OBJECTIVE: To explore the main factors of platelet spreading and provide the foundation for related research. METHODS: Platelets (2×10⁷/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×10⁷/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists. RESULTS: Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing. CONCLUSION The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology* ; Adenosine Diphosphate ; Animals ; Blood Platelets/physiology* ; Fibrinogen ; Humans ; Mice ; Mice, Inbred C57BL ; Platelet Adhesiveness/physiology* ; Thrombin/pharmacology*

Propofol is known to cause vasorelaxation of several systemic vascular beds. However, its effect on the pulmonary vasculature remains controversial. In the present study, we investigated the effects of propofol on human pulmonary arteries obtained from patients who had undergone surgery. Arterial rings were mounted in a Multi-Myograph system for measurement of isometric forces. U46619 was used to induce sustained contraction of the intrapulmonary arteries, and propofol was then applied (in increments from 10–300 µM). Arteries denuded of endothelium, preincubated or not with indomethacin, were used to investigate the effects of propofol on isolated arteries. Propofol exhibited a bifunctional effect on isolated human pulmonary arteries contracted by U46619, evoking constriction at low concentrations (10–100 µM) followed by secondary relaxation (at 100–300 µM). The extent of constriction induced by propofol was higher in an endothelium-denuded group than in an endothelium-intact group. Preincubation with indomethacin abolished constriction and potentiated relaxation. The maximal relaxation was greater in the endothelium-intact than the endothelium-denuded group. Propofol also suppressed CaCl₂-induced constriction in the 60 mM K⁺-containing Ca²⁺-free solution in a dose-dependent manner. Fluorescent imaging of Ca²⁺ using fluo-4 showed that a 10 min incubation with propofol (10–300 µM) inhibited the Ca²⁺ influx into human pulmonary arterial smooth muscle cells induced by a 60 mM K⁺-containing Ca²⁺-free solution. In conclusion, propofol-induced arterial constriction appears to involve prostaglandin production by cyclooxygenase in pulmonary artery smooth muscle cells and the relaxation depends in part on endothelial function, principally on the inhibition of calcium influx through L-type voltage-operated calcium channels.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Arteries ; Calcium ; Calcium Channels ; Constriction ; Endothelium ; Humans* ; Indomethacin ; Myocytes, Smooth Muscle ; Propofol* ; Prostaglandin-Endoperoxide Synthases ; Pulmonary Artery* ; Relaxation ; Vasoconstriction* ; Vasodilation

PURPOSE: Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. RESULTS: SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. CONCLUSION: These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Acetylcholine ; Arginase* ; Arginine ; Blotting, Western ; Endothelium ; Human Umbilical Vein Endothelial Cells ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Nitric Oxide Synthase Type III* ; Oxidation-Reduction ; Peroxynitrous Acid ; Phosphorylation* ; Reactive Oxygen Species

BACKGROUND AND OBJECTIVES: Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels play an important role in myocardial protection. We examined the effects of thromboxane A₂ on the regulation of K(ATP) channel activity in single ventricular myocytes. SUBJECTS AND METHODS: Single ventricular myocytes were isolated from the hearts of adult Institute of Cancer Research (ICR) mice by enzymatic digestion. Single channel activity was recorded by excised inside-out and cell-attached patch clamp configurations at -60 mV holding potential during the perfusion of an ATP-free K-5 solution. RESULTS: In the excised inside-out patches, the thromboxane A₂ analog, U46619, decreased the K(ATP) channel activity in a dose-dependent manner; however, the thromboxane A₂ receptor antagonist, SQ29548, did not significantly attenuate the inhibitory effect of U46619. In the cell-attached patches, U46619 inhibited dinitrophenol (DNP)-induced K(ATP) channel activity in a dose-dependent manner, and SQ29548 attenuated the inhibitory effects of U46619 on DNP-induced K(ATP) channel activity. CONCLUSION: Thromboxane A₂ may inhibit K(ATP) channel activity, and may have a harmful effect on ischemic myocardium.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Adenosine Triphosphate ; Adenosine* ; Adult ; Animals ; Digestion ; Heart ; Humans ; KATP Channels ; Mice* ; Muscle Cells* ; Myocardium ; Perfusion ; Potassium Channels* ; Potassium*

