Antithrombotic effect of PMC, a potent alpha-tocopherol analogue on platelet plug formation in vivo.

Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. PMC (2, 2, 5, 7, 8-pentamethyl-6-hydroxychromane; 20 microg/g, i.v.) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules. When fluorescein sodium was given at 10 microg/kg, PMC (20 microg/g) delayed occlusion time by about 1.7-fold. Furthermore, aspirin (250 microg/g) also showed similar activity in delaying the occlusion time. On a molar basis, PMC was about 14-fold more potent than aspirin at delaying the occlusion time. PMC was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 5 and 10 microg/g. In addition, intravenous injection of PMC (5 microg/g) significantly prolonged bleeding time by about 1.6-fold compared with normal saline in severed mesenteric arteries of rats. Continuous infusion of PMC (1 microg/g/min) significantly increased the bleeding time by about 1.6-fold and the bleeding time was also significantly prolonged for up to 90 min after cessation of PMC infusion. These results suggest that PMC has an effective antiplatelet effect in vivo and may be a potential therapeutic agent for arterial thrombosis, but must be assessed further for toxicity.

Antithrombotic effects of tetramethylpyrazine in in vivo experiments.

In this study, tetramethylpyrazine (TMPZ) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 40 and 80 microg/g. In addition, intravenous injection of TMPZ (10 microg/g) significantly prolonged the bleeding time by approximately 1.5-fold compared with normal saline in severed mesenteric arteries of rats. Continuous infusion of TMPZ (1 microg/g per min) for 10 minutes also significantly increased the bleeding time approximately 1.6-fold, and the bleeding time returned to baseline within 60 minutes after cessation of TMPZ infusion. On the other hand, platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pre-treated intravenously with fluorescein sodium (10 microg/kg). When it was intravenously injected, TMPZ (250 microg/g) significantly prolonged the latent period of the induction of platelet plug formation in mesenteric venules. TMPZ (250 microg/g) prolonged occlusion time approximately 1.4-fold (183 +/- 18 seconds) compared with that of normal saline (132 +/- 14 seconds). Furthermore, aspirin (300 microg/g) showed similar activity in the prolongation of occlusion time in this experiment. In conclusion, these results suggest that TMPZ has effective antithrombotic activity in vivo and may be a potential therapeutic agent for arterial thrombosis but must be assessed further for toxicity.

Melatonin prevents endotoxin-induced circulatory failure in rats.

The pineal secretory product melatonin was found to exert protective effects in septic shock. In a host infected by bacterial lipopolysaccharide (LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNF-alpha) is rapidly increased, suggesting that TNF-alpha is associated with the etiology of endotoxic shock. Recent reports show that the expression of NO synthase (NOS) II and the production of superoxide anion (O2*-) also contribute to the pathophysiology of septic shock. In the present study we demonstrate that melatonin prevents circulatory failure in rats with endotoxemia and improves survival in mice treated with a lethal dose of LPS. The beneficial hemodynamic effects of melatonin in the endotoxemic animal appear to be associated with the inhibition of (i) the release of TNF-alpha in plasma, (ii) the expression of NOS II in liver, and (iii) the production of O2*- in aortae. In addition, the infiltration of polymorphonuclear neutrophils into the liver from the surviving LPS mice treated with melatonin was reduced. Thus, our results support the clinical use of melatonin in endotoxemia.

Modulation of cell cycle regulatory protein expression and suppression of tumor growth by mimosine in nude mice.

Our previous results demonstrated that the plant amino acid mimosine blocked cell cycle progression and suppressed proliferation of human lung cancer cells in vitro by multiple mechanisms. Inhibition of cyclin D1 expression or induction of cyclin-dependent kinase inhibitor p21WAF1 expression was found in mimosine-treated lung cancer cells. However, whether mimosine may modulate the expression of these cell cycle regulatory proteins and suppress tumor growth in vivo is unknown. In this study, we examined the anti-cancer effect of mimosine on human H226 lung cancer cells grown in nude mice. Our results demonstrated that mimosine inhibits cyclin D1 and induces p21WAF1 expression in vivo. Furthermore, results of TUNEL analysis indicated that mimosine may induce apoptosis to suppress tumor growth in nude mice. Collectively, these results suggest that mimosine exerts anti-cancer effect in vivo and might be useful in the therapy of lung cancer.

Antithrombotic effect of rutaecarpine, an alkaloid isolated from Evodia rutaecarpa, on platelet plug formation in in vivo experiments.

