The effects of various smooth muscle relaxing drugs on contractile responses to acetylcholine (ACh), Ba²⁺ and Ca²⁺, and on the tissue cyclic AMP levels were examined in the guinea pig ileum. Papaverine and theophylline caused a decrease both in the maximum height and the slope of dose-response curves induced by the three stimulants, and an increase in the cyclic AMP levels. Diltiazem and D-600 produced a decrease in the maximum and the slope of ACh and Ba²⁺ dose-response curves, shifted the Ca²⁺ dose-response curves to higher concentrations, in a parallel manner, but failed to change the cyclic AMP levels. Etomidoline and benactyzine shifted the curves for the three stimulants in parallel to the right, but at higher concentrations depressed the maximum of ACh and Ba²⁺ responses with a further parallel shift. These drugs exerted little influence on the basal level of tissue cyclic AMP, but etomidoline significantly depressed the Ba²⁺-induced increase in cyclic AMP level. The smooth muscle relaxing drugs used could be classified in three types, thereby suggesting that there are at least three different mechanisms involved in smooth muscle relaxing action.

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Neuroleptic drugs were given to rats and at different time intervals the animals were decapitated and ³H-spiroperidol binding to the striatal homogenates was determined in vitro. Two hours after an intraperitoneal administration of spiroperidol, perphenazine, haloperidol, chlorpromazine or thioridazine in doses of 0.03-30 mg/kg, ³H-spiroperidol binding to the striatal homogenates was inhibited dose-dependently with an ID50 value of 0.11, 0.23, 1.1, 3.7 or 9.4 mg/kg, respectively. After a single intramuscular administration of fluphenazine enanthate (10 mg/kg), a longacting neuroleptic drug, long-lasting inhibitory effect (over 4 weeks) on the binding was also observed. These results indicate that ex vivo ³H-spiroperidol binding method may be useful to measure the duration and the potency of anti-dopaminergic activities of drugs.

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An improved mass fragmentographic assay method for the determination of γ-aminobutyric acid (GABA) in the brain is described. Applicability of the method was examined in a study of the effect of semicarbazide on GABA levels and in a separate study to confirm post-mortem increase in GABA. The method itself is based on Cattabeni's procedure in which GABA is assayed as trimethylsilyl derivative. Three improvements were made: a) application of a more suitable mass spectrometry system for GABA determination; b) use of 6-aminocaproic acid as the internal standard; c) selection of a high intensive ion (m/z 174) for mass fragmentographic analysis. The mass spectrometer used is accurate to as little as 25 pg. GABA levels after semicarbazide treatment decreased 54.4% in rat whole brain and 44.2% in the dosal hippocampus. Rapid post-mortem increases in GABA levels were confirmed by application of the improved assay method; decreases were most clearly observable following microwave irradiation. Post-mortem changes in GABA were observed within 3 min after death, as reported by other researchers.

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Morphine inhibited the adenylate cyclase activity of the crude synaptosomal fraction of the rat caudate nucleus in the presence of GTP, GDP, Gpp(NH)p or ITP. The purine nucleotides themselves had an inhibitory action on the enzyme. β-Endorphin and Met-enkephalin also inhibited the enzyme in the presence of GTP. The GTP-dependent inhibitory action of morphine was blocked by naloxone. Various opiates and opioid peptides inhibited the enzyme by up to approximately 20 per cent in the presence of GTP. The relative potency was in higher order of levorphanol> β-endorphin> Met-enkephalin> morphine> pentazocine. Levorphanol was about 50, 000 times as potent as its biologically inactive enantiomer, dextrorphan. Morphine enhanced the inhibitory actions of GTP and GTPase-resistant Gpp(NH)p on the adenylate cyclase activity. These results suggest that GTP plays an important role in the regulation of adenylate cyclase activity in the rat caudate nucleus and that the occupation of opiate receptor by agonists inhibits the enzyme through an actual increase in the inhibitory action of GTP, rather than a suppression of the enzymatic degradation of GTP.

