Nitric oxide synthase: mRNA expression of different isoforms in human monocytes/macrophages

To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations.