The effects of PG-9 (3α-tropyl 2-(p-bromophenyl)propionate), the acetylcholine releaser, on memory processes and nerve growth factor (NGF) synthesis were evaluated. In the mouse passive-avoidance test, PG-9 (10 - 30 mg/kg, i.p.), administered 20 min before the training session, prevented amnesia induced by both the non selective antimuscarinic drug scopolamine and the M₁-selective antagonist S-(-)-ET-126. In the same experimental conditions, PG-9 (5 - 20 μg per mouse, i.c.v.) was also able to prevent antimuscarine-induced amnesia, demonstrating a central localization of the activity. At the highest effective doses, PG-9 did not produce any collateral symptoms as revealed by the Irwin test, and it did not modify spontaneous motility and inspection activity, as revealed by the hole-board test. PG-9 was also able to increase the amount of NGF secreted in vitro by astrocytes in a dose-dependent manner. The maximal NGF contents obtained by PG-9 were 17.6-fold of the control value. During culture, no morphological changes were found at effective concentrations of PG-9. The current work indicates the ability of PG-9 to induce beneficial effects on cognitive processes and stimulate activity of NGF synthesis in astroglial cells. Therefore, PG-9 could represent a potential useful drug able to improve the function of impaired cognitive processes.

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para-Chlorophenylalanine (PCPA, 100 - 200 mg/kg) was used as a pharmacological tool to characterize the 5-hydroxytryptamine (5-HT) involvement in the emesis occurring 24 hr after the administration of cisplatin (10 mg/kg) in the ferret. PCPA was effective to antagonize the initial 8 hr period of retching and vomiting, but potentiated the emesis that occurred during the remaining 8- to 24-hr observation period. Tissue samples removed from the brainstem at 24 hr post injection of cisplatin alone revealed an elevation of 5-HT, dopamine and homovanillic acid that was antagonized by the injection of PCPA. Cisplatin also induced increases in the urinary levels of 5-hydroxyindoleacetic acid that was similarly antagonized by PCPA. Results are discussed in terms of the relevance of 5-HT to the model of cisplatin (10 mg/kg)-induced emesis in the ferret compared to the problem of acute and delayed emesis in man. The residual or delayed phase of cisplatin-induced emesis may involve a 5-HT-independent mechanism.

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The effects of KRN4884 (5-amino-N-[2-(2-chrolophenyl)ethyl]-N′-cyano-3-pyridinecarboxamidine), a novel K⁺ channel opener, on the electrocardiogram changes caused by the intracoronary administration of endothelin-1 (ET-1) were studied in anesthetized rats and compared with the effects of levcromakalim, a K⁺ channel opener; nilvadipine, a Ca²⁺ antagonist; and propranolol, a β-adrenoceptor antagonist. KRN4884 (50 μg/kg, i.v.) and levcromakalim (300 μg/kg, i.v.) inhibited the ST segment elevation and the development of arrhythmias induced by ET-1 (5 μg, i.c.) and decreased the incidence of death. Nilvadipine (300 μg/kg, i.v.) and propranolol (1000 and 3000 μg/kg, i.v.) each prevented the ST segment elevation, but the suppressions of the occurrence of arrhythmias produced by nilvadipine and propranolol were less than that shown by KRN4884. KRN4884 (30 and 50 μg/kg, i.v.), levcromakalim (100 and 300 μg/kg, i.v.) and nilvadipine (100 and 300 μg/kg, i.v.) significantly decreased the mean blood pressure in a dose-dependent manner, but propranolol did not. The heart rate was decreased by nilvadipine (100 and 300 μg/kg, i.v.) and propranolol (1000 and 3000 μg/kg, i.v.), but was not affected by KRN4884 (30 and 50 μg/kg, i.v.) or levcromakalim (100 and 300 μg/kg, i.v.). These results suggest that pretreatments with KRN4884 and levcromakalim are more effective on ET-1-induced electrocardiogram changes than those with nilvadipine and propranolol.