In this study, platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. Rutaecarpine (200 microg/g) significantly prolonged the latent period of inducing platelet plug formation in mesenteric venules when it was intravenously injected. Rutaecarpine (200 microg/g) prolonged occlusion time by approximately 1.5-fold (control 127 +/- 29 vs. taecarpine 188 +/- 23 s). Furthermore, aspirin (250 microg/g) also showed a similar prolongation of the occlusion time in this experiment. On a molar basis, rutaecarpine was approximately twofold more potent than aspirin at prolonging the occlusion time. Furthermore, rutaecarpine was also effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 25 and 50 microg/g. Intravenous injection of rutaecarpine (50 microg/g) significantly prolonged the bleeding time by approximately 1.5-fold compared with normal saline in the severed mesenteric arteries of rats. Continuous infusion of rutaecarpine (5 microg/g/min) also significantly increased the bleeding time 1. 5-fold, and the bleeding time returned to baseline within 60 min after cessation of rutaecarpine infusion. These results suggest that rutaecarpine has an effective anti-platelet effect in vivo and that it may be a potential therapeutic agent for arterial thrombosis, but it must be assessed further for toxicity.

Terbutaline prevents circulatory failure and mitigates mortality in rodents with endotoxemia.

Septic shock is characterized by a decrease in systemic vascular resistance. Nevertheless, regional increases in vascular resistance can occur that may predispose mammals to organ dysfunction, including the acute respiratory distress syndrome. In the host infected by endotoxin (lipopolysaccharide, LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNFalpha) rapidly increases, and this cytokine production is regulated by agents elevating cyclic AMP. In this report, we present evidence that terbutaline, a beta2-agonist, inhibits TNFalpha production and enhances interleukin-10 (IL-10) release in the anesthetized rat treated with LPS. In addition, an overproduction of nitric oxide (NO, examined by its metabolites nitrite/nitrate) by inducible NO synthase (iNOS, examined by western blot analysis) is attenuated by pretreatment of LPS rats with terbutaline. Overall, pretreatment of rats with terbutaline attenuates the delayed hypotension and prevents vascular hyporeactivity to norepinephrine. In addition, pretreatment of mice with terbutaline also improves the survival in a model of severe endotoxemia. The infiltration of polymorphonuclear neutrophils into organs (e.g., lung and liver) from the surviving LPS mice treated with terbutaline was reduced almost to that seen in the normal controls. These findings suggest that the inhibition of TNFalpha and NO (via iNOS) production as well as the increment of IL-10 production contribute to the beneficial effect of terbutaline in animals with endotoxic shock.

The antihypertensive and cardioprotective effects of (-)-MJ-451, an ATP-sensitive K(+) channel opener.

ATP-sensitive K(+) (K(ATP)) channel openers have been shown to be a potential class of therapeutic agents for the control of cardiovascular diseases, including angina, arrhythmias, and hypertension. In this study, the pharmacological activity of 6-cyano-3S,4R-dihydro-2, 2-dimethyl-2H-3-hydroxy-4-[5S-(1-hydroxymethyl)-2-oxo-1-pyrrolidinyl] -1-benzopyran ((-)-MJ-451), a synthetic K(ATP) opener, was evaluated in anesthetized rat models and in isolated rat thoracic rings. Results demonstrated that intravascular injection of (-)-MJ-451 (0. 02, 0.05 and 0.1 mg/kg) produced an immediate, dose-related reduction in mean arterial blood pressure in anesthetized spontaneously hypertensive rats (SHR), which persisted for more than 3 h and was not accompanied by reflex tachycardia. The hemodynamic changes were completely abolished by pretreatment with glibenclamide (4 mg/kg, i.v. bolus), a selective K(ATP) channel blocker. In isolated thoracic aorta, (-)-MJ-451 (10 nM-3 microM) produced a concentration-dependent vasodilator effect on the phenylephrine (0.3 microM)-induced vasoconstriction. Moreover, (-)-MJ-451 relaxed the thoracic aorta contracted by low (5, 20 and 30 mM), but not high (40 and 60 mM) concentrations of extracellular potassium. In addition, (-)-MJ-451 showed cardioprotective effects in the rat model of 45-min left coronary artery occlusion followed by 1-h reperfusion. In myocardial ischemia, pretreatment with (-)-MJ-451 (2, 5 and 10 microg/kg, i.v. bolus) significantly reduced the incidence of ventricular fibrillation and the mortality, also reducing the total number of ventricular premature contractions, total duration of ventricular tachycardia and ventricular fibrillation. A significant reduction in infarct size was noted in three (-)-MJ-451 (2, 5 and 10 microg/kg)-treated groups. Also, the cardioprotective effects of (-)-MJ-451 were virtually abolished by pretreating the rats with glibenclamide (4 mg/kg, i.v. bolus). In conclusion, (-)-MJ-451, through opening the K(ATP) channel, exerted antihypertensive and cardioprotective effects. Therefore, it is suggested that (-)-MJ-451 has potential in the treatment of hypertension or acute myocardial infarction.

