Early effective drainage in the treatment of loculated tuberculous pleurisy.

The role of early effective drainage in loculated tuberculous (TB) pleurisy treatment remains unclear. Consecutive patients with TB pleurisy subjected to anti-TB treatment and pigtail drainage (n = 64) were divided into three groups: 1) patients with free-flowing effusions irrigated with saline (free-flowing group; n = 20); 2) patients with loculated effusions irrigated with streptokinase (streptokinase group; n = 22); and 3) patients with loculated effusions irrigated with saline (saline group; n = 22). Pleural irrigation was performed for 3 days consecutively and the effusion drained as completely as possible. Outcomes were assessed for 12 months by clinical symptoms, effusion removed, radiological scores for effusion amount, lung function and occurrence of residual pleural thickening. The total effusion volumes removed were significantly greater in the free-flowing (2.36+/-1.62 L) and streptokinase groups (2.59+/-1.77 L) than in the saline group (1.28+/-1.21 L). Compared with the saline group, the free-flowing and streptokinase groups showed significant improvement in radiological scores and forced vital capacity at different time-points during follow-up, and a significantly lower occurrence of residual pleural thickening. All outcome variables were comparable between the streptokinase and free-flowing groups. In summary, early effective drainage and complete anti-tuberculosis treatment may hasten clearance of pleural effusion, reduce residual pleural thickening occurrence and accelerate pulmonary function recovery in patients with symptomatic loculated tuberculous pleurisy.

Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac performance in mouse after acute myocardial infarction.

BACKGROUND: Gene and stem cell therapies hold promise for the treatment of ischaemic cardiovascular disease. However, combined stem cell and angiogenic growth factor gene therapy for acute ischaemic myocardium has not been previously reported. This study hypothesized that combined stem cell and gene therapy would not only augment new vessels formation but also improve myocardial function in acute ischaemic myocardium. METHODS: Human angiopoietin-1 (Ang1) cDNA and VEGF(165) cDNA were ligated into AAV vector. The purified CD34(+) cells were obtained from human umbilical cord blood samples. Cord blood CD34(+) cells were transduced with AAV vector encoding either the human Ang1 (AAV-Ang1) or VEGF(165) (AAV-VEGF) cDNA alone, or both (AAV-Ang1 plus VEGF). Immediately after ligation of the left anterior descending coronary artery in male SCID mice, culture-expanded CD34(+) cells transduced with AAV-Ang1, AAV-VEGF or AAV-Ang1 plus VEGF were injected intramyocardially at the left anterior free wall. RESULTS: Western blot showed that Ang1 and VEGF protein expressions were enhanced in the CD34(+)cells transduced with AAV-Ang1 and AAV-VEGF, respectively. Infarct size significantly decreased and capillary density significantly increased after treatment with CD34(+)/AAV-Ang1 plus VEGF when compared with treatment by CD34(+) only. Combined therapy with CD34(+) and AAV-Ang1, CD34(+) and AAV-VEGF, CD34(+) and AAV-Ang1 plus VEGF, all showed significantly higher cardiac performance in echocardiography than the therapy with CD34(+) alone 4 weeks after myocardial infarction. CONCLUSIONS: Combined therapy with human umbilical cord blood CD34(+) cells and both Ang1 and VEGF genes reduced infarct size, attenuated the progression of cardiac dysfunction and increased capillary density in acute myocardial infarction in mice.

Inhibitory mechanisms of kinetin, a plant growth-promoting hormone, in platelet aggregation.

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 microM) also concentration-dependently inhibited intracellular Ca2+ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 microg/ml). Kinetin (70 and 150 microM) significantly inhibited thromboxane A2 formation stimulated by collagen (1 microg/ml) and arachidonic acid (60 microM) in human platelets. In addition, kinetin (70 and 150 microM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 microM). Rapid phosphorylation of a platelet protein of molecular weight (Mr) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/ml). This phosphorylation was inhibited by kinetin (70 and 150 microM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na+/H+ exchanger. This leads to lower intracellular Ca2+ mobilization, followed by inhibition of TxA2 formation and then increased cyclic AMP formation, followed by a further inhibition of the Na+/H+ exchanger, ultimately resulting in markedly decreased intracellular Ca2+ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.

