Nobuyoshi Iwata

Comparative Studies of the Effects of RS-8359 and Safrazine on Monoamine Oxidase In-vitro and In-vivo in Mouse Brain

Abstract— The effect of RS‐8359, (±)‐4‐(4‐cyanophenyl)amino‐6,7‐dihydro‐7‐hydroxy‐5H‐cyclopenta[d]‐pyrimidine on monoamine oxidase (MAO) has been compared with a hydrazinic MAO inhibitor, safrazine (β‐piperonylisopropylhydrazine hydrochloride,) which is a MAO inhibitor used clinically. In‐vitro radiochemical determination of MAO activity showed that the IC50 of RS‐8359 was 0·52 μM for the deamination of 5‐hydroxytryptamine (5‐HT) in the mouse brain mitochondrial preparation, while β‐phenylethylamine (PEA) deamination was inhibited by only 20% at 100 μM of the drug. 5‐HT deamination in the brain homogenate prepared from mice killed 60 min after administration of RS‐8359 was inhibited significantly by 14 and 48%, at 30 and 100 mg kg−1 (p.o.), respectively, while deamination of PEA was little affected at the same doses. On the other hand, safrazine strongly inhibited both 5‐HT and PEA deaminations, but showed no selectivity toward the substrate used. The extent of MAO inhibition by RS‐8359, measured fluorometrically with kynuramine as a substrate in the brain homogenate, was independent of preincubation up to 80 min. In contrast, the inhibitory potency of safrazine was strengthened by preincubation in a time‐dependent manner. Oral administration of RS‐8359 (3‐30 mg kg−1) caused a dose‐dependent increase in endogenous monoamines in mouse brain, which disappeared a few hours after its administration. Increase in monoamine content caused by safrazine lasted for at least 24 h. These results indicate that RS‐8359 is a reversible and specific inhibitor of MAO‐A, while safrazine is an irreversible and non‐specific MAO inhibitor, in‐vivo and in‐vitro in mouse brain.

S-adenosyl-L-methionine prevents ischemic neuronal death

The effect of S-adenosyl-L-methionine (SAM) on neuronal degeneration induced by transient forebrain ischemia was studied in rats. Bilateral occlusion of the common carotid arteries for 30 min in a 4-vessel occlusion model caused degeneration of CA1 neurons of the hippocampus. When SAM-HCl or SAM sulphate tosylate (SAM-ST, 100 mg/kg as the free form of SAM, i.p.) was administered just after recirculation and every hour for 5 h after recirculation, the degeneration and loss of pyramidal cells were prevented. However, adenosine, a metabolite of SAM, and glycerol, which has the same osmotic pressure as the solution of SAM-ST, did not show any effects on the neuronal damage. The results showed that SAM has a beneficial effect on neuronal damage induced by ischemia.

An experimental model of hyper-irritability in the trigeminal skin field of the rat

&NA; Cutaneous hyper‐irritability was produced in the trigeminal sensory distribution of chronically prepared rats by injecting various central stimulants into the dorsal subarachnoid space overlying caudal medulla. Effective substances were classified into three groups according to the behavioral patterns induced. These were (1) picrotoxin and penicillin G‐K, (2) strychnine and brucine, and (3) d,l‐homocysteic acid. Of these substances, picrotoxin was the most effective. The hyper‐irritability elicited by picrotoxin or penicillin G‐K was associated with a relatively localized trigger zone in the face, while that elicited by strychnine or brucine spread beyond the trigeminal region to the first cervical dermatome. d,l‐homocysteic acid evoked only spontaneous scratching and vocalization. Picrotoxin‐induced hyper‐irritability involved facilitation of impulse transmission from cutaneous afferent terminals to second‐order neurons in the trigeminal nucleus caudalis.

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.

Effects of an anabolic steroid and vitamin B complex upon myophaty induced by corticosteroids

In rats repeatedly treated with dexamethasone, body weight decreased severely, creatine content in urine increased, tetanic contraction of the gastrocnemius muscle decayed easily and twitch tension of the muscle had a slow rise time. In a group of rats given large doses of vitamin B complex along with the anabolic steroid, nandrolone phenylproprionate, all syndromes described above for the dexamethasone-treated rats were prevented, dose dependently by vitamin B complex. The individual components of the vitamin B preparation combined with the nandrolone phenylpropionate partially suppressed the changes induced by dexamethasone. Diphenylhydantoin depressed the slow rise time of twitch tension as effectively as did vitamin B complex with nandrolone phenylpropionate. From the results presented here and facts previously reported it was concluded that the condition of the animals could be well controlled by repeated administration of vitamin B complex with nandrolone phenylpropionate, even though dexamethasone was repeatedly injected.

Is picolinic acid a glycine agonist at strychnine-sensitive receptors?

By means of unit recording and electrophoretic application, the effect of picolinic acid on feline spinal interneurons in situ was studied in comparison with glycine. Picolinic acid inhibited neuronal firing in 60% of neurons and in some cases the inhibitory actions were antagonized by strychnine. Inhibition of firing by glycine, which was also strychnine-sensitive, was reduced in case of concomitant administration of picolinic acid. These results suggest that picolinic acid might act as a glycine agonist at strychnine-sensitive receptors.

