Effects of vitamin B₂-butyrate, nicomol, ML-236B, KF 1492 and pantethine, which are hypolipidemic drugs, on biochemical values and on hepatic peroxisomal enzymes of normolipemic rat. 1) Vitamin B₂-butyrate (100 mg/kg) and nicomol (1g/kg) increased carnitine acetyltransferase and D-amino acid oxidase activities, respectively, while these drugs had no influence on body weight, liver weight, serum and liver triglyceride, and serum and liver cholesterol levels. 2) ML-236B (300 mg/kg) had no influence on biochemical values and on the activities of peroxisomal enzymes containing catalase. 3) KF 1492 (300 mg/kg) had no influence on the biochemical values, but an increase in the activities of fatty acyl-CoA oxdizing system (FAOS) and carnitine acetyltransferase (CAT) participating hepatic lipid metabolism was observed. 4) Pantethine (1g/kg) had no influence on the biochemical values, except a little decrease in the growth rate. However, increase by about 10% in the activities of urate oxidase and D-amino acid oxidase was observed. Catalase activity was decreased to 60% of control level. From these results, it is concluded that, in contrast to clofibrate, vitamin B₂-butyrate, nicomol, ML-236B, KF 1492 and pantethine have little influence on the lipid metabolism of normolipemic animal and on the hepatic peroxisomal enzymes, indicating that the action mechanism of these drugs may be different from that of clofibrate and that the participation of hepatic peroxisomes in hypolipidemic activities of these drugs may be little if any.

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The responses of isolated gall bladder to transmural electrical stimulation with rectangular pulses (50 volt, 0.5 ms) of 30 Hz for 10 s at intervals of 30 min were observed for 5 to 6 hours continuously and compared for different values of resting tonus in Krebs solution at 27°C and 37°C. In 27°C Krebs solution, the resting tonus increased gradually with time and became stable at 0.56±0.07 g (mean±S.D.) after 5 h. In 37°C Krebs solution, the resting tonus increased gradually with time and became stable at 1.48±0.49 g (mean±S.D.) to 1.5±0.19 g (mean±S.D.) after 5 h. Monophasic contraction was observed when the resting tonus was lower than 1.49±0.15 g (mean±S.D.), but above this value, biphasic response consisting of contraction and relaxation was observed. Both monophasic and biphasic responses were blocked by atropine and tetrodotoxin. Physostigmine enhanced monophasic contraction, increased the duration of contraction, and abolished the relaxation of biphasic contractile response. The results suggest that contractile response of isolated guinea-pig gall bladder to transmural electrical stimulation was directly induced by acetylcholine released from postganglionic cholinergic fibers. Transmural electrical stimulation with rectangular pulses (50 volt, 0.5 ms) at 10, 20, 30 or 40 Hz for 10 s induced biphasic response in which the contraction varied with frequency, peaking at about 30 Hz, while the relaxation was almost constant. Biphasic relaxation was reduced or inhibited by 2, 4-dinitrophenol, ouabain, or K⁺-free solution. This suggests that the relaxation phase of the biphasic response induced by transmural electrical stimulation was an active process.

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The physico-chemical properties of a series of hydantoin derivatives and their intestinal absorption from solution were studied. The introduction of the benzenesulfonyl group at the 1-position of the hydantoin ring greatly affects the physico-chemical properties : the acid dissociation constants were increased 1000-fold and the partition coefficients were increased 100- to 1000-fold in the chloroform/water system and 10- to 100-fold in the n-octanol/water system. The solubilities of 1-benzenesulfonylhydantoin derivatives increased with increasing pH of the solution at pH more than 5, but the solubilities of the 1-unsubstituted hydantoin derivatives were scarcely dependent on the pH of the solution in the pH 1 to 8 region. The intestinal absorption from solution was found to be caused by the passive transport according to the first-order kinetics. The rate constants of absorption of 1-benzenesulfonylhydantoin derivatives were rather large even under the condition where they were 99% ionized in the solution than that of the corresponding 1-unsubstituted hydantoin derivatives which exist mainly as the unionized form under the same condition. The intestinal absorption from a solution and the partitioning to chloroform produced linear free energy relationships to each other for the 1-benzenesulfonylhydatoin derivatives and for the 1-unsubstituted hydantoin derivatives independently. However when the partition coefficients in the n-octanol/water system were applied, the hydroxyl derivatives were found to deviate from linear relationships. On the basis of the results, a suggestion was made on the in vivo behavior and the bioavailability.