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Changes in the properties of cyclic AMP-dependent protein kinase were studied in the kidney of hypothyroid rats. The activity of the crude extract was greater in hypothyroid rats than in normal ones. The ratio of type I isozyme to type II isozyme in hypothyroid rats was about twice that in the normal rats with enzymes from both the cortex and medulla. A higher affinity for the substrates was shown in the enzymes of the hypothyroid rat kidney. The effects of actinomycin D and of heat-stable protein kinase inhibitor on the enzyme activity were the same in both normal and hypothyroid rats. These changes in enzyme properties and isozyme distribution may well play an important role in the hypothyroid rat kidney.

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To elucidate the mechanisms of smooth muscle relaxant effects of nitroglycerin, the effects of this substance on the potassium contracture (K⁺-contracture) and the lanthanum-induced contracture (La³⁺-contracture) were studied using smooth muscle preparations of the canine coronary artery and colon and the findings compared with the effects of nifedipine, a representative calcium antagonistic vasodilator. The effects of papaverine, a prototype smooth muscle relaxant, were also studied. La³⁺-contracture was induced in a calcium-free environment with the addition of lanthanum, an effective blocker of calcium influx. In the coronary artery, nitroglycerin produced a relaxation both of the La³⁺-contracture and the K⁺-contracture. However, neither La³⁺-contracture nor K-contracture in the colon was affected. Nifedipine did not relax the La³⁺-contracture in a range of doses at which K⁺-contracture of both types of smooth muscles was relaxed. Papaverine produced a relaxation of La³⁺-contracture as well as K⁺-contracture, in both types of smooth muscles. Unlike the mechanism related to the relaxant effects of nifedipine, which is generally admitted to be an inhibition of calcium influx, the relaxant effect of nitroglycerin was attributed to the suppression of calcium release from the intracellular store sites and/or stimulation of calcium uptake into the intracellular store-sites. Papaverine was assumed to produce a relaxation through augmentation of the calcium binding to the intracellular store sites for calcium as well as through inhibition of the calcium influx.

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The lipids extracted from group A hemolytic streptococci (strain Su, Blackmore and C203U) were examined for their antitumor effect against Ehrlich ascites carcinoma in mice. Total lipids were extracted from streptococcal cells according to the method of Folch et al, and separated into 9 lipid fractions by thin-layer chromatography, using various solvent systems. Three fractions were compound lipids (diphosphatidyl glycerols, monoglucosyl diglycerides and diglucosyl diglycerides), and the remaining 6 fractions were neutral lipids such as free fatty acids, glycerides, sterols and sterol esters. For biological testing, the lipid fractions suspended in physiological saline containing Tween 20 (0.02%) were incubated with Ehrlich tumor cells at 37°C for 90 min, and the cell mixture was given intraperitoneally into mice thereafter. Among 9 lipid fractions, free fatty acids and monoglycerides from the streptococci examined were highly active in suppressing the development of ascites carcinoma in mice. Diphosphatidyl glycerols from two strains of streptococci (Blackmore and C203U) were also effective in suppressing the tumor growth in mice. However, the other lipid fractions had little effect on the tumor growth.

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We examined the pharmacological effects of intracerebroven-tricularly administered aliphatic diamines on ingestive behavior in male rats adapted to a 4 hr per day feeding and drinking schedule. 1, 2-Ethanediamine (ETD), 1, 3-propanediamine (PRD), 1, 4-butanediamine (putrescine, PUT), 1, 5-pentanediamine (cadaverine, CAD) and 1, 6-hexanediamine (HED) suppressed feeding and drinking behavior in a dosedependent manner, but not unless a relatively high dose (over 80 μg) was given. The approximate anorectic potency was HED>CAD=PUT> ETD>PRD. A sedation was also produced in fairly good parallel to these alterations in feeding and drinking behavior. Thus, there appears to be a relationship between the length of the carbon chain and the potency of the pharmacological action, and these inhibitory effects on feeding and drinking behavior are probably not due to a specific action on the regulatory system for ingestive behavior, but rather to a nonspecific action.

