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A newly synthesized agonist for muscarinic acetylcholine (ACh) receptors CS-932, (R)-3-(3-iso-xazoloxy)-1-azabicyclo-[2.2.2]octane hydrochloride, showed a relatively higher affinity for M1 than M2 receptors expressed in Chinese hamster ovary (CHO)-cells in comparison with ACh. CS-932 elevated the intracellular Ca2+ level only in M1-CHO cells, although ACh increased the level in both M1- and M3-CHO cells. CS-932 and ACh reduced forskolin-stimulated accumulation of cAMP in M2-CHO cells by 20% and 80%, respectively. This neurochemical profile of CS-932 indicates that the compound can activate M1-receptor-mediated functions selectively. CS-932 increased firing of cholinoceptive neurons in rat hippocampal slices, and this excitation was antagonized by pirenzepine, but not by AF-DX 116. CS-932 increased awake and decreased slow wave sleep episodes of daytime EEG in free-moving rats. It counteracted scopolamine-induced slow waves in rat cortical EEG. CS-932 also increased the power of alpha- and beta-waves, but decreased delta-wave of the cortical EEG in anesthetized monkeys. It ameliorated scopolamine-induced impairment of working memory in rats. Orally administered CS-932 had the best penetration into the brain among the muscarinic agonists tested and caused the least salivary secretion among the cholinomimetics examined. These results indicate that CS-932 has potential as a cognitive enhancer with fewer side effects in therapy for Alzheimer disease.

This study examined whether the renal kallikrein-kinin system (KKS) is involved with furosemide-induced natriuresis in rats. Intravenous administration of furosemide (10 mg/kg) to anesthetized rats infused with physiological saline (saline) increased renal KK excretion as well as urine volume and urinary excretions of sodium, chloride and potassium. The change in the increase of renal KK excretion by furosemide at a dose of 1.0 mg/kg relative to the control was larger than that of urine volume. Pretreatment with a B2-receptor antagonist, 8-[3-[N-[(E)-3-(6-acetamidopyridin-3-yl)acryloylglycyl]-N-methylamino]-2,6-dichlorobenzyloxy]-2-methylquinoline (FR173657, 100 mg/kg), significantly inhibited the furosemide-induced natriuresis by 58.6%. The effect of FR173657 on the furosemide-induced natriuresis was also examined in hypotonic saline-loading rats. Similar to the saline-loading rats, urinary excretion of sodium collected during the first 8 h in metabolic cages significantly reduced by 22.4% when FR173657 (100 mg/kg) was given concurrently with furosemide (100 mg/kg) and hypotonic saline (5% of body wt.). These results indicate that furosemide increased renal KK excretion through a mechanism different from a washout mechanism and induced natriuresis partly through an augmentation of the renal KKS following the increase in renal KK excretion in both the saline- and hypotonic saline-loading rats.

We evaluated the effects of a new non-steroidal anti-inflammatory drug (NSAID), d-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (M-5011), and indomethacin on the production of arachidonate metabolites and pro-inflammatory cytokines in male Sprague-Dawly rats with monosodium urate crystal (MSU)-induced pleurisy. Levels of tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6 in the pleural exudate were determined by biological assays, while prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and cytokine-induced chemoattractant-1 (CINC-1) levels were quantified by enzyme immunoassays. Orally administered M-5011 (5 mg/kg) decreased the pleural exudate volume at 3 and 4 hr after MSU injection. Indomethacin (10 mg/kg) decreased the volume at 3–5 hr. These drugs reduced the number of leukocytes in the pleural cavity at 6 hr. Both NSAIDs also reduced the content of PGE2 in the exudate without affecting LTB4 levels. Increased productions of both IL-6 and CINC-1 in the exudate were reduced by pretreatment with M-5011 or indomethacin, and TNF levels in the exudate were increased by pretreatment of these drugs. Thus, M-5011 inhibits the production of both IL-6 and CINC-1 at lower doses than those of indomethacin, and the inhibitory effect of M-5011 on CINC-1, but not IL-6, may partly contribute to the inhibition of leukocyte infiltration in rats with MSU-induced pleurisy.

