Recent progress in molecular biology based upon rapidly developing DNA technology is reviewed. Emphasis is placed on the developing human genome project that includes structural as well as functional analyses of the genome. Expression profiling of active genes in the body helps construct a data-base for the functional aspects of the human genome.

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Plasma morphine concentration and naloxone-precipitated withdrawal body weight loss and plasma corticosterone (PCS) increase were determined at 12, 18 and 24 hr after i.v. infusion of morphine at a constant rate of 10 mg/kg/hr for 4 hr in Sprague-Dawley rats. Plasma morphine concentration declined 98.0% within 12 hr and further declined 58.8% during 12-24 hr after morphine infusion. There was a significant difference between plasma morphine concentrations at 12 and 24 hr after the morphine infusion. Naloxone (0.5 and 2.0 mg/kg)-precipitated withdrawal, but not spontaneous withdrawal, was elicited at 12-24 hr after the morphine infusion, and the severity of withdrawal precipitated by 2.0 mg/kg naloxone was the same at 12-24 hr after the morphine infusion. Furthermore, there was no significant correlation between plasma morphine concentration and body weight loss or PCS increase. The results suggest that a constant degree of morphine dependence is sustained during 12-24 hr after the morphine infusion and the severity of naloxone-precipitated withdrawal is not related to the plasma morphine concentration at the time of naloxone injection, that is, the rate of morphine removal from its receptor sites.

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Effects of phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on muscle tension and cytosolic Ca²⁺ ([Ca²⁺]_i) level was investigated in rat anococcygeus muscle in comparison with other smooth muscles. 1) DPB (10^-6 M) induced a large contraction and an elevation of [Ca²⁺]_i level in rat aorta and small and rhythmic changes in tension and [Ca²⁺]_i level in guinea pig ileum. However, DPB did not change either of the parameters in rat anococcygeus muscle. 2) DPB caused tension development without changing the [Ca²⁺]_i level elevated by high K⁺, ionomycin or β-escin in the anococcygeus muscle. 3) In the β-escin permeabilized muscles of guinea pig ileum and urinary bladder, rabbit mesenteric artery and rat anococcygeus muscle, DPB enhanced the Ca²⁺-developed tension. Moreover, the enhancement was inhibited by H-7 (3 x 10-5 M). 4) DPB did not cause muscle tension to develop in the muscle of rat aorta, guinea pig ileum and rat anococcygeus muscle, pretreated with phorbol 12-myristate 13-acetate for 24 hr. In conclusion, DPB showed different contractile effects on the aorta, ileum and anococcygeus muscle, respectively. The initiation of muscle tension by DPB probably requires [Ca²⁺]_i and the DPB-induced enhancement may be due to a Ca²⁺ sensitization of contractile elements in the anococcygeus muscle. Therefore, the difference between the DPB-induced response of the anococcygeus muscle and those of the other muscles seems to be due to a different Ca²⁺ movement caused by DPB. Moreover, it is suggested that DPB develops muscle tension by increasing [Ca²⁺]_i and enhances it through the mediation of protein kinase C in the anococcygeus muscle as well as the other smooth muscles.

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We investigated the effects of YM060 {(R)-5-[(1-methyl-3-indolyl)carbonyl]-4, 5, 6, 7-tetra-hydro-1H-benzimidazole hydrochloride} and YM114 (KAE-393) {(R)-5-[(2, 3-dihydro-l-indolyl)-carbonyl]-4, 5, 6, 7-tetrahydro-1H-benzimidazole hydrochloride} on 5-HT₄ receptors and gastric emptying in normal and cisplatin-treated rats and compared results with those for ondansetron and granisetron. YM060, YM114, ondansetron and granisetron dose-dependently inhibited the specific binding of [³H]-GR113808 { [[1-[(2-methylsulphonyl)amino]ethyl] -4-piperidin-yl]methyl 1-methyl-1H-indole-3-carboxylate} in guinea pig striatum, with pK_i values of 5.53, 5.13, 5.21 and 5.63, respectively. According to the pK_i values reported in 5-HT₃-receptor binding of [³H]GR65630 to rat cortical membranes, the affinity of YM060, YM114, ondansetron and granisetron for 5-HT₄ receptors was approximately 5, 5, 3.5 and 3.5 log units lower than that for 5-HT₃ receptors, respectively. In the guinea pig longitudinal muscle with myenteric plexus and rat esophageal tunica muscularis mucosae, YM060 and YM114 showed neither 5-HT₄-agonistic nor antagonistic properties. Although ondansetron produced concentration-dependent increases in the magnitude of the twitch response in longitudinal muscle, it did not possess 5-HT₃- and 5-HT₄-agonistic activity. Granisetron antagonized 5-HT-induced relaxation of the rat esophagus with an apparent pA2 value of 5.39. Intravenous YM060, YM114, ondansetron and granisetron significantly enhanced gastric emptying of glass beads and improved cisplatin-induced slowing of gastric emptying in rats. These results indicate that the selectivity of YM060 and YM 114 for 5-HT₃ receptors is higher than that of ondansetron and granisetron and that these 5-HT₃ antagonists have gastroprokinetic activity in normal and cisplatin-treated rats without affecting 5-HT₄ receptors.

