The inhibitory effect of Carbidopa (MK-486) on L-3, 4-dihydroxyphenylalanine (DOPA) decarboxylase in various tissues including the brain was investigated as well as the distribution of Carbidopa-¹⁴C in rats. The increase in the brain uptake of radioactive L-DOPA by Carbidopa was demonstrated. Carbidopa, highly distributed in the kindney, lung, and liver, strongly inhibited DOPA decarboxylase activity in the main tissue except the brain in a dose dependent fashion. In spite of a rapid elimination of Carbidopa itself, the inhibitory effect appeared to retain for a long period, i.e., more than 24 hr. With a high dose of Carbidopa, the activity in the brain appeared to be inhibited appreciably. It was proved, however, that this was due to the drug contained in the cerebral blood by the determination of decarboxylase activity or radioactive Carbidopa in the brain perfused with saline, confirming that Carbidopa is a peripheral inhibitor. Carbidopa-¹⁴C was found to be distributed highly in the aorta walls which was presumed to be due to a non-specific binding to elastine in the tissue. Carbidopa enhanced the brain uptake of radioactive L-DOPA linearly to the dose of L-DOPA. The effect depended upon the proportion of oral Carbidopa to L-DOPA, and the mixing ratio around 1 : 10 was suggested to be the most efficient combination to elevate the brain uptake of L-DOPA.

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To study the pulmonary absorption of macromolecular compounds, dextrans, papain, ovalbumin, serratio peptidase, human gamma globulin and catalase, fluorescein isothiocyanate (FITC) labelling of these compounds was carried out. After these FITC labelled macromolecular compounds were administered into the rat trachea, blood samples were collected from zero to 7 hr, and the serum concentration was measured. Almost all macromolecular compounds used except FITC-dextran (MW=150000) were well absorbed after their administration into the bronchia, and it has been suggested that pulmonary route is quite feasible for the administration of macromolecular compounds such as proteins, enzymes and carbohydrates to which oral route is undesirable.

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The metabolic pathway of L-3-methoxy-4-hydroxyphenylalanine (3-O-methylDOPA) in the rat was investigated in vivo and in vitro. When 3-O-methylDOPA-¹⁴C was incubated with rat liver homogenate, 3-methoxy-4-hydroxyphenylpyruvic acid (MHPP) was detected as the major metabolite, on addition of sodium α-ketoglutarate. Without α-ketoglutarate. 3-O-methylDOPA remained unchanged, indicating that the transamination between 3-O-methylDOPA and α-ketoglutarate is the first step in the metabolism of 3-O-methylDOPA. The enzyme responsible for the transamination was identified as tyrosine aminotransferase. After oral administration of MHPP-2-¹⁴C to rats, 3-methoxy-4-hydroxyphenyllactic acid (MHPL) and homovanillic acid (HVA) (the major urinary metabolites of 3-O-methylDOPA) were detected as the major metabolites. Using rat liver homogenate, it was revealed that MHPP-2-¹⁴C is transformed to MHPL with NADH but not with NADPH. Lactate dehydrogenase (LDH) was obtained from rat liver and MHPP was shown to be a substrate of LDH. It was recognized, however, that there is another dominant enzyme reducing MHPP to MHPL. This enzyme was purified from rat liver and identified as aromatic α-keto acid reductase (AKAR). Inhibition of sodium oxamate was over 95% against LDH and below 10% against AKAR, respectively, using MHPP as a substrate. Utilizing this differential inhibition of oxamate, the relative contribution of the two enzymes to the formation of MHPL from MHPP was estimated in rat tissues as 1.2-15.7% for LDH and 84.3-98.8% for AKAR, respectively.

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Styrene showed a weak mutagenic activity towards Salmonella typhimurium TA 100 after being activated by the hepatic 9000g supernatant fraction (S9) obtained from rats pretreated with 3-methylcholanthrene or phenobarbital and fortified with an NADPH-generating system, in the presence of the epoxide hydratase inhibitor, 3, 3, 3-trichloropropene oxide (TCPO). The 3-methylcholanthrene-pretreated rat liver S9 activated styrene more effectively than the phenobarbital treated one. No mutagenic activity was observed when either TCPO or S9 was omitted. As has already been pointed out, phenyloxirane, an intermediate in the major metabolic pathway of styrene by hepatic microsomes, induced mutations in TA 100 and TA 1535 cells in the absence of both S9 and TCPO. Analytical data of phenyloxirane and its hydrolytic product, 1-phenyl-1, 2-ethanediol, formed in the mutation assay system for styrene indicated that the inducers used for hepatic drug-metabolizing enzymes enhanced both microsomal monooxygenase and epoxide hydratase activities. Relative ratios of the enhanced monooxygenase to hydratase activities were twice as high in 3-methylcholanthrene as the control and 1.8 and 1.2 times in phenobarbital and PCB, respectively, although the last enhanced both activities to the highest extent. Phenyloxirane was accumulated in the presence of TCPO in the styrene-activating system most significantly when 3-methylcholanthrene-pretreated rat liver S9 was used. However, the mutagenicity exerted by the metabolic activation of styrene could not necessarily be explained only by phenyloxirane since sums of both metabolites were smaller than the amount of the epoxide required for inducing the mutation in Salmonella and suggested a possibility of the presence of at least one more unknown mutagenic metabolite with an epoxide structure.