To clarify the mechanisms of duodenal ulcerogenic activity of non-steroidal anti-inflammatory drugs (NSAIDs), the effects of indomethacin (IND) on acid-stimulated duodenal bicarbonate secretion and histamine-induced duodenal ulcerogenic responses were studied in comparison with NS-398, a selective cyclooxygenase (COX)-2 inhibitor, in rats. IND (1 and 5 mg/kg, s.c.) significantly decreased duodenal bicarbonate secretion and potentiated duodenal lesion in a dose-dependent manner. On the other hand, NS-398 had no effect on these parameters. These findings suggest that duodenal ulcerogenicity of IND in the presence of histamine is mainly due to the inhibitory action on acid-stimulated bicarbonate secretion mediated by COX-1, but not by COX-2.

The avoidance behavior of rats in a typical step-through passive avoidance task was evaluated by using two types of time measurements that differed from traditional step-through latency (STL). The total stay-time in the light box (TL) was calculated as an index of the dark box avoidance throughout the retention trial, counting the time before and after the first step-through. The total stay-time in the far area of the light box (TF) was calculated as an index of the tendency to keep away from the dark box. TL, TF and the STL increased with electrical shock intensity at acquisition trials and gradually decreased through an extinction procedure. The discrepancy between our new measures and the STL was observed when the effects of a new drug, RS-8359, which was reported to ameliorate ischemic brain damage, were examined in ischemic animals. TL, TF and STL decreased in rats receiving brain ischemia, and we found that after treatment with RS-8359, TL and TF increased, but no effect was observed on STL. The discrepancy observed suggests that a short STL does not necessarily imply a loss of avoidance behavior. Passive avoidance tests can be more revealing about a drug’s effects when stay-time measures are used.

We examined the effect of leminoprazole (an acid pump inhibitor) on reflux esophagitis induced in rats. Intragastrically administered leminoprazole significantly and dose-dependently protected the esophageal mucosa against the reflux of gastric contents, without affecting gastric acid secretion. However, it had no effect on the esophagitis when administered intraduodenally, despite its significant inhibition (about 40%) of gastric acid secretion. Omeprazole significantly prevented the development of esophagitis, most probably through potent inhibition of gastric acid secretion. Indomethacin significantly reduced the synthesis of prostaglandin E2 in the esophagus. Since indomethacin pretreatment had no effect on the esophageal protection by leminoprazole, omeprazole or sucralfate, the involvement of endogenous prostaglandins can be ruled out as a possible underlying mechanism. Intragastrically, but not intraduodenally, administered sucralfate significantly prevented the esophagitis even at a dose not affecting gastric acid secretion. These results strongly suggest that both leminoprazole and sucralfate protect the esophageal mucosa directly.

Changes in local cerebral glucose utilization (LCGU) of the postischemic rat brain were investigated using the rat four-vessel occlusion model. Following 20 or 30 min of ischemia, LCGUs of the cerebral cortices, striatum and hippocampus were decreased at 1 and 3 days postischemia, but were recovered at 7 days postischemia. Effects of repeated administration of RS-8359, (±)-4-(4-cyanoanilino)-7-hydroxycyclopenta(3,2-e)pyrimidine, (30 mg/kg×2/day, p.o., 4 days) were examined at 3 days postischemia following 20 min of ischemia. Compared with the sham-operated group, the LCGUs of 22 out of 34 structures examined in the ischemic-control group were significantly reduced. In the RS-8359-treated group, however, significant reduction was observed in only 9 structures. Compared with the ischemic-control group, RS-8359 significantly ameliorated the reduction of LCGU in 12 structures. These results suggest that RS-8359 has beneficial effects on reduced glucose metabolism in the postischemic brain.

