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Evaluation of nitric oxide production in cultured pulmonary myofibroblasts
Author(s) -
Sharma Bethel,
Clouse Margaret,
Rice Nancy A.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb294
Subject(s) - myofibroblast , nitric oxide , chemistry , nitric oxide synthase , pathogenesis , nitrite , arginine , basal (medicine) , fibrosis , pathology , biochemistry , medicine , nitrate , organic chemistry , insulin , amino acid
Idiopathic pulmonary fibrosis is a destructive disease that stems from collagen buildup in the lungs due to the presence of excess, and/or overactive, myofibroblast cells. Nitric oxide (NO) has been implicated as a key modulator of myofibroblast growth and proliferation. In order to better understand the mechanism by which NO contributes to myofibroblast pathogenesis, a robust functional assay to monitor NO production at submicromolar levels has been developed. Myofibroblasts, isolated from rat lungs, were cultured under normal and pseudo‐pathological conditions for 24 h prior to treatment (10 mM PBS pH 7.4, 100 uM CaCl2, 1 mM L‐arginine, and 1 uM A23187 selected NO synthase inhibitors). Nitric oxide levels were detected in solution amperometrically, using a NO specific electrode. NO levels measured for basal and treated cells were amplified by a nitrite conversion step using a metal‐based catalyst. Treated samples showed a decrease in measured NO as compared to basal levels. The low levels of NO produced by myofibroblasts were detectable using the electrode based system, and the amount of accumulated NO detected was enhanced by the addition of a nitrate reduction step. This system will be used to elucidate additional factors contributing to modulation of NO levels in myofibroblasts. Support for this research has been provided by the NIH (NAR): R15HL087185, P20RR16481.