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Effects of YT-146 [2-(1-octynyl) adenosine], an adenosine A₂ receptor agonist, on cAMP production and noradrenaline (NA) release were investigated in PC12 cells. YT-146 caused a concentration-dependent cAMP accumulation (EC₅₀: 1.2±0.9 nM). In [³H]NA-prelabeled cells, YT-146 increased the basal NA release and enhanced ATP-evoked NA release in a concentration-dependent manner (EC₅₀: 0.23±0.15 nM). YT-146 augmented the maximal response to ATP without affecting the EC₅₀ value of ATP. These effects of YT-146 were inhibited by several adenosine receptor antagonists with a characteristic of adenosine A_2A receptor subtype. The effects of YT-146 were mimicked by forskolin, dibutylyl cAMP and Sp-cAMPS, and inhibited by H-89, a cAMP-dependent protein kinase inhibitor. YT-146 had little effect on ATP-induced increase in intracellular Ca²⁺ concentration. YT-146 enhanced the NA release induced by several different stimuli including Ca²⁺ ionophore A23187. The present results suggest that YT-146 is a potent agonist on adenosine A_2A receptors in PC12 cells and causes a cAMP-dependent enhancement of NA release by affecting the exocytosis process at a point downstream of the intracellular Ca²⁺ increase.

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Wistar strain rats fed low calcium diets (1, 2, 3, 4, 6 and 8 weeks) exhibited changes in secretory function of whole saliva. In particular, there were changes in salivary flow rate, total salivary protein, amylase enzyme activity, salivary amylase content and the level of cyclic AMP in the parotid gland acinar cell. Although there were no changes for the first 3 weeks, the levels increased at week 4 and decreased at week 6. The wet weight of the parotid gland started to decrease at week 4. These results suggest that when fed low calcium diets for long periods of time, rats develop defective salivary secretion.

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The role of a hot water extract of black tea (Camellia sinensis (L). O. Kuntze Theaceae) in the gastric cytoprotective mechanisms was studied using gastric mucosal lesions produced by various ulcerogens in rats as an experimental model. Prior oral administration of black tea extract (BTE) at 20 ml/kg, i.g. once a day for 7 days significantly reduced the incidence of gastric erosions and severity induced by ethanol, diethyldithiocarbamate (DDC) and diethylmaleate (DEM). This treatment also favorably altered the changes in acid and peptic activity of gastric juice in these ulcerogen-treated animals. Singular administration of succimer (60 mg/kg, i.g.), the standard sulfhydryl containing antiulcer drug used as a reference drug, was also effective. The levels of glutathione and glutathione peroxidase were significantly decreased after treatment with ethanol, DDC and DEM, and this decrease was prevented by BTE pretreatment in the aforesaid manner. Other major features of BTE-induced reversal of ulcerogenic agents include a significant decrease in the protein content and a marked increase in hexosamine and sialic acid content. These results suggest a major role for glutathione, an endogenous antioxidant, in the cytoprotection against ulceration afforded by BTE.

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Participation of nitric oxide in the electrical field stimulation-induced nonadrenergic, noncholinergic (NANC) relaxation in various intestinal regions was studied in 2- to 50-week-old Wistar rats. In the jejunum of 2-week-old rats, the extent of the nitric oxide-mediated component of the relaxation of longitudinal muscle was approximately 60 - 70%, whereas the component was 40 - 50% in 4-week-old rats and was absent in 8- and 50-week-old rats. Thus, nitric oxide seems to be the most important mediator at young ages but its significance is lost with age. The same tendency as that in the jejunum was also shown in longitudinal muscle of the ileum, proximal and distal colon, and rectum. The tendency was also shown in the circular muscle of the rectum. Sensitivity of the longitudinal muscle of the jejunum and proximal colon to exogenously added nitric oxide was high in younger rats. Immunoreactive structures for nitric oxide synthase were observed in the circular muscle layer of the rectum. The population of the structures was denser in 4-week-old than that in 50-week-old. The results suggest that NANC relaxation in every region of the intestine at 2-week-old is almost solely mediated by nitric oxide, and its significance as an inhibitory mediator gradually or rapidly decreases with age.

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The mechanism of the prophylactic effect against hyperlipidemia by monatepil maleate was investigated in animal models. Monatepil maleate is an antihypertensive agent with Ca²⁺-channel antagonistic, α₁-adrenergic receptor-blocking, and lipid peroxidation inhibitory activity. In high cholesterol diet-fed rabbits, monatepil maleate (30 mg/kg, p.o., once daily for 9 weeks) showed a prophylactic effect against increases in total cholesterol and β-lipoprotein. Monatepil maleate significantly accelerated the clearance of radioactivity from the blood after intravenous injection of low-density lipoprotein (LDL) labeled with [1α, 2α (n)-³H]cholesterol, increasing biliary excretion of [³H]-bile acids without modifying bile acid composition. Furthermore, monatepil maleate tended to inhibit the absorption of orally administered [1α, 2α (n)-³H]cholesterol from the gastrointestinal tract in these rabbits. In Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of hepatic LDL receptor deficiency, monatepil maleate (30 mg/kg, p.o., once daily for 6 months) did not suppress the increase in plasma lipids. These results suggest that the plasma lipid lowering effect of monatepil maleate requires the presence of hepatic LDL receptors. It is also suggested that monatepil maleate improves plasma lipid metabolism through two mechanisms: enhancement of clearance of plasma LDL, which may be mediated by up-regulation of hepatic LDL receptors, and acceleration of conversion of free cholesterol to bile acids in the liver.

