the closes producing an anti-nociceptive effect (6, 7). DFZ inhibited the acetic acid-induced writhing at low doses (25 and 50 mg/kg) (7) and the heat-induced reflexes were depressed with a high dose (150 mg/kg) (6). Since mice given DFZ, p.o., showed m axinmalanti-nociceptive activity 30--60 min after administration (6), the animals were decapitated 45 min after DFZ or vehicle administration. Brains were dissected within 1.5 min after decapitation according to the method of Glowinski and Iversen (8) and concentration of catecholannines and their metabolites in the brain was determined by the spectrolluorometrical procedure described by Karasavva cat al. (9, 10). Table 1 shows that there were significant differences in catecholamine levels in some regions between the DFZ.-treated and vehicle-treated animals. Striatal DA levels were sig nificantly higher in DFZ (25 and 50 mg/kg)-treated mice than in the controls. DFZ (25 150 mg/kg) produced a dose-dependent increase in dien--r mesencephalic NE in the treated mice. Cortical NE levels increased significantly after the DFZ-administration in a dose of 50 mg/kg. However, there were no significant variations in catecholamine levels in the cortex and the pons medulla oblongata relative to vehicle-treated mice. In Table I , it can be seen that DFZ in doses from 25 mg/ kg to 150 mg/kg produced a significant decrease in striatal 3-methoxytyramine (3-MT) in comparison to vehicle-treated mice. The dien f-mesencephalic normetanephrine (NM) levels were significantly lower in DFZ (25-150 mg/kg)-treated mice as compared to control mice. Significant changes in cortical 3-MT and NM, or pons-1 Medulla oblongatal NM concentrations were not observed. Striatal NM levels and dien mesencephalic and pons-' medulla oblongata) 3-MT levels were below determination limits. Since catechol-0-methyltransferase is known to be mainly located extraneuronally in , 644 (: the brain in contrast to monoamine oxidase (MAO) which is distributed both extra and intraneuronally, the concentrations of cerebral NM and 3-MT have been generally considered to reflect, under certain conditions, the amounts of NE and DA released from these mono aminergic neurons. Therefore, NM and 3-MT should be determined along with NE and DA in the same brain tissue when investigating the dynamics of these neurotransmitters in the brain (9). In our experiment, DFZ in analgesic doses decreased the concentrations of 3-MT in the striatum and NM in the dien -I-mesencephalon (Table 1) and these results suggest that DFZ may depress the release of catecholarnines from these monoaminergic neurons. In addition, we reported that the analgesic action of DFZ was antagonized by intracerebral injection of DA but not by NE (7). As the effect of DFZ was significantly potentiated by 6-hydroxydopamine, phenoxybenzamine and reserpine (7), the analgesic action of DFZ may be more related to the depression of dopaminergic than to the norepin ephrinergic system. Other workers reported that bilateral lesioning of the substantia nigra with a micro injection of 6-hydroxydopamine prolongs the reaction time of the tail flick in rats (11). Iwatsubo and Clouet reported that an increased rate of spontaneous firing of neurons in the substantia nigra was seen after the administration of morphine or haloperidol. The dopamine receptor agonists dopa and apomorphine decreased the firing rate in nigral neurons (12). Kuromi et a/. reported that a brief electrical stimulation of the substantia nigra induced a marked and long lasting inhibition of the somatosensory evoked potentials recorded from the centrum medianum of the thalamus and posterior hypothalamic area following sciatic stimulation in unanesthetized rabbits. All these results suggest that analgesia follows an increased rate of spontaneous firing in the substantia nigral neurons due to the decrease in dopaminergic neuronal activity. As stated above, an analgesic action of DFZ may be related to the dopaminergic system and DFZ may depress the release of DA in the striatum, therefore, DFZ may demonstrate an anlgesic action following depression of dopaminergic neuronal activity in the striatum. Anden et al. reported that CPZ produced a rise in the homovanillic acid (HVA, one of DA metabolites) content of the striatum (14). We gave CPZ (10 mg/kg, p.o.), and found that the concentration of HVA was significantly increased in the striatum (Table 2). CPZ reportedly enhances the accumulation of 3-MT and NM after MAO inhibition (15), however, TABLE 2. Effects of difenamizole and chlorpromazine on contents of dopamine, 3-methoxytyramine and homovanillic acid in mouse striatum in the experiments herein, DFZ decreased not only the concentration of 3-MT but also the concentration of HVA in the striatum. These results suggest that the mechanisms by which CPZ and DFZ depress catecholaminergic neurons differ. REFERENCES 1) KAMEYAMA,T., NIWA, H., KISARA, K. AND IWAIZUMI,H.: Studies on the absorption and distribution of 1,3-diphenyl-5-(2-dimethylaminopropionamide)-pyrazole in rat. Yakugakn Zasshi 90, 829-834 (1970) (Abs. in English)2) NABESHIMA,T. AND KAMEYAMA,T.: Effects of dife namizole on behavior maintained by schedule of positive reinforcement. 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