We investigated the antitumor effects and tissue distribution of adriamycin (ADR) incorporated into a hydroxyapatite (HAP) bead in a cancer rat model bearing Swarm rat chondrosarcoma. The Porous HAP bead (8.48 mm in diameter, 531±0.7 mg in weight) was used as a model bone graft. One ADR-HAP bead (ADR 0.4mg-6.0mg/bead) was implanted s.c. into a Sprague-Dawley rat at 6 days postinoculation of Swarm rat chondrosarcoma. ADR-HAP beads showed strong antitumor activities in a dose dependent manner. The dose of 6.0 mg/bead showed the highest efficacy with no toxic death: It caused a 98% growth inhibition on Day 31 postinoculation and a survival advantage of a 339% increase in life span. After the implantation of the ADR-HAP bead (0.4 mg/bead/body) and the i.v. administration of an equal dose of free adriamycin, we determined the tissue distribution of ADR for up to 90 days. ADR-HAP bead implanted in the tumors released ADR over a 12-week period in the target area. The diffusion of the drug to other organs such as the heart and liver was very low compared with the tumors. The area under the ADR concentration-time curve (AUC) of the tumors was 181.6 μg·day/g and 5.22 μg·day/g after the implantation of the ADR-HAP bead and the i.v. administration of free ADR, respectively. The targeting index of the tumors, defined as the ratio of the AUC after the implantation of the ADR-HAP bead to that after administration of free ADR, was 34.8. The targeting indices of 0.16 and 0.17 for the heart and liver, respectively, indicate that the implantation of the ADR-HAP bead reduced delivery of ADR to these organs. These results demonstrate that this method of administration may be useful in delivering adjuvant chemotherapy in order to prevent local recurrence in the site of the bony defect after the surgical removal of bone tumors.

Levels of monoamines and their metabolites were determined in the cortex, hippocampus, and striatum of rats killed by microwave irradiation. Moclobemide (20 mg/kg, p.o.) and clorgyline (10 mg/kg, p.o.), type A monoamine oxidase (MAO-A) inhibitors, increased the levels of normetanephrine (NM) and 3-methoxytyramine (3MT) and decreased those of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5HIAA) in almost all three regions. Deprenyl (10 mg/kg, p.o.), a type B monoamine oxidase inhibitor, however, little affected monoamine and metabolite levels in all regions. The maximum effects of RS-8359 (10 mg/kg, p.o.) were obtained at 2 to 6 hr after administration, when the levels of norepinephrine (NE), NM, 3MT, and serotonin (5HT) in all regions and dopamine (DA) in the striatum increased, while DOPAC and HVA levels decreased. The levels of monoamines and metabolites had returned to normal by 20 hr after administration. Dose-dependency of the effects of RS-8359 on monoamine metabolites was observed at doses up to 30 mg/kg (p.o.) at 1 and 6 hr after administration. In conclusion, NE, DA, and 5HT are exclusively or preferentially deaminated by MAO-A in the cortex, hippocampus, and striatum of rats, and RS-8359 exhibits a reversible MAO-A inhibitory action in all three regions tested in vivo.

Regional monoamine assays revealed that during veratramine-induced myoclonic movements, the contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the cerebral cortex were reduced with a slight increase in dopamine metabolites in the midbrain and brainstem. A similar tendency to decrease 5-HT and 5-HIAA contents was observed in the hypothalamus and hippocampus without increase in the contents of dopamine and its metabolites. Norepinephrine levels were not modified in any brain region at any time after the administration of the veratrum alkaloid. It was found that the veratramine evoked 3H-5-HT release from the frontal cortical slices was Ca+(+)-independent and persistent, and it continued approximately 20 min after the 2-min exposure to veratramine. The uptake of 3H-5-HT into the frontal cortical slices was inhibited competitively by veratramine. These results suggest that veratramine is both a releaser and uptake inhibitor of 5-HT and that the veratramine-induced involuntary movements may be mediated by serotonergic hyperfunction.

Although possible usefulness of non-selective monoamine oxidase (MAO) inhibitors for Parkinson’s disease therapy has been suggested in the literature, MAO inhibitors whose inhibition is reversible and have dual action to both MAO-A and -B subtypes is not available yet. Subtype selectivity and reversibility of a series of novel MAO inhibitors, 3-(2-aminoethoxy)-1,2-benzisoxazole derivatives, were studied. Several dual MAO inhibitors, which inhibit both MAO-A and -B, were obtained. When administered to mice, their effects were generally reversible. Among the derivatives, RS-1636 and RS-1653 had much longer duration of brain MAO-B inhibition than that of MAO-A. In vitro, the inhibited MAO-A activity by these compounds was partially recovered by buffer change at 4°C, while little MAO-B activity was recovered. Although it is not fully elucidated yet, the reversibility of these inhibitors is probably determined primarily by this dissociation profile. This unique differential reversibility indicates that optimization of the balance of actions can be achieved by differentiating reversibility to each target molecule.