Picolinic acid and indole-2-carboxylic acid: two types of glycinergic compounds modulate motor function differentially.

1. A putative agonist for the strychnine-sensitive glycine receptor picolinic acid was tested for its anticonvulsant activities in mice and muscle-relaxant activities in rats and compared with indole-2-carboxylic acid (I2CA), an antagonist for the strychnine-insensitive glycine receptor. Their effects on segmental reflexes in the cat spinal cord were examined to elucidate their sites of action. 2. Picolinic acid (200 and 400 mg/kg IP) delayed the onsets of strychnine- but not pentylenetetrazole-induced seizures. It delayed the onsets of bicuculline-induced seizures only at the higher dose. I2CA (200 and 400 mg/kg IP) delayed the onsets of these 3 kinds of seizures. Both compounds reduced muscle tone in rat decerebrate rigidity at a dose of 100 mg/kg IV. 3. Picolinate methylester, a picolinate derivative with higher lipophilicity, depressed spinal reflexes in both intact and spinalized cats at cumulative doses of 25 to 200 mg/kg IV. I2CA (50 mg/kg IV) inhibited spinal reflexes only in intact preparations. 4. These results suggest that the anticonvulsant and muscle-relaxant activities of picolinic acid (PA) are due to inhibition of spinal neurons, but that I2CA selectively affects supraspinal structures.

Ischaemia-induced change in clathrin preceding delayed neuronal death.

The mechanism underlying the change in clathrin immunohistochemistry preceding delayed neuronal death (DND) was studied in gerbils. The ischaemic change observed with chc5.9 anti-clathrin antibody in hippocampal CA1 was initially ameliorated by pentobarbital, which blocks DND, but 1 day after ischaemia, no change in the immunoreactivity of the SDS-denatured clathrin molecule was detected by Western blotting and no change in the clathrin content in CA1 was detected by SDS-PAGE. No ischaemia-induced change in immunohistochemistry was observed with another monoclonal anti-clathrin antibody, X-22. The above results imply that some modifications that affect the structure of clathrin molecules around the chc5.9 specific epitope may be a crucial step in the course of DND.

Inhibitory mechanisms of the hyper-irritability caused by picrotoxin in the rat

Abstract Topically applied norepinephrine, dopamine, serotonin, GABA and glycine, and systemically administered clonidine, l‐DOPA (plus carbidopa) and 5‐hydroxytryptophan completely suppressed the cutaneous hyper‐irritability produced in the trigeminal sensory distribution by picrotoxin overlying the caudal medulla. Cholinergic agents and apomorphine were ineffective. Of the positive compounds, norepinephrine, serotonin and GABA showed the shortest latencies and norepinephrine and serotonin required the lowest concentrations in order to inhibit the hyper‐irritability. If l‐DOPA (plus carbidopa) was injected after pre‐treatment with FLA‐63, the effects of l‐DOPA did not appear. Similar depression of the hyper‐irritability was caused by electrical stimulation of the central gray. The inhibitory effects of stimulation of the central gray was suppressed after administration of tetrabenazine, but again produced markedly by injection of l‐DOPA. From these observations it was concluded that the hyper‐irritability could be suppressed by serotonergic as well as noradrenergic fibers terminating at the spinal trigeminal nucleus caudalis. The potential clinical use of l‐DOPA in patients with hyperesthesia is discussed.

Central activation by CS-932, a functionally relative M1 agonist

NOBUYUKI KaSAWAl, KAZUKO WATANABE2, YOKO YAMAWAKI’, IKUKO NAGATSU ‘AND MINORU ONOZUKA3 lDept. of gnat., Fujita Health Univ. Sch. of Med., Toyoake. 470-l 101, Japan; Depts. of 2Physiology and 3Anat., Gifu Univ. Sch of Med., Gifi.r 500-8076, Japan To analyze age-associated changes in monoamine-biosynthesizing ability, we studied senescence-accelerated mice (SAM-P/& 8 months old) and accelerated senescence-resistant mice (SAM-R/l,8 months old) as controls. We administered, intraperitoneahy, 2, 4-diamino-&hydroxypyrimidine (DAHP) or parachlorophenylalanine (PCPA) to both groups of mice and measured the time course of changes in dopamine (DA)immunoreactivity (IR) in the nigrostriatal system, and serotonm (5-HT)IR in the raphe dorsalis in PAP-stained sections using a microphotometry system (Luzex FS, Nicon). While DA-IR in the nigrostriatal system of SAM-R/l was lowest at 3 h after the administration of DAHP and recovered to normal value 24 h after, that of the SAM-p/8 was lowest at 6 h following DAHP administration and did not recover even after 72 h. With respect to 5-HT-IR, while that in SAM-R/l following PCPA administration was lowest at 24 h after the administrauon and recovered 48-72 h after, that in SAM-P/8 showed almost no change at 24 h after and decreased slightly 48-72 h after the administrationThese studies strongly suggest that in SAM-P&while catecholaminergic neurons are significantly affected by monoamine synthesis inhibitors,serotonergic neurons do not show significant changes.