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1.
Cardiovasc Diabetol ; 11: 5, 2012 Jan 16.
Article in English | MEDLINE | ID: mdl-22248260

ABSTRACT

BACKGROUND: Adiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats. METHODS: Male Wistar rats were fed for 10 weeks with standard chow (SCD) or high-fat diet (HFD). ADP (10 µM)- and thrombin (100 mU/ml)-induced washed platelet aggregation were evaluated. Measurement of intracellular levels of ROS levels was carried out using flow cytometry. Cyclic GMP levels were evaluated using ELISA kits. RESULTS: High-fat fed rats exhibited significant increases in body weight, epididymal fat, fasting glucose levels and glucose intolerance compared with SCD group. Platelet aggregation induced by ADP (n = 8) and thrombin from HFD rats (n = 8) were significantly greater (P < 0.05) compared with SCD group. Platelet activation with ADP increased by 54% the intraplatelet ROS production in HFD group, as measured by flow cytometry (n = 6). N-acetylcysteine (NAC; 1 mM) and PEG-catalase (1000 U/ml) fully prevented the increased ROS production and platelet hyperaggregability in HFD group. The NO donors sodium nitroprusside (SNP; 10 µM) and SNAP (10 µM), as well as the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 (10 µM) inhibited the platelet aggregation in HFD group with lower efficacy (P < 0.05) compared with SCD group. The cGMP levels in response to these agents were also markedly lower in HFD group (P < 0.05). The prostacyclin analogue iloprost (1 µM) reduced platelet aggregation in HFD and SCD rats in a similar fashion (n = 4). CONCLUSIONS: Metabolic abnormalities as consequence of HFD cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability that appear to be accompanied by potential defects in the prosthetic haem group of soluble guanylyl cyclase.


Subject(s)
Blood Platelets/metabolism , Diet, High-Fat/adverse effects , Oxidative Stress , Platelet Aggregation , Reactive Oxygen Species/blood , Adenosine Diphosphate , Animals , Antioxidants/pharmacology , Blood Platelets/drug effects , Cyclic GMP/blood , Enzyme Activation , Enzyme Activators/pharmacology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Glucose Intolerance/blood , Glucose Intolerance/etiology , Glucose Tolerance Test , Guanylate Cyclase/blood , Insulin Resistance , Male , Nitric Oxide/blood , Nitric Oxide Donors/pharmacology , Oxidative Stress/drug effects , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Function Tests , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/blood , Signal Transduction , Soluble Guanylyl Cyclase , Thrombin , Time Factors , Weight Gain
2.
Pulm Pharmacol Ther ; 23(4): 327-33, 2010 08.
Article in English | MEDLINE | ID: mdl-20307679

ABSTRACT

There is considerable evidence that platelet activation occurs in allergic airways diseases. In this study we aimed to investigate platelet adhesion to immobilized fibrinogen and intracellular calcium levels in a rat model of allergic inflammation. Male Wistar rats were challenged with ovalbumin (OVA). At 30 min to 24h after OVA-challenge, assays of platelet adhesion to immobilized fibrinogen and intracellular calcium levels using fura 2-AM loaded platelets were performed. The serum levels of IgE were approximately 5-fold greater in OVA-sensitized rats. A marked eosinophil influx in bronchoalveolar lavage (BAL) fluid of OVA-challenged rats at 24h after OVA-challenge was also seen. OVA-challenge resulted in a marked thrombocytopenia, as observed within 12h after OVA-challenge. The agonists ADP (0.5-50 microM) and thrombin (30-100 mU/ml) concentration-dependently increased platelet adhesion to immobilized fibrinogen. At an early time after OVA-challenge (30 min), platelets exhibited greater platelet adhesion compared with the non-sensitized group, whereas at a late time (24h) they exhibited lower platelet adhesion to both agonists. Moreover, at 30 min after OVA-challenge, intracellular calcium levels to ADP (20 microM) and thrombin (100 mU/ml)-activated platelets were greater compared with non-challenged rats. As opposed, at 24h after OVA challenge, a lower intracellular calcium level to ADP- and thrombin-activated platelets was observed. In conclusion, OVA-challenge in rats promotes a biphasic response in platelet adhesion consisting of an increased adhesion and intracellular calcium levels at an early phase (30 min), which progress to a reduction in adhesion and intracellular calcium levels at a late time (24h) after antigen challenge.


Subject(s)
Calcium/metabolism , Inflammation/immunology , Ovalbumin/immunology , Platelet Adhesiveness/immunology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/pharmacology , Animals , Bronchoalveolar Lavage Fluid/immunology , Calcium/immunology , Disease Models, Animal , Dose-Response Relationship, Drug , Fibrinogen/metabolism , Immunoglobulin E/blood , Immunoglobulin E/immunology , Lung/immunology , Lung/physiopathology , Male , Rats , Rats, Wistar , Thrombin/pharmacology , Thrombocytopenia/immunology , Time Factors
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