The effects of extracellular acidosis on gallbladder contraction were investigated using gallbladder strips isolated from guinea pigs. In an acidic medium (pH 6.9), gallbladder contraction induced by histamine and prostaglandin E2 was significantly lower than that in a normal medium (pH 7.4). Acidosis affected neither gallbladder contraction induced by histamine in the absence of extracellular Ca2+ nor that induced by KCl. Acidosis significantly inhibited Ca2+-induced contraction in the presence of sodium fluoride and phorbol 12,13-dibutyrate but not that in the presence of KCl. Staurosporine (30 nM) significantly inhibited gallbladder contraction induced by histamine and prostaglandin E2, but not that by KCl. Histamine-induced contraction in the presence of staurosporine was not affected by acidosis. Acidosis significantly inhibited Ca2+-induced contraction in the presence of histamine but not that in the presence of both histamine and staurosporine. These results suggest that extracellular acidosis selectively inhibits gallbladder contraction mediated by protein kinase C activation.

Effects of beta-blockers (propranolol, arotinolol and nipradilol) and a Ca2+ channel blocker (nicardipine) on oxotremorine-induced tremor were studied in common marmosets. Generalized tremor was elicited by an intraperitoneal administration of 0.25 mg/kg oxotremorine. Intensity of the tremor was classified into 7 degrees, and it was evaluated every 10 min. The total intensity of oxotremorine-induced tremor for each drug was expressed as "points", which were the sum of tremor intensity scores evaluated every 10 min up to 190 min following the administration of oxotremorine. Beta-blockers significantly suppressed the tremor. On the other hand, the Ca2+ channel blocker exacerbated the tremor.

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 dose-dependent 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-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) 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-PGF1 alpha 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 2.24 mg/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.

We measured the number of D1-receptors (Bmax) in the striatum whose dopaminergic terminals were destroyed to various degrees by 6-hydroxydopamine (6-OHDA) to clarify the relationship between the degree of denervation and the change in Bmax for D1-receptor. Rats were sacrificed 28 days after 6-OHDA treatment, and [3H]SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-l-phenyl-2, 3, 4, 5-tetrahydro-1H-3-benzazepine) binding capacity and monoamine content were measured in striatal homogenate. Bmax was significantly upregulated when dopamine content was reduced to less than 5% of that in the vehicletreated striatum. In contrast, it was significantly decreased when dopamine content was 5—25% of that in the vehicle-treated striatum. The D1-receptors showed either upregulation or downregulation depending on the degree of denervation.

We investigated the antitumor effects of adriamycin (ADR)-loaded hydroxyapatite (HAP) beads in a Swarm rat chondrosarcoma model. When one ADR-loaded HAP bead (ADR: 0.8 mg/bead) was implanted into the central portion of a rat bearing tumor, the ADR-loaded HAP beads showed good therapeutic effects, increasing the life span (ILS) by 90%. Significantly, leukopenia and diarrhea were not observed. These results suggest that HAP delivery offers an interesting and a potentially effective method.

The release and synthesis of neuronal histamine are regulated by histaminergic autoreceptors named as histamine H3 receptors. The development of radiolabeled histamine H3 antagonists is needed to characterize the binding of antagonists to these receptors. Here we describe the binding characteristics of a new histamine H3-receptor antagonist, [3H]S-methylthioperamide (SMT), to rat tissues, and compare its binding with that of [3H](R)alpha-methylhistamine ((R)alpha MH), a selective histamine H3-receptor agonist. The binding of [3H]SMT to the membranes of rat forebrain was found to be stereoselective, saturable, reversible and temperature-dependent. Saturation binding experiments indicated a single class of high affinity sites for [3H]SMT in forebrain membranes (KD = 2.1 nM, Bmax = 24.3 pmol/g of tissue at 4 degrees C). The Bmax was approximately 3 times that of [3H](R)alpha MH binding to rat forebrain membranes (KD = 2.5 nM, Bmax = 7.3 pmol/g of tissue at 25 degrees C). Autoradiographic images of [3H]SMT binding in the brain were essentially the same as those of [3H](R)alpha MH. [3H]SMT also bound appreciably to peripheral tissues (the liver, adrenal, stomach, ileum, kidney, lung and bladder), whereas the [3H](R)alpha MH bindings to these peripheral tissues were negligible. These results indicate that [3H]SMT binds to H3 receptors primarily in the central nervous system, and that it also has high affinity toward non-H3 receptors, probably hemoproteins, in peripheral tissues.

