Neurochemical Research (2) 327-349 (1977)

ABSTRACTS OF COMMUNICATIONS OF THE J A P A N E S E N E U R O C H E M I C A L SOCIETY

T h e following are abstracts of 46 c o m m u n i c a t i o n s which were read at the 19th A n n u a l Meeting of the J a p a n e s e N e u r o c h e m i c a l Society held in Niigata from October 14 to 16, 1976. Of the 66 papers read at the meeting, 20 abstracts were not submitted by the authors. The abstracts are presented in greater detail in J a p a n e s e in the Bulletin of the Japanese Neurochemical Society, Vol. 15, pp. 1263 (1976).

SODIUM- AND POTASSIUM-ACTIVATED ADENOSINE TRIPHOSPHATASE FROM YOUNG RAT CEREBRUM: THE MODE OF INHIBITORY ACTION OF BILIRUBIN W I T H R E S P E C T TO M A G N E S I U M A N D S U B S T R A T E . Shigeo Kashiwamata, Shunji Gotoh, Ritsuko K. Semba, and Fujiko Niwa. D e p a r t m e n t of Biochemistry, Institute for D e v e l o p m e n t a l R e s e a r c h , A i c h i Prefecture Colony, Kasugai, Aichi, and D e p a r t m e n t of Obstetrics and Gynecology, University o f N a g o y a School o f Medicine, N a g o y a The m o d e of inhibition by bilirubin of (Na + + K+)-ATPase (ATPase) and K+-p n i t r o p h e n y l p h o s p h a t a s e (PNPPase) activities was investigated with NaI-treated micros o m e s from the cerebra o f 14-25-day-old rats. Bilirubin concentrations causing half m a x i m a l inhibition (I50) at p H 8.5 were approximately 100 and 70 g M for A T P a s e and P N P P a s e , respectively. Bilirubin inhibited A T P a s e activity in a simple linear noncompetitive m a n n e r with A T P (K,~ for ATP, about 0.4 raM) w h e n the molar ratio o f m a g n e s i u m to A T P was maintained at 1 m M and A T P was less than 1 m M . H o w e v e r , a t substrate concentrations higher than 1 raM, the double reciprocal plots appeared to converge to the same point on the vertical axis, showing an apparent competitive inhibition by bilirubin with ATP. At 4 m M ATP, bilirubin inhibition s e e m e d to be noncompetitive with Mg 2+. Without bilirubin, the optimal ratio of m a g n e s i u m to A T P for A T P a s e activity was between 1.5 and 2.5, and was i n d e p e n d e n t o f A T P concentration. A s s u m i n g that the m a g n e s i u m in e x c e s s of a m a g n e s i u m : A T P ratio of l existed as free ion, the activation (Vratio = 1.5 -Vratio = 1) by Mg ~+ o f A T P a s e at a constant ATP-Mg complex level decreased m a r k e d l y with increasing concentrations of bilirubin. An optimal ratio o f m a g n e s i u m to P N P P for P N P P a s e activity varied with P N P P concentration, but K ~ for Mg 2+ was c o n s t a n t (0.62 rnM), suggesting that Mg ~+ acts on the e n z y m e independently of PNPP. Bilirubin inhibited the activity noncompetitively with Mg ~+ or PNPP, E F F E C T O F B O V I N E G R O W T H H O R M O N E O N T H E P R O T E I N S Y N T H E S I S IN THE NEONATAL RAT BRAIN TREATED WITH HYDROCORTISONE ACETATE. Tetsuya Noguchi and Yasuzo Tsukada. D e p a r t m e n t o f Physiology, School o f Medicine, Keio University, Sinjuku-ku, T o k y o We h a v e already reported that the decrease of D N A content per brain o f the neonatally hydrocortisone-treated rat [40 mg/kg of hydrocortisone acetate was administered subcuta-

327 ~) 1977Plenum PublishingCorp., 227 West 17th Street, New York, N.Y. 10011.To promote freer access to published material in the spirit of the 1976 Copyright Law, Plenum sells reprint articles from all its journals, This availability underlines the fact that no part of this publicationmay be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permissionof the publisher. Shipment is prompt; rate per article is $7.50.

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neously once a day from the day of birth until the third day of life (HC group)] was caused by the reduction of the neonatal de novo synthesis of thymidine kinase. The reasons for this were that thymidine kinase activity appeared later in the HC group and its level was lower than that in the control, and that the cerebral protein synthesis in the HC group was less active than that in the control during the period from the third day to the eighth day of life.* In order to make the cerebral protein synthesis of the HC group recover, 10 mg/kg of bovine growth hormone (bGH) was given to the HC and control groups from the day of birth until the tenth day of life.* A bGH increasing effect on the reduced protein synthesis was evident only in the HC group, not in the control. From the sixth day of life, there was no statistical significance between the cerebral protein synthesis in the control and the HC group given bGH. Morphological examination of the thymus from the HC group revealed that its cortex was strongly atrophied and decreased in size. However, the thymus from the HC group given bGH was found to be larger in size and to have a thicker cortex than that of the HC group without bGH treatment. These results suggest that pituitary growth hormone might be indispensable for the neonatal development of the brain, exerting its effect through the thymus. (* Abst. of 5th Int. Meet. of ISN, 433, 1975) D E V E L O P M E N T A L CHANGES IN CEREBRAL CORTEX METABOLISM: POLYPEPTIDE SYNTHESIS BY FREE AND MEMBRANE-BOUND POLYSOMES. Motowo Nagayama, Setsu Komatsu, and Hiroshi Naruse. National Institute of Mental Health, Ichikawa, Chiba In the course of our research on the development of brain and its disturbances, polypeptides synthesized by cerebral polysomes were investigated. Free and membranebound polysomes were prepared from cerebral cortex of developing rats (2, 7, 14, 21, 28, and 45-day-old) by a modification of the method of Blobel and Potter. The polysomes were incubated in a medium including the pH 5 fraction and high-speed supernatant of rat liver; an ATP, GTP, and ATP regenerating system; and a mixture of 15 amino acids labeled with carbon-14. The incubated mixture was centrifuged on a sucrose gradient, and the nascent polypeptides bound to ribosomes were discarded. The supernatant fraction containing terminated polypeptides was dialyzed and analyzed by SDS-disk electrophoresis in 10% polyacrylamide gel slabs. The gels were stained, dried, and cut into 1.5-ram-length pieces which were then digested and counted with a liquid scintillation counter. Patterns of 14Cincorporation into polypeptides separated by electrophoresis showed differences between free and membrane-bound polysomes in each developmental stage examined. In the membrane-bound polysomal system, more carbon-14 was incorporated into faster moving polypeptides, or polypeptides of lower molecular weight, than into heavier ones. On the other hand, in the free polysomal system, more amino acids were incorporated into polypeptides of moderate molecular weight. The number of bands of labeled polypeptides that were distinguished in each slab was about 15. Among these, four bands in the free polysomal system and another four bands in the membrane-bound polysomal system apparently changed with development. In three of them, the rate of amino acid incorporation increased with cerebral maturation, while in one it decreased. In four bands, it increased transiently and then decreased in the course of development. NEUROCHEMICAL STUDIES ON CULTURED CEREBELLA FROM MICE WITH SPECIAL REFERENCE TO MYELIN FORMATION. Kazuhiro Nagaike, Tetsuya Noguehi, and Yasuzo Tsukada. Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo Fragments of newborn mouse cerebellum were explanted and maintained in organotypic culture in a Maximow double coverslip assembly according to the method Of Bornstein and

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Murray. About 95% of the explants formed myelin from the ninth to the sixteenth day in culture. The protein content per four dishes (equivalent to one cerebellum) did not show any developmental changes throughout the period studied. The DNA content, however, decreased continuously until the fifth day, and showed a transient plateau around the seventh day. After the eleventh day in culture, the DNA content remained fairly constant. From the fifth to the ninth day, DNA synthesis was demonstrated by the incorporation of [3H] -thymidine into the DNA fraction. The amino acid uptake and protein synthesis during the period of culture, as reflected by the uptake of [3H]leucine into the acid-soluble fraction and the incorporation of [3H]leucine into the protein fraction, coincided with the appearance and development of myelin. When bovine growth hormone was added to the culture medium, a large number of myelinated axons appeared in the culture, whereas amino acid uptake and protein and DNA syntheses were not changed. With the addition of 2% EAE serum to the culture medium, myelin formation was completely inhibited, and amino acid uptake and syntheses of DNA and protein were also suppressed. When EAE serum was omitted from 15-day cultures, myelination appeared. The addition of bromodeoxyuridine did not affect myelination, amino acid uptake, or protein synthesis, whereas DNA synthesis was strongly inhibited. These results lead to the conclusion that myelin formation in culture is supported by protein synthesis but not by DNA synthesis. ENDOGENOUS PHOSPHORYLATION OF PROTEINS IN MYELIN FROM RAT BRAIN. Eishichi Miyamoto. Department of Biochemistry, Kumamoto University Medical School, Kumamoto The myelin of brain and spinal cord contains a basic protein that is known as an experimental allergic encephalomyelitogenic protein, since it induces the disease in animals when injected with Freund's complete adjuvant. The purified myelin basic protein has been found to serve as a good substrate for the cyclic AMP-dependent protein kinases in cytosol fractions. The incubation of the myelin fraction in the presence of ATP and Mg 2+ resulted in the phosphorylation of the protein. Further, the protein has been shown, by injection of orthophosphate into ventricle of rat brain, to be phosphorylated in vivo and to occur originally in a phosphorylated form. The protein kinase and phosphoprotein phosphatase enzyme systems of rat brain myelin, which are responsible for the phosphorylation and dephosphorylation and myelin basic protein, have also been characterized. The present study demonstrates the endogenous phosphorylation of proteins in the myelin fraction from rat brain, using the method of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two myelin basic proteins and at least five more proteins were phosphorylated after incubation of the myelin fraction in the presence of ATP and Mg2+. The apparent molecular weights of the proteins other than the myelin basic proteins were 120,000, 76,000, 60,000, 41,000, and 38,000, respectively. The proteins of molecular weight 60,000, 41,000, and 38,000 were extracted by treatment with hydrochloric acid, whereas those of molecular weight 120,000 and 76,000 were insoluble in hydrochloric acid and chloroform-methanol. Folch-Lees proteolipid protein was not found to be phosphorylated under the conditions used. The endogenous phosphorylation of the proteins was not stimulated by adenosine 3',5'-monophosphate. STUDIES ON THE TWO MAJOR GLYCOPROTEINS IN BOVINE PERIPHERAL NERVE MYELIN MEMBRANE. Kunio Kitamura, Masaru Suzuki, and Keiichi Uyemura. Department of Physiology, Saitama Medical School, Iruma-gun, Saitama Two major glycoproteins, designated as the BR protein (tool. wt. 28,000) and the PAS-II protein (tool. wt. 13,000), in bovine peripheral nerve myelin membrane were isolated from the acid-insoluble residue of the myelin, by a procedure involving solubilization with