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KT3-671 (2-propyl-8-oxo-1-[(2’-(1H-tetrazole-5-yl)biphenyl-4-yl)methyl]-4, 5, 6, 7-tetrahydrocycloheptimidazole), a structurally new nonpeptide angiotensin AT₁-receptor antagonist, was administered orally and repeatedly to 15-week-old stroke-prone spontaneously hypertensive rats for 7 weeks; and its effects on blood pressure, heart rate, renal function, plasma renin concentration (PRC), plasma aldosterone concentration (PAC) and hypertension-related tissue damage in the brain, heart, kidney and mesenteric artery were investigated. KT3-671 at a dose of 3 or 10 mg/kg, p.o. per day prevented development of hypertension and produced a significant and consistent reduction of blood pressure in a dose-dependent manner. Enalapril at a dose of 10 mg/kg per day produced cardiovascular effects similar to those of KT3-671 at 10 mg/kg. Despite marked reduction in blood pressure, neither KT3-671 nor enalapril affected the heart rate. KT3-671 at 10 mg/kg produced a transient and significant reduction of urinary sodium excretion in the second week, but did not affect renal function at any other time during the experimental period. Both KT3-671 at 10 mg/kg and enalapril at 10 mg/kg produced a significant increase in PRC and showed a tendency to decrease PAC. Repeated administration of KT3-671 reduced the severity of the pathological changes in the kidney. These results suggest that KT3-671 is a potentially useful antihypertensive drug.

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Effects of irsogladine on ionomycin-induced decrease in intercellular communication and increase in intracellular concentration of Ca²⁺ ([Ca²⁺]_i) were investigated in cultured rabbit gastric epithelial cells. Ionomycin (10^-7-10^-6 M) transiently and concentration-dependently inhibited intercellular communication concomitantly with the elevation of [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺. Irsogladine (10^-5M), which has been shown to facilitate intercellular communication, suppressed the ionomycin-induced elevation of [Ca²⁺]_i and decrease in intercellular communication. The suppression of the ionomycin effects by irsogladine was independent of extracellular Ca²⁺. TMB-8 [8-(diethylamino)octyl-3, 4, 5-trimethoxy-benzoate hydrochloride] (10^-6M) also suppressed the ionomycin-induced elevation of [Ca²⁺]_i and decrease in intercellular communication. These results indicate that the ionomycin-induced decrease in intercellular communication may be due to Ca²⁺ mobilization from intracellular stores. Inhibitory effects of irsogladine and TMB-8 on the ionomycin-induced decrease in intercellular communication may be produced by suppressing Ca²⁺ mobilization.

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OPC-18790, (±)-6-[3-(3, 4-dimethoxybenzylamino)-2-hydroxypropoxy]-2(1H)-quinolinone, is a novel positive inotropic agent, and its mechanism of positive inotropic action involves not only phosphodiesterase inhibition, but also a prolongation of action potential duration in ventricular muscle. Prolongation of action potential duration is also a property of class III antiarrhythmic agents; therefore, we examined the cardiohemodynamic effects and arrhythmogenicity of a combination of OPC-18790 and dopamine in halothane-anesthetized dogs. Dopamine (5 μg/kg/min) alone increased the peak of the first derivative of left ventricular pressure (LVdP/dt_max) and cardiac output (CO) by 43-48% and 16-20%, respectively, while OPC-18790 (10 μg/kg/min) increased these parameters by 56% and 22%, respectively. The combination of OPC-18790 (10 μg/kg/min) and dopamine (5 μg/kg/min) and dopamine alone at an increased dose of 10 μg/kg/min further increased LVdP/dt_max and CO by 104-113% and 29-30%, respectively. Thus, positive inotropic effects were equally observed in both groups, and the effects of OPC-18790 and dopamine seemed to be additive. The other hemodynamic effects were similar among all groups. Arrhythmias such as premature ventricular contraction developed in 5 out of 7 dogs (71.4%) in the 10 μg/kg/min dopamine group, while only one premature ventricular contraction was observed in 1 of 7 dogs (14.3%) in the OPC-18790 (10 μg/kg/min) and dopamine (5 μg/kg/min) combination group. These results suggest that the combination of OPC-18790 and dopamine may provide new therapeutic options for the treatment of heart failure.

