The anti-allergic and anti-inflammatory activities of DS-4574, which possesses leukotriene antagonism and inhibits the release of immunologically stimulated mediators such as histamine and leukotrienes, were evaluated in several animal models. DS-4574 had dose-dependent inhibitory effects on IgE-mediated passive cutaneous anaphylaxis and the passive Arthus reaction in rats and the phase I response of Forssman antibody-induced bronchoconstriction. In contrast, this compound had no effect on the phase II response of Forssman antibody-induced bronchoconstriction in guinea pigs, the reverse cutaneous anaphylaxis in rats, complement-dependent hemolysis of sheep erythrocytes and the delayed-type hypersensitivity induced by methylated bovine serum albumin in mice. The results obtained in a double sensitization with two IgE antibodies suggested that DS-4574, as well as disodium cromoglycate, did not impair antigen-antibody combination but prevents the release of chemical mediators such as histamine. DS-4574 also had a weak inhibitory activity on carrageenin paw edema in rats, arachidonic acid ear edema in mice and adjuvant arthritis in rats. In addition, this compound inhibited increased vascular permeability in rat skin induced by leukotriene D4 and platelet activating factor-induced pleurisy in rats in a dose-dependent manner. These results indicate that DS-4574 inhibited type III allergic reactions in addition to type I allergic reactions and some inflammatory reactions. Therefore, DS-4574 could be useful in the treatment of allergic diseases such as asthma.
Effects of aniracetam on whole-cell calcium currents were studied in acutely isolated neuronal cells from postnatal rat ventromedial hypothalamus. There were three types of inward calcium currents, one low-threshold transient current and two high-threshold sustained currents. The nicardipine sensitive L-type current was activated at 20 mV or more depolarized potentials, and the ω-conotoxin sensitive N-type current was recorded at more positive potentials than the L-type. Aniracetam inhibited the N-type current in a dose-dependent manner without affecting the other two types of calcium currents. The effect appeared soon after the addition and lasted for several minutes during washing. Since the N-type current is thought to regulate the release of transmitters, the inhibitory effect may contribute to the nootropic property of aniracetam by modifying the neurotransmission.
This investigation was undertaken to characterize the vasoconstrictor responsiveness in aortas isolated from a rat model of arteriosclerosis induced by vitamin D2 (VD)administration followed by feeding with a high-cholesterol diet. Cumulative contractile responses to KC1, noradrenaline and serotonin in thoracic aortic strips isolated from arteriosclerotic rats were slightly augmented in concentrations lower than the EC50 value of each agent and rather attenuated in their higher concentrations as compared with those from normal rats. Maximum contractions to the agonists were markedly attenuated in arteriosclerotic aortas; the degree of attenuation was greater in rats treated with a combination of VD and cholesterol than in those treated with VD alone. There was a significant negative correlation between the maximum contraction to KCI, noradrenaline or serotonin and the content of calcium or cholesterol ester in aortas. Removal of endothelium markedly enhanced sensitivity and contractility to the agonists in aortic strips from normal rats, whereas the same procedure only slightly enhanced them in aortic strips from arteriosclerotic rats. These results indicate that in arteriosclerotic rat aortas, contractile responsiveness to agonists of vascular smooth muscle cells is impaired with deposition of calcium and cholesterol, and they suggest that augmentation of contractile responses to the agonists in lower concentrations is due to impairment of endothelial function.
Autoimmune MRL-lpr/lpr (MRL/l)mice, with a systemic lupus erythematosus-like disease, were shown to spontaneously develop hyperlipidemia and yet be susceptible to diet-induced hypercholesterolemia and aortic cholesterol deposition. Control animals on a basal diet showed significant increases in the serum total cholesterol, phospholipids, triglycerides, high density lipoprotein (HDL)-cholesterol and lipid peroxide levels, but a significant decline in the serum lecithin: cholesterol acyltransferase (LCAT)activity compared to those of 5-week-old mice. Animals on the high-cholesterol diet showed a rapid rise in serum total cholesterol to a plateau level (800 mg/ 100 ml)that was approximately 2.5 times higher than that in the control animals on a basal diet. However, the levels of serum triglycerides, HDL-cholesterol and lipid peroxides significantly decreased (by 61%, 23% and 53%, respectively)compared to those of the control animals, whereas LCAT activity and phospholipid level were not affected. The aortic contents of total cholesterol, free cholesterol and cholesteryl ester were significantly higher (by 35%, 36% and 31%, respectively)in animals fed the high-cholesterol diet than the control animals. These findings suggest that MRL/l mice are susceptible to diet-induced hypercholesterolemia and aortic cholesterol deposition.