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sodium dodecylsulfate and chromatography on a Sephadex G-200 column with a buffer containing dodecylsulfate. The separation patterns of the proteins on the gel were considerably affected by the dodecylsufate concentration in the elution buffer. At below 0.1% dodecylsulfate concentration in the elution buffer, the two glycoproteins were not separated during the gel chromatography, whereas at above 2%, they could be separated clearly and purified. Each of the purified glycoproteins was homogeneous on dodecylsulfate polyacrylamide gel electrophoresis by periodic acid-Schiff reagent as well as Coomassic Blue staining, The NH2-terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose, and sialic acids, and the PAS-II protein contained glucosamine, mannose, galactose, fucose, and glucose. Neither the BR protein nor the PAS-II protein was a glycosylated derivative of a basic protein, a deduction based on the results of amino acid analysis. The BR protein was considered to be identical with the PO protein reported by Brostoff et al. [Brain Res., 86:449 (1975)], The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits, guinea pigs, and humans on periodic acid-Schiff reagent stained gels after dodecylsulfate gel electrophoresis.

STRUCTURE AND FUNCTION OF A CALCIUM-BINDING PROTEIN: Ca2+-BIND ING SITES AND ACTIVATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE. Hisataka Kasai, Fumio Yamagata, and Tsuneo Okuyama, Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Tokyo, Yoshiko Teshima, and Shiro Kakiuchi, Research Division and Clinical Laboratory, Nakamiya Mental Hospital, Hirakata, Osaka A highly acidic protein (PAPII) with a molecular weight of 16,000 was purified from bovine brain extract by ammonium sulfate precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-75 gel chromatography. Proteins almost identical with PAPII were obtained from human and chick brains, bovine liver, and erythrocytes. Three Ca z+ atoms per molecule of the protein are bound with an apparent dissociation constant of 4 • 10-~ M, as determined by Sephadex G-25 gel filtration. Here we describe the character of the Ca2+-binding sites of the protein and its ability to activate cyclic nucleotide phosphodiesterase in the presence of Ca2+: 1. The estimation of phosphate residues in the protein was performed by the revised method of Eibl and Lands. y-Carbox.yglutamic acid residues were determined with an amino acid analyzer after alkaline hydrolysis. Almost no phosphate or -/-carboxyglutamic acid residues were detected. Therefore, these residues are dispensable for the Ca2+-binding of the protein. 2. The elucidation of the amino acid sequence of PAPII revealed two to three Ca 2+binding sites corresponding with the proposed Ca~+-binding model. Two peptides (A and B sites), each consisting of about 30 amino acid residues, were separated by gel filtration after BrCN cleavage, but lost this Ca~+-binding activity. 3. PAP II protein fractions purified from bovine, human, and chick brains, and bovine liver activated cyclic nucleotide phosphodiesterase of bovine brain with satisfactory efficiency in the presence of Ca 2+. It is suggested that chemically similar protein activators of phosphodiesterase may exist universally in vertebrates. 4. The BrCN peptides as well as A and B sites retained no phosphodiesterase-activating activity and showed no inhibitory effect on the activation. Thus, a specific structure of the protein seems to be necessary for Ca~+-binding and phosphodiesterase activation.

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c~-BUNGAROTOXIN BINDING SUBSTANCE IN MOUSE BRAIN. Akiko Seto, Yasuyoshi Arimatsu, and Takehiko Amano. Mitsubishi-Kasei Institute of Life Sciences, Mechida, Tokyo The biochemical characteristics of a substance in mouse brain that binds c~-bungarotoxin (c~-BGT), a neurotoxin that binds specifically to the peripheral nicotinic acetylcholine receptor (nAchR), was investigated, c~-BGT was purified and labeled with 12~I by the chloramine T method. The homogenates of adult male mouse brain in phosphate-buffered saline were incubated with ['25I]-c~-BGT at 25~ The amount of [IZ~I]-a-BGT binding to the homogenates at saturation was approximately 1.7 pmol/g of original tissue weight. The binding was inhibited by preincubation of the homogenates with d-tubocurarine, but not by atropine, hexarnethonium, or decamethonium at concentrations of less than 1 raM. Preincubation of the homogenates with various concentrations of concanavalin A reduced the binding capacity by 40% at the maximum. This finding corresponds to that of Meunier et al. (1974), who found such inhibition with pure nAchR of electric organ. The toxin binding substance was solubilized with 1% Triton X-100. Upon sucrose density gradient centrifugation, it sedimented with catalase (11.4S). It showed a molecular weight of approximately 7 x 106 by Sepharose 6B column chromatography, and its molecular weight was reduced by trypsin digestion. Concanavalin A bound with this substance. These results clarified that the toxin binding substance in mouse brain was a glycoprotein that resembled the peripheral nAchR except that it was slightly higher than nAchR in molecular weight. EFFECTS OF U N I L A T E R A L EYE ENUCLEATION ON MONOAMINE OXIDASE AND ACETYLCHOLINESTERASE ACTIVITIES IN THE RAT SUPERIOR COLLICULUS. Akihisa Urano. Laboratory of Neurophysiology, Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo The changes in monoamine oxidase (MAO) and acetylcholinesterase (ACHE) activities following unilateral eye enucleation were examined for the superior colliculus (SC), lateral geniculate body (LGB), and nucleus suprachiasmaticus (NSC) in the male albino rat. These enzyme activities were histochemically demonstrated and measured by microspectrophotometry. The most significant and conspicuous change was the decrease in MAO activity in the superficial strata of the SC contralateral to the enucleated eye (10% decrease after 2 days, 15% after 4 days, and about 20% after 1 week); Most of this decrease appeared to occur in the neuropil, with a slight decrease in the cell bodies. The other layers of SC, such as the stratum opticum, the intermediate strata, the deep strata, and the central gray, did not show significant postoperative changes in MAO activity. MAO activity of the LGB and NSC was also not altered significantly. The decrease of MAO activity in the superficial strata of the SC should be located in the degenerating terminals of optic nerves that selectively innervate the SC. The strong AChE activity in the superficial strata, probably derived from the horizontal cells, showed only slight postoperative changes by the thirtieth day after enucleation. In the rat superior colliculus, some groups of optic nerve fibers may be monoaminergic or monoaminoceptive. The monoaminergic mechanism may be profoundly related to the transmission of visual information at the first step of synaptic events in the zone of horiz0ntal cells. Then the intrinsic system of the superior colliculus including the cholinergic mechanism may process the visual input to elucidate the transmitted information, EFFECT OF L-DOPA ON THE BRAIN MONOAMINE METABOLISM OF RATS PRETREATED WITH p-CHLOROPHENYLALANINE. Akira Hori, Michio Makagawara, Akiko Watanabe, Yoichi Yamamura, and Tetsuhiko Kariya. Department of Neuropsychiatry, School of Medicine, Tokyo Medical and Dental University, Tokyo

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The interaction of serotonin (5HT) and catecholamine metabolism under altered concentrations of brain monoamines, and the effect of t-dihydroxyphenylalanine (L-DOPA) on the concentrations of monoamines were investigated in various brain regions of rats pretreated with p-chlorophenylalanine (PCPA). The rats were decapitated 1 h after 200 rag/ kg of L-DOPA injection or 24 h after 250 mg/kg of PCPA injection. The concentrations of 5HT, 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), and norepinephfine (NE) were determined in the following rat brain regions: telencephalon, diencephalon, and mesencephalon plus pons plus medulla oblongata, Our results can be summarized as follows: 1. The levels of 5HT and 5HIAA significantly decreased in all brain areas of rats injected with PCPA. 2. The level of DA markedly increased, and the levels of 5HT and 5HIAA significantly decreased, in telencephalon and diencephalon of rats treated with L-DOPA while the level of NE remained unchanged. 3. The DA level was higher in brain parts of rats injected with PCPA plus L-DOPA, compared with the group treated with 0.9% saline plus L-DOPA. 4. It was noteworthy that the level of 5HT was significantly decreased in teleneephalon and diencephalon, and the level of 5HIAA was significantly decreased in diencephalon, of the rats injected with PCPA plus L-DOPA, compared with the group treated with PCPA plus 0.9% saline. These results suggest that the reduction of 5HT and 5HIAA levels in the brain induced by PCPA injection is enhanced by L-DOPA treatment. EFFECT OF ACUTE AND CHRONIC ADMINISTRATION AND WITHDRAWAL OF ETHANOL ON MONOAMINE METABOLISM IN RAT BRAIN. Hideo Ichihashi, Kenichi Kobayashi, Teruyoshi Kobayashi, and Megumi Odagiri. Matsuzawa Metropolitan Hospital, Tokyo Effects of acute and chronic administration and withdrawal of ethanol on the steadystate levels and turnover rates of monoamines have been investigated in the different regions of rat brain. The levels of serotonin, 5-hydroxyindoleacetic acid, norepinephrine, and dopamine remained unchanged after acute administration of ethanol. However, the turnover rates of serotonin increase transiently after acute ethanol administration. Daily intake of ethanol for 7 weeks induced no significant alterations in the steady-state levels and turnover rates of brain monoamines. Eight hours after ethanol discontinuation, rats showed withdrawal signs, such as tremor, piloerection, hyperexcitability, startling to noise, sleep disturbance, and midriasis. The level of serotonin significantly incleased in all brain regions, while turnover rates of serotonin showed no significant changes. Brain levels of norepinephrine and dopamine remained unchanged 8 h after withdrawal of ethanol. It was reported earlier that, by saturating brain serotonergic centers with excess free serotonin, the rapid and large increase in serotonin counteracted its own action, and consequently excitation was induced by the liberated activity of the adrenergic brain centers. T h e s e results suggest that withdrawal symptoms might be mediated by norepinephrinergic neurons, whose accelerated activity is induced by the blocking of serotonergic neurons in rat brain. CHANGES IN TRYPTOPHAN LEVEL AND BRAIN SEROTONIN METABOLISM AFTER ADMINISTRATION OF L-DOPA. Yasuko Kohno, Tadashi Nishikawa, Takayasu Sano, Nobuyuki Nagasaki, and Tatsuo Furukawa. Department of Pharmacology, Kurume University School of Medicine, Kurume Effects of L-DOPA on tryptophan (Trp) concentration in the brain and peripheral tissues and serotonin metabolism in the brain were studied at various time intervals after i.p. administration to male Wister rats.

