The effect of ibudilast (0.1, 0.3 mg/kg), which has cerebral vasodilating and antiplatelet effects, was evaluated in two models of rat inner ear microcirculation thrombosis by using the photochemical reaction between green light (wave length: 540 nm)and intravenous injection of rose bengal. Furthermore, the inner ear blood flow was measured by a laser-Doppler flowmeter. In the hearing disturbance model, under anesthesia, the compound action potential of the cochlear nerve (AP)was measured by an electrocochleogram. The sound stimulus was an 8-kHz sine wave at 80 dB SPL. The AP was calculated 128 times. In the controls, the AP disappeared about 4 min after the intravenous injection of rose bengal (20 mg/kg). The time required to completely suppress the AP in the animals treated with ibudilast (0.1, 0.3 mg/kg)was significantly prolonged as compared with that in the controls. In the equilibrium dysfunction model, ibudilast (0.1, 0.3 mg/kg)reduced the time of abnormal swimming in the swimming test 24 hr after the completion of photo-illumination. Ibudilast (0.3 mg/kg)increased the inner ear blood flow during the 10-min observation period as compared with the controls, while it did not affect the mean blood pressure. In conclusion, ibudilast increased the inner ear blood flow and was effective in two models of rat inner ear microcirculation thrombosis.
The selectivity of antagonistic effects of nipradilol, its four isomers and denitronipradilol, a major metabolite of nipradilol, on α1-adrenoceptor subtypes in rat heart, brain and spleen were examined by radioligand binding assay with [3H]-prazosin. Pharmacological characteristics of these compounds were determined in isolated aortae from rats and guinea pigs. The order of the pKi values for α1High-affinity sites in the heart, spleen and brain was SR 〉> nipradilol ≥ RR ≥ SS ≈ RS » denitronipradilol, but the order of the pKi values for the α1LOW -affinity sites was different in the heart and brain. There were good correlations between the pKi values of these compounds for the α1High -affinity sites and the pA2 values for the contractile inhibition of the phenylephrine-induced response in rat aorta. There was no correlation between the pKi values of these compounds for the α1LOW -affinity sites and the pA2 values. These results indicate that: 1) α1High -Affinity sites are related to vasoconstriction mediated by α1- adrenoceptors; 2)Nipradilol and its isomers possess low affinity to α1 -adrenoceptors; and 3)The nitroxy group in nipradilol is important for its α1-blocking activity.
Characterization of adrenergic receptors in membranes from the rat seminal vesicle was studied by radioligand binding assay. Seminal vesicle membranes contained saturable and high affinity binding sites for the β-adrenergic receptor antagonist 3H-dihydroalprenolol (3H-DHA)and for the α-adrenergic antagonist 3H-prazosin. The observed order of potency for adrenergic agonists in competing for the 3H-DHA binding sites: isoproterenol > epinephrine ≈ salbutamol > norepinephrine indicates that these membrane receptors have the properties of β2-adrenergic receptors. α1-Adrenergic receptors were defined mainly as α1A subtypes by demonstrating their insensitivity to pretreatment with chlorethylclonidine and the different rank orders of antagonist affinities. No significant binding sites for the α2-adrenergic receptor agonist 3H-clonidine were observed. The GTP-induced reduction in the affinity of α1-adrenergic receptors for epinephrine was significantly reversed by the muscarinic cholinergic agonist carbachol. Atropine effectively antagonized this effect of carbachol on the competitive inhibition of 3H-prazosin binding by epinephrine in the presence of GTP, which suggests that muscarinic cholinergic receptors regulate the affinity of α1-adrenergic receptors by modulating the effect of guanine nucleotides. The effect of GTP on decreasing the affinity of β2-adrenergic receptors was not influenced by the addition of carbachol.
β-Adrenoceptors in pig basilar arteries were investigated by measuring the relaxation responses to norepinephrine and by a radioligand binding assay with [3H]-dihydroalprenolol (DHA). Norepinephrine induced concentration-dependent relaxations. The relaxation responses were independent of the presence of endothelial cells, and they were competitively antagonized by (±)-propranolol, atenolol, butoxamine and ICI 118, 551. Specific [3H]-DHA binding to β-adrenoceptors was saturable, reversible and high affinity (Kd=1.4 nM), with a Bmax of 48.7 fmol/mg protein. Computer analysis of inhibition of [3H]DHA binding by atenolol, butoxamine and ICI 118, 551 gave a β1:β2-adrenoceptor ratio of approximately 65:35. The pA2 values of these antagonists were significantly correlated with the Ki values for β1-adrenoceptor determined by the radioligand binding assay. The present findings indicate that the relaxation responses to norepinephrine are predominantly mediated through the stimulation of β1-adrenoceptors on vascular smooth muscle cells in a pig basilar artery.
