Nitric Oxide Production by Human Peritoneal Mesothelial Cells

Abstract

Nitric oxide (NO) has multiple actions, ranging from immunomodulation to regulation of vascular tone and capillary flow. Thus NO generation within the peritoneum could potentially affect peritoneal transport by increasing capillary vasodilatation, and regulate the response to bacterial invasion. Peritoneal mesothelial cells have a common embryological derivation with endothelial cells. As mesothelial cells are the predominant cell type lining the peritoneal cavity, they could potentially be a major source of locally produced nitric oxide.

Nitric oxide was measured using the Griess reaction, as total nitrite and nitrate, in fresh unused and spent dialysate effluent (SPDE) from both healthy peritoneal dialysis patients, and during episodes of bacterial peritonitis. Whereas fresh CAPD dialysate was nitrite free (5 ± 0.1 μM), SPDE from a standard 4 h day time exchange contained 10.2 ± 0.6 μM/L/h, and that from the overnight dwell 9.1 ±0.7 μM/L/h. During an episode of peritonitis, dialysate nitrite and nitrate increased significantly from 9.0 ± 1.0 μM/L/h, when not infected to 17.5 ± 2.4, from the first CAPD bag drained at presentation, and 15.2 ± 1.8 for the second and 16.0 ± 2.5 for the third exchange (p<0.01). By the following day nitrite levels had returned to baseline, 7.0 ± 1.0 μM/L/h.

Human peritoneal mesothelial cells (HPMC) were cultured and found to produce nitric oxide (261 nmol/mg cell protein), which increased in a dose dependent manner with the addition of spent uninfected CAPD dialysate. The addition of L-arginine, a NO substrate resulted in a 10% increase in nitric oxide production, whereas the addition of the blocker L-NMMA produced a 10% reduction. RNA for inducible nitric oxide synthase (iNOS) was sought using northen blotting technique following combination stimulation with lipopolysaccharide and cytokines (IL-1β, TNFα and γ-INF, and/or spent dialysate from patients with bacterial peritonitis). However, we could not demonstrate RNA production for iNOS.

Peritoneal mesothelial cells may be an important source of locally generated nitric oxide within the peritoneal cavity under basal conditions, but as they do not contain iNOS, the markedly increased NO production observed with episodes of acute bacterial peritonitis is more likely due to a combination of increased NO production by peritoneal macrophages and endothelial cells.