Journal of Leukocyte Biology, Vol 55, Issue 2 253-258, Copyright © 1994 by Society for Leukocyte Biology
JOURNAL ARTICLE |
WO Carter, PK Narayanan and JP Robinson
Purdue University Cytometry Laboratories, Department of Veterinary Physiology and Pharmacology, West Lafayette, IN 47907.
One of the objectives of studying endothelial cells in vitro is to evaluate neutrophil-endothelial cell interactions including potential consequences of oxidant-mediated damage to the endothelial cell. Current understanding of endothelial cell oxidative function is derived primarily from the measurement of extracellular products. We utilized 2 dyes, 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), which measure hydrogen peroxide (H2O2) and superoxide anion (O2-) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH-DA and stimulated with H2O2 exhibited an increase in the fluorescent product 2',7'-dichlorofluorescein (DCF) (measure of intracellular H2O2) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H2O2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O2-) which lasted for approximately 60 min. Superoxide dismutase increased DCF fluorescence in endothelial cells stimulated with H2O2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduced DCF and EB fluorescence by 48% and 37% respectively in endothelial cells stimulated with H2O2. Catalase completely inhibited an increase in DCF or EB fluorescence in endothelial cells stimulated with H2O2. There was a direct correlation between mean DCF and EB fluorescence intensity and the concentration of H2O2 or the number of phorbol 12-myristate 13-acetate-activated neutrophils added to endothelial cells. We conclude from these studies that DCFH-DA and HE can be used to measure intracellular H2O2 and O2- in endothelial cells and that the xanthine oxidase pathway for intracellular O2- production accounts for approximately 40% of the total intracellular O2- generated in endothelial cells after stimulation with H2O2. The combination of image cytometry and flow cytometry will be important for future evaluations of endothelial cell function.
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M. H. Ali, S. A. Schlidt, N. S. Chandel, K. L. Hynes, P. T. Schumacker, and B. L. Gewertz Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction Am J Physiol Lung Cell Mol Physiol, November 1, 1999; 277(5): L1057 - L1065. [Abstract] [Full Text] [PDF]
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Z. Wei, K. Costa, A. B. Al-Mehdi, C. Dodia, V. Muzykantov, and A. B. Fisher Simulated Ischemia in Flow-Adapted Endothelial Cells Leads to Generation of Reactive Oxygen Species and Cell Signaling Circ. Res., October 15, 1999; 85(8): 682 - 689. [Abstract] [Full Text] [PDF]
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D. Nethery, A. DiMarco, D. Stofan, and G. Supinski Sepsis increases contraction-related generation of reactive oxygen species in the diaphragm J Appl Physiol, October 1, 1999; 87(4): 1279 - 1286. [Abstract] [Full Text] [PDF]
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C. Rota, Y. C. Fann, and R. P. Mason Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2',7'-Dichlorofluorescein by Horseradish Peroxidase. POSSIBLE CONSEQUENCES FOR OXIDATIVE STRESS MEASUREMENTS J. Biol. Chem., October 1, 1999; 274(40): 28161 - 28168. [Abstract] [Full Text] [PDF]
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K. K. KININGHAM, T. D. OBERLEY, S.-M. LIN, C. A. MATTINGLY, and D. K. ST. CLAIR Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death FASEB J, September 1, 1999; 13(12): 1601 - 1610. [Abstract] [Full Text]
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D. Nethery, D. Stofan, L. Callahan, A. DiMarco, and G. Supinski Formation of reactive oxygen species by the contracting diaphragm is PLA2 dependent J Appl Physiol, August 1, 1999; 87(2): 792 - 800. [Abstract] [Full Text] [PDF]
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C. Patterson, J. Ruef, N. R. Madamanchi, P. Barry-Lane, Z. Hu, C. Horaist, C. A. Ballinger, A. R. Brasier, C. Bode, and M. S. Runge Stimulation of a Vascular Smooth Muscle Cell NAD(P)H Oxidase by Thrombin. EVIDENCE THAT p47phox MAY PARTICIPATE IN FORMING THIS OXIDASE IN VITRO AND IN VIVO J. Biol. Chem., July 9, 1999; 274(28): 19814 - 19822. [Abstract] [Full Text] [PDF]
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J. A. Hoffman, C. Wass, M. F. Stins, and K. S. Kim The Capsule Supports Survival but Not Traversal of Escherichia coli K1 across the Blood-Brain Barrier Infect. Immun., July 1, 1999; 67(7): 3566 - 3570. [Abstract] [Full Text] [PDF]
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S. R. RÄISÄNEN, P. LEHENKARI, M. TASANEN, P. RAHKILA, P. L. HÄRKÖNEN, and H. K. VÄÄNÄNEN Carbonic anhydrase III protects cells from hydrogen peroxide-induced apoptosis FASEB J, March 1, 1999; 13(3): 513 - 522. [Abstract] [Full Text]
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J. Li, J. H. Pak, F. L. Huang, and K.-P. Huang N-Methyl-D-aspartate Induces Neurogranin/RC3 Oxidation in Rat Brain Slices J. Biol. Chem., January 15, 1999; 274(3): 1294 - 1300. [Abstract] [Full Text] [PDF]
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T. L. Vanden Hoek, L. B. Becker, Z. Shao, C. Li, and P. T. Schumacker Reactive Oxygen Species Released from Mitochondria during Brief Hypoxia Induce Preconditioning in Cardiomyocytes J. Biol. Chem., July 17, 1998; 273(29): 18092 - 18098. [Abstract] [Full Text] [PDF]