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Histamine H₂-receptor antagonistic properties of the anti-ulcer agent T-593, (±)-(E)-1-[2hydroxy-2-(4-hydroxyphenyl)ethyl]-3-[2[[[5-(methylamino)methyl-2-furyl]methyl]thio]ethyl]-2-(methylsulfonyl)guanidine, were investigated on [¹⁴C]aminopyrine accumulation in isolated canine gastric mucosal cells and compared with those of ranitidine and famotidine. The potency of T-593-inhibition of [¹⁴C]aminopyrine accumulation stimulated by 10^-4 M histamine, with an IC₅₀ value of 1.85× 10^-6 M, was approximately 5 times greater than that of ranitidine, but half that of famotidine. T-593 did not affect [¹⁴C]aminopyrine accumulation stimulated by carbachol or dibutyryl-cAMP. T-593 depressed the maximal response of the histamine concentration-response curve with a dose-related displacement to the right, indicating that the nature of the H₂-receptor antagonism of T-593 was insurmountable and included noncompetitive inhibition. The inhibitory efficacy of T-593 was time-dependent and was retained after the cells were washed. The inhibitory potency of (-)-S-T-593, one of the enantiomers, on the [¹⁴C]aminopyrine accumulation stimulated by histamine was approximately twice that of racemic T-593 and it also behaved as an insurmountable H₂-receptor antagonist. However, the potency of (+)-R-T-593 was markedly weak. These results suggest that T-593 has H₂-receptor antagonism that is insurmountable and this agent slowly associates and dissociates with the receptor in isolated canine gastric mucosal cells and that the specific substance causing H₂-receptor antagonism is (-)-S-T-593.

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The cellular mechanisms of norepinephrine (NE)-induced Mn²⁺-dependent contractions were investigated in dispersed smooth muscle cells from guinea pig vas deferens by characterizing the effects of NE and K⁺ on cell length observed by videotape microscopy and on intracellular Mn²⁺ and Ca²⁺ concentration ([Mn²⁺]_i and [Ca²⁺]_i) observed by confocal microscopy. Both stimulants induced slow sustained contraction in Ca²⁺-depleted, Mn²⁺-accumulated cells (Mn²⁺-loaded cells), whereas they induced biphasic contractions in normal cells that were neither Ca²⁺-depleted nor Mn²⁺-loaded. In both conditions, the number of cells responding to NE as well as the magnitude of NE-induced contractions increased in a dose-dependent manner. Contractions induced by K⁺ in Mn²⁺-loaded strip preparations were markedly smaller than those induced by NE. Although individual K⁺-induced contractions in responsive Mn²⁺-loaded cells were as large as those induced by NE, a much smaller percentage of Mn²⁺-loaded cells was responsive to K⁺ than to NE. These results are consistent with the idea that the contractions of strip preparations may reflect the magnitude of the contractions of individual cells as well as the percentage of responsive cells in the preparations. Inconsistent with the contractions, the [Mn²⁺]_i rise induced by K⁺ was larger than that induced by NE, and the percentage of cells responsive to K⁺ was larger than that responsive to NE. These results suggest that NE may increase the Mn²⁺ sensitivity of contractile elements.

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Selectivity of tamsulosin and terazosin to functional α₁-adrenoceptors was examined. Both drugs competitively inhibited the contractile responses to noradrenaline in different tissues where the responses were mediated through the α_1D-, α_1B- or α_1L-subtype. Together with the affinities obtained in the binding study with cloned (α_1a, α_1b, α_1d) and native (α_1A and α_1B) subtypes, the selectivity of tamsulosin was α_1A>α_1L, α_1D>α_1B. Terazosin had lower affinity at various subtypes than tamsulosin, but showed relatively high selectivity to the α_1D-subtype. In the human prostate, tamsulosin was more than 30-fold higher in affinity than terazosin in functional and binding studies.