Anti-leukemic immunity against U937 cells in uremic patients.

To examine anti-tumor immunity in uremic patients undergoing regular hemodialysis, we designed this study using in vitro mononuclear cell (MNC) cultures, with human leukemic U937 cells as the target. MNC were collected and cultured from uremic subjects and age- and gender-matched healthy controls. Conditioned media from the cultures (MNC-CM) were collected after stimulation with various concentrations of phytohemagglutinin (PHA). The proliferation-inhibiting and differentiation-inducing activities of the PHA-MNC-CM on U937 cells were evaluated. The growth inhibition activity of uremic patients' PHA-MNC-CM was lower than that of controls. The differentiation-inducing effects were evaluated by morphological scoring, superoxide production, and monocyte-associated antigen expression (CD14 and CD68). All three parameters demonstrated that the differentiation-inducing effect of MNC-CM increased with increasing doses of PHA. These effects, however, were significantly less in uremic patients compared to controls at higher doses of PHA. The levels of TNF-alpha and IFN-gamma in PHA-MNC-CM increased in a PHA dose-dependent manner and were much higher in the controls. We conclude that the capacity of MNC from uremic hemodialysis patients to produce anti-leukemic immunity is significantly lower than that of healthy controls.

The hyperaggregability of platelets from normal pregnancy is mediated through thromboxane A2 and cyclic AMP pathways.

There is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy, with the risk of developing pre-eclampsia. In this study, platelet function was studied during 28-40 weeks of gestation in a group of women who remained normotensive and in a group of nonpregnant female controls. Platelet aggregation stimulated by thrombin and adenosine diphosphate was markedly enhanced in washed platelets from pregnant subjects. Thrombin (0.04 U/ml)-evoked increases in intracellular Ca+2 mobilization of Fura 2-AM-loaded platelets were also enhanced in pregnant subjects. The binding of fluorescein isothiocyanate (FITC)-triflavin (2 microg/ml) to the glycoprotein IIb/IIIa complex in thrombin-activated platelets did not differ significantly between the nonpregnant and pregnant groups. Thromboxane A2 (TXA2) formation in both resting and thrombin-activated platelets from pregnant subjects was significantly greater than from nonpregnant subjects. Levels of cyclic adenosine monophosphate (cAMP) in both resting and prostaglandin E1-treated platelets (10 micromol/l) from pregnant subjects were significantly lower than those from nonpregnant subjects. There were no significant differences between nonpregnant and pregnant subjects in platelet cAMP levels in the presence of imidazole (600 micromol/l) and indomethacin (500 micromol/l). Intracellular pH values in platelets were measured spectrofluorometrically using the fluorescent probe, BCECF-AM. The increase in intracellular pH stimulated by thrombin (0.04 U/ml) in pregnant subjects was markedly greater than that in observed nonpregnant subjects. We conclude that the agonist-induced hyperaggregability of platelets in normal pregnancy may be due, at least partly, to stimulation of the Na+/H+ exchanger and subsequently to elevated intracellular Ca+2 mobilization, and then to increased TXA2 formation and a lowered level of cAMP, which leads to further increases in intracellular Ca+2 mobilization, and finally to enhanced platelet aggregation.

The protective effects of PMC against chronic carbon tetrachloride-induced hepatotoxicity in vivo.

In this study, PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane), a derivative of alpha-tocopherol, dose-dependently (1-10 mg/kg) ameliorated the increase in plasma aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) levels caused by chronic repeated carbon tetrachloride (CCl4) intoxication in mice. Moreover, PMC significantly improved the CCl4-induced increase of hepatic glutathione peroxidase, reductase, and superoxide dismutase activities. PMC also restored the decrement in the glutathione content of hepatic tissues in CCl4-intoxicated mice. Furthermore, it also dose-dependently inhibited the formation of lipid peroxidative products during carbon tetrachloride treatment. Histopathological changes of hepatic lesions induced by carbon tetrachloride were significantly improved by treatment with PMC in a dose-dependent manner. These results suggest that PMC exerts effective protection in chronic chemical-induced hepatic injury in vivo.