We have developed a simple and reproducible method employing chemiluminescence to measure oxygen-derived free radicals generated in oxygenated myocardium. Isolated perfused rat hearts were frozen in liquid nitrogen during the control perfusion (non-ischemia), after 30 min of global ischemia (ischemia), or after 20 min of reperfusion following 30-min global ischemia (reperfusion). The frozen hearts were ground to a fine powder and then oxygenated by mixing with phosphate-buffered saline (PBS)(pH 7.4 and Po2 183-194 mmHg)containing lucigenin. The mixed solution was injected into a chemiluminescence detection flow cell. The chemiluminescence intensity increased in relation to the lucigenin and myocardium contents of the test solution. However, it was not affected by the temperature of PBS in the range of 25°C to 50°C. The chemiluminescence intensity of ischemic myocardium was 2.5 times larger than that of nonischemic or reperfused myocardium (P<0.01). Recombinant human superoxide dismutase (r-h-SOD)reduced dose-dependently the chemiluminescence intensity induced by oxygenation of ischemic myocardium, while chemically inactivated r-h-SOD did not.
We have previously reported that triacsin C, an acyl-CoA synthetase inhibitor, enhanced the production of platelet-activating factor (PAF)in calcium ionophore-activated rat polymorphonuclear leukocytes (PMNs). In this report, we further demonstrated that the production of PAF by PMNs in response to opsonized zymosan was significantly enhanced by pretreatment with triacsin C and also by the preteatment with merthiolate, which was reported to be an inhibitor of acyl-CoA/lysolecithin acyltransferase. Pretreatment with triacsin C or merthiolate also enhanced the lyso-PAF content in the stimulated PMNs. Addition of lyso-PAF in the incubation mixture of PMNs in the presence of opsonized zymosan augumented the production of PAF. The enhancement of PAF production by lyso-PAF has been reported by several authors, and the importance of lyso-PAF in the remodeling pathway of PAF synthesis has been generally recognized. Therefore, from the above findings, it is assumed that blockades of the reacylation of lyso-phospholipids, by inhibitors such as triacsin C and merthiolate, might lead to accumulation of lyso-PAF and might result in the enhancement of PAF production when the remodeling pathway is active.
The present study was performed to assess the anxiolytic activity of the novel anxiolytic SC-48274 in comparison with those of buspirone and diazepam. Drugs were administered p.o. The experimental anxiety models included gastric lesions of mice induced by socio-psychological stress in the communication box (CB)and the passive avoidance paradigm in rats. In the CB experiments, non-foot-shocked mice (responder)exposed to the emotional responses of foot-shocked mice (sender), 3 hr per day for 3 days, developed gastric lesions. Single treatments of diazepam (1, 2, 5 mg/kg)and SC-48274 (25, 50 mg/kg)prevented gastric lesion formation, but buspirone at 2.5 -10 mg/kg did not. A 3-day treatment with SC48274 at doses over 5 mg/kg prevented gastric lesions; and a 3-day treatment with buspirone at 2, 5 and 10 mg/kg prevented the lesions with a U-shaped dose-response. Diazepam also prevented gastric lesion formation at the doses of 1 and 2 mg/kg. In the passive avoidance response study, rats which had a single acquisition trial for 2 days were used. The step-down latency for rats to enter from the illuminated compartment to the dark one was recorded. Single treatments of SC-48274 (25 mg/kg), diazepam (5, 10 mg/kg)or buspirone (25 mg/kg)shortened the delayed latency. These results suggest that SC-48274 has anxiolytic activity of the same potency as buspirone and repeated-dose administration is needed to induce anxiolytic acitivity.
