FUT-175, 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate (nafamstat mesilate), a novel synthetic protease-inhibiting agent, was studied to determine its in vitro effects against various proteases and other enzymes, as well as to determine its in vivo protease inhibitory effects. FUT-175 was found to inhibit, in an intense, specific and reversible way, the enzyme activities of trypsin, C1r, C1s, thrombin, kallikrein and plasmin with IC50 values of the order of 10^-6-10^-8 M. FUT-175 also inhibited complement-mediated hemolysis, including both classical and alternative pathways, sites of inhibition being on C1r and C1s as evidenced by the intermediate-cell technique. In animal model reactions in which the complement system is known to be involved as pathogenetic factors, e.g., Forssman shock, Forssman cutaneous vasculitis, zymosan-induced paw edema, endotoxin shock and local Shwartzman reaction, FUT-175 was highly effective in that, for example, intravenous dosing at 3 mg/kg could completely protect guinea pigs from the lethal Forssman shock. FUT-175 was also found to be effective in trypsin-induced shock in mice, in lethality due to thrombin-thrombosis in mice and in kinin formation in the inflammatory process in rats.

View full abstract

Direct recording of reduced nicotinamide adenine dinucleotide (NADH) fluorescence was conducted with an organ redoximeter in isolated perfused guinea pig heart. Cross-clamping of the aortic inflow line resulted in an increase in NADH fluorescence. After etafenone (10^-6 M), there was a significant prolongation of the time to the detectable or the maximum increase in NADH fluorescence. The magnitude of the increase in NADH fluorescence tended to be reduced (135% as compared with 145% in the control group). Supplemental chemical analyses revealed a significant increase in creatine phosphate, adenosine triphosphate (ATP) and total adenine nucleotide in the etafenone-pretreated group 15 min after postischemic reperfusion, although the ischemia-induced changes were not improved by this compound. It was suggested that the better recovery of myocardial high energy phosphate levels produced by etafenone was brought about by a decrease in oxygen consumption due to a decrease in mechanical performance of the heart and possibly by a better resynthesis of ATP.

View full abstract

We attempted to determine the existence of a functional serotonergic small intensely fluorescent (SIF) cell in the rat superior cervical sympathetic ganglion (SCG) by evaluating the effects of pargyline, decentralization and electrical stimulation on the preganglionic sympathetic fibers. The objective was to assess changes in serotonin (5-HT) metabolism compared with findings in the case of dopamine (DA) metabolism. The contents (ng/ganglion, n=8, average±S.E.) in the adult male Wistar rat SCG of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) were 1.19±0.11 and 0.11±0.01, respectively, determined by our reverse-phase high-performance liquid chromatographic electrochemical method. A relatively large amount of 5-HT was detected in the capsule of the SCG (0.62±0.16 ng/capsule, n=8) without detection of 5-HIAA. The pargyline injection increased the DA content and decreased the 3, 4-dihydroxyphenylacetic acid (DOPAC) content rapidly. The increase of the 5-HT content was observed without changes in the capsule 5-HT. The mode of pargyline induced-increase in the 5-HT content and decrease of the 5-HIAA content was slow compared to that of DA and DOPAC. Decentralization did not alter the 5-HIAA content, whereas the DOPAC content was markedly reduced. Although electrical stimulation significantly increased the DOPAC content, changes in the 5-HIAA content were not observed. It would thus appear that 5-HT is produced in the rat SCG, and if so, then support for existence of a serotonergic SIF cell in the rat sympathetic ganglion can be obtained. A direct connection between pre- and postganglionic sympathetic neurons through the serotonergic SIF cell was not demonstrated.

View full abstract

Dynorphin is a potent opioid peptide. A synthetic dynorphin-(1-13) (Dyn) induced histamine release accompanied by degranulation from isolated rat mast cells in a dose-dependent manner over the concentration range 10^-7-10^-5 M. Dynorphin-(1-13) -induced histamine release completed within 10 sec at 37°C, and the release was not accompanied by the leakage of lactate dehydrogenase. Calcium (10^-5-10^-3 M) enhanced the release, although higher concentration than 10^-3 M suppressed the release. The pH for the maximum release by Dyn was about 7.3. Disodium cromoglycate (5×10^-6-10^-4 M) inhibited the histamine release by Dyn, but naloxone and leucine-enkephalin did not. These results indicate that Dyn-induced histamine release was not mediated by opioid receptors of the mast cells. Its mode of action appeared to that of anaphylactic histamine release.

