We demonstrated the effect of a novel selective type IV phosphodiesterase (PDE) IV inhibitor, T-440 (1-[1-(2-methoxyethyl)pyrid-2-one-4-yl]-2, 3-bis(hydroxymethyl)-6, 7-diethoxynaphthalene), on antigen- and chemical mediator-induced bronchoconstrictions in anesthetized guinea pigs in vivo. Intravenously (i. v.) administered T-440 inhibited antigen-induced bronchoconstriction dose-dependently in passively sensitized guinea pigs (ED₅₀= 2.3 mg/kg). Histamine-, leukotriene (LT) D₄-, U-46619-, acetylcholine (ACh)-, neurokinin A- and endothelin-1-induced bronchoconstrictions were also inhibited by i. v. injected T-440. Most potent suppression was produced against the bronchoconstriction induced by LTD₄(ED₅₀= 0.89 μg/kg), whereas the effect against ACh was very weak (ED₅₀= 1.8 mg/kg). Additionally, T-440 inhibited histamine-induced bronchoconstriction by intraduodenal and intratracheal administration (ED₅₀and EC₅₀= 1.6mg/kg and 0.50mg/ml, respectively). Bronchoconstrictions induced by antigen and chemical mediators were also suppressed by theophylline. However, all of these anti-spasmolyiic effects of theophylline were less potent than those of T-440 (1.8-110times). Our results indicate the importance of PDE IV in bronchodilation, and PDE IV inhibitors may have potential as anti-asthma drugs.

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Electrophysiological responses to cholecystokinin (CCK) were studied in Xenopus oocyies injected with mRNA from rabbit pancreas or rat hippocampus. CCK-octapeptide(26-33) (sulfated form) (CCK-8) elicited inward currents in both groups. In oocyies injected with pancreatic mRNA, CCK-8-induced currents were composed of two components, fast and slow. However, in oocytes injected with hippocampal mRNA, fast currents disappeared. The potency ranking of the agonists and the antagonist indicated that the receptors expressed by pancreatic and hippocampal mRNA were CCK_A-and CCK_B-subtypes, respectively. Extracellular application of EGTA had little effect on the CCK_B-mediated response, but attenuated the CCK_A-mediated one. Intracellular injection of EGTA abolished the CCK_B-mediated response, whereas small smooth currents remained in oocyies expressing the CCK_A-receptor. The reversal potentials of the CCK_A-and CCK_B-receptor-mediated responses were consistent with that for Cl^- currents. However, the reversal potential of the small smooth currents in EGTA-loaded oocytes expressing the CCK_A-receptors was close to that for a non-selective cation channel. These results suggest that CCK-8 activates at least two different channels, a Ca²⁺-dependent Cl^- channel and a non-selective cation channel in oocytes expressing the CCK_A-receptor, while the CCK_B-receptor elicits only a Ca²⁺-dependent Cl^- channel.

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The aim of this study was to investigate if prostaglandin (PG) metabolism is altered by angiotensin-converting enzyme (ACE) inhibitors as determined in the broncho-alveolar lavage fluid (BALF) of the guinea pig. Enalapril or imidapril was orally administered once a day for 2 weeks to Hartley male guinea pigs. Twenty-four hours after the last treatment, BALF was collected and the concentrations of PGI₂, thromboxane A₂ (TXA₂) and PGE₂ were measured by enzyme immunoassay. Enalapril significantly (P<0.05) increased the TXA₂ content, which was inhibited by indomethacin treatment and significantly (P<0.05) decreased the PGI₂ content. Imidapril, however, did not affect TXA₂ or PGI₂ generation. These findings suggest that altered PG metabolism may be associated with coughing as a side effect of enalapril.

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In the intestinal smooth muscle of guinea pig taenia caeci, acetylcholine and carbachol induced a transient contraction followed by a sustained contraction. The magnitudes of the transient and sustained contractions were similar when muscle was stimulated with acetylcholine (0.1 μM-1 mM) or a lower concentration (0.1μM) of carbachol. However, higher concentrations of carbachol (1-100μM) induced significantly smaller sustained contraction than the transient contraction. In the 45 mM KCl-stimulated strips, addition of 100 μM carbachol induced a transient increase followed by a sustained decrease in the contractile tension. In contrast, acetylcholine (0.1μM-1 mM) showed only weak inhibitory effects on the high K⁺-induced contraction either in the absence or presence of a cholinesterase inhibitor, 0.5 μM diisopropylfluorophosphate. The same concentration of diisopropylfluorophosphate shifted the concentration-response curve for acetylcholine to lower concentrations. In the muscles pretreated with 3 μM phorbol 12-myristate 13-acetate for 24 hr to desensitize protein kinase C, sustained contractions induced by higher concentrations of carbachol (1-100 μM) were significantly greater than those in the strips without the treatment with phorbol ester. However, the transient contraction and the contraction induced by a lower concentration (0.1 μM) of carbachol were not changed by the treatment with phorbol ester. Pretreatment with phorbol ester attenuated the inhibitory effect of carbachol on the high K⁺-induced contraction. These results suggest that the inhibitory effects of carbachol is composed of two phases: protein kinase C-independent transient inhibition and protein kinase C-dependent sustained inhibition.

