Expression of nitric oxide synthase in bladder smooth muscle cells: regulation by cytokines and L-arginine

Abstract

Purpose: The expression and regulation of the different isoforms of nitric oxide synthase (NOS) in bladder smooth muscle cells are controversial and to our knowledge have not yet been studied systematically. Therefore, the expression and regulation of NOS were studied in rat bladder smooth muscle cells after stimulation with cytokines, lipopolysaccharide and L-arginine.

Materials and methods: Primary cell cultures were prepared from rat bladders. The expression of NOS mRNA was examined by reverse transcriptase-polymerase chain reaction and inducible NOS (iNOS) protein expression was studied by Western blot analysis and immunohistochemistry. Nitrite accumulation in the culture medium was determined by the Griess assay. The expression of iNOS was also studied immunohistochemically in whole bladder strips stimulated by cytokines.

Results: NOS mRNA expression was not detected in unstimulated cells. Stimulating bladder smooth muscle cells with a cytokine mixture of interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta induced iNOS mRNA and protein expression. The combination of interleukin-1beta plus tumor necrosis factor-alpha appeared to be crucial for iNOS induction in bladder smooth muscle cells. Exposing bladder smooth muscle cells to lipopolysaccharide did not induce iNOS. Adding L-arginine increased nitrite accumulation in cytokine mixture stimulated bladder smooth muscle cells, while iNOS positive cells were detected in the smooth muscle layer of cytokine mixture stimulated bladder strips.

Conclusions: NOS was not detected in unstimulated bladder smooth muscle cells. However, bladder smooth muscle has the potential to express iNOS when exposed to cytokines known to be produced during urinary tract infection.