Several species of the sulfobromophthalein-binding proteins were isolated from the Y-fraction of rat liver cytosol. Each of these proteins, designated as C_v, C₁, C₂ and C₃ according to the elution order from CM-Sephadex column, migrated to the same position as a single protein band in dodecylsulphate electrophoresis. They are basic proteins of approximately 46000 daltons, composed of two subunits of apparently equal size, and similar to the glutathione S-transferase AA, A, B (ligandin), C purified by Jakoby et al. Each of the C_v, C₁ and C₂ protein forms 1 : 1 primary complex with BSP, with the binding constant of about 5×10⁶ M^-1 or more. The C₁ protein has a somewhat higher binding constant than the others. The binding constant was little influenced by the purification. The binding constants of these proteins for other organic anions were determined by the competitive technique using 1-anilino-8-naphthalenesulfonate. These proteins show broad binding specificity for various organic anions which are all known to be rapidly transported into the liver. From these results, it is concluded that the binding of BSP by the Y-fraction is not due to a single protein like ligandin.

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We studied the activity of one division of the autonomic nervous system, independently of the other, under restraint and water (25°) immersion. For such evaluation, heart rate, gastric motility and catecholamine content of tissues or urine were measured in rats of which the autonomic nervous activity was reduced by adrenal medullectomy (AMX), guanethidine (GNT), atropine (ATR), or their combinations. Heart rate in the controls was increased after restraint or immediately after water immersion and this change was blocked by AMX plus GNT. ATR accelerated the heart rate, but did not affect the extent of the stress-induced increase in the heart rate. With an increase in the duration of water immersion, a decrease in heart rate occurred closely linked with hypothermia in all animal groups, suggesting that heart rate was influenced by hypothermia more prominently than by the autonomic nervous controls. AMX plus GNT significantly shortened the occurrence time of increased gastric motility which was observed after water immersion, while ATR completely inhibited an increase in gastric motility. The determination of catecholamine content showed a stress-induced activation of sympatho-adrenal function and its blockade by the treatment with AMX plus GNT. It is finally suggested that the stress procedures used here may immediately enhance the sympatho-adrenal activity lasting for at least 3 hr and steadily raise the vagal activity reaching the plateau 2 hr later.

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In Ca-free Locke Ringer solution, the contractile responses (mean±S.E. of 8 experiments) induced by acetylcholine (5.5×10^-5 M) and histamine (5.4×10^-4 M) are, respectively, 47.7±9.0 and 29.2±9.9 (%) of those obtained in normal Locke Ringer solution. Furthermore, Ca-blocker, D-600 (10^-7 M) caused a more significant reduction of histamine-induced contraction than that of contraction induced by acetylcholine. Ca-incorporation in a microsomal fraction prepared from the rabbit taenia coli was also studied. Ca-incorporation was increased in the presence of both adenosine triphosphate (ATP) and Mg²⁺, unaffected by azide and enhanced by oxalate. Acetylcholine (5.5×10^-5 M), histamine (5.4×10^-5 M) and atropine (3.5×10^-6 M) were without any effect on Ca-incorporation in the presence and absence of oxalate. The rate of Ca-release from microsomal vesicles was significantly increased by acetylcholine (5.5×10^-5 M), but not by histamine (5.4×10^-5 M). Atropine (3.5×10^-6 M) inhibited this effect due to acetylcholine. Thus Ca-release induced by acetylcholine seems to be due specifically to acetylcholine receptors. The present observations suggest that in rabbit taenia coli, the mechanisms by which acetylcholine and histamine interact with Ca to induce contraction are different.

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The biotransformation of exogenous L-5-hydroxytryptophan (L-5-HTP), an antidepressant agent, was studied in mice, rats, dogs and monkeys after an oral administration of L-5-HTP-3-¹⁴C (5 mg/kg). The major metabolites in urine were 5-hydroxyindole acetic acid (5-HIAA) and its conjugates in mice, dogs and monkeys, accounting for about 60% of the total urinary metabolites. In rats, in contrast to other species, 5-hydroxytryptamine (5-HT) glucuronide was the major metabolite (60%), and 5-HIAA and its conjugates were minor. This species difference in the urinary metabolites was more clearly observed when 5-HT-¹⁴C was orally administered. In monkeys, a specific metabolite was detected in the urine after administration of both L-5-HTP and 5-HT and identified as a conjugate of 3-methyl-5-hydroxyindole. The species difference observed may be related to the fact that a renal toxicity occurs only in rats after an oral administration of L-5-HTP or 5-HT. In order to explain the species difference in L-5-HTP metabolism, the activities of glucuronyltransferase, monoamine oxidase and aromatic amino acid decarboxylase were compared in the liver, kidney and small intestine from the four animal species. The glucuronyltransferase activity in the rat tissues was found to be much higher than that in the other animal species.