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Both analgesic and ulcerogenic activities of d-2-[4-(3-methyl-2-thienyl)phenyl] propionic acid (M-5011), a novel non-steroidal anti-inflammatory drug (NSAID), were compared with those of indomethacin (IND), ketoprofen (KP), diclofenac sodium (DIF), zaltoprofen (ZLT) and tiaprofenic acid (TIA) in mice. All orally administered NSAIDs including M-5011 inhibited kaolin-induced writhing in a dosedependent manner. M-5011 had an effective antinociceptive activity (ED₅₀ Value) of 0.63 mg/kg, being more potent than ZLT (16.80 mg/kg) and TIA (4.78 mg/kg), equipotent to DIF (0.68 mg/kg), and less potent than IND (0.21 mg/kg) and KP (0.28 mg/kg). All drugs tested significantly reduced peritoneal 6-keto-prostaglandin F_1α (6-keto-PGF_1α) levels at the peak kaolin-induced writhing time (7.5 min post-kaolin injection) without affecting peritoneal bradykinin (BK) levels. Antinociceptive effects of all drugs were closely correlated with inhibition of peritoneal 6-keto-PGF_1α levels. Ulcerogenic activities (UD₅₀ value) of M-5011 in the stomach and small intestines were 88.23 and 46.09 mg/kg, respectively. UD₅₀ Values of other drugs in the stomach and small intestines were as follows: 8.96 and 4.78 mg/kg, 20.04 and 10.75 mg/kg, 4.19 and 2.24mg/kg, 62.86 and 46.55 mg/kg, and 110.92 and 54.78 mg/kg for IND, KP, DIF, TIA, and ZLT, respectively. Thus, the safety indexes (UD₅₀/ED₅₀) of the stomach (or small intestine) for M-5011, IND, KP, DIF, TIA and ZLT were 140.05 (73.16), 42.67 (22.76), 71.57 (38.39), 6.16 (3.29), 13.15 (9.74) and 6.60 (3.26), respectively. These findings suggest that M-5011 is a useful NSAID that shows potent antinociceptive effects with low ulcerogenic activities.

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The effects of harmaline on tryptophan-induced 5-hydroxytryptamine (5-HT) syndrome and body temperature changes in pargyline-pretreated rats were investigated. When administered i.p. 60 min after pargyline treatment (50 mg/kg, i.p.), tryptophan, at 100 mg/kg but not 10 mg/kg, induced the 5-HT syndrome. Tryptophan at 100 mg/kg also produced hypothermia followed by hyperthermia in pargyline-pretreated rats. Administration of harmaline (10 mg/kg, i.p.) 30 min after pargyline not only potentiated the 100 mg/kg tryptophan-induced 5-HT syndrome and body temperature changes, but also produced the syndrome following administration of 10 mg/kg tryptophan in pargyline-pretreated rats. In contrast, when administered 30 min before pargyline, 10 mg/kg harmaline completely suppressed the syndrome and body temperature changes caused by 100 mg/kg tryptophan. Tryptophan (100 mg/kg, i.p.) administration significantly increased 5-HT levels and decreased 5-hydroxyindole acetic acid levels and 5-HT turnover in the brain of pargyline-pretreated rats. Harmaline administration 30 min after pargyline did not significantly affect the tryptophan-induced changes in 5-HT Ievels and 5-HT turnover, whereas when administered 30 min before pargyline, harmaline significantly blocked the effect of tryptophan. These results suggest that mechanisms underlying the inhibitory action of harmaline on the tryptophan-induced 5-HT syndrome and body temperature changes in pargyline-pretreated rats differ from those by which harmaline potentiates the effects of tryptophan.