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1. Administration of L-DOPA , 200 mg/kg, elicited a marked reduction of Trp in the plasma (total and free TIP) and kidney, an increase in the liver, and a slight increase in the brain. A significant decrease in brain 5-HT content was accompanied by marked increases in 5-HIAA and dopamine levels. 2. When L-DOPA , 25-75 mg/kg, was administered with Ro 4-4602, the reduction in cerebral 5-HT content was potentiated, while changes in brain 5-HIAA and peripheral Tip levels were reduced. The brain Trp level declined slightly. 3. Dopamine, 100 mg/kg, did not affect brain Tip, 5-HT, and 5-HIAA levels, but exhibited peripheral effects similar to those seen with L-DOPA, 200 mg/kg. 4. After administration of L-DOPA, 20 mg/kg, to newborn rats in which brain tryptophan hydroxylase is considered to be saturated, the changes in brain 5-HT and 5-HIAA levels were similar to those observed with adult rats except that kidney and liver TIP levels were not altered. These results suggest that the decrease of free Trp in the plasma may not contribute to the changes in brain Tip level and that the reduction of brain 5-HT level after L-DOPA administration is not correlated with the changes in the level of Trp. EFFECTS OF DIETARY PROTEIN CONTENT OF THE RAT BRAIN. Masahiko Nomura and Richard J. ogy, School of Medicine, Fujita-gakuen University, Neuroendocrine Regulation, Department of Nutrition bridge, Massachusetts

MONOAMINE SYNTHESIS IN Wurtman. Department of PhysiolToyoake, Aichi; Laboratory of and Food Sciences, MIT, Cam-

In order to determine the effect of dietary protein content on total urinary output of homovanillic acid (HVA), the major metabolite of dopamine, and 5-hydroxyindole acetic acid (5HIAA), the major metabolite of serotonin, rats received diets providing 0, 18, or 40% protein (as casein protein) per day. With increasing dietary protein content, urinary HVA output rose from 22 through 40 to 65 p~g/day, and 5HIAA output also rose from 28 through 41 to 86 >g/day. That is, the urinary output o f H V A and 5HIAA rose more significantly than the increase of dietary protein. Administration of MK-486 (a peripheral decarboxylase inhibitor, 100 mg/kg three times daily throughout) eliminated the increased 5HIAA excretion due to dietary protein. MK-486 obviously inhibited the synthesis of 5HIAA, even in the case of increased dietary protein. The 5HIAA excretion calculated per kilogram of body weight ranged from 140 through 166 to 271/zg/kg/day in the control, and from 109 through 77 to 95 /zg/kg/day in the MK-486 injected group, suggesting that the agent might suppress 5HIAA synthesis in tissues other than brain. The rate of HVA and 5HIAA synthesis by rat tissues increased directly with the protein content of the diet, this acceleration of monoamine synthesis occurring mainly in tissues other than brain. It is suggested that control of dietary protein content is essential in studies relating urinary metabolite output to brain monoamine metabolism. EFFECT OF CHLORPROMAZ1NE ON TYROSINE- AND TRYPTOPHAN HYDROXYLASE ACTIVITY 1N THE RAT LIMBIC SYSTEM. Shuzo Watanabe, Michio Toru, Shigeru Kaneno, Haruo Shibuya, and Yasuo Shimazono. Department of Neuropsychiatry, Tokyo Medical and Dental University, Tokyo The existence of dopamine terminals in the limbic cortex having been demonstrated, the possible involvement of the limbic system in the pathogenesis of schizophrenia was considered, assuming the dopamine hypothesis of the disorder. In the present study the effect of chlorpromazine (CP) was investigated on the dopaminergic function in discrete areas of the rat limbic brain. For this purpose, the synthetic enzymes of catecholamines and indoleamine, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TRH) were estimated. The limbic brain was separated into five structures; hippocampus, gyrus cinguli,

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septal area, amygdaloid-piriform cortex, and the ventral part of frontal 'cortex. From the remaining brain tissue, the dorsal part of the frontal cortex, thalamus, hypothalamus, the mesencephalic part including A 9-10, and striatum were taken. For the assay of TRH activity, the method of Ichiyama et al. (1970) was employed, in which 14CO~ evolved by decarboxylation, following hydvoxylation of tryptophan-l-[14C] with TRH, is trapped. An analogous procedure was applied to the assay of TH activity. In the limbic brain of salinetreated rats, higher TH activity was detected in the septal area, including the nucleus accumbens, and the ventral part of the frontal cortex, while TRH activity was relatively high in the septal area. TH activity in CP-treated rats (10 mg/kg, i.p., 2 h before decapitation), as compared with promethazine-treated vats, significantly increased in the section that contained mainly the nucleus accumbens. TRH activity was unchanged in all sections. A slight increase, however, in TH activity in the striatum was found up to 8 h after CP treatment. EFFECTS OF GONADECTOMY AND THYROIDECTOMY ON THE TYROSINE HYDROXYLASE ACTIVITY IN INDIVIDUAL HYPOTHALAMIC N U C L E I AND LOWER BRAIN STEM CATECHOLAMINERGIC CELL GROUPS OF THE RAT. Tatsuo Nakahara, Hideyuki Uchimura, Makoto Hirano, Masashi Saito, and Masatoshi Ito. Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka; Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga; Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, Fukuoka The tyrosine hydroxylase (TH) activity in individual hypothalamic nuclei, including dopaminergic cell groups ( A l l to A14), in midbrain dopaminergic (A8 to A10) and in pontine noradrenergic (A5 and A6) cell groups of gonadectomized and thyroidectomized rats, was measured using a microtechnique for the precise dissection of nuclei in freezedried sections of brain. Following castration, the TH activity decreased in the median eminence (ME), increased in the nucleus periventficularis and in the medial zone incerta (MZI), and remained unchanged in the other hypothalamic nuclei. The TH activity in dopaminergic (A8, A9, and A10) and noradrenergic (A5 and A6) cell groups did not change after castration. Increased TH activity was found in the ME, nucleus arcuatus, and MZI after thyroidectomy. Although the effect of thyroidectomy on the A8, A9, and A10 dopaminergic and A5 noradrenergic cell groups was not detected, thyroidectomy induced a marked elevation of TH activity in the A6 noradrenergic cell group. TYROSINE HYDROXYLASE ACTIVITIES IN THE HYPOTHALAMIC NUCLEI AND OTHER CATECHOLAMINE N E U R O N SYSTEMS OF THE RAT: DISTRIBUTIONS AND CHANGES BY SOCIAL ISOLATION. Masashi Saito, Makoto Hirano, Hideyuki Uchimura, Tatsuo Nakahara, and Masatoshi Ito. Laboratory of Neurochemistry, Hizen National Mental Hospital, Kanzaki, Saga; Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka; Department of Neuropsychiatry, Faculty of Medicine, Kyushu University, Fukuoka We studied tyrosine hydroxylase (TH) activities in the hypothalamic nuclei and other catecholamine (CA) neuron systems of the rat, using the microdissection technique with freeze-dried sections. A10 and A~ dopamine (DA) cell groups and A~ noradrenaline (NA) cell group in the lower brain stem revealed the highest TH activity among the nuclei examined. In the hypothalamic nuclei, the highest activity was found in the median eminence (ME), and relatively high activity was found in the incertohypothalamic CA cell groups. In addition, the external region of ME had slightly higher TH values than the internal region, but no significant difference was found between the two regions. Isolation of male rats induced three different behavioral states (muricidic, aggressive, and indiffer-

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ent). TH activities in ME and tuberculum olfactorium were elevated in all three behavioral states. In muricidic rats, TH activities increased in the nucleus periventrivularis (A,4 cell group) and nucleus posterior (A~ cell group), and decreased in the medial part of the nucleus periventricularis and nucleus lateralis medialis. In aggressive rats, TH activities increased in the nucleus lateralis posterior, and decreased in the medial zone incerta (At3 cell group) and nucleus dorsomedialis. On the other hand, no specific changes in TH activities were found in indifferent rats. It is of interest that the specific TH activity changes were found in some hypothalamic nuclei of muricidic and aggressive rats. PHENYLETHANOLAMINE-N-METHYLTRANSFERASE AND OTHER CATECHOLAMINE ENZYMES IN H U M A N BRAIN. Toshiharu Nagatsu, Takeshi Kato,