Effect of gastric acid suppression on the cytoprotective effect of a single dose of NC-1300 given intragastrically was studied. NC-1300 given intragastrically prevented gastric mucosal damage caused by absolute ethanol in rats in a dose-related manner, while the drug given subcutaneously did not. Pretreatment with NC-1300 given subcutaneously to suppress acid secretion abolished the protective effect of NC1300 given intragastrically, but not in the presence of 0.1 N HC1. Repeated intragastric administration of NC-1300 for 7 days failed to prevent the ethanol damage. These results suggest that NC-1300 requires gastric acid to exert a protective effect against ethanol in rat gastric mucosa.
The present study was performed to clarify the mechanism of vasodilation of KRN2391 in canine mesenteric artery compared with those of nicorandil and cromakalim. We used the responses of isolated cranial mesenteric artery in vitro and changes in mesenteric blood flow in vivo as indicators reflecting the responses of a conductive artery and resistive arterioles, respectively. In isolated cranial mesenteric artery, KRN2391 (10-8-10-5 M), nicorandil (10-7-10-4 M)and cromakalim (10-7-10-5 M)relaxed contractions caused by 25 mM KCl in a concentration-dependent manner. The concentration-relaxation curve for KRN2391 was shifted to the right by either methylene blue (10-5M)or glibenclamide (10-6M), but the inhibitory effect of methylene blue was more potent than that of glibenclamide. The concentration-relaxation curve for nicorandil was shifted to the right by methylene blue, but not by glibenclamide. In addition, the curve for cromakalim was shifted to the right by glibenclamide, but not by methylene blue. In in vivo experiments, the injections of KRN2391 (0.3-3 μg/kg), nicorandil (10-100 μg/kg)or cromakalim (1-10 μg/kg)into the mesenteric artery increased mesenteric blood flow in a dose-dependent manner. Glibenclamide (5 mg/kg, i.v.)attenuated the increase in mesenteric blood flow caused by KRN2391, nicorandil and cromakalim, but had no effect on that caused by nifedipine (1 μg/kg). The ED20 value increased about 4.7-fold for KRN2391, 3.7-fold for nicorandil and 11.5-fold for cromakalim after administration of glibenclamide, as estimated from the % change to the absolute increase in mesenteric blood flow induced by nifedipine (1 μg/kg). These results suggest that KRN2391 shows nitrate and K channel opening actions in canine mesenteric artery, but the ratio of both actions appears to depend on the segment of mesenteric artery.
The anxiolytic effects of buspirone and its major metabolite, 1-(2-pyrimidinyl)-piperazine (1-PP)have been investigated with a conflict (shock-induced suppression of drinking)paradigm in rats. Buspirone (10 mg/kg, p.o.)showed an anticonflict activity with a bell-shaped dose-response relationship without any effect on spontaneous water consumption. Higher doses of buspirone reduced the punished response. Diazepam (20 and 40 mg/kg, p.o.)also showed an anticonflict activity in a dose-dependent manner, but animals with diazepam showed an increase in spontaneous water consumption at these doses. On the other hand, 1-PP (6.25-200 mg/kg, p.o.)showed a weak anticonflict activity with a significant effect at 25 mg/kg without any effect on spontaneous water consumption. In the 7-day treatment test, buspirone (5 and 10 mg/kg, p.o.), 1-PP (5 and 25 mg/kg, p.o.)and diazepam (10 and 40 mg/kg, p.o.)did not develop the tolerance to the anticonflict activity. Conversely, the anticonflict activity of diazepam was increased by the repeated treatment. Diazepam (10 mg/kg, p.o.)showed an anticonflict activity without any effect on spontaneous water consumption in this test. These results demonstrated that buspirone clearly exhibited an anticonflict effect similar to that of diazepam in a Vogel-type conflict test, and its real anxiolytic effect may not be always based on 1-PP, the main metabolite of buspirone.