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This work addresses the kinetic analysis of the interaction of methotrexate (MTX) with camel retina acetylcholinesterase (AChE, EC 3. 1. 1. 7). It was found that the MTX effect was reversible in nature. The IC₅₀ was determined, by two methods, to be 1.362 mM. The Michaelis-Menten constant (K_s) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.123 mM in the control system, and the MTX-treated systems showed a 10-35% decrease in this value. The V_max was 0.789 μmol/min/mg protein for the control system, while it was decreased by 23-76% in the MTX-treated systems. The Lineweaver-Burk plot, Dixon plot and their secondary replots indicated that the inhibition was a linear mixed type; i.e., uncompetitive and noncompetitive. The values of K_i and K_I were estimated as 0.782 and 0.404 mM, respectively. The use of camel retina as a model for the study of human retina may open new avenues for studying various aspects of AChE.

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Cerebral hypoperfusion was chronically induced in aged rats via permanent bilateral occlusion of common carotid arteries (2VO). Marked reduction of the B_max value of the muscarinic receptors (mAChR) in both the cortex and striatum and the V_max value of choline acetyltransferase (ChAT) activity in the cortex, hippocampus and striatum were observed as compared with those of control aged rats. No significant changes in mAChR and ChAT activity were observed between young control rats and young 2VO rats. One month post-surgery in aged rats, daily doses of bifemelane (10 mg/kg) or aniracetam (50 mg/kg) were administered orally over a 4-week period. Administration of bifemelane significantly increased B_max values and decreased apparent K_d Values for ³H-quinuclidinyl benzilate (QNB) in mAChR in the striatum. Chronic administration of bifemelane or aniracetam also enhanced ChAT activity in the cortex, hippocampus and striatum. In particular, administration of bifemelane resulted in a significant increase in V_max values of ChAT in all three brain regions, while no significant change in K_m values for ChAT was observed. These results suggest that bifemelane is responsible for this activity, thereby enhancing the functioning system of CNS cholinergic neurons of cerebral hypoperfused aged rats.

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We investigated the effects of a phosphodiesterase inhibitor and dibutyryl CAMP (dBcAMP) on the cell injury induced by cephaloridine (CER) in an established renal epithelial cell LLC-PK₁. CER increased the leakage of lactate dehydrogenase (LDH) from LLC-PK₁ cells to the medium and the level of lipid peroxidation in the cells. 3-Isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, increased CAMP content in LLC-PK₁ cells and ameliorated the increase in LDH Ieakage induced by CER. dBcAMP reduced the cell injury induced by CER. Our results suggest that a signalling pathway of CAMP protects against CER-induced renal cell injury, which is probably due to generation of oxygen radicals.

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Hoelen is used to treat gastric disease in Eastern traditional medicine. The efferent activity on the gastric vagus nerve was studied in the rat, and the activity was found to increase after administration of Hoelen into the duodenum. In addition, we examined the effect of Hoelen fractions. Both the fraction containing polysaccharide and the triterpenoid-rich fraction increased activity significantly. Hoelen would thus appear useful for treating gastric disease by activating efferent activity of the gastric vagus nerve through the action of triterpenoid and polysaccharide.

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Removal of external Ca²⁺ facilitated endogenous taurine release in cultured rat astrocyies. The stimulated release was not affected by furosemide, sucrose, tetrodotoxin and 3, 4-dichlorobenzamil, but partially inhibited by nifedipine. Omission of external Na+ increased basal taurine release, and the effects of Na⁺ removal and Ca²⁺ depletion on the release were additive. The Na⁺-free condition did not affect Ca²⁺ paradox-induced cell death in astrocyies. These findings suggest that Ca²⁺ depletion facilitates taurine release in a mechanism independent of volume and the Na+ gradient and that the release is not involved in Ca²⁺ paradox-induced delayed cell toxicity in astrocytes.

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The effects of toborinone ([(±)-6-[3-(3, 4-dimethoxybenzylamino)-2-hydroxypropoxy]-2(1H)-quinolinone], OPC-18790), milrinone and E-4031 (1-(2-(6-methyl-2-pyridil)-1-ethyl)-4-(4-methanesulfonyl-amino-1-benzoyl)piperidine dihydrochloride) on membrane potential were examined in isolated guinea pig sinoatrial node preparations. Toborinone, a new positive inotropic agent, prolonged cycle length (CL), depolarized maximum diastolic potential (MDP) and decreased maximum upstroke velocity (V_max) and action potential amplitude (APA). On the other hand, milrinone, a peak III phosphodiesterase (PDE III) inhibitor, increased V_max and APA and shortened CL and action potential duration. E-4031, an I_K blocker, prolonged CL, depolarized MDP and decreased V_max and APA. These results suggest that toborinone modulates the action potential like an I_K blocker rather than a PDE III inhibitor in a sinoatrial node.

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