Yukiko Numata(Sudo), K. Ikuta, Masao Sano, Ikulo Nagatsu, Yukari Kondo, Shinobu Inagaki, Reiji Iizuka, A. Hori, and Hirotaro Narabayashi. Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagatsuda, Tokyo Institute of Technology, Tokyo; Department of Biochemistry and Department of Anatomy, School of Dentistry, Aichi-Gakuin University, Nagoya; Department of Anatomy, School of Medicine,, Fujita-Gakuen University, Toyoake, Aichi; Department of Psychiatry and Department of Neurology, School of Medicine, Juntendo University, Tokyo The activities of tyrosine hydroxylase (TH), DOPA decarboxylase (DDC), dopamine-/3hydroxylase (DBH), phenylethanolamine-N-methyltransferase (PNMT), and monoamine oxidase (MAO) with serotonin (A type) and phenylethylamine (B type) as substrate were measured in catecholaminergic regions of human brain from 10 controls and three patients of parkinsonism. PNMT activity was demonstrated in the hypothalamus, thalamus, and cerebellar nucleus of control and parkinsonian human brains and was reduced in the hypothalamus of parkinsonian cases. MAO-A and MAO-B activities were higher as compared with activities of catecholamine-synthesizing enzymes, with little individual variations in all brain regions of both controls and parkinsonian cases. TH activity was markedly decreased in substantia nigra, caudate nucleus, putamen, and pallidum in all three cases of parkinsonism as compared with that in controls. DDC activity in these dopaminergic regions was also decreased in two parkinsonian patients, but one parkinsonian case had only decreased TH activity with normal DDC activity. DBH activity in hypothalamus was also reduced in the parkinsonian cases. ADRENERGIC MECHANISM AND INTRACRANIAL VASOSPASM AFTER EXPERIMENTAL SUBARACHNOID HEMORRHAGE. Hiroshi Morooka, Kenji Shibata, Toshihiko Miyamoto, Takashi Ohmoto, and Aldra Nishimoto. Department of Neurological Surgery, Okayama University Medical School, Okayama It has been postulated that cerebral ischemia secondary to arterial spasm is one of the principal causes of morbidity and mortality in patients with subarachnoid hemorrhage (SAH). Recently, we found the concentration of noradrenaline (NA) and dopamine-/3hydroxylase (DBH) activity markedly elevated in human cerebrospinal fluid (CSF) after SAH. It was clarified that the adrenergic mechanism plays an important role in the production of cerebral vasospasm following SAH. Results are as follows: 1. Experimental SAH was produced by injection of fresh blood into the cisterna magna in cats. The cerebral vasospasm was angiographically shown to be biphasic in nature: an immediate constriction lasting for 1 h and a marked prolonged spasm occurring on the third to fifth day after SAH. The amount of NA and DBH activity decreased in the vessel walls of both the basilar artery and locus cemleus within 24 h and then gradually increased, reaching the maximum on the third day after SAH.

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2. Direct application of spasmogenic substances (NA, blood) produced marked constriction of the basilar artery, which can be considered a hypersensitive vessel, on the third day after bleeding. 3.6-Hydroxydopamine injection into the cisterna magna eliminated the hypersensitivity of the intracranial vessels to spasmogenic substances and produced prolonged vasodilatation. 4. Various spasmogenic substances (such as serotonin, NA, prostaglandins, and methemoglobin) were measured in the mixture of blood and CSF incubated at 37~ Their contents became very low within 3 days, except for methemoglobin. S p a s m o g e n i c substances that contain heine components, such as catalase, hemoglobin, and methemalbumin, elevated the DBH activity of arterial walls in vitro. It is suggested that the prolonged late spasm following experimental SAH is caused by the elevation of DBH activity within arterial walls. BEHAVIORAL CHANGES UNDER INTENSE STIMULI AND THE BIOCHEMICAL MECHANISM IN SOCIALLY ISOLATED RATS. Tadashi Nishikawa, Yumiko Karlwara, Yasuko Kohno, Takayasu Sano, Masatoshi Tanaka, and Nobuyuki Nagasaki. Department of Pharmacology, Kurume University School of Medicine, Kurume We hypothesized that prolonged isolation during the early postnatal period results in an altered composition of brain macromolecules, affecting properties such as receptor sensitivity or enzyme activity, following alteration of the monoamine turnover rate. Consequently, the behavior in such isolated rats changes upon exposure to stress situations. To prove this hypothesis, rats isolated immediately after weaning were exposed to electric foot shock of various intensities. The shock-elicited jumping behavior was calculated every 10 min for 1 h. The frequency of shock-elicited jumping in isolated rats was lower than that in grouped animals and the difference between the two groups was the greatest with the most intense shock. This behavioral difference was almost absent at the third week of isolation, occurred at the ninth week, continued up to the fifteenth week, and diminished after the rats were placed into grouped housing. Under these experimental situations, there was no significant difference in monoamine turnover rate between the two groups, while noradrenaline turnover increased remarkably in isolated rats. Consequently, it was postulated that changes in the receptor sensitivity of catecholaminergic neurons play a role in this behavioral change in isolated rats. The changes in shock-elicited jumping behavior induced by administration of the stimulant (methamphetamine) and the blocker (chlorpromazine) of catecholaminergic neurons were examined. These results made it partially clear that receptor supersensitivity was involved in this behavioral change. To confirm the involvement of this factor, the similarity of behavior under foot shock situations and the denervation supersensitivity of catacholaminergic neurons by 6-hydroxydopamine were examined in isolated and grouped rats. The results suggested that the behavioral change in isolated rats involves factors other than supersensitivity of catecholaminergic neurons. On the basis of these findings, we consider the shock-elicited jumping behavior in rats to be a potentially useful new screening test for psychotropic drugs. RELATIONSHIP BETWEEN EMOTIONALITY AND BRAIN 5-HYDROXYTRYPTAMINE (5-HT) IN RATS. Toshitaka Nabeshirna, Masahiko Suzuki and Tsutomu Kameyama. Department of Chemical Pharmacology, Faculty of Pharmaceutical Science, Meijo University, Nagoya The open-field (OF) test has been used for many years to study emotional behavior in experimental animals. The novel situation of the OF evokes a pattern of behavior characterized by defecation, urination, ambulation, rearing, and grooming. To determine

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whether or not a biochemical mechanism is responsible for the defecation in the OF, we investigated the correlation between the defecation and 5-HT. Experiments were carried out according to the method describd in previous reports (Jpn. J. Pharmacol., 25, Suppl., 47-, and 48p., 1975). In t h e OF, 5-hydroxytryptophan (5-HTP, i.p.) and 5-HT (i.v.) significantly decreased defecation in rats, while p-chlorophenylalanine (p-CPA, i.p.) and 5,6-dihydroxytryptamine (5,6-DHT, i.v.) treated animals tended to show an increase in the number of fecal boluses excreted. 5-HTP was effective in diminishing the OF performance ofp-CPA treated animals, while reserpine (RSP, i.p.) had no influence on defecation. The inhibitory effects of 5-HTP on defecation were observed also in RSP treated rats. On the contrary, among the rats reared in home cages, those pretreated with 5-HTP and 5-HT tended to show an increase in defecation, and those with p-CPA a significant decrease. The 5-HT content in various brain sites was increased by 5-HTP treatment, but decreased by p-CPA, 5,6-DHT, and RSP in both situations. 5-HTP in combination with p-CPA or RSP increased 5-HT content. The accumulative rate for brain stem 5-HT was increased in OF as compared with home cage rats. These results suggest that the defecation in OF is due to brain stem 5-HT. The mechanism that regulates defecation in OF tests may be different from that operative in the home cage rats. BLOOD PRESSURE REGULATION AND CENTRAL NORADRENERGIC NEURONS IN THE RAT. Masashi Ogawa, Yuhzo Fukita, and Masayori Ozaki. Second Department of Pharmacology and Department of Neurosurgery, Nagasaki University, School of Medicine,, Nagasaki The role in blood pressure regulation of noradrenergic neurons originating fi'om the locus coeruleus (LC) was studied by stereotaxic administration of 6-hydroxydopamine (6OHDA) into the LC in Kyoto-Wistar Rats (from which a spontaneously hypertensive rat strain was bred) weighing 250-400 g. Intracerebral administration of 6-OHDA (912/xg/6/xl) into the bilateral LC resulted in hypertension and tachycardia, which lasted for 12 days and gradually returned to the level before administration. The blood pressure and heart rate of the bilaterally 6-OHDA administered group were 172 mm Hg and 460 beats/min, on the mean, respectively, l day after administration. The hypertension and tachycardia were accompanied by the depletion of norepinephrine content in the cortex and medulla-pons 3 days after administration. A weak inverse correlation was realized between blood pressure and norepinephrine content in the medulla-pons. Furthermore, 3 days after administration, an increase of the serotonin content in the spinal cord was observed even when the blood pressure returned to the level before administration. Three weeks after administration, the degenerative changes in noradrenergic cell bodies of the LC and the pile up of catecholaminergic fibers surrounding the LC were histochemically observed. It is suggested, therefore, that the regional chemical denervation of the bilateral LC brings about an increase in blood pressure and heart rate caused by the degeneration of,inhibitory ascending noradrenergic neurons associated with a beta adrenergic receptor of the hypothalamus. Furthermore, it is thought that the decompensation mechanism after the hypertension and tachycardia might be due to serotonergic neurons in the spinal cord. A C C U M U L A T I O N OF CATECHOLAMINE IN THE CENTRAL CATECHOLAM1NERGIC CELL BODIES AFTER INTRAVENTRICULAR ADMINISTRATION OF COLCHICINE..Masaru Sorimachi. Department of Physiology, Ehime University Medical School, Ehime Antimitotic agents are known to inhibit the axoplasmic transport of noradrenaline (NE) and its synthesizing enzymes, probably through their actions on the neurotubules in the peripheral adrenergic nerves. However, little is known about the actions of these agents in

