The combined effects of paeoniflorin (PF), a main component of paeony roots, and glycyrrhizin (GLR), a main component of licorice roots, were investigated on isolated sciatic nerve-sartorius muscle preparations in frogs, or on isolated or ^{in situ} phrenic nerve-diaphragm muscle preparations in mice, intending to explain the effects of "Shakuyaku-Kanzô-Tô, composed of both these Chinese drugs, on clinical neuropathy. PF and GLR used together blocked indirectly stimulated twitchings at concentrations which when used alone induced no blocking effects. PF and GLR at a combining ratio of 1:2 (weight concentrations) corresponding to the amounts contained in "Shakuyaku-Kanzô-Tô, was more potent than when they were used at the ratio of 1:1 or 2:1. The synergistic effects induced by GLR were also confirmed for the other components, paeoniflorigenone or oxypaeoniflorin, which are contained in paeony roots, and for succinylcholine. The blocking effect of d-tubocurarine were not increased by GLR. Concludedly, PF and GLR were found to cause the pharmacological blend effect. The two combined compounds were mainly therapeutic components in "Shakuyaku-Kanzô-Tô".

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The effect of a new anti-platelet agent, KF4939, on thromboxane A₂ (TXA₂)-induced vasocontraction was studied in superfused rabbit mesenteric and dog coronary arteries, in comparison with the effects on the contractions evoked by KCI, noradrenaline, serotonin, angiotensin II and histamine. The calcium sources involved in the TXA₂-induced vasocontraction were also examined. The TXA₂-induced contraction of the rabbit mesenteric artery was partly attenuated after exposure to the calcium-free medium, but was not attenuated by nifedipine. The TXA₂-induced contraction of the dog coronary artery was markedly attenuated by nifedipine. These results indicate that TXA₂ utilizes both intracellularly stored calcium and an extracellular source of calcium for its vasocontraction, and the voltage-dependent calcium channel plays an important role in the dog coronary artery, but in the rabbit mesenteric artery. KF4939 inhibited the TXA₂-induced contraction in both arteries. In the rabbit mesenteric artery, three times and more higher concentration than that to inhibit TXA₂-induced one were required to inhibit other agonist induced contractions, KF4939 caused no alteration in the KCI-induced contraction of both arteries. Thus, KF4939 seems to be a selective inhibitor of TXA₂-induced vasocontraction, and the receptor-linked mechanism may be a possible site of the TXA₂ antagonistic action of KF4939.

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The effects of a novel compound, 6-(10-hydroxydecyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (idebenone, CV-2619), on cerebral and renal vascular changes were examined in stroke-prone spontaneously hypertensive rats (SHRSP) and in rats with experimentally induced hypertension. CV-2619 (35 mg/kg/day, p.o.) significantly inhibited the onset of cerebrovascular lesions (stroke) and the elevation of blood pressure in SHRSP with mild hypertension . A higher dose (2×50 mg/kg/day, p.o.) clearly delayed the onset of both stroke and proteinuria without any effect on the blood pressure in SHRSP with severe hypertension. In DOCA-salt hypertensive rats, CV-2619 (2×5 or 2×25 mg/kg/day, p.o.) dose-dependently inhibited decreases in body weight and water balance and the development of cerebral and renal vascular changes. These results suggest that CV-2619 inhibits the development of stroke and renal vascular lesions in hypertensive rats.

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In order to evaluate both the hypotensive and uric acid-retaining effects of thiazide diuretics in an animal model with hypertension, the effects of trichlormethiazide were studied using spontaneously hypertensive rats (SHR) under dietary sodium restriction. Trichlormethiazide was dosed daily for two weeks at 0.05, 0.5, 3 and 10 mg/kg, p.o. All doses caused obvious natriuresis, while an increase of urine volume was observed only at 3 and 10 mg/kg. The hypotensive effect, which was estimated at day 6 and 13, was recognized at doses of more than 0.5 mg/kg. At the end of the dosing, the hematocrit value of all medicated groups rose, and both the uric acid excretory capacity, estimated by the clearance values of inulin and uric acid, and the plasma potassium level clearly decreased at 3 and 10 mg/kg. A detailed study using a dose of 3 mg/kg showed shifts of the cumulative sodium and potassium balances to negative directions against the control group. Thus, trichlormethiazide-treated SHR under dietary sodium restriction showed both a hypotensive effect which might be due to the natriuresis and a tendency toward undesirable side effects such as hypokalemia and hyperuricemia. As there is no practical method in animal studies for simultaneously proving hypotensive and uric acid-retaining effects of diuretic antihypertensives, the findings of the present study might aid in the evaluation of diuretics more useful than the thiazides.

