title: Detection of nitric oxide production in cell cultures by luciferin-luciferase chemiluminescence.
abstract: A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin-luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca(2+) ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 10(2)-10(4) cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).
authors.affiliation: Department of Chemistry, Valdosta State University, Valdosta, GA 31698, USA. Electronic address: ywoldman@valdosta.edu.
authors.forename: Yakov Y
authors.initials: YY
authors.lastname: Woldman
authors.affiliation: Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Tim D
authors.initials: TD
authors.lastname: Eubank
authors.affiliation: Department of Biology, Valdosta State University, Valdosta, GA 31698, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Mock
authors.affiliation: Department of Biology, Valdosta State University, Valdosta, GA 31698, USA.
authors.forename: Natalia C
authors.initials: NC
authors.lastname: Stevens
authors.affiliation: Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Saradhadevi
authors.initials: S
authors.lastname: Varadharaj
authors.affiliation: Department of Biology, Valdosta State University, Valdosta, GA 31698, USA.
authors.forename: Jenifer
authors.initials: J
authors.lastname: Turco
authors.affiliation: Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Gavrilin
authors.affiliation: Department of Chemistry, Connecticut College, New London, CT 06320, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Branchini
authors.affiliation: Davis Heart & Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Valery V
authors.initials: VV
authors.lastname: Khramtsov
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001681:Biological Assay; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D049454:Firefly Luciferin / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D049449:Luminescence; D008163:Luminescent Measurements / Q000592:standards*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis* / Q000096:biosynthesis; D020030:Nitric Oxide Donors / Q000378:metabolism / Q000494:pharmacology; D009573:Nitrites / Q000737:chemistry / Q000378:metabolism; D009603:Nitroso Compounds / Q000378:metabolism / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D012680:Sensitivity and Specificity; D000071756:Soluble Guanylyl Cyclase; D013430:Sulfate Adenylyltransferase / Q000378:metabolism
chemical_list: D011756:Diphosphates; D008070:Lipopolysaccharides; D020030:Nitric Oxide Donors; D009573:Nitrites; D009603:Nitroso Compounds; D018160:Receptors, Cytoplasmic and Nuclear; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009569:Nitric Oxide; C107241:diphosphoric acid; D049454:Firefly Luciferin; D006160:Guanosine Triphosphate; D008156:Luciferases; D013430:Sulfate Adenylyltransferase; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP; D001920:Bradykinin
keywords: Endothelial cells; Guanylyl cyclase; Macrophages; Nitric oxide; Pyrophosphate; Thermostable luciferase