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the catecholamine neurons in the central nervous system. Administration of colchicine into the lateral ventricles of rats caused the time-dependent accumulation of catecholamines (NE, dopamine), measured by the radiochemical methods in the locus coeruleus and the A8-10. The histochemical study also demonstrated a more marked accumulation of NE fluorescence in the LC of treated rats than in that of control. The tyrosine hydroxylase activity (TH) in the locus increased markedly (up to 150% of the control) 2 days after treatment, but the activity in A8-10 remained unaltered. Cold-restraint stress for 6 h significantly increased the TH activity (131% of control), but failed to alter the NE contents in the LC, suggesting that the increase of TH, but not NE accumulation, is, at least partially caused by the general stress caused by colchicine administration. This is reflected by both animal behavior (paraparesis and urinary tract disorder) and increased adrenal TH activity. On the other hand, no significant increase of NE was observed in the LC after treatment with lumicolchicine, an isomer of colchicine, which has a much lower microtubule binding capacity. This suggests that NE accumulation after colchicine administration is due to the blockade of axonal transport resulting from the actions of neurotubules. Dopamine-/3-hydroxylase (DBH), another neurochemical marker of NE neurons, decreased markedly 3 days after colchicine administration. Available evidence on the peripheral adrenergic nerves suggests that the decrease of DBH is indicative of chromatolysis in the LC. If this were so, the results would indicate that the axonal flow of some substances is essential for the maintenance of functions concerned with transmitter synthesis in the cell bodies. AN IMPROVED IMMUNOCYTOCHEMICAL PROCEDURE AND ITS APPLICATION TO THE VISUALIZATION OF DOPAMINE-/3-HYDROXYLASE IN THE LOCUS COERULEUS AND HYPOTHALAMUS OF THE RAT. Kihachi Saito, Donald L. Cimarusti, and Eugene Roberts. Division of Neuroscience, City of Hope Medical Center, Duarte, California A soluble complex of peroxidase was prepared with the Fab fragment obtained by papain digestion of antiperoxidase IgG and used in immunocytochemical studies of dopamine-/3-hydroxylase (DBH). The complex prepared by this new method has many advantages over other procedures used in immunocytochemical studies. At the light microscopic level, specific staining for DBH was obtained in the cytoplasm of the somata of neurons in the locus coeruleus. The localization of DBH-specific reaction product in Golgi complex and cisternae of the granule endoplasmic reticulum was confirmed at the ultrastructural level. In the hypothalamus, DBH-specific staining was associated with dense punctate structures in the area of neuronal somata located in the interstitial nucleus of the stria terminalis. At the electron microscopic level, the DBH specific reaction product was observed to be associated with synaptic vesicles in the axon terminals. A NEW FLUOROMETRIC ASSAY FOR TYPE B MONOAMINE OXIDASE ACTIVITY. Osamu Suzuki, Etsuko Noguchi, and Kunio Yagi. Institute of Biochemistry, Faculty of Medicine, university of Nagoya, Nagoya A new fluorometric assay for monoamine oxidase (MAO) activity toward/3-phenylethylamine (PEA) was devised. This method, which is simple and quite sensitive, makes it possible to determine type B MAO activity, even with small amounts of samples (1-10 mg of wet tissue). Phenylacetaldehyde (PAA), the product of the reaction of PEA catalyzed by MAO, is measured fluorometrically after the reaction with ninhydrin in the presence of Lleucyl-L-alanine. This ninhydrin reaction is carried out at pH 5.7 to avoid the reaction of PEA, the substrate of MAO, with ninhydrin. The assay mixture contains choral hydrate to protect PAA from being degraded by aldehyde dehydrogenase. Methanol is added after the

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enzyme reaction to inhibit the adsorption of PAA to macromolecules. MAO activity showed a linear relationship, with the amount of tissue in the 2.5-10 mg range in the assay mixture, and with the incubation time within 45 min. As an application of the method, the developmental change in MAO activity toward PEA of chick retina was investigated and compared with that toward 5-hydroxytryptamine (5-HT). The pattern of the developmental change in MAO activity toward PEA was similar to that toward 5-HT, even though the former activity was higher. The MAO activity on the twelfth day of incubation was rather low, after which it increased remarkably until the eighteenth day of incubation; then it reached a plateau and remained unchanged until the seventh day after hatching, but decreased significantly between the seventh and the fourteenth day after hatching. The MAO activities toward both PEA and 5-HT in chick retina were less than one-tenth of those in chick cerebral hemisphere. EFFECTS OF SUBSYNAPTOSOMAL FRACTIONS ON THE RELEASE OF [3H]DOPAMINE FROM PLAIN SYNAPTIC VESICLES. Minoru Takeda and Ryo Tanaka. Center for Brain Research, Medical Center, University of Rochester; Department of Biochemistry, Faculty of Medicine, Nagoya City University, Nagoya As an approach to an understanding of the transmitter release mechanism, the mode of [3H]dopamine uptake and release by plain synaptic vesicles was investigated, especially with regard to the influence of synaptic membranes. It is generally accepted that catecholamines are released from synaptic vesicles directly into the extracellular space without being released into the nerve terminal cytosol. If this were the case, then an interaction between, at least, synaptic vesicles and synaptic membranes should be required for the release: It also seems reasonable that plain synaptic vesicles are older in their life cycle than coated ones and are ready to release transmitters. A partially purified fraction of plain synaptic vesicles was obtained by differential centrifugation (Kadota and Kadota, J. Cell. Biol. 58, 135, 1973). The vesicles (100-150 ~g protein) were incubated in 200/zl of solution containing 156 mM KC1, 5 mM NaCI, 2 mM MgClo, 0.2 mM CaCI2, 2 mM ATP, 10 /~M [3H]dopamine (1 b~Ci), and 50 mM Tris-HC1, pH 7.4. Approximately 40% of the incorporated radioactivity was observed to be released within 5 rain after addition of synaptosomal membrance (20/~g). The degree of release was dependent on the quantity of the added membranes, the partially purified synaptic junction being more effective for the rlease. Only a small portion of the dopamine taken up was released by interaction with the isolated myelin fraction. STIMULATORY EFFECT OF DOPAMINE ON ADENYLATE CYCLASE AND NaAND K-ATPase IN N E U R A L MEMBRANE. Kirnio Akagawa and Yasuzo Tsukada. Department of Physiology, Keio University School of Medicine, Shinjuku-ku, Tokyo The molecular basis of dopamine binding to neural membrane has been reported previously; the relation between dopamine (DA) binding and activation of Na- and KATPase (Na, K) and adenylate cyclase (AC) in neural membrane obtained from rat caudate nucleus was discussed. Both enzymes were apparently present in the synaptosomal and microsomal membrane fractions, but scarcely present in the myelin fraction. The stimulatory effect of DA on Na and K reached its maximum at the concentration of 10-4 M. The activity of Na and K was stimulated by the addition of DA, noradrenaline, isoproterenol, or L-DOPA, whereas AC was stimulated only by DA or noradrenaline. Catechol, tyrosine, or tyramine had no effect on either enzyme. The DA effect on Na and K activity was suppressed in the presence of catechol. These results showed that the catechol ring was essentially necessary for the stimulatory effect on the enzymes to occur, and that the stimulatory effects varied depending on the structure of the side chains. In the kinetic

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study, DA decreased the Km value of Na and K, while it increased the Vmax of AC. This finding indicated that DA activated the inactive AC molecules and enhanced the affinity between Na and K and ATP. Calcium ions inhibited the activities of both enzymes in the presence or absence of DA. EGTA activated the Na and K without DA, there being no increase in this effect by the addition of DA. However, EGTA inhibited the activities of AC in the presence or absence of DA. FORMATION OF y-GLUTAMYLATED COMPOUNDS FROM p-TYRAMINE, NORADRENALINE, AND SEROTONIN IN RAT BRAIN. Motohiro Tsuji, Yukio Matsuoka, Tetsunori Tanaka, Hiroyuki Konishi, and Teruo Nakajima. Department of Neurology, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka Various y-glutamyl peptides in brain have been isolated and identified in our laboratory. Recently, the occurrence of y-glutamylputrescine and formation of y-glutamylhistamine were demonstrated in brain. These findings aroused our interest in the possibility that biogenic amines, i.e., p-tyramine, dopamine, noradrenaline, and serotonin, might be coupled with glutamic acid to form y-glutamylamines. Radioactive p-tyramine, noradrenaline, or serotonin was intraventricularly injected into rat brains and the brains were taken out 30 min after the injection, y-Glutamylamines were purified from the TCA extract of the brains by a combination of ion-exchange chromatography on Dowex 50 x 2, Amberlite IR120, and Amberlite CG 50. The identification of y-glutamylated compounds are based on a comparison of behavior in ion-exchange chromatography in Amberlite CG 50, paper chromatography, and high-voltage paper electrophoresis with that of the authentic compounds, which were synthesized by coupling of N-carbobenzoxy-3,-glutamylhydrazide and the amines, followed by catalytic hydrogenation of the products. Acid hydrolysis of the purified compounds in 1 N HC1 at 100~ for 1 h confirmed the identification of these compounds. Radioactivities associated with these compounds after acid hydrolysis under the conditions described above were located only in the amines. IMPAIRED CEREBELLAR DNA SYNTHESIS CAUSED BY PURKINJE CELL DAMAGE. Noriko Yamada, Yoshio Sawasaki, and Hiroshi Nakajima. Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo Homozygous (jj) Gunn rat shows marked cerebellar hypoplasia due to hereditary hyperbilirubinemia, impaired DNA synthesis being the apparent cause of this abnormality (Sawasaki, Yamada, and Nakajima, J. Neurochem. 27, 577, 1976; Brain Res., in press.) In jj cerebellum, DNA content showed no increase after 10 days, and thymidine kinase (TK) activity decreased from 6 days. Purkinje c e l l s were almost selectively affected in jj cerebellar cortex, but cerebellar lobuli that develop earlier, such as nodulus, were less affected, the Purkinje cells being only slightly damaged. These findings have suggested that Purkinje cell lesion might somehow influence the proliferation of the external granular cells. In the present study, this possibility was investigated in developing Gunn rat cerebellum. Photodegradation of bilirubin in jj cerebellum exhibited no improvement in TK activity, and the presence of enzyme inactivator was not suggested. Therefore, the induction of TK might be decreased in Gunn rat cerebellum. TK activity was decreased in dissected jj culmen, one of the late-developing cerebellar lobuli with damaged Purkinje cells, from 4 days (15%), though no decline was found in nodulus throughout development. Both total and proliferative areas of jj culmen decreased from 12 days in the saggital section. In contrast, TK activity was not affected in the jj olfactory bulb. The correlation between Purkinje cell damage and impaired DNA synthesis seems to suggest the possibility mentioned above. This effect of Purkinje cells on DNA replication in external

