An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts
Affiliation(s)
Department of Chemistry, Sewanee: The University of the South, Sewanee, TN, USA. Department of Biology, Western Kentucky University, Bowling Green, KY, USA.
Department of Chemistry, Sewanee: The University of the South, Sewanee, TN, USA. Department of Biology, Western Kentucky University, Bowling Green, KY, USA.
ABSTRACT
Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.
Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NOX) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.
Cite this paper
Sharma, B. , Rowland, N. , Clouse, M. and Rice, N. (2014) An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts. Advances in Biological Chemistry, 4, 214-221. doi: 10.4236/abc.2014.43026.
Sharma, B. , Rowland, N. , Clouse, M. and Rice, N. (2014) An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts. Advances in Biological Chemistry, 4, 214-221. doi: 10.4236/abc.2014.43026.
References
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http://dx.doi.org/10.1038/ki.1992.75 [2] Powell, D.W., et al. (1999) Myofibroblasts. I. Paracrine Cells Important in Health and Disease. American Journal of Physiology, 277, C1-C9.
http://dx.doi.org/10.1111/j.1469-7793.1999.001af.x [3] Phan, S.H. (2002) The Myofibroblast in Pulmonary Fibrosis. Chest, 122, 286S-289S.
http://dx.doi.org/10.1378/chest.122.6_suppl.286S [4] Gabbiani, G. (1999) Some Historical and Philosophical Reflections on the Myofibroblast Concept. Current Topics in Pathology, 93, 1-5.
http://dx.doi.org/10.1007/978-3-642-58456-5_1 [5] Gabbiani, G. (1996) The Cellular Derivation and the Life Span of the Myofibroblast. Pathology—Research and Practice, 192, 708-711.
http://dx.doi.org/10.1016/S0344-0338(96)80092-6 [6] Schmitt-Graff, A., Desmouliere, A. and Gabbiani, G. (1994) Heterogeneity of Myofibroblast Phenotypic Features: An Example of Fibroblastic Cell Plasticity. Virchows Archiv, 425, 3-24.
http://dx.doi.org/10.1007/BF00193944 [7] Schurch, W., Seemayer, T.A. and Gabbiani, G. (1998) The Myofibroblast: A Quarter Century after Its Discovery. The American Journal of Surgical Pathology, 22, 141-147.
http://dx.doi.org/10.1097/00000478-199802000-00001 [8] Gentiloni Silveri, N., Mazzone, M., Portale, G. and Carbone, L. (2001) Nitric Oxide. A General Review about the Different Roles of This Innocent Radical. Minerva Medica, 92, 167-171. [9] Moncada, S. and Higgs, A. (1993) The L-Arginine-Nitric Oxide Pathway. The New England Journal of Medicine, 329, 2002-2012.
http://dx.doi.org/10.1056/NEJM199312303292706 [10] Knowles, R.G. and Moncada, S. (1994) Nitric Oxide Synthases in Mammals. Biochemical Journal, 298, 249-58. [11] Forstermann, U., Boissel, J.P. and Kleinert, H. (1998) Expressional Control of the “Constitutive” Isoforms of Nitric Oxide Synthase (NOS I and NOS III). The FASEB Journal, 12, 773-790. [12] Arnal, J.F., Dinh-Xuan, A.T., Pueyo, M., Darblade, B. and Rami, J. (1999) Endothelium-Derived Nitric Oxide and Vascular Physiology and Pathology. Cellular and Molecular Life Sciences, 55, 1078-1087.
http://dx.doi.org/10.1007/s000180050358 [13] Vyas-Read, S., Shaul, P.W., Yuhanna, I.S. and Willis, B.C. (2007) Nitric Oxide Attenuates Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells. American Journal of Physiology—Lung Cellular and Molecular Physiology, 293, L212-L221.
http://dx.doi.org/10.1152/ajplung.00475.2006 [14] Kordes, C., Sawitza, I., Müller-Marbach, A., Ale-Agha, N., Keitel, V., Klonowski-Stumpe, H. and Häussinger, D. (2007) CD133+ Hepatic Stellate Cells Are Progenitor Cells. Biochem. Biochemical and Biophysical Research Communications, 352, 410-417.
http://dx.doi.org/10.1016/j.bbrc.2006.11.029 [15] Mookerjee, I., Hewitson, T.D., Halls, M.L., Summers, R.J., Mathai, M.L., Bathgate, R.A., Tregear, G.W. and Samuel, C.S. (2009) Relaxin Inhibits Renal Myofibroblast Differentiation via RXFP1, the Nitric Oxide Pathway, and Smad2. The FASEB Journal, 23, 1219-1229.