BACKGROUND AND OBJECTIVES: Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels play an important role in myocardial protection. We examined the effects of thromboxane A₂ on the regulation of K(ATP) channel activity in single ventricular myocytes. SUBJECTS AND METHODS: Single ventricular myocytes were isolated from the hearts of adult Institute of Cancer Research (ICR) mice by enzymatic digestion. Single channel activity was recorded by excised inside-out and cell-attached patch clamp configurations at -60 mV holding potential during the perfusion of an ATP-free K-5 solution. RESULTS: In the excised inside-out patches, the thromboxane A₂ analog, U46619, decreased the K(ATP) channel activity in a dose-dependent manner; however, the thromboxane A₂ receptor antagonist, SQ29548, did not significantly attenuate the inhibitory effect of U46619. In the cell-attached patches, U46619 inhibited dinitrophenol (DNP)-induced K(ATP) channel activity in a dose-dependent manner, and SQ29548 attenuated the inhibitory effects of U46619 on DNP-induced K(ATP) channel activity. CONCLUSION: Thromboxane A₂ may inhibit K(ATP) channel activity, and may have a harmful effect on ischemic myocardium.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Adenosine Triphosphate ; Adenosine* ; Adult ; Animals ; Digestion ; Heart ; Humans ; KATP Channels ; Mice* ; Muscle Cells* ; Myocardium ; Perfusion ; Potassium Channels* ; Potassium*

OBJECTIVETo determine the effect of resveratrol on constrictions of isolated human intrapulmonary arteries and its mechanisms.

METHODSIntrapulmonary arteries (1-1.5 mm in diameter) were dissected and cut into rings (1.8-2.0 mm in length) under microscope, and were then mounted in a Multi Myograph system. The rings were stimulated with 100 nmol/L U46619, 30 nmol/L endothelin-1, or 60 mmol/L KCl to produce sustained contraction of the intrapulmonary arteries, after which resveratrol was applied cumulatively. Endothelium denudation, L-NAME and indomethecin were used to investigate the effect of resveratrol on constrictions of the isolated arteries, suing DMSO as the control.

RESULTSResveratrol induced concentration-dependent relaxations in endothelium-intact rings that contracted in response to stimulations with U46619, ET-1 and KCl, with pD2 of 3.82±0.20, 3.84±0.57, and 3.68±0.27, Emax of (99.58±0.83)%, 100%, and (99.65±0.98)%, respectively. Treatment of the arterial rings with the eNOS inhibitor L-NAME, but not with indomethecin or endothelium denudation, obviously affected the relaxant effects of resveratrol.

CONCLUSIONResveratrol can concentration-dependently produce relaxant effect on human intrapulmonary arteries independent of the endothelium possibly by promoting synthesis and release of NO.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; pharmacology ; Humans ; In Vitro Techniques ; Pulmonary Artery ; drug effects ; Stilbenes ; pharmacology ; Vasoconstriction ; drug effects

Coronary artery disease is a common occurrence in human, and causes enormous social cost. Poncirus fructus (PF), the dried immature fruits of Poncirus trifoliata Rafinesquem, is used in the treatment of womb contraction and dyspepsia, as a prokinetic, and in improving blood circulation. This study was performed to investigate the effects of PF and some of its flavonoids components on the coronary from the pig. The arterial ring was suspended by a pair of stainless steel stirrups in an organ bath. The end of the upper stirrup was connected to an isometric force transducer. A dose-dependent induction of relaxation was observed by both water and 70% ethanol extracts of PF in the porcine coronary artery precontracted with U46619 (100 nM), a stable analogue of the potent vasoconstrictor thromboxane A2. The 70% ethanol extract showed more efficacy than the water extract. Pretreatment of the artery with L-NAME (100 microM), a nitric oxide synthase inhibitor, resulted in a significant reduction in the relaxation induced by PF extract. In addition, ODQ (10 microM), a soluble guanylate cyclase inhibitor, also significantly reduced the effects of PF extracts. Hesperidin, a flavonoid present in PF, induced very weak relaxation of the porcine coronary artery at a high concentration (100 microM), while its aglycone, hesperetin, demonstrated a dose-dependent relaxation. In conclusion, PF extracts induced relaxation in the porcine coronary artery, partially through the nitric oxide-cGMP pathway, and the aglycones of flavonoids might be also involved in the relaxation of the same artery.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Arteries ; Baths ; Blood Circulation ; Coronary Artery Disease ; Coronary Vessels* ; Dyspepsia ; Ethanol ; Flavonoids ; Fruit ; Guanylate Cyclase ; Hesperidin ; Humans ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase ; Poncirus* ; Relaxation ; Stainless Steel ; Thromboxane A2 ; Transducers ; Water