Muscarinic cholinoceptor subtypes in the rat prostatic membrane were characterized by using [3H]-methyl-quinuclidinyl benzilate (QNB)in ligand binding studies. [3H]-Methyl-QNB saturation experiments showed the existence of a homogeneous population of binding sites with a high affinity (KD value)of 0.24 ± 0.04 nM and a maximum binding site number (Bmax)of 219 ± 65 fmol/mg protein. Inhibition of [3H]-methyl-QNB binding by nonlabelled compounds was in the following order of effectiveness in rat prostate: atropine > 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP)> hexahydro-sila-difenidol hydrochloride, p-fluoroanalog (p-F-HHSiD)> pirenzepine > methoctramine > [1 l-((2-((dimethylamino)methyl)-1-piperidinyl)acetyl)-5, 11-dihydro-6H-pyrido(2, 3-b)(1, 4)benzodiazepine-6-one] (AF-DX 116). This ranking order was similar to that for the salivary gland (M3 subtype), but not for the brain (M1 subtype)or the heart (M2 subtype). These results indicate that the muscarinic cholinoceptors in the rat prostate belong mainly to the M3 subtype. Furthermore, Bmax values for muscarinic cholinoceptors in the aged rat prostate (approximately 1-year-old)were smaller than those in the young rat prostate (6 to 8-week-old)(87 ± 13 vs. 183 ± 32 fmol/mg protein). However, KD values for muscarinic cholinoceptors, and Bmax and KD values for β-adrenoceptors showed no change. These results suggest that the number of prostatic muscarinic cholinoceptors decreases with aging.
Effects of rat α-calcitonin gene-related peptide (α-CGRP)microinjected into various hypothalamic nuclei on plasma levels of catecholamines and arterial blood pressure were investigated in urethane-anesthetized rats. α-CGRP (0.05 and 0.25 nmol)microinjected into the hypothalamic paraventricular nucleus (PVN)increased the plasma level of noradrenaline (NA), but not that of adrenaline (AD), in a dose-dependent manner. A similar increase in plasma level of NA was also observed by α-CGRP (0.05 nmol)microinjected into the preoptic area (POA), anterior hypothalamus (AH), dorsomedial hypothalamus (DMH)and ventromedial hypothalamus (VMH). A significant increase in arterial blood pressure was observed by microinjection of a-CGRP (0.05 nmol)into the PVN, POA, AH and DMH, and the most prominant increase was caused by its microinjection into the PVN. Microinjection of the same dose of this peptide into the VMH, lateral hypothalamic area and posterior hypothalamus was without effect. The increase in plasma level of NA induced by α-CGRP (0.05 nmol)into the PVN was not affected by bilateral adrenalectomy. Electrical stimulation of the PVN elicited increases in plasma levels of both NA and AD. This increase in NA was abolished by chemical sympathectomy with 6-hydroxydopamine (50 mg/kg, i.v., 3 days before experiments). These results suggest that activation of the PVN by electrical stimulation elicits both sympathetic and adrenomedullary outflow. α-CGRP microinjected into the PVN selectively activates the sympathetic outflow.
We attempted to observe the dentate gyrus field potential evoked by low-frequency stimulation of the perforant path in Mg2+-free medium and identify the N-methyl-D-aspartate (NMDA)receptormediated synaptic potential using rat hippocampal slices. When perfusing solution was changed from normal medium (1.3 mM Mg2+)to Mg2+-free medium, the evoked potential was greatly increased and secondary population spikes appeared following a primary population spike. The evoked potential recorded in Mg2+-free medium was only partly blocked by 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), a nonNMDA receptor antagonist. The CNQX-resistant component of the evoked potential in Mg2+-free medium was completely abolished by 30 μM DL-2-amino-5-phosphonovalerate, a NMDA receptor antagonist, indicating that the NMDA receptor-mediated synaptic response can be isolated by masking the non-NMDA receptor-mediated component of the evoked potential under the Mg2+-free condition. The isolated NMDA receptor-mediated synaptic potential was also blocked by 7-chlorokynurenate, an antagonist at the glycine site of the NMDA receptor, and restored by the concomitant presence of glycine. Observation of pharmacologically isolated NMDA receptor-mediated synaptic potential is a simple and good method for studying the direct effects of drugs on NMDA receptor-mediated responses.
SUT-8701 is a cholecystokinin octapeptide (CCK8)analog and a more lipophilic peptide than CCK8. We previously demonstrated that intra-ventricularly administered CCK8 protected against the degeneration of the cholinergic neurons in the cortex of the nucleus basalis magnocellularis (nbm)-lesioned rat. We determined whether SUT-8701 and CCK8 have the ability to protect against cholinergic degeneration in the cerebral cortex of nbm-lesioned rats. Systemically administered SUT-8701 (0.1-1 μg/day/animal, s.c.)preserved choline acetyltransferase activity and K+-evoked acetylcholine release in nbm-lesioned rats. SUT-8701 was more potent than CCK8. However, SUT-8701 was much less potent than CCK8 in satiety action. The affinity of SUT-8701 to the cholecystokinin (CCK)receptors assessed by using [125I]-CCK8 was almost the same as that of CCK8 in the mouse cerebral cortex, but was 107 times less than that of CCK8 in guinea pig pancreas. These results suggest that SUT-8701 may be effective in slowing down the degenerative processes in Alzheimer''s disease by preserving the integrity of cholinergic neurons in the nucleus basalis.