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Endomorphin-1 and endomorphin-2 are newly identified endogenous peptides and have high affinity and selectivity for μ-opioid receptors. The present experiments were conducted to determine whether intracisternal injection of these peptides would produce an itch-associated response and antinociception and to compare their effects to that of morphine. Endomorphin-1 and endomorphin-2 (0.3 - 3 nmol/mouse) elicited facial scratching characterized by bell-shaped dose-response curves with a peak effect at endomorphin-1 at 0.3 nmol/mouse and endomorphin-2 at 1 nmol/mouse. Their peak effects were inhibited by subcutaneous pretreatment with naloxone (1 mg/kg). Morphine (0.3 - 30 nmol/mouse) produced facial scratching, and its dose-response curve was also bell-shaped. Scratching of the body trunk, head and ears were not elicited by these doses of endomorphins and morphine. Endomorphin-1 and -2 at doses of 0.3 - 3 nmol/mouse produced dose-dependent antinociception, as measured with the tail-pressure test. The potency and duration of actions of these peptides were comparable to those of morphine. The results suggest that endomorphin-1 and endomorphin-2 are involved in itch-signaling and pain-inhibiting functions of the brain.

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A bolus injection of theophylline produced a significant increase in peripheral blood flow in anesthetized rat ear, monitored by laser-Doppler flowmetry, with increases in arterial blood pressure and heart rate. These effects were attenuated by previous treatment with reserpine, but reserpine had no effect on the blood flow increase produced by acetylcholine. A dose of propranolol, which caused attenuation of the theophylline-induced increase in heart rate, did not change the peripheral blood flow. The higher dose of propranolol, which nearly canceled the increases in blood pressure and heart rate, caused attenuation of the blood flow increase but did not cancel it. However, the theophylline-induced flow increase was completely reversed by a nitric oxide synthase inhibitor, N^G-nitro-L-arginine methyl ester, which alone had no effect, without any change in arterial blood pressure and heart rate. Treatment of the rats with the dose of inhibitor slightly and significantly reduced the response of peripheral blood flow to acetylcholine. The other isomer, N^G-nitro-D-arginine methyl ester, and the other inhibitor, N^G-monomethyl-L-arginine, did not have such an effect. These results suggest that the flow increase is due to an independent effect on the heart with modification by autonomic reflexes and involves the adrenergic and nitrergic pathways.

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Pharmacological characterization of NTE-122 (trans-1, 4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was performed with both in vitro and in vivo assay systems. NTE-122 inhibited microsomal ACAT activities of various tissues (liver of rabbit and rat, small intestine of rabbit and rat, and aorta of rabbit) and cultured cells (HepG2 and CaCo-2), with IC₅₀ values from 1.2 to 9.6 nM. The inhibition mode of NTE-122 was competitive for HepG2 ACAT. NTE-122 had no effect on other lipid metabolizing enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA synthetase, cholesterol esterase, lecithin:cholesterol acyltransferase, acyl-CoA:sn-glycerol-3-phosphate acyltransferase and cholesterol 7α-hydroxylase up to 10 μM. When NTE-122 was administered to the cholesterol diet-fed rats, serum and liver cholesterol levels were markedly reduced with an ED₅₀ of 0.12 and 0.44 mg/kg/day, respectively. In the cholesterol diet-fed rabbits, NTE-122 significantly lowered plasma and liver cholesterol levels at more than 2 mg/kg/day. These results indicate that NTE-122 is a potent, selective and competitive inhibitor of ACAT, making it a worth while therapeutic agent for hypercholesterolemia and atherosclerosis.

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Inhibitory activity and the mechanism of action of JTE-522 (4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide), a novel selective cyclooxygenase (COX)-2 inhibitor, on human COX-1 and COX-2 were investigated and compared with those of reference compounds. In an enzyme assay, JTE-522 inhibited yeast-expressed human recombinant COX-2 with an IC₅₀ value of 0.085 μM. In contrast, JTE-522 did not inhibit human COX-1 prepared from human platelets at concentrations up to 100 μM. In a cell-based assay, JTE-522 diminished lipopolysaccharide-induced prostaglandin E₂ production in human peripheral blood mononuclear cells (COX-2) (IC₅₀ value = 15.1 nM). On the other hand, JTE-522 was less potent at inhibiting calcium ionophore-induced thromboxane B₂ production in washed human platelets (COX-1) (IC₅₀ value = 6210 nM). JTE-522 showed highly selective inhibition of human COX-2, and its activity was more selective than that of other COX-2 inhibitors (NS-398 and SC-58635). Human recombinant COX-2 activity was attenuated by JTE-522 in a dose-dependent and time-dependent manner. In contrast, the inhibitory activity of JTE-522 on human COX-1 was not affected by preincubation time. COX-2 inhibition by JTE-522 could not be recovered by gel filtration. These results indicate that JTE-522 is a highly selective, time-dependent and irreversible inhibitor of human COX-2.