Mechanisms involved in the antiplatelet activity of Staphylococcus aureus lipoteichoic acid in human platelets.

In this study, gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose-dependently (0.1-1.0 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LTA also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. LTA (0.5 and 1.0 microg/ml) also significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LTA (0.1-1.0 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. Rapid phosphorylation of a platelet protein of Mr. 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (30 nM). This phosphorylation was markedly inhibited by LTA (0.5 and 1.0 microg/ml) within a 10-min incubation period. These results indicate that the antiplatelet activity of LTA may be involved in the following pathways: LTA's effects may initially be due to induction of conformational changes in the platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in gram-positive septicemic and endotoxemic patients.

Tetramethylpyradizine prevents inducible NO synthase expression and improves survival in rodent models of endotoxic shock.

This study is to investigate the possible mechanism of beneficial effects of tetramethylpyrazine (TMP) on endotoxic shock which we showed in our preliminary study (Liao et al. 1998; Proc Natl Sci Counc Repub China B 22:46-54). Here, we have confirmed the beneficial effects of TMP on the hypotension, vascular hyporeactivity to noradrenaline (NA), release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide, LPS). In addition, we further examined the expression of inducible NO synthase in the lung and in the aorta from these rats and evaluated the effect of TMP on the 36-h survival rate in a murine model of endotoxaemia. Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure (MAP) and heart rate (HR). Injection of LPS (10 mg kg(-1), i.v.) resulted in an acute fall followed by a substantial fall in MAP within 4 h and an increase in HR. In contrast, animals pretreated with TMP (10 mg kg(-1), i.p.; at 30 min prior to LPS) maintained a significantly higher MAP but the tachycardia was further enhanced at 1-2 h when compared to rats given only LPS (LPS rats). The pressor effect of NA (1 microg kg(-1), i.v.) was also significantly reduced after the treatment of rats with LPS. Similarly, the thoracic aorta obtained from rats at 4 h after LPS showed a significant reduction in the contractile responses elicited by NA (1 microM). Pretreatment of LPS rats with TMP partially, but significantly, prevented this LPS-induced hyporeactivity to NA in vivo and ex vivo. The injection of LPS resulted in a bell-shaped change in plasma TNF-alpha level which reached a maximum at 1 h, whereas the effect of LPS on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner. This increment of both TNF-alpha and nitrate levels was significantly reduced in LPS rats pretreated with TMP. Endotoxaemia for 4 h caused a significantly increased protein expression of iNOS in the lung and the aorta. In LPS rats pretreated with TMP, iNOS protein expression in lung and aorta homogenates was attenuated by 75+/-3% and 57+/-6%, respectively. In addition, the lack of evidence of pressor effect of TMP on rats with endotoxaemia for 4 h suggested that TMP inhibits the induction of iNOS rather than directly inhibiting NOS activity. Treatment of conscious ICR mice with a high dose of endotoxin (60 mg kg(-1), i.p.) resulted in a survival rate of only 15% at 36 h (n=20). However, therapeutic application of TMP (10 mg kg(-1), i.p.; at 0, 6, 15 and 24 h after LPS) increased the 36 h survival rate to 55% (n=20). Thus, TMP inhibits the expression of iNOS and mitigates the delayed circulatory failure caused by endotoxic shock in the rat. In addition, TMP also improves survival in a murine model of severe endotoxaemia.

Antiplatelet effect of amlodipine: a possible mechanism through a nitric oxide-mediated process.