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granular cells cannot be explained by transsynaptic degeneration in such an early stage of development. LOCALIZATION OF OLIGO(A) IN POLY (A) CONTAINING RAPIDLY LABELED DNA-LIKE RNA FROM MOUSE BRAIN. Hiroshi Onishi, Koichi Mori, Nobuo Kiriike, Sakae Yamagami, and Yukio Kawakita. Department of Neuropsychiatry, Osaka City University Medical School, Osaka One hundred 50-day-old mice were injected with 20 ~tCI of [3H]adenosine intracranially. Rapidly labeled DNA-like RNA fractions were obtained at 63 and 85~ by the method of Markov and Arion (Eur. J. Biochem., 35, 186, 1973). The T1RNAase-digested RNA sample was subjected to poly(U) sepharose chromatography by the method of Molloy and Jelinek (Cell, 1, 43, 1974). The bound material emerged in two peaks (peak I and peak II), peak I appearing at a position estimated to be 3-5% formamide, and peak II at approximately 50-65%. Each peak was analyzed by gel filtration on Sephadex G50 (Stanley's method) and 15% polyacrylamide gel electrophoresis containing 8 M urea. Peak I consisted of 20- and 30-unit adenylate-rich nucleotides, while about 230 nucleotides of poly(A) were identified from peak II. Cordycepin interfered with the incorporation of radioactivity into peak II but not peak I. Rapidly labeled RNA was sedimented through a 4.6-22% sucrose density gradient containing 70% formamide (SW40 rotor, 39000 rmp, 960 rain, 25~ The "larger than 20S," "20-11S," and "smaller than 1IS" components were collected for further fractionation of poly(A) + RNA, the digested material was separated on poly(U) sepharose, the oligo(A) and poly(A) eluting out together with 90% formamide; the eluate was then subjected to electrophoresis on 15% polyacrylamide gel containing 8 M urea. The ratios of oligo(A) per poly(A) were calculated from the radioactivities, assuming the segments of oligo(A) to be 20 and 30 nucleotides long, and poly(A) 200 nucleotides long. The ratios of oligo(A~0) and oligo (A:3;30) per poly(A~00) in the "larger than llS portion" were 6 and 8, respectively, while neither of the oligo(A) segments were present in the "smaller than 11S" fraction. PUTRESCINE METABOLISM IN THE BRAIN OF CHICK EMBRYO AND CULTURED N E U R O N A L AND GLIAL CELLS. Kenji Sobue, Tadashi Noto, Motoharu Hirai, and Teruo Nakajima. Department of Neurology, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka Metabolism of polyamines in the brain of chick embryo and cultured neuronal and glial cells was studied using radioactive [1,4-~4C]putrescine to clarify the physiological significance of polyamines in the central nervous system. Very active formation of y-aminobutyric acid from putrescine was ~bserved at the early stage (fourth to eighth day of incubation) of ontogenesis, while the formation of spermidine from putrescine was active at the early and the late stages (fourth to sixth and eleventh to fiftee~ath day of incubation). Since proliferation and differentiation of cells occur in the early stage, and proliferation of glial and endothelial ceils in the late stage, it was speculated that the formations of yaminobutyric acid and spermidine are related. To clarify this relationship more precisely, the formations of y-aminobutyric acid and spermidine from putrescine were analyzed during cultivation of neuroblastoma (N-18) and glioblastoma cells (C-6). In the N-18 clone, the formation of y-aminobutyric acid was low during the log phase, becoming active at the beginning of the stationary phase. In the C-6 clone, the formation of y-aminobutyric acid was low during all phases. The formation of spermidine was more active at the early stage of the log phase and then decreased gradually in both cell lines. Since it has been reported that in the N-18 clone the stationary phase was compatible with the biochemically,

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morphologically, and electrophysiologically differentiated stage of the cells, the formation of y-aminobutyric acid might be related to the differentiation of nerve cells. The formation of spermidine might be related to the proliferation of neuronal and glial cells. STUDIES OF C E L L U L A R INTERACTION BETWEEN NEUROBLASTOMA CLONES AND MYOBLAST CLONE L6. Takehiko Amamo. Mitsubishi-Kasei Institute of Life Sciences, Machida, Tokyo Proteins released from neuroblastoma clones and rat myoblast clone L6 into the culture medium were studied using SDS-polyacrylamide gel electrophoresis according to the method of Truding et al. (1975). Proteins released from MNI8 adapted to serum-free Dulbecco's modified Eagle's medium for 3 years were also analyzed by the same method, and their protein pattern was compared with those from N18 clone, L6, and rat glioma C6. In proteins released from MN18, the three main bands had molecular weights of 86,500, 72,000, and 62,000, respectively. Cocultivation of L6 myoblast cells with neuroblastoma clone NI8 induced a twofold increase in acetylcholinesterase activities compared with neuroblastoma cells cultivated separately. Cocultivation of C6 glioma cells with neuroblastoma cells had no effect on the acetylcholinesterase activity of neuroblastoma cells. Used medium or conditioned medium of L6 cells also increased AChE activities in MN18 cells. These results confirm the existence of an interaction between neuroblastoma cells and myoblast cells, which is mediated by factors diffused from muscle cells. NEUROTROPHIC FACTORS REGULATING ACTION POTENTIALS OF ORGANCULTURED MOUSE SKELETAL MUSCLE: PARTIAL PURIFICATION AND CHARACTERIZATION. Shuji Hasegawa, Tohru Gonoi, and Hiroshi, Kuromi. Brain Research Institute, School of Medicine, Chiba University, Chiba Some properties of the action potential in skeletal muscle are controlled by neurotrophic factors. Decreases in the maximal rising rate of action potentials and in its tetrodotoxin (TTX) sensitivity after denervation were partially reversed during cultivation of denervated muscle in a medium supplemented with spinal cord extract. Preliminary attempts were made to isolate and characterize the trophic factor(s) for TTX sensitivity from mouse spinal cord. The extensor digitorum longus muscle of adult mouse was excised 3 or 4 days after denervation and cultured with spinal cord extract for 3 days. Membrane potentials or organ-cultured muscle were measured by a standard glass-microelectrode method. Mouse spinal cord was homogenized and centrifuged, and supernatant was obtained as crude spinal cord extract. The trophic factor(s) in crude extract was (were) separated from macromolecules by gel-filtration through a Biogel P2 column. Biogel fractions having trophic effects on TTX sensitivity were further purified by CM Sephadex column. Chromatographic behavior showed that the trophic factor(s) was (were) a low-molecularweight substance and had a positive charge in neutral solution. Trophic factor(s) was (were) heat-stable, acid-stable, and alkali-labile. It was not adsorbed on charcoal and was inactivated during lyophilization. Trophic activity was abolished by pronase digestion, but not by trypsin, chymotrypsin, or thermolysin treatment. These results indicate that trophic factor(s) for TTX sensitivity of action potentials in mouse skeletal muscle differs (differ) from those substances reported to be trophic factor(s) for cholinesterase activity of chick and newt skeletal muscle. ISOLATION OF THE PLASMA MEMBRANE FROM CRAYFISH NERVOUS TISSUES WITH AN AQUEOUS TWO-PHASE POLYMER SYSTEM. Seiji Uehara and Keiichi Uyemura. Department of Physiology, Saitama Medical School, Moroyama, Saitama A method using an aqueous two-phase polymer system for the isolation of a neuronal

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plasma membrane fraction from crayfish nervous tissues has been developed. The pellet obtained by 30-rain centrifugation, at 50,000g, of the homogenate was suspended in a solution containing 6.6% polyethylene glycol 4000 and 4.8% dextran 300. After low-spin centrifugation, the membrane fraction (IP), which formed a thin white sheet at the interphase, was obtained. Successively, the IP was subjected to a discontinuous sucrose density gradient centrifugation. The plasma membrane fraction (PM) was obtained at the interphase between the 0.9 and 0.5 M layers. Another membrane fraction (A-2) obtained directly by density gradient centrifugation without using the two polymer system was also analyzed for comparison. (Na + and K+)-dependent ATPase showed a 22-fold enrichment in PM over the homogenate compared to a 10-fold enrichment in both IP and A-2. All three fractions, PM, IP, and A2, were enriched 3- to 6-fold in acetylcholinesterase activity. On the other hand, the specific activities of succinate- and NADH-cytochrome c reductase of both PM and IP were lower than those of the homogenate. By electron microscopic observations, it was observed that the membrane structures were highly concentrated in PM with a few contaminants. It was concluded that the aqueous two-phase polymer system described in this communication can be used to advantage in the preparation of the plasma membrane fraction of nervous tissues. ENZYMATIC ACTIVITIES AND METABOLIC REACTION IN K+-R1CH MEDIUM OF ETHYLNITROSOUREA-INDUCED GLIOMA TISSUE. Shusuke Hirano, Yukifumi Noda, Tomoyuki Kanamatsu, Hiroaki Asou, and Kenji Nakai. Department of Physiology and Section of Central Laboratory, Toho University School of Medicine, Ohmori, Tokyo In order to clarify the biochemical specificity of glial cells, the enzyme activities and the metabolic reactions in K+-rich medium of an experimental glioma tissue were investigated. The experimental glioma was made to multiply in nude mouse by subcutaneous transplantation of ethylnitrosourea-induced glioma from rat brain. Analysis of the enzyme activities of the glioma tissue revealed that the choline acetylase activity was 1.5%, and the acetylcholinesterase activity 5-fold, that in rat brain homogenate; the activities of CNPase, pyruvate kinase, and Na and K-dependent ATPase were lower, and that of carbonic anhydrase 50% higher. The free amino acid constituents of brain tissue in ddY were quite different from those in both BALB/c and nude mice, the contents of aspartic acid, glycine, histidine, and arginine being higher in both BALB/c and nude mouse brain. A stimulating effect on oxygen consumption by K+-rich medium was observed in BALB/c brain cortex, but not in glioma slices. In the glycolytic pathway, the oxidative portion of the hexose monophosphate pathway was essentially operative in glioma tissue, though inoperative in mature brain slicens. The utilization of glucose, especially [6-14C]glucose, by glioma slices decreased in medium higher in potassium concentration. UPTAKE AND UTILIZATION OF D-GLUCOSE ANOMERS BY THE BRAIN CELLS IN VITRO. Yutaka Nagata, Tsuneatsu Nanba, Masato Ando, Ichitomo Miwa, and Jun Okuda. Department of Physiology, School of Medicine,, Fujita-Gakuen University, Toyoake, Aichi; Department of Clinical Biochemistry, Faculty of Pharmaceutical Science, Meijo University, Nagoya D-Glucose is an important substrate maintaining the functional activity of brain cells. It is known to exist as an equilibrium mixture of its two anomers in aqueous solution: 36.5% a-D-glucose and 63.5% r-D-glucose. It was found that/g-glucose was more actively taken up and metabolized by the cerebral cortex slices than a-glucose when incubated for 5 rain during which the anomeric equilibrium was not attained. The preferential utilization of r glucose to a-glucose by the brain slices did not appear before the fifteenth day after birth. The anomeric specificity of glucose uptake was demonstrated after the twentieth day and

