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.
Highlights
The origin of myofibroblasts in the lung is not clear, the favored model is that resident adventitial fi-How to cite this paper: Sharma, B.V., et al (2014) An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts
Nanomolar Concentrations of nitric oxide (NO) Are Produced by Pulmonary Myofibroblasts
In this report we describe an enhanced assay that provides a straightforward method for measuring relative changes of NO produced by cultured pulmonary myofibroblasts
Summary
The origin of myofibroblasts in the lung is not clear, the favored model is that resident adventitial fi-How to cite this paper: Sharma, B.V., et al (2014) An Improved Assay for Measuring Low Levels of Nitric Oxide in Cultured Pulmonary Myofibroblasts. Three different forms of NOS have been identified and named based upon tissue of origin: neural NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), it is known that many cells types express multiple isoforms [10]. Both nNOS and eNOS are constitutive, Ca2+dependent enzymes, which produce NO in the pM range, while the activity of iNOS is induced in response to activated macrophages, is Ca2+-independent, and results in nM levels of NO [11]. While constitutively produced NO is most commonly associated with maintenance of vascular tone in the endothelium [12], it is implicated as a key modulator of myofibroblast cell growth and activity in numerous tissues including lung [2] [13], liver [14], kidney [15], heart [16] [17], and urogential [18] tissues
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