The growth of mouse mastocytoma P-815 cells in culture (37°, 42 hr) was inhibited by exogenous adenosine (0.2 to 1.0 mM) and more effectively by AMP (0.01 to 0.1 mM), but not by adenine. The inhibited growth (a 25% inhibition by 0.5 mM adenosine and a 80% inhibition by 0.25 mM AMP) was restored to a near control level by the addition of uridine (0.5 mM) to the medium. The pretreatment (37°, 3 hr) of the cells with adenosine or AMP caused a 60% inhibition of incorporation (37°, 2 hr) of [U-¹⁴C] aspartate into uracil nucleotides, accumulating ¹⁴C-orotate and orotidine. Both dipyridamole, an inhibitor of adenosine uptake, and exogenous adenosine deaminase suppressed the growth inhibition induced by not only adenosine but also AMP. 2-Chloroadenosine, which is resistant to the action of adenosine deaminase, was a more potent growth inhibitor, while 3'AMP and 2'-AMP, which are not hydrolyzed to adenosine by membrane 5'-nucleotidase, were ineffective. Adenosine 5'-sulfate and other 5'-substituted adenosines were also ineffective. These observations indicate that AMP inhibits the growth of mastocytoma P-815 cells as a result of its continuous conversion to adenosine and a constant exposure of the cells to a low concentration of adenosine which readily permeates the cell membrane. In addition, adenosine, AMP and their agarose-linked forms rapidly (37°, 20 min) elevated cellular levels of cAMP. This effect was not suppressed by dipyridamole. Apparently adenosine and AMP also act extracellularly for growth inhibition by regulating cAMP levels.

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Growth-inhibited mouse mastocytoma P-815 cells at stationary phase contained more histamine, serotonin and adenosine 3', 5'-monophosphate (cAMP), and higher activities of histidine decarboxylase and adenylate cyclase than the cells during exponential growth. The elevation of endogenous cAMP levels induced by several growth-inhibiting agents such as N⁶, O^2'-dibutyryl cAMP (Bt₂cAMP), prostaglandin E₁, AMP and 2-chloroadenosine stimulated several functions characteristic of mastocytoma P-815 cells in culture, elevating the synthesis of histamine and serotonin, the activity of chymotrypsin-like protease, and the incorporation of [³⁵S] sulfate into acidic glycosaminoglycans. 1-Methyl-3-isobutyl-xanthine (MIX), a potent inhibitor of cAMP phosphodiesterase, potentiated stimulatory effect of these agents. The results indicate that cAMP regulates the growth and functions of mastocytoma P-815 cells. [³⁵S]-Sulfated acidic glycosaminoglycans synthesized in cells at stationary phase or in cells treated with Bt₂cAMP plus MIX mainly localized in the 3000-10000×g sedimentable fraction of cell homogenates, and had a molecular weight of 200000 to 400000 based on gel filtration. This acidic glycosaminoglycan was resistant to condroitinase ABC and the heparindegrading enzyme present in the 20000×g sedimentable fraction of the cells, and was identified as a highly sulfated macromolecular heparin based on behaviors on DEAE-cellulose column and on acidic electrophoresis. Cycloheximide suppressed the stimulatory effect of Bt₂cAMP on the synthesis of histamine and [³⁵S]-sulfated acidic glycosaminoglycan.

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Acute effects of methylmcrcuric chloride and mercuric chloride on thyroidal functions were examined. The organic mercurial concentration of 4×10^-5 M inhibited by 50% of Na⁺K⁺ ATPase in the membraneous preparation from the hog thyroid, and 6×10^-7 M of the inorganic mercurial showed the same extent of the inhibition. The Mg²⁺ATPase activity in the preparation was neither affected by CH₃HgCl up to a concentration of 2×10^-3 M, nor by HgCl₂ up to 1×10^-4 M. After an intraperitoneal injection to mice of 5 μg of mercurial per gram body weight daily for 2 consecutive days, the 4-hour and the 24-hour uptakes of ¹³¹I by the thyroids were partially reduced by both organic and inorganic mercurials. A significant reduction in percentages of labeled iodothyronines was demonstrated to suggest that mercurial may cause a coupling defect in the synthesis of iodothyronines. Incubation of hog thyroglobulin with 8×10^-3 M of methylmercuric chloride caused no observable aberration in slab disc electrophoreogram, but the protein was apparently denatured by the same concentration of mercuric chloride suggesting that thyroglobulin may carry a large binding capacity against either mercurial, but the inorganic mercurial can be more potent denaturant of the protein. The in vitro lysosomal hydrolysis of the mercurialpretreated rat thyroglobulin which was labeled with ¹²⁵I in vivo and fortified with the carrier hog thyroglobulin was not affected, but the direct addition of either mercurial in the medium resulted in a significant inhibition of the proteolytic action. Iodotyrosine deiodinase in the thyroid was inhibited by both mercurials in in vitro and in vivo systems. A partial reduction in the serum bound ¹³¹I-iodide in both mercurial treated groups was observed at 4 hours and 24 hours after the radioiodide administration. The blood thyroxine levels estimated by radioimmunoassay were quite reduced in the inorganic mercurial treated group and also moderately reduced in the methylmercurial treated group, indicating that the hormone secretion was affected by mercurials.

