The sites of action of many chemical agents that modify the contraction of smooth muscle are in the smooth muscle membrane. However, a few agents, such as calmodulin inhibitors and protein kinase inhibitors, interact directly with contractile elements of the actomyosin system so as to modify smooth muscle contraction. Here, we describe experimental procedures that are applicable for the screening of smooth muscle relaxants with this mode of action. Myosin B was extracted from chicken gizzard smooth muscle. Because myosin B was a crude preparation of smooth muscle actomyosin, it consisted of regulatory proteins of calmodulin, myosin light chain kinase and protein phosphatase in addition to the contractile proteins of actin and myosin. Interaction of chemical agents with these proteins could be detected by measuring the Mg-ATPase activity of the myosin B preparation. Then we examined whether the agents that altered the ATPase activity was associated with changes in phosphorylation of myosin light chain. If the levels are altered, the agents may interact with the regulatory protein(s). If not, the site of their action was in the cotractile proteins. The analysis with these respective proteins will be also described.

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We examined the special characteristics of various kinds of fixatives and tried to find the most suitable method for simultaneous histological estimation of the mucous gel layer and mucous cells in rat gastric paraffin sections stained with Alcian blue (pH2.5)-periodic acid Schiff (AB-PAS). The tested fixatives were (group in parentheses) : absolute ethanol at -80°C (A), absolute methanol at -80°C (B), Carnoy's solution (C), formalin-ethanol (D) and formalin-Tyrode (E). The thickness of the mucous gel layer (ML) and the numbers of AB- and PAS-positive cells (AB cells, PAS cells) of both the fundic gland area (F. area) and pyloric gland area (P. area) were measured microscopically (× 200). ML in the F. area was found in the order of (A)>(B)>(C)>(D)=(E). AB cells were in the order of (A)>(E)>(B)>(C)>(D); and PAS cells were in the order of (B)>(A)>(E)>(C)>(D). Effects of various fixatives in the P. area showed the same trend as the F. area. Apart from above mentioned procedures, we observed the effects of ethanol at various temperatures: -80°C, -25°C, -4°C and 20°C. ML, AB and PAS cells showed the highest level at -80°C. Moreover, to confirm that these fixatives retain the mucus, we evaluated the hexose and hexosamine contents contained in the fixative solution after fixation. As a result of various fixations, the hexose and hexosamine values in the fixative solution were the smallest for ethanol at -80°C. In conclusion, ethanol at -80°C was the most suitable fixative for histological estimation of mucous gel and mucous cells in rat gastric surface mucosa.

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We examined the effects of FRG-8813, a new histamine H₂-receptora ntagonist with potent antisecretory activity, on gastric mucus in male SD rats (7w). In this study, the effects of FRG-8813 (1, 3, 10 mg/kg), given orally twice a day for 7 days, were investigated by histochemical and biochemical methods in comparison with those of cimetidine (CM, 30 mg/kg) and famotidine (FM, 1 mg/kg) in the fundic gland area (F. area) and pyloric gland area (P. area). In the histochemical study by alcian blue (pH 2.5)-PAS (AB-PAS) or high iron diamine-alcian blue (HID-AB) staining, the CM group showed a significant decrease in PAS and tended to show decreases in HID-AB positive mucus and mucous gel layer in the F. area; the FM group also showed a decrease in AB positive mucus in the P. area. On the other hand, the AB-PAS and HID-AB positive mucus of the FRG-8813 group were not affected. In the biochemical study, FRG-8813 increased the gastric mucosal hexose, hexosamine and sialic acid composing the mucus in a dose-dependent manner in the F. area. These results suggest that FRG-8813 does not cause a decrease in gastric mucus, unlike CM or FM, and it may be able to promote mucus secretion through increasing the mucous component in the F. area.

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We examined the effects of daily injections of doxorubicin, an anti-tumor drug, on unimpeded eruption rates, pulp cells, odontoblasts, periodontal ligament and dentine formation in the rat mandibular incisor. The experimental animals were intraperitoneally injected with 5 mg of doxorubicin/kg body weight/day for a period of 7 days. The eruption rates decreased gradually; and on the seventh day, the mean eruption rate had decreased to 14% of the control value. In addition, we noticed marked destruction of pulp cells and periodontal cells at the basal region of the incisor. The amount of dentine formation was measured using the tetracycline labeling method, but significant decreases in dentine formation were not observed. These results suggest that destruction of the basal tissues is related to inhibition of incisor eruption rates, but that tooth eruption is not related to dentine formation.

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Hemodialysis was performed on dogs, following intravenous bolus injections of LHG at dosage levels of 50, 100 and 200 IU/kg and heparin at the levels of 100 and 200 IU/kg. LHG exerted dose-dependent anticoagulant effects and prolonged the hemodialysis time, compared to heparin with similar anti-Xa activity. When LHG was administered, the half-life of plasma anti-Xa activity was longer than that of heparin at similar anti-Xa activity. LHG prolonged the activated partial thromboplastin time (APTT) linearly and dose-dependently. However, the prolongation was much less than that of heparin, and the anticoagulant activity of LHG continued even after the APTT returned to the value before LHG administration. When LHG was administered, whole blood Xa activated coagulation time (XCT) and plasma Xa activated coagulation time (PXCT) were prolonged in a significantly greater degree compared to APTT. Therefore, XCT and PXCT were considered to be appropriate parameters for monitoring LHG. In the groups administered with LHG at 100 and 200 IU/kg, where hemodialysis could be continued for 8 hr, the tissue factor pathway inhibitor (TFPI) activity in the plasma tended to show a sustained increase. These findings suggested the possibility that not only the antithrombin III dependency mechanism but also the TFPI mechanism contributed to a longer LHG hemodialysis duration compared to heparin administration.

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The effect of single or daily oral administration of hot water extracts (HWE) from Byakushi or Ogon on rat hepatic drug-metabolizing enzymes were investigated in vivo. Enzymes were measured for 3 to 72 hr after single oral administration of HWE at the dose of 1.0 g/kg or 5.0 g/kg. Administration of 1.0 g/kg of Byakushi and 5.0 g/kg of Ogon inhibited aniline hydroxylase activity, while 5.0 g/kg of Byakushi inhibited it in the early phase, but increased it in the late phase. Byakushi inhibited aminopyrine N-demethylase activity, while 5.0 g/kg of Ogon increased it. Byakushi and Ogon decreased the amount of cytochrome P-450. Byakushi and Ogon increased the amount of cytochrome b₅. Byakushi increased cytochrome c reductase activity 3 hr after administration and decreased it 6 and 12 hr after administration. In contrast, 1.0 g/kg of Ogon decreased cytochrome c reductase activity, and 5.0 g/kg increased it 6 hr after administration and decreased it 12 hr after administration. At 24 hr after the last administration to animals treated with a regimen of once a day administration of the HWE (0.1 or 1.0 g/kg) of Byakushi or Ogon for 14 days, the enzymes were measured. Byakushi decreased aminopyrine N-demethylase activity, the amount of cytochrome P-450, and cytochrome c reductase activity. Ogon decreased cytochrome c reductase activity. Byakushi altered the composition of cytochrome P-450 isozyme after daily administration.

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