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Two series of 8-acetyl-12-hydroxyalkadienoic acids and 14-hydroxy-9-oxoalkadienoic acids which can be regarded as 11, 12-and 8, 12-secoprostaglandin E₂ were synthesized and evaluated for their biological properties. Key members of each series, 11, 12-(8Ac-HAD) and 8, 12-seco-11-norprostaglandin E₂ (14H-OAD), were found to induce platelet aggregation which were inhibited by preincubation of platelet rich plasma with prostaglandin I₂ but not inhibited by indomethacin. 8Ac-HAD produced dose-dependent potent contraction of rabbit aorta. Injection of 8Ac-HAD (1 mg/kg) into vein of rat induced sudden death of the animal. Both compounds were stable and platelet aggregating activity did not decrease at least for four hours at 0°C. Structure-activity relationship study of the series were carried out. Reduction of the acetyl carbonyl and methoxime formation of 8Ac-HAD lowered platelet aggregating acrivity, and 8-propionyl substituent and 12-deoxy derivative of 8Ac-HAD showed no activity. 12 (R)-Isomer and dl 12-methyl derivative of 8Ac-HAD retained the platelet aggregating activity. Modification of ω-chain did not cause any essential effect on the activity. Unlike 8Ac-HAD, several modification of 14H-OAD failed to maintain the aggregating activity.

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Effect of disodium cromoglycate (DSCG) on hemolysis of rat erythrocytes was studied. The heat-induced hemolysis was remarkably inhibited by DSCG. This effect was not affected by normal rat serum and was maintained for longer than 60 min similar to the activity in clinical use, although the inhibitory effect of DSCG on reagin-induced histamine release from mast cell disappeared rapidly in 5 min after administration. Non-steroidal antiinflammatory drugs such as flufenamic acid or phenylbutazone which are known to inhibit the hemolysis depending on their protein stabilizing activities inhibited both of the heatinduced hemolysis and albumin denaturation, but DSCG did not inhibit the albumin denaturation. Furthermore, it was also observed that DSCG inhibited the hemolysis induced by lipophilic agents such as saponin, linoleic acid or phospholipase C. These findings suggest that DSCG has a certain membrane stabilizing activity, in addition to the inhibitory effect on reagin-induced histamine release, and that the activity may be probably related to membrane-lipids rather than membrane-proteins.

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The electrical stimulation of the caudate nucleus could induce a caudate spindle in the rat as in case of the cat and monkey. The spindle was enhanced by atropine (3-10 mg/kg, i.v.) or morphine (3-10 mg/kg, i.v.), and the effects of these drugs were completely abolished by eserine (0.3 mg/kg, i.v.), but not by methysergide (1 mg/kg, i.v.), PCPA (400 mg/kg, i.p. for 3 days) or reserpine (3 mg/kg, i.p. for 2 days). An intraventricular injection of atropine (5-10 μg/rat) or morphine (10 μg/rat) exerted a facilitatory action on the spindle, while an intrathalamic and intracaudate injection of atropine (5-50 μg/rat) showed a suppressive action. The spindle was also markedly enhanced by the microinjection of atropine (15-30 μg/rat) or morphine (30 μg/rat) into the reticular formation, and these actions were completely antagonized by eserine (0.3 mg/kg, i.v.), but only partially by methysergide (4 μg/rat, intraventricular administration.) These results suggest that the cholinergic system may play an important role in the regulation of the caudate spindle.

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Involvement of divalent cations was examined in the activation of catecholaminesensitive adenylate cyclase in Ehrlich ascites tumor cells. In the presence of various concentrations of either Mg²⁺ or Mn²⁺, both basal and norepinephrine-stimulated adenylate cyclase activities were measured. Both activities were gradually increased with the increase in Mg²⁺ concentration. On the other hand, with Mn²⁺, both activities were maximum at the low (2.5mM) concentration, and decreased at the higher concentrations, the decrease being marked in the norepinephrine-stimulated activity. Similar difference between the two divalent cations was also observed in the effect on the binding of the β-adrenergic ligand to the membrane receptor.