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Intraventricular administration of nicotine produced a biphasic effect, consisting of an initial rise then a slight fall in blood pressure in unanaesthetized rats. Spontaneously hypertensive rats (SHR) showed increased pressor responses, but these responses were within normal limits in renal hypertensive and DOCA-saline hypertensive rats. The blood pressure response to nicotine in SHR was abolished by intraventricular administration of hexamethonium, but not by atropine given via the same route. Central phentolamine and 6-hydroxydopamine did not affect the pressor response to nicotine. The pressor effect of nicotine in SHR was markedly diminished after removal of the adrenals and abolished after bilateral adrenalectomy plus peripheral 6-hydroxydopamine. These results indicate that the pressor response to intraventricular nicotine is increased in SHR. The pressor effect of nicotine may result from the activation of the central nicotinic receptor sites, which may cause the release of catecholamines both from the adrenal medulla and adrenergic nerve terminals.

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The effect of calcium hydroxide on the maximal seizure threshold was studied in 18-day-old rats using the up-and-down method. The maximal seizure was induced by administering an electric shock through the eyes. When calcium hydroxide was given orally once a day (0.04 m moles/kg) for 10 days from the 8th to 18th day after birth, the maximal seizure threshold was raised by 4.8 mA, which corresponded to 16% of the threshold current in the control. The serum calcium concentration was not significantly altered after the treatment. When calcium chloride was given intraperitoneally, the maximal seizure threshold markedly increased with the increase in serum calcium. It is suggested that the mechanism of the action of calcium hydroxide is different from that induced by increasing the serum calcium.

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Effects of gonadotropin and estrogen on the levels of prostaglandin (PG) in the rat ovary were investigated. PGF content in rat ovary, as measured by radioimmunoassay, was slightly higher than that by bioassay using rat stomach fundus, although there was no statistically significant difference. PGE and PGF levels in rat ovary during the estrous cycle were lowest on the day of diestrus, PGE was of the highest level on the day of estrus, and PGF on the day of proestrus. Both PGE and PGF were increased 24 hours after treatment with pregnant mare serum gonadotropin on the first day of diestrus. PGE was increased about 2.5 fold and PGF about 2.3 fold. PGF was significantly increased 24 hours after treament with estradiol on the first day of diestrus. These results suggest that gonadotropin may directly or indirectly regulate changes in ovarian PG content via actions of estrogen.

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To assess the effects of pH on the cytotoxicity of carboquone (CQ), use was made of the mouse tail skin and HeLa cells in culture. CQ had the most potent cytotoxic effect at pH 6 rather than at pH 7 or 8. Regarding the interaction between ¹⁴C-CQ and HeLa cells, both the intracellular accumulation of free ¹⁴C-CQ and the ratio of bound ¹⁴C-CQ to total ¹⁴C-CQ uptake were enhanced at pH 6. Among the fractionated biomolecules of DNA, RNA and protein, DNA was the most active in binding CQ, under the same conditions of pH. The ¹⁴C-CQ binding to nucleic acids at pH 6 was more apparent than was the binding to protein. Thus, the enhancement of CQ cytotoxicity at low pHs is probably due to an increase in the intracellular accumulation of free CQ as well as to an enhanced reactivity of CQ with DNA, within the ranges of a lower pH.

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Effects of dopamine, N-methyl-, ethyl and propyl-derivatives of dopamine, and alpha and beta-adrenoceptor stimulants on catch contraction of anterior byssus retractor muscle of Mytilus edulis were tested. The test drugs except the beta-adrenoceptor stimulants relaxed catch contraction. Dopamine was most active and substitution of amino group in dopamine with ethyl and propyl decreased activity considerably. The concentration-action curves of dopamine, its derivatives and norepinephrine shifted in parallel with application of haloperidol but were not influenced by the alpha and beta-adrenoceptor antagonists. These results suggest that relaxation of catch contraction by catecholamines is mediated through a dopamine receptor. This muscle is considered to be suitable for a study of the dopamine receptor.

