The activity of silipide, a silybin-phosphatidylcholine complex (IdB 1016), was tested in different models of liver damage in rodents. After oral administration, silipide exhibited a significant and dose-related protective effect against the hepatotoxicity induced by CCl₄, praseodymium, ethanol and galactosamine. The ED₅₀ values for inhibition of the rise in ASAT and ALAT levels caused by CCl₄ and praseodymium and for antagonism of the increase in liver triglycerides caused by ethanol ranged from 93 to 156 mg/kg (as silybin). At a dose of 400 mg/kg (as silybin), silipide was also active in protecting against paracetamol-induced hepatotoxicity. Silybin and phosphatidylcholine at doses equivalent to those contained in the active doses of silipide failed to show any significant protective activity in these models. The liver protective effect of silipide is probably related to its antioxidant activities and to a stimulating effect on the hepatic synthesis of RNA and proteins.

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Daily variation in the effects of furosemide, a loop diuretic agent, was examined in Wistar rats maintained under conditions of light from 7 a.m. to 7 p.m. and dark from 7 p.m. to 7 a.m. Furosemide (30 mg/kg) was given orally at 12 p.m., 4 a.m., 8 a.m., 12 a.m., 4 p.m. or 8 p.m. Urine was collected for 8 hr after furosemide administration, and urinary excretions of sodium and furosemide were determined. There were significant daily variations in the urine volume and urinary excretions of sodium and furosemide with a peak at 8 a.m. and a trough at 12 p.m. Significant correlations were observed between the urinary amount of furosemide and its diuretic effects (urine volume and urinary sodium excretion). These results suggest that the diuretic effects of furosemide show daily variations which are, at least in part, caused by the daily variation in the urinary excretion of furosemide.

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Prostaglandins (PGs) have modulatory effects on spontaneous and nicotine-induced release of acetylcholine (ACh) from the myenteric plexus of guinea pig ileum. To determine whether cyclic AMP is involved in the mechanisms of these effects, we studied ACh release under conditions that inhibit PG synthesis. Indomethacin (IND), a cyclooxygenase inhibitor, inhibited ACh release concentration-dependently. The effect of the maximally inhibitory concentration of IND (2.8 μM) on nicotine-induced ACh release were reversed concentration-dependently by PGE₂, forskolin, 3-isobutyl-1-methylxanthine (IBMX) and 8-bromo cyclic AMP. These compounds caused concentration-dependent reversal of the inhibition of spontaneous ACh release by IND, but their concentrations for restoration of spontaneous release were higher than those for restoration of nicotine-induced release. The effects of PGE₂ and forskolin or IBMX were not additive in reversing the inhibition of nicotine-induced ACh release by IND. Neither forskolin nor 8-bromo cyclic AMP alone had any significant effect on either release. These results showed that increase in the level of cyclic AMP in myenteric cholinergic neurons restored ACh release from the tissue whose PG level had been lowered by IND and indicated that endogenous PGs may modulate the level of intraneuronal cyclic AMP.

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DS-4574 is a peptidoleukotriene antagonist with mast cell stabilizing activity. In the present study, we studied the effects of this compound on gastric secretion and various acute gastric lesions in rats. Intraduodenal administration of DS-4574 at doses of 5 to 10 mg/kg significantly and dose-dependently inhibited gastric acid secretion in pylorus-ligated rats, but a further increase in the dose up to 50 mg/kg did not cause any further inhibition. Shay ulceration in response to pylorus ligation was dose-dependently prevented by DS-4574 (10-25 mg/kg, i.d.). Water-immersion restraint stress- and aspirin-induced gastric ulcers were also significantly prevented in a dose-related manner by oral pretreatment with DS-4574 (10-50 mg/kg). The lower doses of DS-4574 (1-10 mg/kg, p.o.) significantly and dose-dependently protected the gastric mucosa against the necrotizing action of either absolute ethanol or concentrated hydrochloric acid, indicating that this compound possesses a potent gastro protective activity. These antiulcer and gastric protective effects of DS-4574 were more potent than those of cimetidine used as a reference drug. These findings suggest that DS-4574 is useful for peptic ulcer therapy, as well as for the therapy of various allergic diseases, including asthma.