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NCO-650 and its congeners and two other antiallergics, disodium cromoglycate and tranilast, were studied to determine the degree of protection of rat erythrocytes against hypotonic hemolysis, the reduction of the surface tension of dipalmitoylphosphatidylcholine (DPPC) monolayer and the depression of the phase-transition temperature of DPPC liposome bilayers. NCO-650 was found to show the greatest hemolysis protection, reduction of the surface tension and depression of the phase-transition temperature, indicating that it possesses a significant affinity to cell membranes and a significant ability to stabilize cell membranes. Disodium cromoglycate and tranilast showed neither cell membrane affinity nor hemolysis protection, although they inhibit histamine release from mast cells like NCO-650 and its congeners. The significant membrane stabilizing action of NCO-650 must be related, at least in part, to its extraordinarily high lipid solubility.

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A new anti-anxiety drug, CM6912 (ethyl loflazepate, ethyl 7-chloro-2, 3-dihydro-5-(2-fluorophenyl)-2-oxo-1H-1, 4-benzodiazepine-3-carboxylate), was investigated for its effects on the a and r-motor systems and on the cooperative muscular motions and for its interactions with other CNS drugs. The results obtained are as follows: Muscular discharges (EMG) induced by decerebrate rigidity were unaffected by 10 mg/kg (p.o.) of CM691 2, but the amplitudes of the EMG were reduced by 50% for 3 hr by 30 mg/kg of CM691 2 at 30 min after administration. Diazepam (10 mg/kg) also decreased the amplitudes of the EMG even at 5 min after administration, indicating that diazepam had a stronger than CM6912. Both monosynaptic spinal reflex (MSR) and polysynaptic spinal reflex (PSR) were unaffected by CM6912 (100 mg/kg). Dorsal root reflex potential was slightly enhanced by CM6912 (100 mg/kg), but not at a dose of 30 mg/kg. Diazepam (10 mg/kg) did not decrease MSR, but slightly reduced PSR. Dorsal root reflex potential was almost doubled by diazepam. The frequency of spontaneous discharges of Gla spindle afferent fiber of the extensor muscle of the hindlimb of anesthetized cats was unchanged by 10 mg/kg CM6912, but was suppressed by diazepam at the same dose while at a dose of 30 mg/kg, it was reduced mildly by CM6912, and markedly by diazepam. ED50 values for the antagonistic action on bemegride-induced convulsions were 0.30 mg/kg for CM6912 and 0.49 mg/kg for diazepam at 1 hr after administration, and they were 0.30 mg/kg and 0.67 mg/kg for CM6912 and diazepam, respectively, at 4 hr. The potentiating action of CM6912 on chlorprothixene-induced anesthesia was far weaker than that of diazepam. The suppressive potency of CM6912 on the adaptability to the rotarod was about half that of diazepam, and the muscle relaxant action of CM6912, examined by the inclined board test and the hanging test, was found to be similar to that of diazepam. These results suggest that CM6912 is less potent than diazepam in reducing muscular tone and in inducing sleep, while it has a stronger and longer-lasting anti-anxietic activity than diazepam.