Repeated administration of indomethacin delays the healing of acetic acid-induced gastric ulcers in rats, but the underlying mechanism remains unclear. We examined the effect of indomethacin on the contraction of the connective tissue isolated from the base of 1-week-old ulcers. The tissue samples, suspended in an organ bath, were contracted by serotonin, bradykinin and carbachol and also slightly contracted by prostaglandin (PG)F1α. The effect of serotonin was the most potent. However, 6-keto-PGF1α, PGE2 and histamine had little or no contraction inducing effect. The contractile response to serotonin of the tissue in the indomethacin-treated group was significantly less than that in the control group (without indomethacin treatment). After a 2-week treatment with indomethacin, a histological study of the ulcers showed that the length of rupturing of the muscularis mucosa was significantly greater than that observed in the control group. However, indomethacin had no effect on the length of the regenerated mucosa and the thickness of the ulcer base. We conclude that the connective tissue at the ulcer base has the ability to contract and that the prevention of the contraction of the tissue by indomethacin might be involved in the mechanism underlying delayed ulcer healing.
Repeated administration of indomethacin or prednisolone for 2 weeks apparently delayed the healing of initial gastric ulcers induced by acetic acid injection in rats. While indomethacin significantly interfered with the healing of partially healed 2-week-old ulcers, prednisolone did not. Indomethacin had no effect on the healing of “unhealed” 4-week-old ulcers. However, prednisolone significantly enhanced the healing of such “unhealed” ulcers, and it decreased the weight of the connective tissue at the ulcer base and the collagen content. These results suggest that prednisolone enhances the healing of “unhealed” gastric ulcers by degrading the fibroplasia that had amply developed in the connective tissue.
Chronopharmacokinetic profiles of a new immunosuppressive agent, FK 506, were examined in mice. FK 506 (1 mg/kg)was given orally at 10 AM (day trial)or 10 PM (night trial)once a day for 7 days. Blood samples for measurement of FK 506 concentration in whole blood were obtained just before and at 1, 2, 3, 4, 6, 8 and 12 hr after the final dosage. The time to maximum concentration was shorter and the maximum concentration was greater in the night trial than in the day trial. These findings suggest that absorption of FK 506 is faster and its blood concentration is higher in the night trial.
Effects of nerve growth factor (NGF)on the basal forebrain (BF)lesion-induced amnesia in rats were investigated. When NGF infusion was begun immediately after the formation of BF lesions, NGF ameliorated amnesia in a water maze task and showed a tendency to increase choline acetyltransferase (CAT)activity in the fronto-parietal cortex. The amnesia and the decrease of CAT activity were not ameliorated when NGF infusion was begun 4 weeks after BF lesion formation. These observations suggest that NGF may act as a trophic and/or a protective factor on partially damaged cholinergic neurons and that the efficacy of NGF was influenced by the phase of neuronal damage.
The role of protein kinase C (PKC)in the muscarinic excitation of chromaffin cells freshly isolated from rat adrenal medullae was examined by the patch-clamp recording method. Acetylcholine and McN-A-343, a M1-receptor agonist, depolarized the cell and induced action potentials. Phorbol 12, 13dibutyrate (PDBu), an activator of PKC, increased acetylcholine-induced firing concomitant with a persistent depolarization. Under voltage-clamp recording, both McN-A-343 and PDBu decreased the cesium-sensitive K+ current, which was induced by shifting the membrane potential between -140 mV and -40 mV. These results suggested that the stimulation of muscarinic M1-receptors by cholinergic drugs activated phospholipase C to degrade phosphoinositide, consequently producing diacylglycerol, and diacylglycerol activates PKC to induce excitation of adrenal chromaffin cells.
The modification of the stimulant and motor-incoordinate actions of ketamine induced by repeated administration was investigated by means of ambulatory activity and rota-rod performance in mice, respectively. Ketamine (12.5, 25 and 50 mg/kg, s.c.)increased the mouse''s ambulation in a dosedependent manner, and the repeated 5-times administration at 3 to 4-day intervals enhanced the increment effect. However, a disruption of the rota-rod performance by ketamine was not modified by the repeated treatment. These results suggest that a reverse tolerance to the stimulant action of ketamine is produced, and that a tolerance to its motor-incoordinate action may not cause the enhancement.
A cDNA (PST-1)isolated from a rat liver cDNA library was expressed in COS-1 cells and found to encode a form of arylsulfotransferase (termed ST1A1), which sulfated p-nitrophenol, α-naphthol, minoxidil, β-estradiol and dopamine, but not dehydroepiandrosterone and cortisol. ST1A1 showed a mobility identical with a major immunodetectable sulfotransferase contained in the livers of male and female rats in Western blots. In addition, ST1A1 mRNA was detected in the liver and extrahepatic tissues by Northern blots.