Effects of hirsuteine, an indole alkaloid extracted from Uncaria genus, on nicotine and high K-induced responses were investigated in rat pheochromocytoma PC12 cells. Hirsuteine (300 nM-10 μM)inhibited dopamine release evoked by 100 μM nicotine in a concentration-dependent manner. Hirsuteine did not produce a parallel shift of the concentration-response relationship curve for nicotine, but reduced maximal dopamine release. Dopamine release evoked by 60 and 155 mM KCl was also inhibited by hirsuteine, but the concentration necessary for significant inhibition was higher (more than 10 μM). Under whole cell voltage-clamp, hirsuteine reversibly inhibited inward currents activated by 100 μM nicotine. The current inhibition was slightly accelerated by hyperpolarization. The results suggest that hirsuteine non-competitively antagonizes nicotine-evoked dopamine release by blocking ion permeation through nicotinic receptor channel complexes. The blockade of Ca channels, which are activated during nicotine-evoked depolarization, may not play a major role in the antagonism.
A transient decrease in cytosolic pH ([pH]i)in rat parotid cells was evoked by the addition of carbachol (CCh), phenylephrine, or substance P, whereas isoproterenol and dibutyryl cyclic AMP had little or no effect on [pH]i. The decrease in [pH]i induced by the Ca2+-mobilizing agonists was also observed in Ca2+-free medium, but not when the intracellular Ca2+ stores were previously depleted. lonomycin and thapsigargin elicited a decrease in [pH]i with an increase in cytosolic Ca2+ concentration ([Ca2+]i). The protein kinase C activator and inhibitor had no effect on the agonist-induced decrease in [pH]i. These results suggest that the cytosolic acidification is associated with an increase in [Ca2+]i.
The effects of the novel calcium channel antagonist KB-2796, other calcium channel antagonists, an N-methyl-D-aspartate (NMDA)antagonist, non-NMDA antagonists, Mg2+, a Ca2+ -chelator and a calcium channel agonist on neurotoxicity induced by a 10-min application of 100μM glutamate were studied in rat hippocampal primary cell cultures. KB-2796 (0.1 and 1 μM), flunarizine (1 μM), nimodipine (10 μM), MK-801 (0.01-1 μM), Mg2+ (10 mM)and EGTA (10 mM)significantly prevented the neurotoxicity, but 6-cyano-7-nitro-quinoxalinedione (CNQX)(10 μM)and 6, 7-dinitro-quinoxalinedione (DNQX)(10 μM)did not. Bay K 8644 (10 and 100 nM)enhanced the neurotoxicity. These findings indicate that KB2796 protects the neuronal cell from the glutamate-induced neurotoxicity, presumably by blocking the Ca2+ influx into brain neurons.
Effect of an extract of Ginkgo biloba leaves (EGb) on oxidative metabolism was studied using rat brain neurons and 2'', 7''-dichlorofluorescin fluorescence. lonomycin (100 nM to 1 μM), a Ca2+ -ionophore, dose-dependently augmented the 2'', 7''-dichlorofluorescin fluorescence in the presence of external Ca2+, but not under the external Ca2+ -free condition. Preincubation of neurons with EGb (3 μg/ml) greatly reduced the ionomycin-induced increase in 2'', 7''-dichlorofluorescin fluorescence. Results suggest that EGb may reduce the Ca2+ -induced increase in the oxidative metabolism of brain neurons.
We investigated the effects of class III antiarrhythmic agents, d-sotalol, E-4031 and MS-551, on the currents of two cloned K channels, Kvl.2 (IKv1.2)and Kvl.4 (IKv1.4), by using the Xenopus oocyte expression system. Both IKv1.2 and IKv1.4 were sensitive to 4-aminopyridine and quinidine, but insensitive to tetraethylammonium, d-sotalol, E-4031 and MS-551. The results suggest that some types of structural proteins may be necessary for class III agents to inhibit the cardiac cloned K channels.
Effects of a nitric oxide (NO)-producing agent, sodium nitroprusside, on N-methyl-D-aspartate (NMDA)receptor activation in the cultured retinal neurons of rats were examined. NMDA in a Mg2+ free medium evoked inward currents at the resting membrane potential. Inward currents were also evoked by kainate. Sodium nitroprusside markedly reduced the NMDA-induced currents without affecting those induced by kainate. These results suggest the possible existence of a negative feed back system of NO which serves to regulate the activation of NMDA receptors in retinal neurons.