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Regional vascular responses to the thromboxane A₂ analogue U46619 and effects of the selective thromboxane receptor blocking drug vapiprost on these responses were examined in anesthetized dogs. Hemodynamic responses to U46619 (0.5 μg/kg into the left atrium), norepinephrine (NE, 0.3 μg/kg, i.v.) and angiotensin II (All, 30 or 60 ng/kg, i.v.) were periodically tested before and after administration of vapiprost (10, 30 or 100 μg/kg, i.v.) or its vehicle. In the absence of vapiprost, U46619 increased total peripheral (TPR), vertebral (VR), coronary (CR) and renal (RR) vascular resistance by 60.1 ± 4.7%, 33.6 ± 4.9%, 15.3 ± 1.3% and 120.8 ± 17.4%, respectively, indicating that vasoconstrictor responses to U46619 were most prominent in the renal vascular bed as compared to those in the vertebral or coronary vasculatures. Vapiprost as well as the vehicle did not affect the baseline hemodynamics. However, vapiprost apparently inhibited the U46619-induced vasoconstriction in all measured vascular beds in a dose-related manner without attenuating vasoconstrictor responses to NE and All, although significantly larger inhibition of U46619-induced increases in RR was observed as compared to the inhibitions of VR and CR. These results demonstrate that there was a regional difference both in the vasoconstrictor responses to U46619 and in the blocking effects of vapiprost, and indicate that vapiprost is a potent and selective antagonist for thromboxane receptors in vivo.

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Lipid microspheres have been used as carriers of drugs such as prostaglandin E₁ (lipo-PGE₁) and corticosteroid (liposteroid). Lipo-PGE₁ is used for the treatment of chronic arterial occlusive diseases because its activity is far greater than that of free PGE₁ in vivo. To verify the fact that the drug carriers, lipid microspheres, are preferentially taken up by endothelial cells, we labeled lipid microspheres with a fluorescent probe, DiI (DiI-LM), and observed them in some in vitro models. Stoichiometric fluorescence was obtainable, and the fluorescence was stable between pH 3.3 and pH 8.9. Human umbilical vein endothelial cells and cells of a human endothelial cell line, ECV304, showed increased uptake of DiI-LM, 81% and 61%, respectively. In contrast, uptakes were less than 7% in human skin fibroblasts, 3T3 cells, and human neutrophils. Prominent perinuclear fluorescence was also observed in endothelial cells by fluorescence microscopy. DiI-LM and flow cytometric analysis will be useful for studies to elucidate the precise mechanism of the selective accumulation of lipid microspheres by cells in blood vessel walls.

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Monkey cerebral artery strips partially contracted with prostaglandin F_2α responded to histamine with biphasic patterns of relaxation. The delayed and sustained relaxation was suppressed by cimetidine, whereas the phasic response was abolished by treatment with chlorpheniramine and N^G-nitro-L-arginine (L-NA), a nitric oxide (NO) synthase inhibitor. The inhibition by L-NA was reversed by L-arginine. D-NA was without effect. Endothelium denudation abolished the phasic relaxation. We hypothesized that endothelium-dependent, phasic relaxations caused by histamine are mediated by NO that is released by H₁-receptor stimulation, whereas the sustained relaxation is associated with the activation of H₂-receptors in the smooth muscle of monkey cerebral arteries.

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We have studied the effects of intrathecally-injected drugs that act on α-adrenoceptors in the urinary bladder reflex contractile activity evoked by continuous infusion of fluid into the bladder of anesthetized rats. Clonidine (10 and 30 μg) facilitated and yohimbine (100 μg) abolished the bladder contractile activity, and pretreatment with yohimbine (30 μg) inhibited the effect of clonidine (10 μg). Phenylephrine (60 μg) abolished the bladder contractile activity, but prazosin (40 μg) had no significant effect on it. The bladder contractions induced by electrical stimulation of the pontine micturition center were inhibited by yohimbine in a dose-dependent manner. These results suggest that transmission in the descending neurons from the pontine micturition center to the sacral parasympathetic neurons that control bladder motility is mediated by α₂-adrenoceptors in rats.

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The anti-proliferative effects of heparin on subcultured endothelial cells (EC) of rat aorta were investigated to determine whether heparin inhibits the competence phase or the progression phase in the cell cycle using the starting time and the rate of proliferation. Fetal bovine serum (FBS)-stimulated EC proliferation increased to 1.5-times in cell number, but decreased to 0.5-times in ³H-thymidine incorporation, compared with the proliferation of rat primary cultured smooth muscle cells (SMC). The FBS-effects on EC proliferation were attributed to the progression phase rather than the competence phase. Heparin (1, 10 or 100 μg/ml) significantly inhibited the proliferation of FBS (5%)-stimulated EC, and the extent of inhibition was the same in both cell number and ³H-thymidine incorporation. In the ³H-thymidine incorporation every 3 hr, heparin reduced the rate of incorporation into G₀-arrested EC, but did not affect the starting time of DNA synthesis. When the index of competence phase, starting time, was plotted against that of progression phase, rate of proliferation, the inhibition of heparin was attributed to the progression phase. These results demonstrate that heparin selectively inhibits the progression phase in subcultured EC of rat aorta.