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Effects of nonspecific smooth muscle relaxants, papaverine and Aspaminol (1, 1-diphenyl-3-piperidinobutanol hydrochloride) on Ca-uptake by a microsomal fraction and mechanical activity in the rabbit taenia coli were studied. Papaverine which inhibits cyclic AMP phosphodiesterase increased Ca-uptake by the microsomal fraction in a concentration of 10^-5 M but not in a concentration of 10^-4 M. Potentiation of Ca-uptake by papaverine is considered to be due to the increase of cyclic AMP as the results of the inhibition of phosphodiesterase. Aspaminol which is found to have little inhibitory action on phosphodiesterase activity inhibited Ca-uptake by the microsomal fraction. This inhibition by Aspaminol was abolished by the increase of Ca-concentration in the reaction medium. Noncompetitive antihistaminic activity of Aspaminol was considerably reduced by an increase of external Ca-concentration, while the increase of external Ca-concentration slightly decreased antihistaminic activity of papaverine. Furthermore, contraction of KCl-depolarizd taenia coli induced by CaCl₂ was inhibited competitively by Aspaminol and noncompetitively by papaverine. These results suggest that Aspaminol competes with Ca ions at a surface site of muscle membrane concerned with Ca-uptake process, thus decreasing the supply of Ca ions to the contractile elements and that papaverine may have inhibitory actions on Ca-uptake by the microsomal fraction in addition to stimulatory action which seems to be mediated through cyclic AMP. These actions on Ca-uptake may induce smooth muscle relaxation.

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In order to elucidate whether the effects of bleomycin on the production or accumulation of intercellular substances by rat carrageenin granuloma are specific to bleomycin or a general response of the granulomatous tissue to the substances interfering with DNA synthesis, we examined the wet (or dry) weight and various components (DNA and glycosaminoglycans) in the granulomatous tissue of rats treated with bleomycin, neocarzinostatin, or hydroxyurea. The changes in various parameters of the granuloma after withdrawal of the inhibitors of DNA synthesis were different with different inhibitors tested. The highest recovery rate of DNA contents in the granulomatous tissue observed after withdrawal of bleomycin. It was also found that bleomycin caused much higher accumulation of hyaluronic acid in rat carrageenin granuloma than neocarzinostatin or hydroxyurea. This accumulation mechanism was indicated to be the results of the inhibition of mucopolysaccharidase activities via cells rather than those of the stimulation of glycosaminoglycan synthesis. These findings obtained in our experimental granuloma-system are of particular importance in considering the mechanism of the side effects of bleomycin.

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Three new antibiotics isolated from broth cultures of a Pseudomonas were evaluated for antitumor activity against murine leukemias L1210 and P388. The antibiotic with the dichloromethyl group at the 3 position of actinobolin, an antibiotic produced by a Streptomyces, is the major product (Y-12278), and two analogs of Y-12278 are minor. When these antibiotics were administered i.p. on days 1 to 4 to mice bearing ascitc leukemias, the most effective was Y-12278, which increased the lifespan of mice implanted with leukemias L1210 and P388 by 88 and 110% over controls, respectively. On the same treatment schedule, less than 60% ILS (increase in lifespan) was obtained by oral and s.c. administration of Y-12278 to mice implanted i.p. with these leukemias, and by its i.p. injection to mice implanted i.c., i.v. and s.c. with leukemia L1210. With i.p. administration of Y-12278, a single injection on day 1 only was less effective in increasing the lifespan of mice bearing ascitic leukemias L1210 and P388 than the prolonged treatment schedules such as daily on days 1 to 4. Y-12278 administered i.p. on days 3 to 6 was shown to possess antitumor activity against i.p. implanted rat hepatomas. More than 200% ILS was seen in hepatomas AH44 and AH7974F.

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