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We investigated effects of (±)-cis-2-methylspiro[1, 3-oxathiolane-5, 3′-quinuclidine] hydrochlo- ride, hemihydrate (SNI-2011, cevimeline hydrochloride), a rigid analogue of acetylcholine, on saliva and tear secretions in rats and mice to evaluate its therapeutical efficacy for xerostomia and xerophthalmia in patients with Sjögren’s syndrome and X-ray exposure in the head and neck. Intraduodenal administrations of SNI-2011 increased saliva secretion in a dose-dependent manner at doses ranging from 3 to 30 mg/kg in normal rats and mice, two strains of autoimmune disease mice and X-irradiated saliva secretion defective rats. The salivation elicited by SNI-2011 was completely inhibited by atropine. A similar atropine-sensitive response was observed in tear secretion. In rat submandibular/sublingual gland membranes, [³H]quinuclidinyl benzilate (QNB) binding was saturable, and Scatchard plot analysis revealed a single population of binding sites with a K_d of 22 pM and a maximal binding capacity of 60 fmol/mg protein. The competitive inhibition curve of the [³H]QNB binding by SNI-2011 was obtained, and its dissociation constant value calculated from IC₅₀ was 1 - 2 μM. These results suggest that SNI-2011 increases saliva and tear secretions through a direct stimulation to muscarinic receptors in salivary and lacrimal glands, and they suggest that SNI-2011 should be beneficial to patients with Sjögren’s syndrome and X-ray exposure in the head and neck.

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Characteristics of the binding of [³H]ramosetron to cloned human 5-hydroxytryptamine₃ (5-HT₃) receptors were investigated and directly compared to those of [³H]granisetron binding. Saturation studies revealed that [³H]ramosetron labeled more sites with high affinity (K_d=0.15±0.01 nM, B_max=653±30 fmol/mg protein) than [³H]granisetron (K_d=1.17±0.25 nM, B_max=427±43 fmol/mg protein). Kinetic studies revealed that dissociation of [³H]ramosetron was slower than that of [³H]granisetron. These results suggest that ramosetron is a highly potent 5-HT₃-receptor antagonist.

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Effects of GTS-21 [3-(2, 4-dimethoxybenzylidene)-anabaseine dihydrochloride], a selective nicotinic agonist, on locomotor activity and dopamine turnover were examined and compared to those of nicotine to test if GTS-21 exhibits side effects similar to those of nicotine. GTS-21 had no effect on locomotor activity in mice or dopamine turnover in rats. In contrast, nicotine produced a biphasic effect on locomotor activity. It also enhanced dopamine turnover rates in the striatum and cerebral cortex, suggesting the involvement of dopaminergic systems in the nicotine-induced changes in locomotor activity. GTS-21 exhibits fewer adverse effects, suggesting that it has therapeutic potential for cognitive disorders related to central cholinergic dysfunction.

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We tested the effect of calcium dobesilate (DOBE) in aorta from spontaneously diabetic (BB/wor) rats. The contraction induced by 10^-6 M noradrenaline (NA) in BB/wor rats was smaller than that induced in control rats (1.21±0.11 vs 0.82±0.02 g, P<0.01, n=8, respectively) in arteries with intact endothelium. Incubation with DOBE (10^-4 M) impaired the contractions induced by NA in BB/wor rats (1.21±0.11 vs 0.67±0.01 g, P<0.01, n=8). The effect of DOBE was reversed by 10^-6 M propranolol (0.67±0.01 vs 1.20±0.60 g, P<0.001, n=8, with 10^-4 M DOBE and 10^-4 M DOBE plus 10^-6 M propranolol, respectively). DOBE increased the endothelium-dependent relaxation in arteries from diabetic rats. These findings suggest that DOBE might improve vascular reactivity in BB/wor rats.

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We studied the hypoglycemic effect of 4-hydroxybenzoic acid, a constituent of the root of Pandanus odorus Ridl. (Pandanaceae, Thai name: Toei-hom), in streptozotocin-diabetic rats. Oral administration of 4-hydroxybenzoic acid caused a decrease in plasma glucose levels dose-dependently in the diabetic rat. The constituent did not affect serum insulin level and liver glycogen content in the diabetic model, but increased glucose consumption in normal and diabetic rat diaphragms. These results suggest that 4-hydroxybenzoic acid produces a hypoglycemic effect mediated by an increase in the peripheral glucose consumption.

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