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Eurystatins A and B, which are produced by Streptomyces eurythermus R353-21, potently inhibited Flavobacterium prolyl endopeptidase (PED) with IC₅₀ values of 0.004 and 0.002 μg/ml, respectively, while no inhibition was observed against another 5 proteases, even at 100 μg/ml. The protective effect of eurystatins A and B against scopolamine (3 mg/kg, i.p.)-induced amnesia in rats was evaluated by the step-through one-trial passive avoidance method. When administered i.p. 30 min prior to the acquisition trial, both eurystatins A, at 2-8 mg/kg, and B, at 4-8 mg/kg, significantly protected rats from the amnesic effect of scopolamine without behavioral side effects.

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The convulsive movements, blood levels of corticosterone and pharmacokinetics of nicotine after an acute intraperitoneal injection of nicotine (5 mg/kg) were examined in young (6-week-old) and old (2-year-old) rats. In pharmacokinetic study, blood nicotine levels during the elimination phase were significantly higher in old rats than in young rats. However, the duration of convulsions and the elevation of corticosterone levels after the nicotine injection showed significant decreases in old rats compared with those in young rats. These differences of nicotine-induced responses between young and old rats may be involved in the decrease in nicotine sensitivity.

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The effect of Ginkgo biloba extract (GBE) on the content of hydrogen peroxide was estimated in cerebellar neurons dissociated from rats, by means of a flow-cytometer and 2'', 7''-dichloro-fluorescin (DCF) diacetate, a fluorescent dye for intracellular hydrogen peroxide. The GBE started to reduce the DCF fluorescence of the neuron at 0.1 μg/ml to 0.3 μg/ml. Further increases in the GBE concentration (up to 3 μg/ml) produced a dose-dependent decrease in the DCF fluorescence, suggesting that GBE reduces the content of hydrogen peroxide or suppresses the reactive oxygen species (ROS) formation of cerebellar neurons. The present technique may be useful for preliminary evaluations of agents affecting the ROS formation in mammalian brain neurons.

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The inhibitory effect of 1, 9-dideoxyforskolin (DFK) on the contraction of rat aorta was compared with that of forskolin. DFK inhibited the contraction induced by high K⁺ more strongly than that induced by norepinephrine, whereas forskolin more strongly inhibited the norepinephrine-induced contraction. The inhibitory effect of DFK on high K⁺-induced contraction was antagonized by an increase in extracellular Ca²⁺ concentration. DFK inhibited the increase in cytosolic Ca²⁺ level and contraction in parallel whereas forskolin inhibited the contraction more strongly than the cytosolic Ca²⁺ level. These results suggest that DFK, but not forskolin, inhibits vascular smooth muscle contraction by a Ca²⁺ channel blocker-like action.

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Single cells were prepared from guinea pig tracheal smooth muscle and used in binding studies of [³H]p-aminoclonidine. Specific binding of [³H]p-aminoclonidine was saturable to a single class of receptors, with an equilibrium dissociation constant (K_D) of 1.62 ± 0.17 nM and a density (B_max) of 6.20 ± 0.78 × 10⁴ sites/cell. Competition studies were carried out with several adrenergic antagonists. The rank order of potency was idazoxan = phentolamine > yohimbine > prazosin = corynanthine. These results suggest that the α₂-adrenoceptor in guinea pig tracheal smooth muscle represents a single pharmacological subtype, which is designated as α_2D.

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Antiproliferative effects of the Japanese-Sino medicine Shimotsu-to (a combined prescription of cnidium rhizome, angelica root, peony root and rehmannia root) were investigated in the primary culture of smooth muscle cells (SMC) of mouse aorta. Fetal bovine serum (10%)-induced proliferation of primary cultured SMC was inhibited by Shimotsu-to at 4, 20, 100 or 500 μg/ml. The inhibitory effect was selective on SMC and due to cnidium rhizome or angelica root. The IC₅₀ values of senkyunolide H, senkyunolide A, ligustilide and butylidenephthalide derived from cnidium were below 0.1, 1.52, 1.68 and 3.25 μg/ml, respectively. These results indicate that the antiproliferative effect of Shimotsu-to may depend on these cnidium-derived phthalides.

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In Ca²⁺-free solution containing endothelin-1 (ET-1), depolarization by high KCl increased the force of contraction without any changes in intracellular calcium concentration ([Ca²⁺]_i) in canine coronary artery. The [Ca²⁺]_i-force relationships were examined in the absence or presence of ET-1 in 5 or 90 mM KCl. The relation curve in the presence of ET-1 in 90 mM KCl-PSS was shifted to the left and upward compared with that in 5 mM KCl. These results suggest that KCl-depolarization potentiates the Ca²⁺ sensitization by ET-1.

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