http://dx.doi.org/10.1096/fj.08-120857 [16] Lu, L., Chen, S.S., Hassid, A. and Sun, Y. (2008) Cardiac Fibrogenesis Following Infarction in Mice with Deletion of Inducible Nitric Oxide Synthase. American Journal of the Medical Sciences, 335, 431-438.
http://dx.doi.org/10.1097/MAJ.0b013e3181571f97 [17] Pessanh, M.G. and Mandarim-de-Lacerda, C.A. (2000) Myofibroblast Accumulation in Healing Rat Myocardium Due to Long-Term Low-Dosage Nitric Oxide Synthesis Inhibition. Experimental and Toxicologic Pathology, 52, 192-194.
http://dx.doi.org/10.1016/S0940-2993(00)80027-5 [18] Gonzalez-Cadavid, N.F. and Rajfer, J. (2009) Experimental Models of Peyronie’s Disease. Implications for New Therapies. Journal of Sexual Medicine, 6, 303-313.
http://dx.doi.org/10.1111/j.1743-6109.2008.01104.x [19] Hall, C.N. and Garthwaite, J. (2009) What Is the Real Physiological NO Concentration in Vivo? Nitric Oxide, 21, 92-103.
http://dx.doi.org/10.1016/j.niox.2009.07.002 [20] Taha, Z.H. (2003) Nitric Oxide Measurements in Biological Samples. Talanta, 61, 3-10.
http://dx.doi.org/10.1016/S0039-9140(03)00354-0 [21] Räthel, T.R., Leikert, J., Vollmar, A.M. and Dirsch, V.M. (2003) Application of 4,5-Diaminofluorescein to Reliably Measure Nitric Oxide Released from Endothelial Cells in Vitro. Biological Procedures Online, 5, 136-142.
http://dx.doi.org/10.1251/bpo55 [22] Leikert, J.F., Räthel, T.R., Müller, C., Vollmar, A.M. and Dirsch, V.M. (2001) Reliable in Vitro Measurement of Nitric Oxide Released from Endothelial Cells Using Low Concentrations of the Fluorescent Probe 4,5-Diaminofluorescein. FEBS Letters, 506, 131-134.
http://dx.doi.org/10.1016/S0014-5793(01)02901-5 [23] Boo, Y.C., Tressel, S.L. and Jo, H. (2007) An Improved Method to Measure Nitrate/Nitrite with an NO-Selective Electrochemical Sensor. Nitric Oxide, 16, 306-312.
http://dx.doi.org/10.1016/j.niox.2006.09.004 [24] Berkels, R.S., Purol-Schnabel, S. and Roesen, R. (2001) A New Method to Measure Nitrate/Nitrite with a NO-Sensitive Electrode. Journal of Applied Physiology, 90, 317-320. [25] McIntosh, J.C., Hagood, J.S., Richardson, T.L. and Simecka, J.W. (1994) Thy1 (+) and (-) Lung Fibrosis Subpopulations in LEW and F344 Rats. European Respiratory Journal, 7, 2131-2138.
http://dx.doi.org/10.1183/09031936.94.07122131 [26] Grinnell, F. (1994) Fibroblasts, Myofibroblasts, and Wound Contraction. Journal of Cell Biology, 124, 401-404.
http://dx.doi.org/10.1083/jcb.124.4.401 [27] Rice, N.A. and Leinwand, L.A. (2003) Skeletal Myosin Heavy Chain Function in Cultured Lung Myofibroblasts. Journal of Cell Biology, 163, 119-129.
http://dx.doi.org/10.1083/jcb.200303194 [28] Griffith, O.W. and Kilbourn, R.G. (1996) Nitric Oxide Synthase Inhibitors: Amino Acids. Methods in Enzymology, 268, 375-392.
http://dx.doi.org/10.1016/S0076-6879(96)68040-9 [29] Jagnandan, D., Sessa, W.C. and Fulton, D. (2005) Intracellular Location Regulates Calcium-Calmodulin Dependent Activation of Organelle-Restricted eNOS. American Journal of Physiology: Cell Physiology, 289, C1024-C1033.
http://dx.doi.org/10.1152/ajpcell.00162.2005 [30] Hunter, R.A., Storm, W.L., Coneski, P.N. and Schoenfisch, M.H. (2013) Inaccuracies of Nitric Oxide Measurement Methods in Biological Media. Analytical Chemistry, 85, 1957-1963.