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Pharmacological properties of the facilitatory presynaptic β-adrenoceptor mechanism were studied in superfused spiral preparations of guinea-pig pulmonary arteries preloaded with ³H norepinephrine. (-) Isoproterenol (0.3 μM)-induced increases in total ³H efflux per pulse evoked by transmural field stimulation (1, 5, 10 and 20 Hz, 10 V, 2 msec pulse width, 100 pulses and 30 min intervals) were neither dependent on impulse-frequencies nor selective at lower frequencies. Isoproterenol increased ³H efflux at 5 Hz in a concentration-dependent manner (1 nM to 1 μM): pD² was 7.7. Salbutamol increased ³H efflux in a similar manner to isoproterenol: pD² was 7.4. Prenalterol at 3 μM only slightly increased ³H efflux. Tazolol (10 nM to 3 μM) produced no increases. Atenolol (3 μM) and practolol (3 μM) did not antagonize isoproterenol (0.3 μM)-induced increases in ³H efflux. Butoxamine (3 μM) and H 35/25 (3 μM) did antagonize this parameter. (-)-Epinephrine (1 nM to 0.1 μM) decreased ³H efflux at 5 Hz and concentration-dependently increased this parameter in the presence of 10 μM phentolamine. (-)-Norepinephrine (10 nM to 1 μM) concentration-dependently inhibited evoked ³H efflux and did not increase the parameter in the presence of 10 μM phentolamine, 10 μM cocaine and 10 μM normetanephrine. Thus, there exist presynaptic β²-subtype receptors on noradrenergic nerve endings innervating guinea-pig pulmonary arteries.

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The alterations of local glucose utilization in 101 cerebral nuclei following the subcutaneous administration of the specific opioid antagonist naloxone were measured using the quantitative autoradiographic ¹⁴C-deoxy-D-glucose technique in conscious rats. Met⁵-enkephalin-like immunoreactivity (MELI) and ME receptor binding (MERB) levels in 82 cerebral nuelci were assayed quantitatively by microdensitometry of fluorescence micrographs and autoradiographs of the brain slices. Naloxone administration significantly reduced glucose utilization rate in 18 lower brainstem nuclei including the n. tractus solitarii, n. dorsalis nervi vagi, substantia grisea centralis, n. parabrachialis dorsalis and ventralis, and n. interpeduncularis. These nuclei contained ME perikarya with high levels of MELI and MERB. However, naloxone did not alter glucose utilization of other lower brainstem nuclei containing ME neurons and all thalamic, epithalamic, hypothalamic and limbic nuclei with ME perikarya. The distribution and magnitude of the neuronal response of cerebral nuclei to naloxone were apparently not related to the distributions of ME neurons. The present study offers direct evidence for the selective action of naloxone per se in some lower brainstem nuclei with ME perikarya.

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An experimental model of amnesia induced by cerebral ischemia after one-trial passive avoidance learning was established to test the effects of a novel compound, 6-(10-hydroxydecyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (idebenone, CV-261 9), and some commonly used drugs in rats. One day after the vertebral artery was electrocauterized bilaterally, the common carotid artery was transiently occluded bilaterally to produce cerebral ischemia. The amnesia was estimated by the response latency for a rat to step from a light safety compartment to a dark compartment in which a foot-shock was given. The results of the retention test given 24 hr after the ischemia indicated that amnesia was successfully produced when the 200-600 sec ischemia was provided within 20 min after the avoidance learning. The effects of drugs on the amnesia induced by a 200-sec ischemia immediately after the avoidance learning were as follows: CV-2619 (10, 30 mg/kg, i.p. or p.o.) given before the retention test significantly increased the response latency, indicating a reversal effect on the amnesia. Physostigmine (0.1, 0.2 mg/kg, i.p.) and arginine-vasopressin (10 μg/kg, s.c.) were also effective, and calcium hopantenate (500 mg/kg, p.o.) showed a slight reversal action. Furthermore, CV-2619 (10 mg/kg, i.p.), given before or after the ischemia, significantly inhibited the appearance of amnesia. These findings suggest that CV-2619 exerts an ameliorating effect on memory disturbance induced by cerebral ischemia in rats.