Sites of the muscle relaxant action of CS-722 were investigated in rats. Doses injected intra-4th ventricularly (i.c.v.) or intrathecally (i.t.) were determined according to measurements of CS-722 concentration in the brain stem and the spinal cord after systemic administration. When administered i.c.v. (50 and 100 micrograms) or i.t. (200 and 400 micrograms), CS-722 reduced the radio frequency decerebrate rigidity, although the effect after i.c.v. injection was transient. These results indicate that CS-722 exerts its muscle relaxant action by affecting both the supraspinal structure and the spinal cord. Spinal reflexes were not affected by CS-722 injected i.c.v. It seems that muscle relaxation is not always accompanied by impairment of the spinal reflex.

We investigated the effects of disulfiram (DS) on ovarian carbonyl reductase activity in rats to determine the influence of DS on female reproductive function. Three consecutive treatments with DS significantly inhibited ovarian carbonyl reductase activity as well as ovulation, dose-dependently. Single treatment with DS had no effect on ovarian carbonyl reductase activity. Our observations indicate that consecutive treatment with DS has an inhibitory action on female reproductive function, although DS is well-known to inhibit aldehyde dehydrogenase and dopamine beta-hydroxylase.

Changes in monoamine levels during and after ischemia and effects of RS-8359, a type A monoamine oxidase (MAO-A) inhibitor, were studied in the cerebral cortex, hippocampus, and striatum of rats killed by microwave irradiation. The patterns of the changes in norepinephrine (NE), dopamine (DA), and serotonin (5HT) levels were similar during ischemia: All these monoamines decreased in all three regions. After reperfusion, however, the three monoamines showed different patterns of changes: NE, except in the striatum, decreased further; DA increased over the controls; 5HT remained suppressed in all three regions. With regard to the metabolites of the monoamines, the changes during and after reperfusion were almost similar in all regions: O-methylated metabolites, normetanephrine and 3-methoxytyramine, markedly increased during ischemia; After reperfusion, the elevated levels of normetanephrine and 3-methoxytyramine returned to normal, while deaminated metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid, homovanillic acid (HVA), and 3-methoxy-4-hydroxy-phenylethyleneglycol clearly increased. RS-8359 pretreatment (30 mg/kg, p.o.) at an hour prior to ischemia elevated the levels of NE in the cortex and hippocampus during ischemia and inhibited the increases in DOPAC and HVA levels and the decrease in 3MT levels at 30 min after reperfusion. These results suggest that deamination of NE, DA, and 5HT is activated by the increases in the substrates for MAO in all three regions, except the noradrenergic system in the striatum, and that MAO-A participates in the activated deamination after reperfusion.

The average values of the local cerebral glucose utilization (LCGU) were approx. 35-110 mumols/100 g/min in various nuclei in the brain of sham-operated animals. The values, however, were decreased by 25-75% in all areas examined at 24 hr after 4-vessel occlusion (forebrain ischemia) for 0.5 hr. Particularly, the LCGU values in the forebrain nuclei such as the cerebral cortices and thalamus were severely reduced, while that in the nuclei in the midbrain and hindbrain such as the red nucleus, chochlear nucleus and vestibular nucleus were slightly reduced. In rats treated with S-adenosyl-L-methionine (SAM, 100 mg/kg, i.p.) every 1 hr for 6 times from the recirculation, however, the LCGU values were increased in all brain structures by 35-195% in the forebrain ischemic rat. A significant increase was observed in the cerebral neocortices, caudate-putamen, lateral septal nucleus, thalamic nuclei, substantia nigra, cerebellum and some other nuclei. In some forebrain structures whose LCGU were mildly reduced to approx. 60-70% by the ischemia, SAM recovered the LCGU to more than 90% of the value in the sham-operated group. Thus it was concluded that SAM ameliorated widely ischemia-induced reduction of LCGU in the rat.