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The mode of the inhibition of purified rat brain adenylate cyclase by the βγ-subunits of G protein (βγ) was studied. These subunits inhibited the catalytic activity of the cyclase with the maximal inhibition of 8501o and the half-maximal inhibition at about 0.7 nM βγ. The complex of βγ and adenylate cyclase isolated by density gradient centrifugation contained 1.8 - 2.0 mol βγ per mol of the cyclase when βγ was assayed by immunoblotting and by its inhibitory activity on adenylate cyclase. However, the βγ concentration-inhibition curves suggest that one of the two βγ molecules bound may be essential for the inhibition. The role for the second βγ molecule is unknown. As a tentative estimate, 70010 of the adenylate cyclase activity remained inhibited by βγ when the complex was isolated. The inhibition was not dependent on Gα_s or calmodulin. Although purified adenylate cyclase contained a protein (0.06 - 0.08 mol/mol of adenylate cyclase) that reacted with anti-Gα_s antibody, this protein was not liberated from the cyclase when it formed a complex with βγ. In addition, guanine nucleotide analogs little affected the cyclase activity or the inhibition by βγ. The inhibition by βγ was reversed by the dilution of the complex, and the following re-addition of βγ suppressed the enzyme activity to about 15010 of the initial activity again. These findings provide strong evidence that Rr inhibits adenylate cyclase directly and reversibly through the formation of the complex.

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AZO02 (L-threo-(3, 4-dihydroxy phenyl)-N-methyl serine methyl ester) is a newly synthesized adrenaline derivative. AZO02 caused relaxation of rat jejunum β₃-receptors) (ED₅₀=18μM), but did not affect the atrial rate (β₁) or tracheal relaxation (β₂) at a concentration of 0.3 mM. The pA₂ values for propranolol in inhibiting the isoproterenol- and AZOO2-stimulated relaxation of rat jejunum were 6.27 and 6.33, respectively. Thus, AZO02 is a selective agonist for β₃-adrenoceptor. AZO02 stimulated lipolysis (ED₅₀=10 μM) and glucose uptake (ED₅₀=1 μM) in rat adipocytes. In both cases, stimulation was antagonized by high concentrations of the β-adrenoceptor antagonist propranolol, but not by the α-adrenoceptor antagonist phentolamine. The effect of AZO02 on glucose uptake was synergistic with that of insulin. AZO02 was also assessed in vivo by using genetically obese mice (KK/Ay strain) with hyperglycemia. Administration of AZO02 in the diet for a week decreased blood glucose and non-esterified fatty acids.

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Effects of famotidine on neuronally evoked acid secretion were investigated by means of vagal (at the lower esophagus level) and field-electrical stimulation (around the stomach) in the isolated mouse whole stomach preparation. Each of the electrical stimulations caused a frequency-dependent (1 to 20 Hz) increase in acid output, and the secretory response was abolished by tetrodotoxin or atropine. In the case of field stimulation, the acid secretion was not completely inhibited by hexamethonium. When 10 Hz frequency was applied with either vagal or field-electrical stimulation, the acid secretion was only partly inhibited by famotidine at doses of up to 30 μM. In contrast, the acid response to 2 Hz stimulation was almost completely inhibited by 1 μM famotidine. In the presence of neostigmine (30 nM), the 2 Hz vagally stimulated acid secretion became partly resistant to the effect of famotidine (10 μM). These results suggest that both vagally and field-electrically stimulated acid secretions have essentially the same characteristics and that the secretory mechanism through histamine release is exclusively dominant with weak stimulation, while the cholinergic mechanism on parietal cells is sufficient for reaching the maximal secretory response with strong stimulation.