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in the adult brain. Depolarizing conditions of the brain cells, such as the addition of potassium to or omission of calcium from the incubation medium, resulted in a much higher rate of increase of not only glucose utilization but also oxygen uptake and lactate output by the brain slices when a-glucose rather than/3-glucose was used as the substrate. On the contrary, in sodium-free medium or in the medium with added phlorizin or ouabain, the aerobic glycolysis of the brain slices decreased. From these results, it may be concluded that anomeric specificity in o-glucose metabolism by rat brain cortex slices was preferential for the /3-anomer rather than the a-anomer. This preferential uptake and utilization of /3-glucose over a-glucose was also shown in synaptosomes and in bulkseparated neuronal and glial cell fractions obtained from adult rat brains. A SPECTROPHOTOMETRIC ASSAY OF 2',3'-CYCLIC-NUCLEOTIDE 3'-PHOSPHOHYDROLASE (E.C. 3.1.4.37). Yoshiko Nishizawa, Tadashi Kurihara, and Yasuo Takahashi. Department of Neuropharmaeology, Brain Research Institute, Niigata University, Niigata A simple and rapid spectrophotometric assay of brain 2',3'-cycllc-nucleotide 3'-phosphohydrolase (E.C. 3.1.4.37) was reported. Both sample and reference cuvettes in a Hitachi 323 spectrophotometer contained a mixture consisting of bromothymol blue (pKa 7.0), cetyltrimethylammonium bromide, imidazole-HC1, pH 6.6 (PKa 7.0), 2-mercaptoethanol, and 2',3'-cyclic AMP. The reaction was started by adding an enzyme solution to the sample cuvette, and extinction at 420 nm was recorded for 1-2 min with appropriate scale expansion, E n z y m e activity was determined by a linear slope taken from the initial 20-sec reaction. The method is based on the use of an acid-base indicator and a buffer having identical pKa values. REGIONAL DIFFERENCES IN SPHINGOLIPID COMPOSITION IN THE NERVOUS SYSTEM. Takeshi Ishibe, Akira Yamamoto, and Mitsuo Nishikawa. Second Department of Internal Medicine, Osaka University Medical School, Osaka The lipid composition in different parts of the nervous system was determined in suckling (13-day-old) and adult rabbits. Tissues were homogenized and extracted with 20 vol of chloroform-methanol 2 : 1 (v/v) three times, and lipid analysis was performed by quantitative thin-layer chromatography. The ratio of cerebrosides having a-hydroxy fatty acid to those having unsubstituted fatty acid (CH/CN) was higher in rostral than in caudal parts on the brain. The gray matter had a higher CH/CN value than the white. Among the cranial nerves, the optic nerve showed the highest CH/CN value, which was almost the same as that in cerebrum. The CH/CN ratio in the oculomotor nerve was the lowest, being the same as that in sciatic nerve. The CH/CN ratio had an inverse relation with the ratio of total shingolipids (galactolipid + sphingomyelin) to phosphatidylcholine. There was a positive correlation between the CH/CN ratio and the ratio of galactolipid to sphingomyelin in both central and peripheral nervous tissues. Newly developed areas of the nervous system contained much larger amounts of galactolipids, especially cerebrosides with ahydroxy fatty acids, than did less differentiated areas. The major fatty acids in cerebrosides were the saturated and monounsaturated ones with carbon chain length 24. In the central nervous system, there was a negative correlation between the CH/CN and Cz4:1/ C24:0 ratios. LONG-CHAIN-BASE COMPOSITION OF GANGLIOSIDES IN AMAUROTIC FAMILY IDIOCY OF LATE INFANTILE FORM (BIELSCHOWSKY-JANSKY DISEASE). Nariko Kawamura and Tarnotsu Taketomi. Department of Biochemistry, Institute of Adaptive Medicine, Shinshu University, Matsumoto

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Total lipids extracted from the brain of a patient with Bielschowsky-Jansky disease (9 years) and other human subjects as controls were directly subjected to column chromatography on Silica Gel G-Silica Guhr G (1 : 1, w/w) slurried with chloroform-methanol-28% ammonia (32 : 8 : 1, by vol) without the Folch partition. After the total lipids excluding gangliosides were eluted with the same solvent, total gangliosides were eluted with chloroform-methanol-water (60 : 35 : 8, by vol). After the total gangliosides were subjected to alkaline methanolysis and dialyzed against deionized water, the ganglioside fraction thus obtained was rechromatographed in the same way. The individual gangliosides were then separated by preparative TLC using plates coated with Silica Gel G-Silica Guhr G (1 : 1, w/w). The content ofgangliosides and their C20/C18 sphingosine ratio were determined by our new ozonolytic procedure. The gray and white matter total gangliosides in controls, respectively, contained N400-500/zg and ~150-200/xg of long chain base per gram fresh tissue. The total gangliosides in the patient, respectively, contained 393 and 135 /xg of long chain base and gave normal composition patterns of individual gangliosides. The C2o/C18 sphingosine ratio in controls seemed to increase with age in order of G~3 < G~2 < G~I < GD1~ < GDlb -- GT1. But the ratios in patient individual gangliosides decreased 2- to 3-fold compared with those in age-matched controls. This disease also seemed to be related to an abnormal metabolism of long chain bases of gangliosides. THE METABOLISM OF HORMONES IN CNS: 5a-OXIDOREDUCTASE AND 3c~DEHYDROGENASE IN RAT DIENCEPHALON AND THE EFFECTS OF CENTRALLY ACTING DRUGS. Mutsutoshi Kohsaka, Toshikiyo Shohmori, Takao Kaneyuki, and Misako Okita. Division of Clinical Neurochemistry, Institute for Neurobiology, Okayama University Medical School, Okayama Sex hormones often have been shown clinically to be related to psychic excitation in humans. The present experimental study investigated sex-hormone .metabolism in the animal brain for the purpose of developing some preliminary concepts on the possible relationship between sex-hormone metabolism and psychic states. Rat diencephalon homogenates were incubated with [4-14C]testosterone (T) or [4-14C]dihydrotestosterone (DHT) and NADPH. The findings indicated that DHT was a metabolite of T and that 5c~androstan-diol was a metabolite of DHT. Therefore, the activities of 5a-oxidoreductase and 3c~-dehydrogenase appear evident in the rat diencephalon. The characteristics of 5c~oxidoreductase included an optimal pH of 6.8 and a K,~ value of 2.2 • 10-6 M, whereas 3adehydrogenase had an optimal pH of 7.0 and a K,~ value of 5.9 x 10-6 M. Furthermore, diphenylhydantoin, phenobarbital, reserpine, chlorpromazine, imipramine, and dipiperone were injected intraperitoneally into rats, and the enzyme activities in the diencephalon were examined. Adrenalectomized and gonadectomized male rats were also used in these experiments. EFFECTS OF BIOLOGICALLY ACTIVE PEPTIDES ON THE EXCITABILITY OF IDENTIFIABLE MOLLUSCAN GIANT NEURONS (Achatina fulica Frrussac). Hiroshi Takeuchi, Michiko Matsurnoto, Akinori Sakai, and Akitane Mori. Institute for Neurobiology, Okayama University Medical School, Okayama. Examined were the effects of biologically active peptides obtained in an analytically pure state, including vasoactive peptides, hypothalamic hormonal peptides, and peptides analogous to neurohypophyseal hormones, on the excitability of two giant neurons (the TAN, tonically autoactive neuron, and the PON, periodically oscillating neuron) identified in the subesophageal ganglia of Achatina fulica Frrussac. Of these peptides, only physalaemin at 10-4 kg/liter (bath application) showed an excitatory effect on the TAN. On the other hand, deamino-dicarba-(d-d)-oxytocin and d-d-Arg-vasotocin at a concentration

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of 10-4 kg/liter had an excitatory effect on the PON, while d-d-vasopressin did not. We measured the current-voltage relationships (the I - V curve) of identifiable neurons as an indicator of their electrical resistances by application of a transmembrane triangular current of long duration. We conclude that physalaemin at 2 • 10-4 kg/liter (bath application) does not affect the TAN I - V curve, and that d-d-oxytocin at 2 • 10-4 kg/liter does not affect the PON I - V curve, based on the results of a superimposition of the I - V curve measured in the presence of the peptides on that of the physiological state, using the firing level of the neuron as the common standard. Physalaemin lost its excitatory effect on the TAN by the treatment of trypsin. This substance, when treated with chymotrypsin for 6 h, showed an inhibitory effect on the same neuron, opposite to that of untreated physalaemin. Deamino-dicarba-(d-d)-oxytocin and d-d-Arg-vasotocin continued to show their excitatory effects on the PON after treatment with chymotrypsin for 6 h. However, dd-Arg-vasotocin, when treated by trypsin, lost its effect on the PON. INFLUENCE OF SODIUM AND POTASSIUM IONS ON GLUTAMATE UPTAKE INTO RAT CEREBRAL SYNAPTOSOMES. Genkichiro Takagaki, Tokyo Metropolitan Institute for Neuroscience, Fuchu-shi, Tokyo Kinetic studies on glutamate uptake were undertaken, using labeled substrate and the Millipore filtration technique. The uptake rate of L-glutamate was almost saturated at the Na + concentration of the standard medium (132 mM), but that of D-glutamate, the Km for which was much higher than for the r-isomer, was increased at higher Na / concentrations. Reducing the concentration of Na + in the medium increased the K,, with no significant effect on the V~ax for L- and D-glutamate uptake. However, for the uptake of GABA, reducing the concentration of Na / increased the K,~ and reduced the Vmax. Removing K + from the medium increased the Km without altering the Vmaxfor L- and D-glutamate uptake. Ouabain was a competitive inhibitor for L- and D-glutamate uptake, but the inhibition of GABA uptake by ouabain was of the mixed type. The requirement for Na / in the uptake of L- and D-glutamate as well as of GABA seemed to be specific, as it could not be substituted by NH4+, Li >, Cs ), or Rb +. Rb + could completely substitute for K > in the uptake of L- and D-glutamate as well as of GABA. NH4 ~ and T1 + could partly substitute for K > in the uptake of GABA, but not in that of glutamate. Thus, the mechanisms for Land D-glutamate uptake seemed to be the same. These observations are in conflict with the Na ~ concentration gradient hypothesis for the transport. Rather, Na and K ATPase may participate in the transport, presumably by facilitating the binding of Na + and glutamate with the carrier. The mechanisms of GABA uptake were found to differ from those of glutamate. ACCELERATION BY A23187 AND X537A OF CRUDE SYNAPTOSOMAL FRACTION AND Tanaka. Department of Biochemistry, Nagoya Nagoya; Center for Brain Research, University Dentistry, Rochester, New York