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F. J. Miller, D. D. Gutterman, C. D. Rios, D. D. Heistad, and B. L. Davidson Superoxide Production in Vascular Smooth Muscle Contributes to Oxidative Stress and Impaired Relaxation in Atherosclerosis Circ. Res., June 29, 1998; 82(12): 1298 - 1305. [Abstract] [Full Text] [PDF]
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G. W. De Keulenaer, D. C. Chappell, N. Ishizaka, R. M. Nerem, R. W. Alexander, and K. K. Griendling Oscillatory and Steady Laminar Shear Stress Differentially Affect Human Endothelial Redox State : Role of a Superoxide-Producing NADH Oxidase Circ. Res., June 1, 1998; 82(10): 1094 - 1101. [Abstract] [Full Text] [PDF]
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J. E. Hong, L. A. Santucci, X. Tian, and D. J. Silverman Superoxide Dismutase-Dependent, Catalase-Sensitive Peroxides in Human Endothelial Cells Infected by Rickettsia rickettsii Infect. Immun., April 1, 1998; 66(4): 1293 - 1298. [Abstract] [Full Text] [PDF]
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Z. Spolarics and J.-X. Wu Role of glutathione and catalase in H2O2 detoxification in LPS-activated hepatic endothelial and Kupffer cells Am J Physiol Gastrointest Liver Physiol, December 1, 1997; 273(6): G1304 - G1311. [Abstract] [Full Text] [PDF]
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R. G. Allen, B. P. Keogh, M. Tresini, G. S. Gerhard, C. Volker, R. J. Pignolo, J. Horton, and V. J. Cristofalo Development and Age-associated Differences in Electron Transport Potential and Consequences for Oxidant Generation J. Biol. Chem., October 3, 1997; 272(40): 24805 - 24812. [Abstract] [Full Text] [PDF]
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A. Martin and B. Frei Both Intracellular and Extracellular Vitamin C Inhibit Atherogenic Modification of LDL by Human Vascular Endothelial Cells Arterioscler Thromb Vasc Biol, August 1, 1997; 17(8): 1583 - 1590. [Abstract] [Full Text]
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H. Suzuki, A. Swei, B. W. Zweifach, and G. W. Schmid-Schonbein In Vivo Evidence for Microvascular Oxidative Stress in Spontaneously Hypertensive Rats : Hydroethidine Microfluorography Hypertension, May 1, 1995; 25(5): 1083 - 1089. [Abstract] [Full Text]
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K. A. Pritchard Jr., A. W. Ackerman, E. R. Gross, D. W. Stepp, Y. Shi, J. T. Fontana, J. E. Baker, and W. C. Sessa Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase J. Biol. Chem., May 18, 2001; 276(21): 17621 - 17624. [Abstract] [Full Text] [PDF]
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N. Nieto, P. Greenwel, S. L. Friedman, F. Zhang, A. J. Dannenberg, and A. I. Cederbaum Ethanol and Arachidonic Acid Increase alpha 2(I) Collagen Expression in Rat Hepatic Stellate Cells Overexpressing Cytochrome P450 2E1. ROLE OF H2O2 AND CYCLOOXYGENASE-2 J. Biol. Chem., June 23, 2000; 275(26): 20136 - 20145. [Abstract] [Full Text] [PDF]
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N. S. Chandel, D. S. McClintock, C. E. Feliciano, T. M. Wood, J. A. Melendez, A. M. Rodriguez, and P. T. Schumacker Reactive Oxygen Species Generated at Mitochondrial Complex III Stabilize Hypoxia-inducible Factor-1alpha during Hypoxia. A MECHANISM OF O2 SENSING J. Biol. Chem., August 11, 2000; 275(33): 25130 - 25138. [Abstract] [Full Text] [PDF]
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S. Sanlioglu, C. M. Williams, L. Samavati, N. S. Butler, G. Wang, P. B. McCray Jr., T. C. Ritchie, G. W. Hunninghake, E. Zandi, and J. F. Engelhardt Lipopolysaccharide Induces Rac1-dependent Reactive Oxygen Species Formation and Coordinates Tumor Necrosis Factor-alpha Secretion through IKK Regulation of NF-kappa B J. Biol. Chem., August 3, 2001; 276(32): 30188 - 30198. [Abstract] [Full Text] [PDF]
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K. Nakamura, V. P. Bindokas, D. Kowlessur, M. Elas, S. Milstien, J. D. Marks, H. J. Halpern, and U. J. Kang Tetrahydrobiopterin Scavenges Superoxide in Dopaminergic Neurons J. Biol. Chem., September 7, 2001; 276(37): 34402 - 34407. [Abstract] [Full Text] [PDF]
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V. H. Guaiquil, J. C. Vera, and D. W. Golde Mechanism of Vitamin C Inhibition of Cell Death Induced by Oxidative Stress in Glutathione-depleted HL-60 Cells J. Biol. Chem., October 26, 2001; 276(44): 40955 - 40961. [Abstract] [Full Text] [PDF]
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R. S. Arnold, J. Shi, E. Murad, A. M. Whalen, C. Q. Sun, R. Polavarapu, S. Parthasarathy, J. A. Petros, and J. D. Lambeth Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1 PNAS, May 8, 2001; 98(10): 5550 - 5555. [Abstract] [Full Text] [PDF]
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N. Li, F.-X. Yi, J. L. Spurrier, C. A. Bobrowitz, and A.-P. Zou Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney Am J Physiol Renal Physiol, June 1, 2002; 282(6): F1111 - F1119. [Abstract] [Full Text] [PDF]
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S. Rajagopalan, D. Duquaine, S. King, B. Pitt, and P. Patel Mineralocorticoid Receptor Antagonism in Experimental Atherosclerosis Circulation, May 7, 2002; 105(18): 2212 - 2216. [Abstract] [Full Text] [PDF]
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