We investigated the mechanism of the elevation of serum corticosterone level by 4-ethoxy-2-methyl-5-morpholino-3 (2H)-pyridazinone (M73101) and its participation in the anti-inflammatory action. M73101 when given to rats intraperitoneally at doses of 50-200 mg/kg, caused an increase in serum and adrenal corticosterone in a dose-dependent manner. Oral administration of 200 mg/kg of M73101 also elevated the serum corticosterone level. Such action of M73101 was completely abolished by adrenalectomy, hypophysectomy, or the pretreatment with pentobarbital and morphine. In vitro experiment demonstrated that M73101 had no direct effect on adrenocortical function. These results indicate that the response to M73101 must be mediated through the release of ACTH from the adenohypophysis, which is probably due to the secretion of corticotropin releasing factor from the hypothalamus. M73101 at an oral dose of 200 mg/kg significantly decreased the volume of exudative fluid and the number of leucocytes in carrageenin-induced pleurisy of intact rats. However, the inhibitory action of this drug on cell mobilization decreased by adrenalectomy but not on exudative fluid, indicating that anti-inflammatory actions of M73101 may be due in part to pituitary-adrenocortical stimulation.

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The effect of zinc on serum calcium was investigated after a single oral administration of zinc sulfate in rats. Zinc (5, 10, and 20 mg Zn/100 g body weight) administration produced a significant decrease of serum calcium. This effect of zinc was not inhibited by thyroparathyroidectomy. Zinc administration resulted in a significant increase in calcium content of lungs and muscle but it was not significant in liver, kidneys, spleen and heart. Intestinal calcium absorption and bile calcium excretion was not affected by zinc administration. However, the urinary calcium excretion after zinc administration decreased markedly. On the other hand, zinc administration caused a remarkable elevation of calcium content in gastric secretion. This effect was dose-dependent (5 and 10 mg Zn/100 g). However, an increase in gastric calcium after zinc administration was completely prevented by atropine (0.1μg/100g) treatment, which also showed a marked inhibition of hypocalcemic effect of zinc. Meanwhile, acetylcholine (4.0μg/100g) administration caused a significant decrease of serum calcium and a significant increase in gastric calcium. These results suggest that the hypocalcemic effect by zinc administration is mainly based on an increase in gastric calcium secretion.

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Effects of age and sex on microsomal heme oxygenase activity and cytochrome P-450 content were investigated in Wistar rats. heme oxygenase activity declined with an increase in age, namely, its activity in 100 days old (young) rats was 58% in male and 72% in female rats as compared with respective 30 days old (immature) rats, and in 300 days old (old) rats it was 32% in male and 39% in female rats. In male rats, cytochrome P-450 content based on 100 g body weight decreased only in old rats as compared with immature rats, whereas a significant reduction was observed in female rats in the content based on mg microsomal protein as well as based on 100 g body weight in young and old animals. In littermate rats at the age of 56 days which obtained by inbreeding in our laboratory, sex difference was apparent in heme oxygenase activity and cytochrome P-450 content, and further a good reciprocal correlation between that activity and content (r=-0.9730 in male, and r=-0.8252 in female) was observed.

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Single smooth muscle cells were isolated from taenia coli of guinea pigs and degrees of responses of acetylcholine, histamine and prostaglandin E₂, and velocity of acetylcholinecontraction were compared with those of whole tissue. The contraction of single cells by these agonists were dose-dependent and ED₅₀ of acetylcholine, histamine and prostaglandin E₂ were 0.3-1.1×10^-6, 5-11×10^-7 and 1-3×10^-9M, respectively. These values were quite similar to those obtained with whole tissue. It took 2.0±0.2s for the single cells to be contracted completely by 10^-4 M acetylcholine while it took 6.9±0.2s for the isotonic contraction of whole tissue. Times for a half maximum contraction, assuming that the whole tissue and the single cells were contracted at the maximum velocity, were 1.43±0.05 and 0.92±0.12s, respectively when 10^-4M acetylcholine was applied. These results revealed that response of muscle cells in the tissue to agonists was reflected on magnitude of the contraction of whole tissue and suggested that pharmacological properties of smooth muscle cells of taenia coli were not changed during the isolation procedure, whereas velocity of contraction of single cells was faster than that of whole tissue. Reasons for the difference in velocity were discussed.