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The concentrations of acetylcholine (Ach) and choline in various brain regions of rats with and without cerebral ischemia and the effects of 6-(10-hydroxy-decyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (idebenone, CV-2619) on the levels of these parameters were investigated. Cerebral ischemia was induced by a 200-sec occlusion of both common carotid arteries in animals in which both vertebral arteries had been permanently cauterized. The concentrations of Ach and choline in the brain were determined by means of pyrolysis gas chromatography. In normal rats, CV-2619 (10, 30 and 100 mg/kg, i.p.) did not alter the concentrations of Ach and choline in the brain regions examined. In the ischemic rats, a significant decrease in Ach and a marked increase in choline were observed in the cerebral cortex, hippocampus, striatum, and diencephalon. A slight increase in the concentration of choline was also observed in the cerebellum and brain stem. Pretreatment with CV-2619 (10 mg/kg, i.p.) inhibited the decrease in Ach and the increase in choline in the forebrain regions. Moreover, the same dose of CV-2619 inhibited the increment of lactate content and tended to inhibit the decrement of ATP content in the cerebral cortex. These results indicate that CV-2619 inhibits alterations of the concentrations of Ach and choline in the brain of the ischemic rats; this inhibition may be due to the ameliorating effect of CV-2619 on the disturbance of cerebral energy metabolism under ischemic conditions.

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The effect of the endogenous prostaglandin E (PGE) level in the vascular wall on the vasoconstrictor response to noradrenaline (NA) was examined using the perfused central artery of an isolated rabbit ear. The endogenous PGE level in the ear artery was estimated from that in the venous perfusate, which was measured by radioimmunoassay. Pretreatment with indomethacin, a cyclooxygenase inhibitor, was followed by a significant decrease in the PGE level and a significant increase in the vasoconstrictor response to exogenous NA. In addition, there was a significant correlation between the response to NA and the PGE level in the venous perfusate in combination with the results during perfusion of indomethacin and of PGE₁ plus indomethacin, although the former did not correlate with the latter under the basal conditions alone. From these results, it seems possible to draw the conclusion that as far as vasoconstrictor responsiveness is concerned, there is some antagonism between NA and endogenous and/or exogenous PGE in the rabbit ear artery.

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Effects of verapamil on electrophysiological alterations induced by lysophosphatidylcholine (LPC), a toxic membrane-derived phospholipid potentially responsible for ischemia-induced arrhythmias, were evaluated in canine Purkinje fibers. LPC (10-160 μM) induced concentration-dependent decreases in resting membrane potential, action potential amplitude and maximum upstroke velocity of phase 0. The depolarization was not associated with the reduction of the potassium equilibrium potential, which was calculated from the intracellular potassium ion activities measured by ion-selective electrodes. A graded increase in LPC concentration invariably induced abnormal automaticity arising from depolarized membrane potentials. Although verapamil did not prevent LPC-induced changes in action potential characteristics, it suppressed the appearance of rapid spontaneous activity at reduced membrane potential. These results suggest that verapamil may modify the dysrhythmias induced by LPC in ischemic myocardium.

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OPC-8212 is a new positive inotropic drug which scarcely produces an increase in heart rate and vasodilatation. The positive inotropic effect of OPC-8212 was investigated in relation to cyclic AMP metabolism in canine isolated ventricular muscle. The positive inotropic effect of OPC-8212 was accompanied by accumulation of cyclic AMP in the tissue: the cyclic AMP level was elevated before the increase in force of contraction and elevated in a concentration-dependent manner in the presence of a β-adrenoceptor antagonist, atenolol (10^-6 M). The OPC-8212-induced increases in force of contraction and cyclic AMP level were abolished by carbachol (3×10^-6 M). The relationship between the force of contraction and cyclic AMP level in the presence of OPC-8212 was changed by neither carbachol nor isoproterenol. OPC-8212 enhanced the positive inotropic effect of isoproterenol as cyclic AMP phosphodiesterase inhibitors did. OPC-8212 shortened the time to peak tension and had a tendency to shorten the relaxation time of single contractions. These results altogether suggest that change in cyclic AMP metabolism is involved at least in the positive inotropic effect of OPC-8212 on canine ventricular muscle.