Propofol is a widely used anesthetic. Many studies have shown that propofol has direct effects on blood vessels, but the precise mechanism is not fully understood. Secondary intrapulmonary artery rings from male rats were prepared and mounted in a Multi Myograph System. The following constrictors were used to induce contractions in isolated artery rings: high K+ solution (60 mmol/L); U46619 solution (100 nmol/L); 5-hydroxytryptamine (5-HT; 3 micromol/L); or phenylephrine (Phe; 1 micromol/L). The relaxation effects of propofol were tested on high K+ or U46619 precontracted rings. Propofol also was added to induce relaxation of rings preconstricted by U46619 after pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). The effects of propofol on Ca2+ influx via the L-type Ca2+ channels were evaluated by examining contraction-dependent responses to CaCl2 in the absence or presence of propofol (10 to 300 micromol/L). High K+ solution and U46619 induced remarkable contractions of the rings, whereas contractions induced by 5-HT and Phe were weak. Propofol induced dose-dependent relaxation of artery rings precontracted by the high K+ solution. Propofol also induced relaxation of rings precontracted by U46619 in an endothelium-independent way. Propofol at different concentrations significantly inhibited the Ca2+-induced contractions of pulmonary rings exposed to high K+-containing and Ca2+-free solution in a dose-dependent manner. Propofol relaxed vessels precontracted by the high K+ solution and U46619 in an endothelium-independent way. The mechanism for this effect may involve inhibition of calcium influx through voltage-operated calcium channels (VOCCs) and receptor-operated calcium channels (ROCCs).
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Animals ; Arteries* ; Blood Vessels ; Calcium ; Calcium Channels ; Endothelium ; Humans ; Male ; NG-Nitroarginine Methyl Ester ; Nitric Oxide Synthase ; Phenylephrine ; Propofol* ; Pulmonary Artery ; Rats* ; Relaxation ; Serotonin

OBJECTIVETo investigate the reactivity of intrapulmonary arterial rings to vasoactive substances as thromboxane A2 and endothelin-1 in patients with chronic obstructive pulmonary disease (COPD).

METHODSIntrapulmonary arterial rings isolated from patients with normal lung function and COPD were mounted in a Multi Myograph system to determine the reactivity of the intrapulmonary arterial rings to 60 mmol/L KCl, thromboxane A2 analogue U46619 and endothelin-1 before and after preconditioning with the COX synthase inhibitor indomethacin.

RESULTSThe reactivity of intrapulmonary arterial rings to U46619 and endothelin-1 was significantly decreased in patients with COPD. The reactivity to U46619 was dramatically decreased in patients with normal lung function after application of indomethacin.

CONCLUSIONThe reactivity of intrapulmonary arterial rings is significantly decreased in patients with COPD.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; pharmacology ; Aged ; Endothelin-1 ; metabolism ; Female ; Humans ; In Vitro Techniques ; Indomethacin ; pharmacology ; Male ; Middle Aged ; Pulmonary Artery ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; Thromboxane A2 ; metabolism

Hypoxic pulmonary vasoconstriction (HPV) is physiologically important response for preventing mismatching between ventilation and perfusion in lungs. The HPV of isolated pulmonary arteries (HPV-PA) usually require a partial pretone by thromboxane agonist (U46619). Because the HPV of ventilated/perfused lungs (HPV-lung) can be triggered without pretone conditioning, we suspected that a putative tissue factor might be responsible for the pretone of HPV. Here we investigated whether HPV can be also observed in precision-cut lung slices (PCLS) from rats. The HPV in PCLS also required partial contraction by U46619. In addition, K+ channel blockers (4AP and TEA) required U46619-pretone to induce significant contraction of PA in PCLS. In contrast, the airways in PCLS showed reversible contraction in response to the K+ channel blockers without pretone conditioning. Also, the airways showed no hypoxic constriction but a relaxation under the partial pretone by U46619. The airways in PCLS showed reliable, concentration-dependent contraction by metacholine (EC50, ~210 nM). In summary, the HPV in PCLS is more similar to isolated PA than V/P lungs. The metacholine-induced constriction of bronchioles suggested that the PLCS might be also useful for studying airway physiology in situ.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Animals ; Bronchioles ; Constriction ; Contracts ; Lung ; Perfusion ; Pulmonary Artery ; Rats ; Relaxation ; Thromboplastin ; Thromboxane A2 ; Vasoconstriction ; Ventilation