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The metabolic pathway of SQ 14225 (Captopril) and the mechanism of the mixed disulfide formation with endogenous sulfhydryl compounds were studied in in vitro cell free systems. In the rat liver 9000×g supernatants, SQ 14225-¹⁴C was metabolized to one major metabolite, glutathione-SQ 14225 mixed disulfide (GSSQ), and two minor metabolites including SQ 14551, a symmetrical disulfide of SQ 14225. The formation of GSSQ was markedly accelerated by the addition of oxidized glutathione (GSSG), but not affected by the addition of reduced glutathione (GSH), indicating that GSSQ was formed by the thiol-disulfide interchange between SQ 14225 and GSSG. Although the thiol-disulfide interchange was also observed between SQ 14225 and L-cystine, L-homocystine and SQ 14551 as well as GSSG, only the formation of GSSQ was a rapid reaction and markedly decreased by heat treatment of the liver 9000×g supernatants. These findings demonstrate that the formation of GSSQ is catalyzed by a GSSG specific enzyme which is supposed to be thioltransferase (Glutathione : Disulfide Oxidoreductase). Although GSSQ was stable in the rat liver 9000×g supernatants, it was rapidly hydrolyzed to cysteine-SQ 14225 mixed disulfide (CySSQ) in the rat kidney 9000×g supernatants. A specific inhibitor of γ-glutamyltranspeptidase, anthglutin, inhibited the hydrolysis. GSSQ-¹⁴C administered to a beagle dog was excreted into the urine in the form of CySSQ. Thus, it was speculated that CySSQ excreted into the urine as a major metabolite of SQ 14225 was derived from GSSQ formed in the liver followed by hydrolysis in the kidney.

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Participation of histamine H₁-and H₂-receptors in the bronchial musculature and vasculature of the dog was investigated by using the method for evaluating airway responses. A peristaltic pump and a Starling pneumatic resistance were used for a constant pressure perfusion. Dogs given 0.1-10μg histamine by a close intraarterial injection showed increases in ventilation overflow (bronchoconstriction) and in blood flow (bronchial vasodilatation) in a dose-dependent manner. The bronchoconstriction produced by histamine was antagonized strongly by chlorpheniramine, a H₁-receptor antagonist, but not modified by cimetidine, a H₂-receptor antagonist. The bronchial vasodilatation produced by histamine was antagonized by both chlorpheniramine and cimetidine. These results suggest that histamine evokes bronchoconstriction through H₁-receptors and bronchial vasodilatation through H₁-and H₂-receptors.

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In anesthetized dogs, pressor and hyperglycemic effects of Ephedra aqueous extract as well as its alkaloid fraction and absorption of alkaloid from the digestive tract were studied in order to investigate how closely effects of the alkaloid fraction relate to those of the Ephedra extract itself. Blood pressure, heart rate, and blood glucose concentration were increased by intraduodenal administration of the extract and the alkaloid fraction on the bases of the same alkaloid content (20 mg/kg). In respect to the pressor and hyperglycemic effects, changes in time course of the effects produced by these two drug preparations were similar and in either of the two physiological parameters the maximal value given by the extract was about a half that given by the alkaloid fraction. A maximal level of blood pressure and blood glucose concentration was reached 10-15 min after the administration of the two drug preparations, while the amount of alkaloid absorbed in the portal vein until this time was about 2-3 times as much under administration of the alkaloid fraction as under administration of the extract. Absorption of alkaloid in the extract occurred slowly and lasted longer compared with that of the alkaloid fraction itself. On the contrary, the alkaloid-free extract administered intraduodenally showed no virtual effects on blood pressure and blood glucose concentration except producing tachycardia. It seems that the effect of Ephedra extract on blood pressure and blood glucose concentration was mostly qualitatively and quantitatively represented by that of alkaloid contained and that effect of the extract mainly depended on that of alkaloid absorbed early after its application.

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Controlling the brain level of pentobarbital and the duration of hypnosis at the initial treatment in relation to development of acute hypnotic tolerance was studied. Simultaneous treatment of bemegride or TRH with pentobarbital attenuated the hypnotic effect of pentobarbital in a dose dependent manner, but neither the brain level of pentobarbital nor the development of tolerance was modified by this treatment. The effect of TRH was further demonstrated in rats by concomitant intracarotidal infusion with pentobarbital maintaining the brain concentration of pentobarbital and also the duration of exposure of the brain to pentobarbital under a constant condition. On the other hand, THC significantly prolonged the hypnosis induced by pentobarbital but did not potentiate the effect of pentobarbital to develop actute tolerance. Thus, the brain level of pentobarbital at the initial treatment is the primary determinant for the development of acute tolerance and the duration of hypnosis is not the essential factor in this mechanism.