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Esterase purified from human intestinal mucosa was investigated for its substrate specificity. Then the intestinal esterase was applied to the development of salicylic acid derivatives as prodrugs. The purified esterase hydrolyzed ester-type drugs. In all tested estertype drugs, aspirin was hydrolyzed most rapidly. So, the esters of salicylic acid were chosen as prodrugs. The purpose of this study was to seek salicylic acid derivatives as prodrugs which would be nonirritant, rapidly absorbed, and rapidly hydrolyzed. In the studies of hydrolysis of the esters of salicylic acid by the purified intestinal esterase and hepatic esterase, the esters were cleaved after absorption to exert their pharmacological activities due to the resultant salicylate. Of all synthesized esters, n-heptanoylsalicylic acid proved to be the most effective prodrug, because it exhibits higher analgesic activity, and its pharmacological profiles is quantitatively similar to that of aspirin. Besides, the acute toxicity and the gastric irritation potential of n-heptanoylsalicylic acid are less than those of aspirin.

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The structure of two sulfur-containing metabolites of p-dichlorobenzene (p-DCB), the distribution of p-DCB and the metabolites in the blood and some tissues, and the excretion of the metabolites in the urine and feces of rats were studied. After the oral administration of p-DCB, two metabolites emerged in the blood. And these metabolites were found for many hours even after p-DCB had almost disappeared from the blood. It was concluded by comparison of spectral data, thin layer chromatogram and gas chromatogram with those of authentic samples, that the metabolites, M-1 and M-2, are 2, 5-dichlorophenyl methyl sulfoxide and 2, 5-dichlorophenyl methyl sulfone, respectively. The concentration of M-1 in the blood was higher than that of M-2 for 12 hr after dosing p-DCB, but the blood level of M-2 prevailed thereafter, and maintained an appreciable level even after 120 hr. The storage of p-DCB in the adipose tissue and the slow excretion rates of M-1 and M-2 may be responsible for the existence of the metabolites in the blood for long hours. The highest concentration of M-1 was found in the kidney and that of M-2 was in the blood for the period observed. The excretion of M-1 and M-2 in the urine was much less than that of 2, 5-dichlorophenol, the major metabolite.

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Guinea pigs were given ²⁰³Hg-labeled methylmercuric chloride or mercuric chloride and then the distribution of mercury in the blood, brain and liver was investigated. The methylmercury in the blood and brain was extracted with a chloroform-methanol mixture. Most of the extractable mercury from the blood was detected in the proteolipid fraction by further fractionation. The mercury in the chloroform-methanol extract was also dialyzable against the same solvent. These findings suggest the occurrence of loosely bound mercury in the blood after administration of methylmercury.

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Effect of surfactants on the absorption of p-aminobenzoic acid (PABA) and p-acetamidobenzoic acid (Ac-PABA) from the rat intestine was investigated using the in situ recirculation technique. The surfactants tested were polyoxyethylene-(20)-sorbitol monooleate (Tween 80), polyoxyethylene-(9-10)-p-t-octylphenol (Triton X-100), sodium dodecylsulfate (SLS) and cetyltrimethylammonium bromide (CTAB). The micellar interaction of PABA and Ac-PABA with these surfactants were almost negligible except CTAB. The absorption of Ac-PABA from the small intestine was increased by these four surfactants. The order of magnitude of the absorption enhancing effect was as follows : Triton X-100>SLS≤CTAB>>Tween 80. The absorption of PABA and Ac-PABA from the large intestine was also increased by 20 mM SLS. In addition, the exsorption of PABA to the small intestinal lumen was greatly increased by the addition of SLS to the perfusate. In spite of such a general increase in the membrane permeability, the absorption of PABA from the small intestine was significantly inhibited by these four surfactants. The extent of inhibition was dependent on the concentration of surfactants and the exposure time of intestine to surfactants. SLS greatly accelerated the release of protein from the small intestine and an inverse correlation was found between the amount of released protein and the absorption of PABA in the presence of SLS. The specific inhibitory effect of surfactants may be attributed to the solubilization and the following release of protein, which is responsible for the absorption of PABA.

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Therapeutic effect of FU was improved by the combination with orotic acid. This combination exhibited a marked schedule dependency. The most suitable schedule was to give orally 30 mg/kg/day of orotic acid 30 minutes before FU administration. In combination of FU and orotic acid in this schedule, ILS₃₀ was decreased to 2.1 from 3.1 mg/kg/day and the optimal dose was increased to 30 from 20 mg/kg/day. Maximum ILS was also increased to 81 from 54% and the therapeutic ratio was improved to 14 from 5.1.

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