The effect of amlodipine, a novel calcium channel blocker of the dihydropyridine type, on rabbit platelet aggregation, and the possible antiaggregatory mechanisms of amlodipine, especially on the nitric oxide (NO) guanosine 3',5'-cyclic monophosphate (cyclic GMP)-mediated pathway, were investigated. Other effects of amlodipine on thromboxane B2 (TXB2) formation in platelets also were examined. Amlodipine concentration-dependently inhibited rabbit platelet aggregation induced by collagen (10 microg/mL) or thrombin (0.1 U/mL) with an IC50 range of 32-69 microM. Along with this inhibition, our results also demonstrated that in the presence of L-arginine (100 IM), amlodipine (50 microM) increased nitric oxide synthetase (NOS) activity (from the resting activity of 2.05+/-0.36 to 7.11+/-0.95 pmol/mg protein/min) and NO release (by 80%), accompanied by an elevation of the cyclic GMP level (from the resting platelet level of 1.27+/-0.12 to 6.21+/-0.55 pmol/10(9) platelets) induced by collagen (10 microg/mL). However, the antiaggregatory effect of amlodipine (50 microM) could be attenuated significantly by oxyhemoglobin (5 microM), a NO scavenger, or N(G)-nitro-L-arginine methyl ester (100 microM), a specific NOS inhibitor. In addition, the TXB2 production in platelets induced by collagen or thrombin was concentration-dependently inhibited by amlodipine. Therefore, we propose that the antiaggregatory mechanisms of amlodipine might be mediated, in part, by a NO-cyclic GMP process accompanied by the inhibition of TXB2 formation in platelets.

Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms.

The plant amino acid mimosine has been reported to block cell cycle progression in the late G1 phase. A recent study showed that mimosine might induce growth arrest by activating the expression of p21CIP1, a cyclin-dependent kinase inhibitor (CDKI), and by inhibiting the activity of cyclin E-associated kinases in human breast cancer cells. However, mimosine at higher concentrations also blocked proliferation of p21-/- cells by unknown mechanisms. In this study, we investigated the effect of mimosine on the expression of cyclins and CDKIs in human lung cancer cells. We found that mimosine specifically inhibited cyclin D1 expression in H226 cells. The expression of another G1 cyclin, cyclin E, was not regulated by mimosine in all lung cancer cell lines examined. Moreover, mimosine induced p21CIP1 expression in H226 and H358 cells, while it activated p27KIP1 expression in H322 cells. However, mimosine does not affect transcription of these genes directly because significant changes in cyclin D1 or CDKI expression were observed at 12-24 h after drug addition. Our results indicate that mimosine may block cell proliferation by multiple mechanisms and this amino acid is a useful agent for the study of cell cycle control.

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.

Pentoxifylline improves circulatory failure and survival in murine models of endotoxaemia.

Pentoxifylline, a methylxanthine derivative, has been widely used to improve erythrocyte deformability and capillary blood circulation in patients with claudication and cerebrovascular disorders as well as in animals with sepsis. Here, we investigate the effects of pentoxifylline on the hypotension, vascular hyporeactivity to noradrenaline, release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), and inducible NO synthase protein expression in a rat model of circulatory shock induced by bacterial endotoxin (Escherichia coli lipopolysaccharide). In addition, we have evaluated the effect of pentoxifylline on the 36-h survival rate in a murine model of endotoxaemia. Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure and heart rate. Injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a significant fall in mean arterial pressure and an increase of heart rate. In contrast, animals pretreated with pentoxifylline (3 mg/kg, i.v., at 30 min prior to lipopolysaccharide) maintained a significantly higher mean arterial pressure but showed no effect on the tachycardia when compared to rats given only lipopolysaccharide (lipopolysaccharide-rats). The pressor effect of noradrenaline (1 microg/kg, i.v.) was also significantly reduced after the treatment of rats with lipopolysaccharide. Similarly, rings of thoracic aorta obtained from lipopolysaccharide-rats showed a significant reduction in the contractile responses elicited by noradrenaline (1 microM). Pretreatment of lipopolysaccharide-rats with pentoxifylline partially, but significantly, prevented this lipopolysaccharide-induced hyporeactivity to noradrenaline in vivo and ex vivo. The injection of lipopolysaccharide resulted in bell-shape changes in plasma TNF-alpha level which reached a peak at 60 min, whereas the effect of lipopolysaccharide on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner. This increase of both TNF-alpha and nitrate levels induced by lipopolysaccharide was significantly reduced in lipopolysaccharide-rats pretreated with pentoxifylline. Endotoxaemia for 240 min caused a significantly increased protein expression of inducible NO synthase in the lung. In lipopolysaccharide-rats pretreated with pentoxifylline, inducible NO synthase protein expression in lung homogenates was attenuated by 48 +/- 5%. Treatment of conscious mice with a high dose of endotoxin (60 mg/kg, i.p.) resulted in a survival rate of only 10% at 36 h (n = 20). However, therapeutic application of pentoxifylline (3 mg/kg, i.p. at 0, 6, 15 and 24 h after lipopolysaccharide) increased the 36-h survival to 35% (n = 20). Thus, pentoxifylline protects against circulatory failure and improves survival in rodents with severe endotoxaemia. These effects may be due to inhibition of the release of TNF-alpha and of the induction of inducible NO synthase.