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We examined the effects of KW-5092 ({1-[2-[[[5-(piperidinomethyl)-2-furanyl]methyl]-amino]ethyl] -2-imidazolidinylidene}propanedinitrile fumarate), a novel gastroprokinetic agent, on gastric mucosal blood flow (GMBF) in anesthetized rats. Intravenous infusion of KW-5092 (0.1 mg/kg/min for 30 min), which did not affect the basal GMBF, reversed the norepinephrine (1 μg/kg/min, i.v. infusion for 30 min)-induced decline of GMBF in the corpus and the antrum. The improvement by KW-5092 of the GMBF was abolished by atropine (0.1 mg/kg/min, i.v. infusion for 30 min). These results suggest that KW-5092, via cholinergic activation, could counteract the decline of GMBF induced by adrenergic activation.

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We investigated the effects of a novel prolyl endopeptidase inhibitor, JTP-4819 ((S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl)-1-pyrrolidinecarboxamide), on thyrotropin-releasing hormone (TRH)-like immunoreactivity (TRH-LI) in the cerebral cortex and hippocampus of aged rats. The TRH-LI content of both brain regions in aged rats was significantly lower than that in young rats. A single oral dose of JTP-4819 (3 mg/kg) restored the cortical TRH-LI content in aged rats, while doses of 0.3-3 mg/kg restored it in the hippocampus. Repeated oral administration of JTP-4819 at a dose of 1 mg/kg for 21 days produced a significant increase of TRH-LI in the cerebral cortex, while it did so in the hippocampus at doses of 0.3 and 1 mg/kg. Our findings suggest that JTP-4819 may improve the functioning of TRHergic neurons, which deteriorate with senescence.

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A phosphodiesterase (PDE) III inhibitor, amrinone, inhibited both the negative inotropic actions of verapamil and nicardipine in guinea pig ventricular papillary muscle; this effect was canceled by the protein kinase A inhibitor H-89. The PDE IV inhibitor 1, 3-di-n-butyl-7-(2’-oxopropyl)xanthine (denbufylline), which elicited a negative inotropic action by itself, attenuated the action of verapamil up to 10 μM, without any interaction, with nicardipine. The attenuation by denbufylline was not influenced by H-89. This suggests that in the ventricular papillary muscle, denbufylline acts on some verapamil-sensitive site(s) in the membrane and interferes with the calcium channel function without involvement of its PDE inhibitory activity.

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Effects of paeoniflorin (PF) and glycyrrhizin (GLR), contained in paeony and licorice roots, respectively, on contractile and non-contractile Ca²⁺ mobilization were examined by measuring the Ca²⁺-aequorin luminescence (Ca²⁺ transients) of the nerve-stimulated skeletal muscle of mice in the presence of neostigmine (0.3 μM). PF (0.1-1 mM) prolonged the duration of non-contractile Ca2²⁺ transients, which may induce the desensitization of nicotinic acetylcholine receptor, but did not affect contractile Ca2²⁺ transients. GLR (0.3-1 mM) depressed contractile Ca²⁺ transients without affecting non-contractile transients. These results suggest that PF and GLR may have complementary effects on intracellular Ca²⁺ mobilization to block the neuromuscular transmission.

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Agmatine was about as potent as aminoguanidine to inhibit the activity of the inducible form of nitric oxide synthase (iNOS) in isolated rat aorta. Like aminoguanidine, agmatine was devoid of significant activity on the constitutive form of NOS. Agmatine inhibited the conversion of [³H]L-arginine in [3H]L-citrulline in partially purified iNOS from macrophages (IC₅₀=262±39.9 μM). Thus, our data suggest that agmatine may act as endogenous inhibitor of iNOS.

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We investigated the induction of cyclooxygenase (COX) in rat endometrium during the estrus cycle. The estrus cycle was divided into five stages (proestrus, estrus, diestrus-1, diestrus-2, diestrus-3) by cytological observation of vaginal smears. COX-1 was detected in the endometrium during all five stages of the estrus cycle. In contrast, COX-2 in the endometrium was detectable only in three stages (diestrus-3, proestrus, estrus) and showed a peak at diestrus-3, coinciding with the peak in serum estradiol level, suggesting the estradiol-dependent induction of COX-2.

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