Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthes es (NOS), are essential to analyze the mechanism of action of NO in this pr ocess. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO product ion using 4,5-diamino-fluorescein-diacetate (DAF-2/DA). The presence of NOS -3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed tha t the how cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assay s, both under basal conditions and after Ad.NOS-3 transduction. Simultaneou s analysis of NOS-3 protein and NO production, made possible by the here-de scribed novel how cytometric assays, is of significant value to those inves tigating NOS-3 and NO. (C) 2001 Academic Press.