Reduction in lipopolysaccharide-induced thrombocytopenia by triflavin in a rat model of septicemia.

BACKGROUND: Thrombocytopenia frequently occurs early in the course of Gram-negative bacterial infections. Triflavin, an Arg-Gly-Asp-containing disintegrin, has been suggested to interfere with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. The present study was undertaken to determine whether triflavin could prevent thrombocytopenia in lipopolysaccharide (LPS)-treated rats. METHODS AND RESULTS: In this study, 51Cr-labeled platelets were used to assess blood and tissue platelet accumulation after LPS challenge. The administration of LPS (4 mg/kg IV bolus) for 4 hours induced a reduction in radiolabeled platelets in blood and an obvious accumulation of platelets in liver. Triflavin (500 microg/kg) but not GRGDS (20 mg/kg) significantly prevented the alteration of radiolabeled platelet distribution in blood and liver when induced by LPS. Furthermore, triflavin but not GRGDS markedly suppressed the elevation in plasma thromboxane B2 concentration within the 4-hour period of LPS administration. In LPS-treated rats, the 5-hydroxytryptamine level was lower in the blood and higher in the liver compared with levels in normal saline-treated rats. Pretreatment with triflavin (500 microg/kg) significantly reversed the 5-hydroxytryptamine concentration in blood and liver of LPS-treated rats. In histological examinations and platelet adhesion assay, triflavin markedly inhibited the adhesion of platelets to subendothelial matrixes in vivo and in vitro. CONCLUSIONS: The results indicate that triflavin effectively prevents thrombocytopenia, possibly through the following 2 mechanisms: (1) Triflavin markedly inhibits platelet aggregation, resulting in decreased thromboxane A2 formation. (2) It inhibits the adhesion of platelets to subendothelial matrixes, thereby leading to a reversal in the distribution of platelets in blood and liver in LPS-treated rats.

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation.

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.

Cytotoxic isoprenylated flavans of Broussonetia kazinoki.

Two new prenylflavans [kazinols Q (1) and R (2)] and five known compounds [kazinols D (3), K (4), and H, 7,4'-dihydroxyflavan (5), and oleanolic acid] were isolated from the root bark of Broussonetia kazinoki. The cytotoxic activity of 1-5 was evaluated against several different cell lines.

Comparison between effects of dantrolene and nifedipine on lipopolysaccharide-induced endotoxemia in the anesthetized rats.

Intracellular calcium is an important mediator for regulating the cellular response in endotoxemia. In this study, we investigated the effects of dantrolene and nifedipine, two agents of reducing intracellular calcium levels, on bacterial endotoxin (lipopolysaccharide, LPS; 10 mg/kg i.v.)-induced production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) as well as hemodynamic changes in the anesthetized rat. Injection of LPS (i) induced biphasic changes of blood glucose and rectal temperature: an initial increased phase (<180 min after injection of LPS) followed by a decreased phase (at 240 or 360 min), (ii) caused a significant fall in mean arterial blood pressure from 119+/-3 mmHg (at time 0) to 73+/-67 mmHg (at 360 min) with a concomitant increase of heart rate, (iii) resulted in a substantial hyporeactivity to norepinephrine (NE) (1 microg/kg i.v.), (iv) increased plasma nitrate (an indicator of NO formation) in a time-dependent manner, and (v) induced bell-shape changes in plasma TNF-alpha levels which reached a peak at 60 min. Pretreatment of animals with dantrolene (1 mg/kg i.v. at 20 min prior to LPS) or nifedipine (20 microg/kg i.v. infusion for 20 min at 20 min prior to LPS) not only attenuated the delayed circulatory failure (e.g. delayed hypotension and vascular hyporeactivity to NE), but also prevented the overproduction of NO caused by LPS in the rat. However, the prevention of NO overproduction by dantrolene, but not by nifedipine, was associated with an inhibition of TNF-alpha production elicited by LPS. Thus, both dantrolene and nifedipine have beneficial hemodynamic effects, although through different mechanisms, in animals with endotoxic shock.

Mechanisms involved in the antiplatelet activity of Escherichia coli lipopolysaccharide in human platelets.

In this study, Escherichia coli lipopolysaccharide (LPS) dose-dependently (100-300 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LPS also dose-dependently inhibited the phosphoinositide breakdown and the intracellular Ca+2 mobilization in human platelets stimulated by collagen. LPS (300 microg/ml) also significantly inhibited the thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LPS (100-300 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. In addition, LPS (200 and 300 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) also significantly increased the production of nitrate within a 30 min incubation period. Rapid phosphorylation of a platelet protein of Mr 47,000, a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by LPS (200 microg/ml) within a 30 min incubation period. These results indicate that the antiplatelet activity of LPS may be involved in two important pathways. (1) LPS may induce conformational changes in the platelet membrane, leading to change in the activity of phospholipase C. (2) LPS also activated the formation of nitric oxide (NO)/cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicaemic and endotoxaemic patients.

Inhibitory mechanisms of naloxone on human platelets.

1. In the present study, naloxone was tested for its antiplatelet activities in human platelet-rich plasma (PRP). In human PRP, naloxone (0.1-0.5 mmol/L) inhibited aggregation stimulated by a variety of agonists (i.e. collagen, adenosine diphosphate (ADP), U46619 and adrenaline). 2. Naloxone (0.1-0.5 mmol/L) did not significantly affect cyclic adenosine monophosphate and cGMP levels in human washed platelets, whereas naloxone (0.5 mmol/L) significantly inhibited thromboxane B2 formation stimulated by collagen (5 micrograms/mL) in human washed platelets. 3. Naloxone (0.5 mmol/L) significantly inhibited [3H]-inositol monophosphate formation of [3H]-myoinositol-loaded platelets stimulated by collagen and U46619. Moreover, naloxone did not influence the binding of 125I-triflavin to platelet membranes. Triflavin is an Arg-Gly-Asp-containing specific fibrinogen receptor antagonist. 4. Addition of naloxone (0.5 mmol/L) to platelet preparations tagged with diphenylhexatriene (DPH) resulted in a considerable decrease in relative fluorescence intensity. 5. It is suggested that the anti-platelet effects of naloxone may be caused, at least partly, by the induction of conformational changes in the platelet membrane initially, followed by the inhibition of thromboxane A2 formation and phosphoinositide breakdown of platelets stimulated by agonists.

Pyrrolidine dithiocarbamate improves the septic shock syndromes in spontaneously hypertensive rats.

1. The aim of the present study was to evaluate the effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor-kappa B (NF-kappa B), on septic shock induced by Escherichia coli lipopolysaccharide (LPS) in spontaneously hypertensive rats (SHR). 2. After injection of LPS in SHR, a marked decrease in blood pressure was observed at 3 h and vascular hyporeactivity to noradrenaline (NA) was observed after 1 h. A marked increase in plasma levels of tumour necrosis factor-alpha (TNF-alpha) and nitrite (an indicator of nitric oxide) was also observed in SHR. 3. The delayed hypotension and hyporeactivity to NA induced by LPS were significantly reserved by pretreatment of rats with PDTC (10 mg/kg). The increase in plasma levels of TNF-alpha and nitrite in LPS-treated groups was also significantly suppressed by PDTC pretreatment. In addition, the survival time of SHR treated with LPS was significantly prolonged by PDTC pretreatment. 4. The present ex vivo study demonstrates that the NA-induced contraction is attenuated and the L-arginine-induced relaxation is enhanced in aortic rings obtained from LPS-treated SHR. Both the reduction of the NA-induced contraction and the increase of L-arginine-induced relaxation were reversed by pretreatment with PDTC. However, the relaxation elicited by acetylcholine (ACh) was not affected in LPS-treated SHR when compared with sham-operated SHR. In addition, the ACh-induced relaxation in LPS-treated SHR was not affected by PDTC pretreatment. 5. In normotensive Wistar-Kyoto (WKY) rats, LPS had mild effects on blood pressure, vascular hyporeactivity and plasma levels of TNF-alpha and nitrite. At a higher dose, PDTC (10 mg/kg) also prolonged survival time and improved haemodynamics in LPS-treated WKY rats. In the ex vivo study, it was noted that the relaxation elicited by ACh was significantly (P < 0.05) attenuated in LPS-treated WKY rats. This attenuation of the ACh-induced relaxation by LPS in WKY rats was significantly reversed by pretreatment with 10 mg/kg PDTC. 6. In conclusion, PDTC prolongs survival time in rats with endotoxaemia and improves the septic shock syndromes both in vivo and ex vivo. Thus, we propose that PDTC may be of use in septic patients.