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A smooth muscle relaxant (suloctidil) relaxed a contractile response of taenia coli to acetylcholine and noncompetitively depressed a dose response curve of CaCl₂. Ca-uptake by the microsomal fraction was inhibited by suloctidil. Its noncompetitive antiacetylcholine activity and inhibitory action on Ca-uptake were not influenced by an increase of external Caconcentration. Ca-release from the Ca-incorporated microsomal fraction was not significantly influenced by suloctidil. These results suggest that suloctidil inhibit Ca-influx noncompetitively, thus inducing smooth muscle relaxation.

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The effect of a new aspirin derivative, aspirin-isopropylantipyrine (AIA), with very little gastric ulcerogenic activity and very slight acute toxicity and with analgesic, antipyretic anti-inflammatory, and platelet aggregation inhibitory activities was evaluated in vitro and ex vivo and compared with those of aspirin and isopropylantipyrine (IA). In vitro, AIA, aspirin and IA (50-200 μM) caused concentration-dependent inhibition of collagen-induced aggregation in rabbit platelets although AIA was several-fold more active than the others. Arachidonic acid-induced aggregation was inhibited by all three agents (200 μM) in the following magnitude ; IA > aspirin > AIA. Three agents did not influence primary ADP-induced aggregation. The in vitro effects on the release-inducing aggregants were confirmed by ex vivo experiments in rats. These demonstrated that AIA and aspirin (50 mg/kg) exhibited almost identical inhibitory potencies in the extent and the rate of collagen-induced aggregation 4 h after subcutaneous injection. AIA was still effective 24 h after administration as well as aspirin. IA was less effective, differing from the results in vitro. AIA had no effect on plasmin activity and blood flow through the common carotid artery. AIA (1 mM) maintained spreading and beating of myocardial cells in a serum-free culture. As special toxicity trials on AIA mutagenecity tests were made by the Rec-assay with Bacillus subtilis, by the plate culture with Escherichia coli, and by the Ames system with Salmonella typhimurium. AIA was found to have no mutagenic effect under any of those methods and to have no effect on the mutagenic action of 3, 4-benzopyrene under the liver microsome test using the Ames system.

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A novel convenient procedure was introduced for enzyme labelling of neocarzinostatin (NCS) with β-D-galactosidase using a new hetero-bifunctional reagent N-(γ-maleimidobutyryloxy) succinimide (GMBS). With the enzyme labelled NCS and a rabbit anti-NCS serum which was elicited in a rabbit immunized with NCS in complete Freund's adjuvant, a highly sensitive enzyme immunoassay which can quantify a femto mol order NCS was developed. Accuracy and precision of the assay as well as a comparison with the immunodiffussion method were studied with satisfactory results. This enzyme immunoassay of NCS was applicable to monitoring serum NCS levels of patients in clinical treatment of the antitumor agent.

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Changes in bile composition during cholesterol gallstone formation were investigated in hamsters. Gallstones were found in the gall-bladder and/or bile duct after feeding on a lithogenic diet for two weeks or more. Treatment with the lithogenic diet caused a marked increase in cholesterol secretion and a significant decrease in bile acid secretion, without affecting phospholipid secretion. The increase in cholesterol secretion was greater in gallstone-present than in gallstone-absent animals. Analysis of bile acids in the bile revealed that cholic acid, a major primary bile acid, was markedly decreased by the lithogenic diet. A decrease in glycine-conjugated bile acids and a relative increase in taurine-conjugated bile acids were also observed. The critical period for gallstone formation produced by the lithogenic diet was one to two weeks after the start of the lithogenic diet. From the above findings, the following mechanisms for cholesterol gallstone formation are suggested. A decrease in bile acids and a marked increase in cholesterol in the bile may result in the reduction of cholesterol solubility in micells consisting of bile acids, phospholipids and cholesterol. Subsequently, insoluble cholesterol forms gallstones. In addition, an alteration of glycine-conjugation capability may also play a part in gallstone formation.

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Oral administration of 500 mg/kg of butylated hydroxytoluene (BHT) daily for three days elevated the total glutathione (GSH) level and the activities of GSH-S-transferase for CDNB and DCNB and GSSG reductase in the rat liver. BHT-alcohol, which is a metabolite of BHT, also elevated the level or activity of these components. The induction of these components with BHT-alcohol was slightly less than that with BHT. However, BHT or BHT-alcohol in doses used had no effect on GSH peroxidase activity.

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