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The intestinal absorption and the site for hydrolysis of pivampicillin were compared with those of ampicillin by in situ ligated loop method in rats, using the radioactive compounds labeled at the phenylglycyl and the oxymethylene moieties. It was shown that pivampicillin is well absorbed from all parts of intestine, while ampicillin is absorbed from a more limited part of intestine. Pivampicillin was found to be rapidly transferred from the lumen into the intestinal wall as an intact ester and was hydrolyzed rapidly in the tissue. Ampicillin thus formed appeared to be transitorily accumulated in the tissue before being transferred into the portal venous blood gradually. Ampicillin was absorbed slowly without any accumulation in the intestinal wall. Microautoradiography of pivampicillin-¹⁴C in the intestine demonstrated that the radioactivity is accumulated in the epithelial cells in a high concentration. Histochemical and cytochemical detection of non-specific esterase revealed that a high activity is located specifically in the epithelial cells of villi and that the activity is localized in the membranes of endoplasmic reticulum as well as the cytoplasm, while there was almost no activity in the microvilli and terminal web region. It is considered therefore that pivampicillin is penetrated into the epithelial cells without any cleavage of the ester group and hydrolyzed by non-specific esterase in the apical cytoplasm of the cells. When the esterase activity was compared histochemically in rats, dogs and monkeys, it was found that the activity was extremely weak in dog intestine, providing an explanation for the fact that intact pivampicillin was detected in the portal venous blood only in dogs after oral administration. A possible relation between these results and the fact that a hepatic toxicity of pivampicillin was observed only in dogs among these animals was pointed out and discussed.

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Among the twenty-three compounds isolated from bracken, kaempferol showed noticeable mutagenicity to Salmonella typhimurium, strains TA 100 and TA 98, with S-9 mix. With pretreatment of 'hesperidinase' the mutagenic activity of the boiling water extract was concentrated to the fraction containing astragalin. Several extracts and fractions were prepared for test of mutagenicity and the presence of other mutagens in bracken besides flavonoids was evidenced.

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Effects of centrally acting muscle relaxants on the tonic and phasic tension of the forelimbs were studied in the rigid rats. Anemic decerebrate rigidity of rats was obtained by ligation of both basilar and common carotid arteries. Tension recorded from forelimbs was employed as measures of the intensity of the rigidity. In addition to the rigidity developed in forelimbs (tonic component), phasic tension (phasic component) was induced by mechanical stimulation given to the hindlimbs. On the basis of the effects on the tonic and phasic components of rigidity, the drugs examined were classified into three types of groups : (1) drugs which depressed both tonic and phasic components (mephenesin and tolperisone), (2) drugs which depressed tonic and augmented phasic components (orphenadrine and chlorpromazine), and (3) drugs which depressed phasic but had almost no effects on the tonic components (diazepam, nitrazepam and clonazepam).

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Responses to ACh, pilocarpine, BaCl₂ and KCl in isolated duodenum from SART stressed mice (specific stress caused by alternating rhythm in temperature) were investigated using the Magnus's method, and the results were compared with those seen in non-stressed mice. We found that the ACh response was significantly decreased, while the BaCl₂ response was not decreased in SART stressed mice duodenum, as compared with the non-stressed controls. And it is recognized that pretreatment with adrenergic and anticholinergic drugs inhibited the decrease of ACh response. These results suggest that SART stressed mice may be unbalanced in the autonomic nervous system.

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A hypoalbuminemic substance was purified from mouse Ehrlich ascites carcinoma cells by ammonium sulfate fractionation, adsorption on DEAE-cellulose, gel filtration on Sephadex G-200, treatment with butanol, ultracentrifugation, and gel filtration on Sephadex G-75. Electrophoresis of the substance on polyacrylamide gel containing sodium dodecylsulfate gave a single band which corresponded to a molecular weight of 53000. It showed hypoalbuminemic, hypoproteinemic, reticuloendothelial system-stimulating, and leukocyte-lowering effects when given to mice by intraperitoneal injection. Of these effects, only the leukocyte-lowering effect disappeared when it was administered to adrenalectomized mice. The vascular permeability-increasing activity concentrated into the fraction unadsorbed on DEAE-cellulose and distinctly separated from the hypoalbuminemic activity. The purified substance was composed of 28.9% of DNA, 24.9% of hexose, 24.6% of RNA, 14.6% of amino acid residues, and 1% of hexosamine. The hypoalbuminemic activity was decreased markedly by the partial digestion with Pronase, trypsin, or DNase, whereas the treatment with RNase or the butanol extraction exerted no influence on the activity.

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