http://dx.doi.org/10.1021/ac303787p [31] Dudzinski, D. and Michel, T. (2007) Life History of eNOS: Partners and Pathways. Cardiovascular Research, 75, 247-260.
http://dx.doi.org/10.1016/j.cardiores.2007.03.023
[1] Gabbiani, G. (1992) The Biology of the Myofibroblast. Kidney International, 41, 530-532.
http://dx.doi.org/10.1038/ki.1992.75 [2] Powell, D.W., et al. (1999) Myofibroblasts. I. Paracrine Cells Important in Health and Disease. American Journal of Physiology, 277, C1-C9.
http://dx.doi.org/10.1111/j.1469-7793.1999.001af.x [3] Phan, S.H. (2002) The Myofibroblast in Pulmonary Fibrosis. Chest, 122, 286S-289S.
http://dx.doi.org/10.1378/chest.122.6_suppl.286S [4] Gabbiani, G. (1999) Some Historical and Philosophical Reflections on the Myofibroblast Concept. Current Topics in Pathology, 93, 1-5.
http://dx.doi.org/10.1007/978-3-642-58456-5_1 [5] Gabbiani, G. (1996) The Cellular Derivation and the Life Span of the Myofibroblast. Pathology—Research and Practice, 192, 708-711.
http://dx.doi.org/10.1016/S0344-0338(96)80092-6 [6] Schmitt-Graff, A., Desmouliere, A. and Gabbiani, G. (1994) Heterogeneity of Myofibroblast Phenotypic Features: An Example of Fibroblastic Cell Plasticity. Virchows Archiv, 425, 3-24.
http://dx.doi.org/10.1007/BF00193944 [7] Schurch, W., Seemayer, T.A. and Gabbiani, G. (1998) The Myofibroblast: A Quarter Century after Its Discovery. The American Journal of Surgical Pathology, 22, 141-147.
http://dx.doi.org/10.1097/00000478-199802000-00001 [8] Gentiloni Silveri, N., Mazzone, M., Portale, G. and Carbone, L. (2001) Nitric Oxide. A General Review about the Different Roles of This Innocent Radical. Minerva Medica, 92, 167-171. [9] Moncada, S. and Higgs, A. (1993) The L-Arginine-Nitric Oxide Pathway. The New England Journal of Medicine, 329, 2002-2012.
http://dx.doi.org/10.1056/NEJM199312303292706 [10] Knowles, R.G. and Moncada, S. (1994) Nitric Oxide Synthases in Mammals. Biochemical Journal, 298, 249-58. [11] Forstermann, U., Boissel, J.P. and Kleinert, H. (1998) Expressional Control of the “Constitutive” Isoforms of Nitric Oxide Synthase (NOS I and NOS III). The FASEB Journal, 12, 773-790. [12] Arnal, J.F., Dinh-Xuan, A.T., Pueyo, M., Darblade, B. and Rami, J. (1999) Endothelium-Derived Nitric Oxide and Vascular Physiology and Pathology. Cellular and Molecular Life Sciences, 55, 1078-1087.
http://dx.doi.org/10.1007/s000180050358 [13] Vyas-Read, S., Shaul, P.W., Yuhanna, I.S. and Willis, B.C. (2007) Nitric Oxide Attenuates Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells. American Journal of Physiology—Lung Cellular and Molecular Physiology, 293, L212-L221.
http://dx.doi.org/10.1152/ajplung.00475.2006 [14] Kordes, C., Sawitza, I., Müller-Marbach, A., Ale-Agha, N., Keitel, V., Klonowski-Stumpe, H. and Häussinger, D. (2007) CD133+ Hepatic Stellate Cells Are Progenitor Cells. Biochem. Biochemical and Biophysical Research Communications, 352, 410-417.
http://dx.doi.org/10.1016/j.bbrc.2006.11.029 [15] Mookerjee, I., Hewitson, T.D., Halls, M.L., Summers, R.J., Mathai, M.L., Bathgate, R.A., Tregear, G.W. and Samuel, C.S. (2009) Relaxin Inhibits Renal Myofibroblast Differentiation via RXFP1, the Nitric Oxide Pathway, and Smad2. The FASEB Journal, 23, 1219-1229.
http://dx.doi.org/10.1096/fj.08-120857 [16] Lu, L., Chen, S.S., Hassid, A. and Sun, Y. (2008) Cardiac Fibrogenesis Following Infarction in Mice with Deletion of Inducible Nitric Oxide Synthase. American Journal of the Medical Sciences, 335, 431-438.
http://dx.doi.org/10.1097/MAJ.0b013e3181571f97 [17] Pessanh, M.G. and Mandarim-de-Lacerda, C.A. (2000) Myofibroblast Accumulation in Healing Rat Myocardium Due to Long-Term Low-Dosage Nitric Oxide Synthesis Inhibition. Experimental and Toxicologic Pathology, 52, 192-194.
http://dx.doi.org/10.1016/S0940-2993(00)80027-5 [18] Gonzalez-Cadavid, N.F. and Rajfer, J. (2009) Experimental Models of Peyronie’s Disease. Implications for New Therapies. Journal of Sexual Medicine, 6, 303-313.
http://dx.doi.org/10.1111/j.1743-6109.2008.01104.x [19] Hall, C.N. and Garthwaite, J. (2009) What Is the Real Physiological NO Concentration in Vivo? Nitric Oxide, 21, 92-103.
http://dx.doi.org/10.1016/j.niox.2009.07.002 [20] Taha, Z.H. (2003) Nitric Oxide Measurements in Biological Samples. Talanta, 61, 3-10.
http://dx.doi.org/10.1016/S0039-9140(03)00354-0 [21] Räthel, T.R., Leikert, J., Vollmar, A.M. and Dirsch, V.M. (2003) Application of 4,5-Diaminofluorescein to Reliably Measure Nitric Oxide Released from Endothelial Cells in Vitro. Biological Procedures Online, 5, 136-142.
http://dx.doi.org/10.1251/bpo55 [22] Leikert, J.F., Räthel, T.R., Müller, C., Vollmar, A.M. and Dirsch, V.M. (2001) Reliable in Vitro Measurement of Nitric Oxide Released from Endothelial Cells Using Low Concentrations of the Fluorescent Probe 4,5-Diaminofluorescein. FEBS Letters, 506, 131-134.
http://dx.doi.org/10.1016/S0014-5793(01)02901-5 [23] Boo, Y.C., Tressel, S.L. and Jo, H. (2007) An Improved Method to Measure Nitrate/Nitrite with an NO-Selective Electrochemical Sensor. Nitric Oxide, 16, 306-312.
http://dx.doi.org/10.1016/j.niox.2006.09.004 [24] Berkels, R.S., Purol-Schnabel, S. and Roesen, R. (2001) A New Method to Measure Nitrate/Nitrite with a NO-Sensitive Electrode. Journal of Applied Physiology, 90, 317-320. [25] McIntosh, J.C., Hagood, J.S., Richardson, T.L. and Simecka, J.W. (1994) Thy1 (+) and (-) Lung Fibrosis Subpopulations in LEW and F344 Rats. European Respiratory Journal, 7, 2131-2138.
http://dx.doi.org/10.1183/09031936.94.07122131 [26] Grinnell, F. (1994) Fibroblasts, Myofibroblasts, and Wound Contraction. Journal of Cell Biology, 124, 401-404.
http://dx.doi.org/10.1083/jcb.124.4.401 [27] Rice, N.A. and Leinwand, L.A. (2003) Skeletal Myosin Heavy Chain Function in Cultured Lung Myofibroblasts. Journal of Cell Biology, 163, 119-129.
http://dx.doi.org/10.1083/jcb.200303194 [28] Griffith, O.W. and Kilbourn, R.G. (1996) Nitric Oxide Synthase Inhibitors: Amino Acids. Methods in Enzymology, 268, 375-392.
http://dx.doi.org/10.1016/S0076-6879(96)68040-9 [29] Jagnandan, D., Sessa, W.C. and Fulton, D. (2005) Intracellular Location Regulates Calcium-Calmodulin Dependent Activation of Organelle-Restricted eNOS. American Journal of Physiology: Cell Physiology, 289, C1024-C1033.
http://dx.doi.org/10.1152/ajpcell.00162.2005 [30] Hunter, R.A., Storm, W.L., Coneski, P.N. and Schoenfisch, M.H. (2013) Inaccuracies of Nitric Oxide Measurement Methods in Biological Media. Analytical Chemistry, 85, 1957-1963.
http://dx.doi.org/10.1021/ac303787p [31] Dudzinski, D. and Michel, T. (2007) Life History of eNOS: Partners and Pathways. Cardiovascular Research, 75, 247-260.
http://dx.doi.org/10.1016/j.cardiores.2007.03.023