Nitric oxide synthase isoforms expression in fibroblasts isolated from human normal peritoneum and adhesion tissues.

OBJECTIVE

To determine the expression of nitric oxide synthases (NOSs) and their modulation by hypoxia in human peritoneal (NF) and adhesion fibroblasts (ADF).

DESIGN

Prospective experimental study.

SETTING

University medical center.

PATIENT(S): Fibroblasts from peritoneum and adhesion tissues.

INTERVENTION(S): Hypoxia and silencing inducible NOS (iNOS) gene expression in fibroblasts.

MAIN OUTCOME MEASURE(S): We used reverse-transcriptase polymerase chain reaction to quantify messenger RNA (mRNA) levels of NOS isoforms. Griess assay was used to measure NO levels.

RESULT(S): The mRNA copies/mug RNA of neuronal NOS (nNOS) and endothelial NOS (eNOS) were 6.6 x 10(3) in NF, 5.7 x 10(3) in ADF and 7.0 x 10(3) in NF, 6.1 x 10(3) in ADF, respectively. The mRNA copies/mug RNA of iNOS were 31.3 x 10(3) in NF and 33.0 x 10(3) in ADF. Hypoxia increased iNOS mRNA copies/mug RNA from 31.3 x 10(3) to 61.3 x 10(3) in NF and from 33.0 x 10(3) to 63.9 x 10(3) in ADF, whereas there were no changes in mRNA levels of nNOS and eNOS in NF and ADF. Nitric oxide levels were lower in ADF (0.94 micromol/L) than NF (1.97 micromol/L). Silencing iNOS decreased NO levels in NF (from 1.97 micromol/L to 0.41 micromol/L) and in ADF (from 0.94 micromol/L to 0.27 micromol/L).

CONCLUSION(S): Nitric oxide synthases are differentially expressed in NF and ADF, with iNOS being the most expressed and the main source of NO. Hypoxia was shown to alter the expression of NOSs and NO in NF and ADF.