Human dermal fibroblasts produce nitric oxide and express both constitutive and inducible nitric oxide synthase isoforms

Nitric oxide (NO) is produced by a variety of human and animal cells and is involved in a broad array of physiological and pathophysiological processes. It can cause vasodilation, serve as a neurotransmitter, and have anti-neoplastic, anti-microbial, and antiproliferative effects. In this study, we have demonstrated that fibroblasts derived from human skin spontaneously produce NO and that this production can be enhanced by stimulating the cells with interferon-gamma and lipopolysaccharide. The production of NO by human dermal fibroblasts can be blocked by N-G-monomethyl-L-arginine (L-NMMA). The inhibitory effect of L-NMMA on NO production was restored by addition of L-arginine but not D-arginine, By measuring the rate of conversion of [C-14]L-arginine to [C-14]L-citrulline, we show that unstimulated cells expressed only Ca2+-dependent NO synthase (NOS) activity (1.36 +/- 0.57 pmol/mg/min; n = 4) whereas stimulated cells expressed both Ca2+-dependent (2.60 +/- 0.54 pmol/mg/min; n 4) and -independent (1.59 +/- 0.14 pmol/mg/min; n = 4) NOS activities. With reverse transcription polymerase chain reaction (RT-PCR), the 422-bp RT-PCR product for human endothelial constitutive NOS and the 462-bp RT-PCR product for human hepatocyte inducible NOS were detected in proportion to the amount of mRNA-related RT-cDNA added to the reaction mixture. Further evidence by immunocytochemistry demonstrated that human dermal fibroblasts express both constitutive and inducible NOS proteins. These data collectively suggest that in addition to macrophages and other inflammatory cells, nitric oxide production by dermal fibroblasts could be important during the inflammatory stages of wound healing and possibly also in the later stages of proliferation and tissue remodeling after skin injury in humans.