Induction of cyclooxygenase-2 protein by lipoteichoic acid from Staphylococcus aureus in human pulmonary epithelial cells: involvement of a nuclear factor-kappa B-dependent pathway.

1. This study investigated the role of protein kinase C (PKC) and transcription factor nuclear factor-kappaB (NF-kappaB) in cyclooxygenase-2 (COX-2) expression caused by lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, in human pulmonary epithelial cell line (A549). 2. LTA caused dose- and time-dependent increases in COX-2 expression and COX activity, and a dose-dependent increase in PGE(2) release in A549 cells. The LTA-induced increases in COX-2 expression and COX activity were markedly inhibited by dexamethasone, actinomycin D or cyclohexamide, but not by polymyxin B, which binds and inactivates endotoxin. 3. The phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D-609) and the phosphatidate phosphohydrolase inhibitor (propranolol) reduced the LTA-induced increases in COX-2 expression and COX activity, while phosphatidylinositol-phospholipase C inhibitor (U-73122) had no effect. The PKC inhibitors (Go 6976, Ro 31-8220 and GF 109203X) and NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), also attenuated the LTA-induced increases in COX-2 expression and COX activity. 4. Treatment of A549 cells with LTA caused an increase in PKC activity in the plasma membrane; this stimulatory effect was inhibited by D-609, propranolol, or Go 6976, but not by U-73122. 5. Exposure of A549 cells to LTA caused a translocation of p65 NF-kappaB from the cytosol to the nucleus and a degradation of IkappaB-alpha in the cytosol. Treatment of A549 cells with LTA caused NF-kappaB activation by detecting the formation of NF-kappaB-specific DNA-protein complex in the nucleus; this effect was inhibited by dexamethasone, D-609, propranolol, Go 6976, Ro 31-8220, or PDTC. 6. These results suggest that LTA might activate PC-PLC and phosphatidylcholine-phospholipase D to induce PKC activation, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE(2) release in human pulmonary epithelial cell line.

Magnolol reduces myocardial ischemia/reperfusion injury via neutrophil inhibition in rats.

The accumulation of oxygen-free radicals and activation of neutrophils are strongly implicated as important pathophysiological mechanisms mediating myocardial ischemia/reperfusion injury. It has been proven that various antioxidants have cardioprotective effects. Magnolol, an active component extracted from the Chinese medicinal herb Magnolia officinalis, possesses potent antioxidant and free radical scavenging activities. In this study, the cardioprotective activity of magnolol was evaluated in an open-chest anesthetized rat model of myocardial ischemia/reperfusion injury. The results demonstrated that pretreatment with magnolol (0.2 and 0.5 microg/kg, i.v. bolus) at 10 min before 45 min of left coronary artery occlusion, significantly suppressed the incidence of ventricular fibrillation and mortality when compared with the control group. Magnolol (0.2 and 0.5 microg/kg) also significantly reduced the total duration of ventricular tachycardia and ventricular fibrillation. After 1 h of reperfusion, pretreatment with magnolol (0.2 and 0.5 microg/kg) caused a significant reduction in infarct size. In addition, magnolol (0.2 microg/kg) significantly reduced superoxide anion production and myeloperoxidase activity, an index of neutrophil infiltration in the ischemic myocardium. In addition, pretreatment with magnolol (0.2 and 0.5 microg/kg) suppressed ventricular arrhythmias elicited by reperfusion following 5 min of ischemia. In vitro studies of magnolol (5, 20 and 50 microM) significantly suppressed N-formylmethionyl-leucyl-phenylalanine (fMLP; 25 nM)-activated human neutrophil migration in a concentration-dependent manner. It is concluded that magnolol suppresses ischemia- and reperfusion-induced ventricular arrhythmias and reduces the size of the infarct resulting from ischemia/reperfusion injury. This pronounced cardioprotective activity of magnolol may be mediated by its antioxidant activity and by its capacity for neutrophil inhibition in myocardial ischemia/reperfusion.

Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger.

The antioxidant properties of cinnamophilin were evaluated by studying its ability to react with relevant reactive oxygen species, and its protective effect on cultured cells and biomacromolecules under oxidative stress. Cinnamophilin concentration-dependently suppressed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 8.0+/-0.7 microM and iron ion/ADP/ascorbate-initiated rat liver mitochondrial lipid peroxidation with an IC50 value of 17.7+/-0.2 microM. It also exerted an inhibitory activity on NADPH-dependent microsomal lipid peroxidation with an IC50 value of 3.4+/-0.1 microM without affecting microsomal electron transport of NADPH-cytochrome P-450 reductase. Both 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azo-bis(2-amidinopropane) dihydrochloride-derived peroxyl radical tests demonstrated that cinnamophilin possessed marked free radical scavenging capacity. Cinnamophilin significantly protected cultured rat aortic smooth muscle cells (A7r5) against alloxan/iron ion/H2O2-induced damage resulting in cytoplasmic membranous disturbance and mitochondrial potential decay. By the way, cinnamophilin inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity and thiobarbituric acid-reactive substance formation in a concentration-dependent manner. On the other hand, it was reactive toward superoxide anions generated by the xanthine/xanthine oxidase system and the aortic segment from aged spontaneously hypertensive rat. Furthermore, cinnamophilin exerted a divergent effect on the respiratory burst of human neutrophil by different stimulators. Our results show that cinnamophilin acts as a novel antioxidant and cytoprotectant against oxidative damage.

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.

Mechanisms in the inhibition of neointimal hyperplasia with triflavin in a rat model of balloon angioplasty.

RGD-containing peptides are able to inhibit the binding of ligands to certain beta3 integrins, such as alpha(IIb)beta3 and alpha(v)beta3, both of which are involved in neointimal hyperplasia. The present study was designed to elucidate the detailed mechanisms involved in the inhibition of neointimal hyperplasia with triflavin in a rat model of balloon angioplasty. Triflavin (0.25 mg x kg(-1) x d(-1)), an RGD-containing disintegrin, time dependently inhibited both neointimal hyperplasia and lumen occlusion after angioplasty in carotid arteries of rats. Furthermore, electron micrographs highlighted that SMCs were phenotypically different from the typical contractile, spindle-shaped SMCs normally seen in uninjured vessel walls. PDGF-BB was strongly produced in thrombus formation and neointimal SMCs after angioplasty, and triflavin significantly reduced PDGF-BB expression in vessel lumens and neointimal SMCs after angioplasty. Balloon angioplasty caused a significant increase of nitrate and cyclic guanosine monophosphate levels compared with levels found in sham-operated rats, and these were not significantly changed with infusion of triflavin (0.25 mg x kg(-1) x d(-1)). Furthermore, the plasma level of TXB2 obviously increased after angioplasty, and triflavin markedly suppressed the elevation of plasma TXB2 concentration. The results indicate that triflavin effectively prevents neointimal hyperplasia, possibly through the following 2 mechanisms. First, triflavin binds to alpha(IIb)beta3 integrin on platelet membranes, resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, followed by inhibition of TXA2 formation and PDGF-BB release from platelets. Second, triflavin may also bind to alpha(v)beta3 integrin on SMCs, thus subsequently inhibiting cell migration and proliferation. These results provide new insights into the mechanisms of neointimal hyperplasia and have significant implications for disintegrin therapy for the treatment of restenosis and atherosclerosis.

Mechanisms involved in the inhibition of neointimal hyperplasia by abciximab in a rat model of balloon angioplasty.

Monoclonal antibodies raised against beta(3) integrin are able to inhibit the binding of ligands to certain beta(3) integrins such as alpha(IIb)beta(3) (glycoprotein IIb/IIIa complex) and alpha(v)beta(3) (vitronectin receptor) and as such are inhibitors of platelet aggregation and smooth muscle cell (SMC) migration, both of which are involved in neointimal hyperplasia. The present study was designed to explore the detailed mechanisms of abciximab (Reopro), a monoclonal antibody (mAb) raised against alpha(IIb)beta(3) integrin in neointimal hyperplasia. In this study, carotid arteries of Wistar rats were damaged, and neointimal hyperplasia and lumen occlusion was determined at different time points. Abciximab was administered intravenously by an implanted osmotic pump. Abciximab (0.25 mg/kg/day) time-dependently inhibited both neointimal hyperplasia and lumen occlusion after angioplasty in carotid arteries of rats. Furthermore, the electromicrographs highlighted that SMCs were phenotypically different from the typical contractile, spindle-shaped SMCs normally seen in uninjured vessel walls. Platelet-derived growth factor (PDGF)-BB was strongly produced in thrombus formation and neointimal SMCs after angioplasty, while abciximab significantly reduced PDGF-BB expression in vessel lumens and neointimal SMCs after angioplasty. Balloon angioplasty caused a significant increase of nitrate and cyclic GMP as compared with sham-operated rats. Infusion of abciximab (0.25 mg/kg/day) did not significantly change. Furthermore, the plasma level of thromboxane B(2) (TxB(2)) obviously increased after angioplasty, while abciximab markedly suppressed the elevation of plasma TxB(2) concentration. The results indicate that abciximab effectively prevents neointimal hyperplasia, possibly through the following 2 mechanisms: (1) Abciximab binds to alpha(IIb)beta(3) integrin on platelet membranes resulting in inhibition of platelet adhesion, secretion, and aggregation in injured arteries, followed by inhibition of thromboxane A(2) formation and PDGF-BB release from platelets. (2) Abciximab may also bind to alpha(v)beta(3) integrin on SMCs, thus, subsequently inhibiting cell migration and proliferation.

The antiplatelet activity of tetramethylpyrazine is mediated through activation of NO synthase.

Tetramethylpyrazine (TMPZ) is an active ingredient of a Chinese herbal medicine (Ligusticum wallichii Franchat). In this study, TMPZ (50-200 microM) significantly increased production of nitrate and cyclic GMP in human platelets within a 15-min incubation period. TMPZ concentration-dependently inhibited intracellular Ca2+ mobilization in human platelets stimulated by collagen (5 microg/ml). Furthermore, TMPZ concentration (50 and 200 microM)- and time (15 and 30 min)-dependently triggered endothelial-type constitutive nitric oxide synthase (ecNOS) protein expression in human platelets. These results indicated that TMPZ at micromolar concentrations stimulated nitric oxide production in human platelets via a novel mechanism that activated ecNOS protein expression.

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.

Antiplatelet activity of Staphylococcus aureus lipoteichoic acid is mediated through a cyclic AMP pathway.

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 (i.e., thrombin and collagen). LTA also dose dependently inhibited intracellular Ca(2+) mobilization in human platelets stimulated by collagen. In addition, LTA (0.5 and 1.0 microg/mL) dose dependently increased the formation of cyclic AMP but not cyclic GMP in platelets. LTA (0.5 and 1.0 microg/mL) did not significantly increase the production of nitrate within a 10-min incubation period. Rapid phosphorylation of a platelet protein of M(r) 47,000, a marker of protein kinase C activation, was triggered by PDBu (0.03 microM). This phosphorylation was dose dependently inhibited by LTA (0.5 and 1.0 microg/mL) within a 10-min incubation period. Furthermore, LTA (0.5 and 1.0 microg/mL) also inhibited platelet aggregation induced by PDBu (0.03 microM) in human platelets. These results indicate that the antiplatelet activity of LTA may be involved in the increase of cyclic AMP, leading to inhibition of intracellular Ca(2+) mobilization and protein kinase C activity. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.

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.

Involvement of protein kinase C-gamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells.

The signaling pathway of protein kinase C (PKC) is known to play a role in mediating the action of various cytokines. Here we examined the signal transduction pathway of PKC activation and the role of PKC isoforms in interleukin-1beta (IL-1beta)-mediated cyclooxygenase-2 (COX-2) expression in human pulmonary epithelial cell line (A549). The tyrosine kinase inhibitors (genistein and tyrphostin AG126) and phosphatidylcholine-phospholipase C inhibitor (D-609) prevented IL-1beta-induced prostaglandin E(2) (PGE(2)) release and COX-2 expression, whereas U-73122 (a phosphatidylinositol-phospholipase C inhibitor) and propranolol (a phosphatidate phosphohydrolase inhibitor) had no effect. The PKC inhibitors (Go 6976 and Ro 31-8220) and NF-kappaB inhibitor, pyrrolidine dithiocarbamate, also attenuated IL-1beta-induced PGE(2) release and COX-2 expression. Western blot analysis using PKC isoenzyme-specific antibodies indicated that A549 cells expressed PKC-alpha, -gamma, -iota, -lambda, -zeta, and -micro. IL-1beta caused the translocation of PKC-gamma but not other isoforms from cytosol to the membrane fraction. Moreover, the translocation of PKC-gamma was inhibited by genistein or D-609, but not by U-73122. IL-1beta caused the translocation of p65 NF-kappaB from cytosol to the nucleus as well as the degradation of IkappaB-alpha in cytosol. Furthermore, the translocation of p65 NF-kappaB was inhibited by genistein, Go 6976, Ro 31-8220, or pyrrolidine dithiocarbamate. These results indicate that in human pulmonary epithelial cells, IL-1beta might activate phosphatidylcholine-phospholipase C through an upstream tyrosine phosphorylation to elicit PKC activation, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE(2) release. Of the PKC isoforms present in A549 cells, only activation of PKC-gamma is involved in regulating IL-1beta-induced responses.

The effects of a newly synthesized ATP-sensitive potassium channel opener, MJ-355, on blood pressure and myocardial ischemia-reperfusion injury in rats.

ATP-sensitive potassium (K(ATP)) channel openers, exerting a potent vasodilatory action, are useful in the treatment of cardiovascular disorders; e.g., hypertension and angina pectoris. This study was designed to evaluate the effect of MJ-355 (6-cyano-3,4-trans-3,4-dihydro-2,2-dimethyl-2H-3-hydroxy-4-[2-oxo-5S-(1- ethoxyethoxymethyl)-1-pyrrolidinyl]-1-benzopyran), a newly synthesized K(ATP) channel opener, on hemodynamics in spontaneously hypertensive rats and on myocardial ischemia-reperfusion injury in a rat model of 45 min left coronary artery occlusion followed by 1-h reperfusion. Intravascular injection of MJ-355 (0.005, 0.05 and 0.1 mg/kg) produced a dose-related reduction in mean arterial blood pressure. The depressor effect started 10-15 min after the administration and persisted for more than 3 h and was not accompanied by a reflex tachycardia. In myocardial ischemia, pretreatment of MJ-355 (0.02 mg/kg) significantly reduced the total number of ventricular premature contractions and ventricular tachycardia, total duration of ventricular fibrillation and the mortality. Additionally, a significant reduction in infarct size was noted in all of the MJ-355-treated groups. The hemodynamic and cardioprotective effects of MJ-355 were virtually abolished by pretreating the rats with glibenclamide (4 mg/kg, i.v. bolus), a selective K(ATP) channel blocker. In conclusion, MJ-355, through the activation of K(ATP) channels, exhibited antihypertensive and cardioprotective effects. It is suggested that MJ-355 should be useful in the treatment of hypertension and/or acute myocardial infarction.

The antiplatelet activity of PMC, a potent alpha-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase.

PMC, a potent alpha-tocopherol derivative, dose-dependently (5-25 microM) inhibited the ATP-release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP). PMC also dose-dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen. PMC (10 and 25 microM) significantly inhibited collagen-stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100 microM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200 microM) did not affect the thromboxane synthetase activity of aspirin-treated platelet microsomes. PMC (10 and 25 microM) markedly inhibited the exogenous arachidonic acid (100 microM)-induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600 microM) in washed human platelets, indicating that PMC inhibits cyclo-oxygenase activity. We conclude that PMC may exert its anti-platelet aggregation activity by inhibiting cyclooxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP-release.