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Effect of methylxanthines (theophylline, theobromine and caffeine) on urinary prostaglandin E (PGE) excretion in male rats was studied. Oral administration of xanthines significantly increased the urinary excretion of PGE. Dose-response studies showed that the maximal excretion of urinary PGE and water was obtained by administration of theophylline (50 mg/kg), where the increase in PGE was about 20 times that of the control. The excretion of urinary sodium, potassium and chloride was also markedly increased by xanthines, particularly, theophylline. Increases in urinary PGE excretion, urine volume and electrolytes excretion were inhibited by 10 mg/kg of indomethacin administered prior to theophylline. The increase of urinary PGE excretion after theophylline administration (50 mg/kg) preceded increases in water and sodium excretion. These results suggest that renal PGE mediates, at least in part, the diuretic effect of theophylline.

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Both in vivo and in vitro experiments were performed to elucidate the relationship between the action of bradykinin (BK) and the possibility of release of pharmacologically active fragments from BK in the rat brain. In in vivo experiments, the activities of electroencephalogram (EEG) increased immediately after the intracerebral administration of 5 nmole BK. The effect was prolonged by intracerebral pretreatment of o-phenanthroline which inhibits plasma kininases. In vitro experiments, o-phenanthroline inhibited partially purified enzyme of rat brain which released fragments of BK possessing phenylalanine, serine, proline and/or arginine as N-terminal amino acids. Only arginine and minute amounts of phenylalanine were observed after the incubation with o-phenanthroline. The evidence suggests that the inhibition of the enzyme by o-phenanthroline resulted in a prolongation of the excited phase produced by BK on the EEG and on behavior. Supporting evidence indicates that Ser-Pro containing frag-ments derived from BK are concerned with the sedative phase of BK which was observed after the excitative stage.

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Using oxyntic and duodenal rat tissues, we investigated the effects of cimetidine, an antisecretagogue and a H₂-receptor antagonist, on deoxyribonucleic acid (DNA) biosynthesis, determined according to the ³H-thymidine incorporation into DNA fraction. Serum gastrin concentration and DNA biosynthesis in the tissues increased significantly in the fed rats, and administration of cimetidine significantly increased both serum gastrin concentration and the DNA biosynthesis in the oxyntic tissues. There was a significant correlation between the oxyntic DNA biosynthesis and the serum gastrin concentration. Cimetidine in concentrations of 0.01 to 1.0 mM, added to the incubation mixture, had no stimulatory effect on the DNA biosynthesis in the oxyntic tissues. These results support the hypothesis that DNA biosynthesis in oxyntic mucosa of rats may be regulated by the circulating gastrin.

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We investigated the influence of electroconvulsive shock (ECS), regarded to possess an antidepressant effect clinically, on muricide in olfactory bulbectomized rats (0B rats). Muricide in these rats was markedly inhibited by ECS treatment. Five and 10 min after the termination of ECS-induced convulsions, muricide was inhibited by 100%. Even after intervals of 20 and 60 min, inhibition rates of 80% and 30% were obtained, respectively. ECS-induced muricide inhibition was remarkably antagonized by pretreatment with the α-blocker phenoxybenzamine but not by pretreatment with the β-blocker sotalol. ECS-induced suppression of muricide was potentiated by repeated ECS treatment once daily for 10 days. After several applications of ECS treatment, muricide was inhibited in muricide tests done 24 hours after ECS treatment; this state persisted for up to 10 days thereafter. The results of this experiment demonstrated that ECS treatment specifically inhibited muricide in OB rats and further suggested that the cerebral noradrenergic α-receptor system plays an important role in this ECS-induced inhibition of muricide. Similar to findings in the case of antidepressant administration, inhibition of muricide was potentiated by chronic ECS treatment. Specific inhibition of muricide in OB rats by antidepressants indicated that this phenomenon may serve as an animal model for the evaluation of antidepressant activity. Our results reiterate the usefulness of muricide in OB rats as an excellent experimental model for the assessment of antidepressant activity.

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