calcimycin and dihydrorhodamine-123

Methods currently employed for measuring reactive oxygen species production can lead to both cellular depletion and in artifactual activation. The objective of this study was to design a methodology allowing the measurement of oxidative burst (OB) with minimal sample manipulation.. To that purpose a flow cytometry technique developed in our laboratory, based on nucleic acid staining to discriminate erythrocytes and debris, was employed. DRAQ5 dye and PECy5-CD45 monoclonal antibody (MoAb) were simultaneously used in FL3 to identify the leukocyte population and the PE-CD14 MoAb emission was detected in FL2 for monocytes. OB was measured by using the fluorogenic probe dihydrorhodamine 123, a marker of hydrogen peroxide production. Phorbol myristate acetate (PMA), Opsonized Zymosan (OZ), fMLP and calcium ionophore A23187 activators were also used in this study. For OB assays, dose-response curves were performed for each activator. In addition, the effect of activator concentration on annexin V binding, as a measure of phosphatidylserine translocation, was evaluated.. With this method no-lysis and no-wash steps are required, thus avoiding an unwanted damage to leukocytes. PMA and Zymosan produced an increase in annexin V binding, while fMLP and calcium ionophore did not.. This study reports a feasible and reproducible new flow cytometry assay for assessing OB of neutrophils and monocytes with minimal sample manipulation. In addition, under PMA and OZ conditions, the number of neutrophils showing annexin V binding was strikingly increased. This effect is not related with a phagocytic overstimulation, but with an increased neutrophil-platelet complexes formation.

Topics: Anthraquinones; Antibodies, Monoclonal; Blood Platelets; Calcimycin; Carcinogens; Flow Cytometry; Humans; Ionophores; Leukocyte Common Antigens; Lipopolysaccharide Receptors; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Reactive Oxygen Species; Respiratory Burst; Rhodamines; Sensitivity and Specificity; Tetradecanoylphorbol Acetate; Zymosan

HMGCoA reductase inhibitors (statins) can have effects outside the target tissue, liver, including serious side-effects such as rhabdomyolysis as well as beneficial pleiotrophic effects. One such effect is upregulation of endothelial nitric oxide synthase (e-NOS) which generally leads to vasorelaxation. However, changing the balance between localized NO and O(2-) fluxes can also lead to oxidant stress and cellular injury through formation of reactive secondary oxidants such as peroxynitrite. We compared different statins for e-NOS subcellular localization, formation of pro-oxidants, and endothelial-dependent vascular function. Vascular relaxation in aortas of statin-dosed rats was inhibited with simvastatin (sevenfold higher EC50 for acetyl-choline induced relaxation) and atorvastatin (twofold increase) but not pravastatin. Ex vivo oxidation of the fluorescent redox probe dihydrorhodamine-123 (DHR-123) was increased in aortas from simvastatin treated rats, indicating increased reactive nitrogen and oxygen species. Human aortic endothelial cells incubated with simvastatin exhibited up to threefold higher intracellular oxidation of DHR-123 along with a twofold increase in total e-NOS protein. The elevated e-NOS was found in the Golgi/mitochondrial fraction and not in the plasma membrane, and using immunofluorescence greater e-NOS was observed proximal to Golgi and cytoskeletal structures and away from plasma membrane in simvastatin-treated cells. The data suggest that the action of lipophilic statins in endothelium can shift e-NOS localization towards intracellular domains, thereby increasing the encounter with metabolically generated O(2-) to produce peroxynitrite and related oxidants. Thus, under some conditions the direct action of lipophilic HMGCoA reductase inhibitors may unbalance NO and O(2-) fluxes and promote oxidant stress, compromising potentially beneficial vascular effects of e-NOS upregulation and increasing the potential for damage to muscle and other tissues.

Topics: Animals; Aorta; Atorvastatin; Bradykinin; Calcimycin; Cells, Cultured; Endothelium, Vascular; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Immunohistochemistry; In Vitro Techniques; Lipids; Microscopy, Fluorescence; NAD; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxygen; Pyrroles; Rats; Rats, Sprague-Dawley; Rhodamines; Simvastatin; Subcellular Fractions; Superoxide Dismutase; Up-Regulation; Vasodilation