TRANSMITTER RELEASE FROM A SLICES. Kuniko Okumura and Ryo City University School of Medicine, of Rochester School of Medicine and

We investigated the effects of calcium ionophores on the release of [~H]5-hydroxytryptamine (5HT) and [3H]dopamine (DA) which had been incorporated into either a crude synaptosomal fraction of rat cerebrum or slices prepared from rat cerebral cortex and striatum. Both A23187 and X537A increased the release rate of radioactive materials from the crude synaptosomal fraction. The acceleration effects of A23187 were almost totally eliminated under conditions suppressing the production of metabolic energy, whereas the X537A effects were only partially affected under identical conditions. The radioactivity released from cerebral cortex slices was also enhanced by these two types of ionophores

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except when metabolic inhibitors were present. A23187 decreased the number of synaptic vesicles per unit area of synaptosome sections. Using thin-layer chromatography, we examined the radioactive materials released in the presence of ionophores from either the cortex synaptosomal fraction or slices that had incorporated [ZH]DA. The examination revealed that most of the materials were DA and NA, and the remainder were metabolized product(s) of DA. In the exploratory experiment, the presence of the ionophores in the incubation medium increased 4~Cae+ uptake into both the crude synaptosomal fraction and the slices. The results suggest the following: (1) The process of transmitter release that is accelerated by calcium ionophores requires both the production of metabolic energy and the presence of Ca 2+ in the medium; (2) however, there is a difference in the mechanism of acceleration by ionophores A23187 and X537A, with the latter not necessarily needing metabolic energy for the acceleration; (3) the materials released in the presence of ionophores are incorporated DA and NA, a transmitter derived from DA. Only a small portion of the materials consisted of DA metabolite(s). (Supported in part by USPHS NIH Grants NS11951 and NS06827.) DISTRIBUTIONS OF GABA AND GAD ACTIVITY IN RAT THALAMUS AND THEIR MODIFICATION WITH MORPHINE ADMINISTRATION. Yukio Yoneda and Kinya Kuriyama. Department of Pharmacology, Kyoto Prefecture, University of Medicine, Kyoto Rectangular blocks (750 x 750 x 320 p~m) of rat thalamus were made from frozen sagittal sections obtained 5.4, 4.6, 3.2, and 2.6 mm anterior to the interaural line (according to the stereotaxic atlas of de Groot). In the anterior part of the thalamus (A 5.4), it was found that areas having high GABA content and GAD activity coincided with the ventrolateral part of the ventral nucleus of the thalamus (VM), entopeduncular nucleus (EP), and nucleus reuniens thalami (RE), respectively. In the medial part of the thalamus (A 4.6), it was found that GABA content was relatively low in all tissue blocks examined. In the posterior part of the thalamus (A 3.2), the areas surrounding the central gray matter, dorsal fasciculus of Schfitz, and nucleus parafascicularis thalami (PF) showed high GABA content and GAD activity. GABA content in the VM, PF, EP, RE, and interpeduncular nucleus (IP) increased significantly following the administration of morphine (20-30 mg/kg, i.p.), and these increases were antagonized by pretreatment with levallorphan (5 mg/kg, i.p.). GAD activities in the EP, VM, and RE were also elevated by the morphine treatment. On the other hand, the administration of sodium salicylate (80 mg/kg, i.p.) and of pentazocine (45 mg/kg, i.p.) failed to induce any change in GABA content in any of the areas mentioned above except RE. The GABA content in RE also showed an increase following the administration of these two drugs. These results suggest that functional alterations of GABA-containing neurons involved and/or associated with well-known pathways for pain perception may have an important role in inducing morphine analgesia, possibly by increasing inhibitory inputs at the level of the thalamus. STIMULATION OF BRAIN ACETYL-CoA HYDROLASE BY ATP AND ATP ANALOGUES. Tomohiro Matsuda, Norifumi Yonehara, and Hiroshi Yoshida. Department of Pharmacology, Osaka University School of Medicine, Osaka The effects of ATP and ATP analogues on brain acetyl-CoA hydrolase (E.C. 3.1.2.1) were studied. The enzyme was stimulated reversibly by ATP-Mg v+, the presence of Mg ~ being absolutely required for the stimulation. The stimulatory effect of ATP was highly specific, since adenine nucleotides other than ATP had no stimulatory effects. Stimulation by nucleoside triphosphates other than ATP was much less than for ATP, decreasing in the following order: ATP > ITP ~ CTP, UTP ~> GTP. A nonphosphorylating analogue of

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ATP, AMP-P(NH)P, had a stimulatory effect similar to that of ATP, indicating that the mechanism of ATP stimulation is caused neither by a phosphorylation of the enzyme nor by a reaction involving the hydrolysis of the terminal phosphate group of ATP. The other ATP analogues, such as AMP-P(CH~)P and AMP(CH~)POP, were less stimulatory than ATP, decreasing in the following order: ATP > AMP-P(HN)P > AMP-P(CHz)P > AMP(CH2)POP. The concentrations needed for half-maximal stimulation of ATP, AMPP(NH)P, and AMP-P(CH~)P were 0.11 mM, 0.22 mM, and 0.22 raM, respectively. Double reciprocal plots demonstrated that ATP as well as AMP-P(NH)P produced a significant decrease in the apparent Km value for acetyl-CoA and an increase in Vmax, indicating that these nucleotides increase the affinity for acetyl-CoA by binding at a site other than the catalytic site. These data suggest that the rate of hydrolysis of acetyl-CoA may be regulated by the concentration of ATP in the microenvironment of the enzyme. THE RESTORATION OF PHOSPHODIESTERASE ONTO PHOTORECEPTOR DISK MEMBRANES AND SUBSEQUENT RECOVERY OF LIGHT SENSITIVITY. Naomasa Miki and Mark W. Bitensky. Department of Pharmacology, Kyoto Prefecture University of Medicine, Kyoto; Department of Pathology, Yale University Medical School, New Haven, Connecticut Disk membranes prepared from frog rod outer segments contained a light-sensitive and ATP-dependent cyclic GMP phosphodiesterase (PDE), which is firmly bound to disk membranes and is maximally stimulated (8- to 15-fold) in the presence of 0.75-1 mM ATP. The half-maximal activation of PDE was achieved b y bleaching with 0.05% rhodopsin. PDE was released from disk membranes by washing with 10 mM Tris-HCl buffer (pH 7.4) not containing Mg 2+. The eluted PDE had a markedly reduced activity and was no longer stimulated by light and ATP. It was, however, activated by polycations such as protamine or by mild trypsin digestion. In the presence of Mg 2+, the eluted PDE was reassociated with the disk membranes from which PDE had been removed (depleted membranes), thus restoring their light sensitivity. The PDE restored to the disk membranes had a markedly decreased sensitivity to light, and approximately 15% of bleached rhodopsin was required to produce half-maximal stimulation of the restored PDE. The molar ratio of restored PDE to rhodopsin was estimated to be l : 250 to 1 : 500. Mildly trypsin-digested PDE did not bind to the depleted membranes in the presence of Mg 2+. These results suggest that the close association of the PDE molecule with disk membranes is indispensable for the activation of PDE by light and ATP. THE MODE OF ACTIVATION OF CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES FROM THE SUPERNATANT FRACTION OF RAT BRAIN. Tsunesada Sakai, Hiroko Yamanaka, Hideo Makino, and Ryo Tanaka. Department of Biochemistry, Nagoya City University School of Medicine, Department of Medicine, Fujita Gakuen Medical School, Toyoake, Aichi The 100,000g supernatant fraction of rat brain contains cyclic-AMP and cyclic-GMP phosphodiesterases that are known to be stimulated by activator protein and Ca 2+. The supernatant enzymes were separated into discrete active fractions by DEAE-cellulose chromatography. One of these fractions was markedly stimulated by activator protein that had been purified from bovine brain. Sodium c~-tocopherylphosphate (TPNa), one of the vitamin E derivatives, mimicked the stimulatory effects of activator protein, with a comparable magnitude of stimulation (5- to 10-fold over the basal activity) and a halfmaximum effect of 0.1 mM when 1 p~M cyclic AMP was used as substrate. Although the TPNa activated both cyclic-AMP and cyclic-GMP PDE's, the extent of stimulation was greater for cyclic-AMP PDE activity. The stimulation by TPNa was partly dependent on

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Ca 2+. The activation of cyclic-AMP PDE by both TPNa and activator protein appeared primarily in the Vmax without affecting the Km value (50 mM for cyclic-AMP). Activator protein and TPNa decreased the thermal stability of cyclic-AMP PDE, suggesting that the PDE appears to undergo a structural modification upon interaction with the activators, and this is associated with increased catalytic activity. TPNa activation of PDE was not further enhanced by activator protein. This result indicates that the action of TPNa may be similar to that of activator protein and that an understanding of its mode of action may be crucial to the clarificar of the regulatory properties of PDE.

Abstracts of communications of the Japanese Neurochemical Society.

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