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 (NO X ) 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.
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.
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