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A convenient and precise method by an application of a highly sensitive enzyme immunoassay of penicillin to detect the veterinary penicillin residue in milk was studied. The procedures of the assay to detect more than 0.01 μg/ml of ampicillin in milk were developed. Five commercial milk samples were tested by the assay procedure, and no veterinary ampicillin residue was detected in any of them.

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The activity of palmitoyl-CoA hydrolase (EC 3. 1. 2. 2.) in the liver from clofibric acid (p-chlorophenoxyisobutyric acid)-treated rats was studied. The study for subcellular localization showed that palmitoyl-CoA hydrolase was found in both cytosolic and microsomal fractions. Feeding of diet containing 0.5% clofibric acid for 6 days, the specific activities of palmitoyl-CoA hydrolase in homogenates, microsomes and cytosol were increased 5, 2 and 22 times, respectively, compared with that in control rats. Gel filtration of cytosolic fraction showed two new peaks, which have the activity capable of hydrolyzing palmitoyl-CoA and different molecular weights compared with the peaks in cytosol of control rat liver. Acyl-CoA hydrolases in cytosolic fraction showed different kinetic properties with each other.

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Atherosclerotic lesions of the aorta and coronary arteries were induced in rats fed a diet supplemented with cholesterol and vitamin D₂ for 8 weeks. Rats fed a purified diet supplemented with 1% cholesterol and 1.25 million units of vitamin D₂/kg of diet showed cholesterol deposition in the aorta but no abnormal findings were observed either macroscopically or microscopically in the aorta and coronary arteries. Rats fed a diet supplemented with 1.5% cholesterol and 1.8 million units of vitamin D₂/kg of diet showed not only marked deposition of cholesterol and calcium but also extensive macroscopic lesions of atherosclerosis in the aorta and coronary arteries. No significant deposition of cholesterol and calcium in the aorta of rats fed the diet without the vitamin D₂ supplement was observed in either experiment.

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In this work, the choleretic properties and mechanism of coumarin compounds and phenolic compounds were studied by examining their effects on parameters such as bile flow, bile acids, electrolytes, and biliary metabolites. Choleretic intensity and property of each sample was far different. The choleretic efficacy of 6, 7-dimethylesculetin (6, 7-DME) was far weaker than that of 4-methylumbelliferone (4-MU). 4-Hydroxybenzyl alcohol (4-HBA), isoeugenol, vanillin, paeonol, and phenolphthalein accelerated bile secretion, but 4 (β-D-glucopyranosyloxy)-benzyl alcohol, safrol, and arbutin did not. 4-MU and 4-HBA underwent conjugation in the liver to give mainly a glucuronide and their metabolites were rapidly excreted into bile, but 6, 7-DME was converted into some metabolites which were excreted little by little over a long period of time. The biliary gap between cations (Na⁺, K⁺) and anions (Cl^-, HCO₃^-, bile acids) produced after intravenous administration of 4-MU, 6, 7-DME, and 4-HBA was substantially offset by biliary concentrations of their metabolites. It was suggested that a hydroxyl group which can be mainly converted into a glucuronide is necessary in exerting a strong choleretic action. The choleretic mechanism of coumarin compounds and phenolic compounds is considered to be as follows : Their metabolites (mainly glucuronide) are actively secreted into the biliary tree as an organic anion coupled with Na⁺ or K⁺, and water is passively excreted.

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Inclusion complex of digoxin with γ-cyclodextrin (γ-CyD) in 1 : 4 molar ratio was prepared, and its dissolution and absorption behaviors were compared with those of digoxin alone. The dissolution rate of digoxin in water was found to be markedly in creased by γ-CyD complexation. Bioavailability of digoxin following the oral administration of γ-CyD complex to dogs was 5.4 times as much as that of digoxin alone. The present data did indicate the improvement of dissolution and absorption characteristics of digoxin by inclusion complexation, suggesting the decrease in dose in oral digoxin therapy.

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The microsomal fraction derived from the taenia caecum of guinea pigs was used as a particulate guanylate cyclase preparation. The guanylate cyclase activity was not influenced by butyltrimethylammonium bromide and Ca²⁺ (4.0 to 24×10^-7M). The results suggest that the particulate guanylate cyclase is not activated by the drug-receptor interaction and also by the increase of intracellular Ca²⁺ concentration. Therefore, the increase of tissue level of cyclic GMP brought about in the guinea pig taenia caecum might be due to activation of soluble guanylate cyclase following the increase in the intracellular Ca²⁺ concentration.

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