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To elucidate the mechanisms of fever response by bacterial pyrogen (lipopolysaccharide, LPS), we investigated both pyrogenicity and Limulus amoebocyte lysate (LAL) gelation activity of cerebrospinal fluid (CSF) withdrawn from febrile rabbits induced by i.v. injection of LPS. One ml of CSF was withdrawn from donor animals 2 hr after i.v. injection of E. coli LPS at graded doses of 0, 1, 10 and 50 μg/kg, and its pyrogenicity was checked by administration into cisterna magna of recipient animals. Pyrogenicity was revealed only in the CSF withdrawn from the group injected with 50 μg/kg of LPS. Differences of both protein content and concentrations of ions (Na, K and Ca) were not obtained among the CSF withdrawn from control and LPS-injected groups. All of the above CSF had no LAL gelation activity, but the activity could be detected in the CSF withdrawn from animals injected with a higher dose of LPS (500 μg/kg, i.v.). LAL gelation activity of LPS dissolved in normal CSF was 2 orders less potent in comparison with that of LPS dissolved in saline, which suggested the presence of inhibitor(s) in CSF for LAL assay. Pyrogenicity was not revealed in the CSF withdrawn from hyperthermic rabbits induced by administration of reserpine after pretreatment with a monoamine oxidase inhibitor. These findings suggest that the central action of LPS is involved in pyrogen fever and that monoamines are not closely related to pyrogen fever.

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Effects of 5'-guanylylimidodiphosphate (Gpp(NH)p) and sodium on the inhibition by various opioids of [³H]-naloxone binding to guinea-pig brain membrane preparations were studied. The ratio of the concentration required to produce a 50% inhibition of [³H]-naloxone binding in the presence of both Gpp(NH)p and sodium to that in the absence of both Gpp(NH)p and sodium (IC50 ratio) was less than 1 for antagonists, from 3 to 10 for mixed agonist-antagonists, from 16 to 85 for either kappa, delta, or peptide mu agonists, and more than 200 for morphine-like non-peptide mu agonists. Exceptionally, the IC50 ratio of N, N-diallyl-[D-Ala², D-Leu⁵]-enkephalin, an opioid which had been shown not to have an agonist activity in guinea-pig ileum but to have a naloxone-reversible agonist activity in mouse vas deferens, was less than 1. The significance of the different IC50 ratio among opioids employed in the present study was discussed.

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In urinary bladder and gall bladder of guinea-pig, the application of hyperosmotic 65.4 mM K solution induced a tonic contraction and increased the rate of oxygen consumption two or three times. The increased O₂ consumption maintained steady level during 120 min. In isosmotic 154 mM K, Na-deficient solution, the tonic contraction gradually decreased and oxygen consumption also showed a transient increase followed by a small one. In the urinary bladder, the addition of pyruvic acid completely prevented the decline of the tonic contraction by iso-154K solution at 120 min, but this addition partially prevented it in the gall bladder. Hyperosmotic application of sucrose partially prevented it in the urinary bladder, but completely recovered it in the gall bladder. When NaCl was applied to iso-154K solution, the decrease of tonic contraction was prevented by half in both the muscles. The application of pyruvic acid and NaCl prevented the reduction of oxygen consumption by iso-154K solution in the urinary bladder, but in the gall bladder, this had little effect. There was a close correlation (r=0.950) between the muscle tension and the rate of oxygen consumption in the urinary bladder at 120 min under various conditions. In the gall bladder, a correlation (r=0.875) was also found between both when the results by hyperosmotic addition of NaCl and sucrose were omitted. In the urinary bladder, tension cost, a ratio of O₂ consumption rate (μmol O₂/g/min) to developed tension (kg force/cm²), was large (0.206) and similar to that of taenia coli; and that of gall bladder was small (0.130) and almost similar to that of vascular smooth muscle. In summary, the inhibition of contraction and rate of oxygen consumption induced by high K, Na-deficient solution in the urinary bladder is probably caused by inhibition of Na-glucose symport and similar to that in taenia coll. On the other hand, that of the gall bladder seems to be mainly caused by cell swelling, similar to that of vascular smooth muscle, but partially caused by inhibition of the glucose symport.

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