NO_x generation by cultured small intestinal epithelial cells

The effect of cytokines, growth factors, mitogens, and bacterial products on nitric oxide (NO) generation by monolayers of small intestinal epithelial cells-6 (IEC-6) cells was evaluated. Subconfluent IEC-6 cells were maintained in DMEM containing 5% fetal calf serum and after 16-24 hr of incubation, the medium was replaced with fresh medium in the presence or absence of calcium ionophore (CaI), l-NAME, l-NNA, individual growth factors, cytokines, or mitogens. After 72 hr of culture, the media supernatant was collected and NO_x generation was determined. NO synthase activity was determined in sonicated supernatants of IEC-6 cells by [¹⁴C] arginine conversion to citrulline. NO_x generation in subconfluent cultures was greater than in fully confluent cultures, suggesting contact inhibition. NO_x generation by IEC-6 cells was significantly increased by CaI and inhibited by l-NAME and l-NNA. LPS, IL-1β, IL-2, IL-8, IFN-8, TFN-α, EGF, TGF-α, bFGF, and PHA significantly increased NO_x generation. NO synthase activity in IEC-6 cells (4.2±1.7 pmol/min/10⁶ cells) was NADPH dependent. These results suggest that stimulation of NO_x generation by intestinal epithelial cells through cytokine bacterial products and mitogens may be one of the mechanisms responsible for their effects in the intestinal tract.