View full abstract

The reactivity and specificity of commonly used anti-inflammatory agents with lysosomal cysteine proteinases cathepsins B and H purified from rat spleen have been investigated. Of the different agents tested, flufenamic acid and indomethacin were known to be potent inhibitors of cathepsin B. A half-maximal inhibition of the activity of cathepsin B was achieved at drug concentrations of 7.6×10^-5 M of flufenamic acid and 4.0×10^-4 M of indomethacin. The inhibition by these two agents was of a non-competitive type with benzyloxy-carbonyl-phenylalanyl-arginine-4-methyl-7-coumarylamide (Z-Phe-Arg-MCA) as a substrate. The maximal inhibitory potencies of these agents for the cathepsin B activity were observed at pH 7.0. At pH values between 4.5 and 6.5, the inhibitory potencies were less than at pH 7.0. No preincubation time was needed for the reaction between these agents and cathepsin B. In contrast, cathepsin H was not affected by these two drugs even at the drug concentration of 10^-3 M at pH values between 4.5 and 8.0. Other anti-inflammatory agents including aspirin, sodium salicylate, phenylbutazone and prednisolone were found to be poorly or scarcely inhibitory for both cathepsins B and H.

View full abstract

The close interrelation between endogenous prostaglandin E (PGE) and the vasoconstrictor response to noradrenaline (NA) was evaluated using a perfused central arterial segment of a rabbit ear. The endogenous PGE level, measured radioimmunologically, was estimated from that in the perfusate. Pretreatment with arachidonic acid (AA) caused a decrease in the response to NA which was accompanied by a rise in the PGE level. The response to NA (% control) correlated significantly and negatively with the PGE level (ng/ml) in the perfusate. In addition, repetitively applied NA elicited gradual augmentation in the response to NA, which was accompanied by a reduction in the PGE level in the perfusate. These results seem to suggest that the endogenous PGE in the perfused central arterial segment of the rabbit ear is related to the inhibitory action on the response to NA.

View full abstract

Effects of short-chain fatty acids (SCFA) on the contractile response of rat ileum were studied in vivo. The contractile response was estimated by means of changes in the intraluminal pressure under the isometric condition. Intravenous administration of sodium salts of propionate, butyrate, valerate or caproate produced biphasic contractions : an initial phasic contraction and a subsequent tonic contraction. The effect of propionate was studied in detail. A sigmoid dose-response curve was obtained for the phasic contraction. Atropine, hexamethonium and tetrodotoxin inhibited the phasic contraction, while neostigmine vigorously enhanced it. On the other hand, the tonic contraction was not inhibited by atropine, hexamethonium or tetrodotoxin. Repeated administration of propionate at intervals of less than 3 min led to tachyphylaxis, and this tachyphylaxis disappeared by about 10 min. These results suggest that SCFA induced the biphasic contraction of the rat ileum, probably by neurogenic and myogenic mechanisms.

View full abstract

The behavioral effect and acute toxicity of methamphetamine were tested alone and in combination with quinine in rats. Quinine prolonged and increased the effect of methamphetamine. The enhancement of methamphetamine-induced stereotyped behavior was very marked when quinine was given prior to or simultaneously with methamphetamine. However, the time for onset of methamphetamine-induced stereotyped behavior was not affected by quinine. The mortality of methamphetamine was also potentiated markedly by quinine. The enhancement of the behavioral effect and the toxicity of methamphetamine may be due to inhibition of the metabolism of methamphetamine by quinine.

View full abstract

The synthetic activity of rat gastric sulfated glycoproteins (SGP) in vitro was investigated at various time intervals after water immersion stress using ³⁵S-sulfate as a precursor. More than 90 percent of the total radioactivity was incorporated into mucosal SGP, and the rest was incorporated into glycosaminoglycans in the gastric muscular layer. The incorporation of ³⁵S-sulfate into SGP increased at 2 hr and decreased at 6 hr after the onset of stress. The incorporating activity again increased markedly at 12 hr and then recovered to the normal level at 24 hr after the onset of stress. An anti-ulcer agent, N-(N-acetyl-β-alanyl)-L-histidine aluminum complex (AAHA), significantly increased the SGP synthetic activity at 12 hr and at 24 hr after the onset of stress. It was indicated from the elution patterns on the DEAE-cellulose column that AAHA increased the amount of highly sulfated glycoproteins compared with the stress control at 12 hr after the onset of stress. The uronic acid content in the gastric muscular layer of the rat was unchanged with stress. These results in the in vitro experiment indicate that the SGP synthetic activity does not decrease with stress load, but rather increases at 2 hr and at 12 hr after the onset of stress when a sufficient amount of ³⁵S-sulfate is supplied. Accordingly, it is suggested that SGP facilitates the restoration of the gastric mucosal damage caused by stress.

View full abstract

The behavioral effect of brotizolam was investigated in mice and rats, in comparison with those of diazepam, nitrazepam and estazolam. Locomotor activity of rats in an open field situation was slightly increased with smaller doses of brotizolam and estazolam and with larger doses of nitrazepam, while it was decreased with large doses of brotizolam and estazolam. The anticonflict effect of brotizolam in rats was approximately as potent as that of diazepam and was augmented following chronic administration for 10 days. In suppressing hyperemotionality and muricide of olfactory bulbectomized rats, brotizolam was more potent than diazepam, being approximately equipotent to nitrazepam and estazolam. Brotizolam, diazepam, nitrazepam and estazolam prevented both maximal electroshock and pentetrazol convulsions in mice, the effects on the latter being much more potent than those on the former. In impairing rotarod performance, brotizolam was as potent as estazolam and nitrazepam and was much more potent than diazepam in mice, but was less potent than estazolam and nitrazepam in rats. These results indicate that brotizolam possesses pharmacological properties characteristic to benzodiazepines and that the activity is more potent than that of diazepam and approximately as potent as those of nitrazepam and estazolam.

View full abstract

The indexes for agonistic activity (pD₂ values) and for antagonistic activity (pA₂ values) of some beta-adrenergic partial agonists were estimated in the isolated taenia caecum and tracheal preparation of guinea pig. The pK_i values, negative log of dissociation constants, were also estimated by photoinactivation of the beta-adreneoceptor in the taenia caecum. The pD₂ values of the partial agonists were significantly different from their pA₂ values, but equal to their pK_i values. If the partial agonists interacted with one site in their receptor, the pD₂ values would be equal to the pA₂ values because the partial agonists had little receptor reserve. These results suggest that the properties of the binding site for agonistic action were different from those for antagonistic action.

View full abstract

The effects of trimebutine maleate (TM-906) on electrical and mechanical activities of smooth muscles of the guinea-pig stomach were investigated using a microelectrode and isometric tension recording methods. TM-906 (2×10^-5 M) depolarized the membrane of smooth muscles in the antrum to about 10 mV. From the current-voltage relationship and changes in membrane potentials in various [K]_o, the TM-906-induced depolarization is considered to be mainly due to a decrease in the K-conductance. TM-906 increased the amplitude of the first spike potential and regularized the rhythm of slow waves. These excitatory effects are presumably due to the K-channel-blocking action during the repolarizing phase of the spikes and to the depolarization. TM-906 reduced the amplitudes of mechanical activities and slow waves. These inhibitory effects are presumably due to the inhibition of Ca-release from storage sites and to the block of Ca-influx. The biphasic effects are possibly due to the local anesthetic properties. TM-906 modified neither the membrane potential nor the membrane conductance of circular muscles in the fundus. This may mean that the circular muscles in the fundus lack the K-channel sensitive to TM-906.

View full abstract

Ritodrine hydrochloride (ritodrine) is a beta₂-adrenoceptor stimulant which has been effectively prescribed for the prevention of premature labor. The present studies were carried out to investigate the effects of ritodrine on uterine motility in rats and rabbits during gestation, as compared with those of isoproterenol and isoxsuprine. The results were as follows : 1) Spontaneous movements and evoked contractile responses of isolated rat uterus (19-20th days of gestation) were suppressed by 10^-9-10^-6 M ritodrine. The potency of ritodrine was approximately 10 times more than that of isoxsuprine and 100-1, 000 times less than that of isoproterenol. 2) When these drugs were administered to pregnant rats or rabbits intravenously, the tocolytic potency was in the following order : isoproterenol>ritodrine>isoxsuprine. 3) Ritodrine induced hypotension and tachycardia, but these effects were less than those of isoproterenol and isoxsuprine. 4) The effects of isoproterenol and ritodrine were almost prevented by pretreatment with propranolol, but those of isoxsuprine were only partially or not affected. These results suggest that ritodrine is effective in preventing the uterine contractions in rats and rabbits and that it has less effect on the circulatory system than isoproterenol and isoxsuprine. It is also concluded that ritodrine produces these effects through activation of beta-adrenoceptors.

View full abstract