ABSTRACT-We studied whether reactive oxygen species (ROS) generated by normal colonic mucosa affect 5-hydroxytryptophan (5-HTP)-evoked 5-HT formation (measured as the sum of 5-HT plus 5-hydroxyindole acetic acid (5-HIAA) accumulation) of guinea pig's isolated colonic mucosa. Catalase (3000 -6000 U /ml), a hydrogen peroxide (H 2O2) scavenger or diphenylene iodonium (DPI, 10 -100 mM), an NADPH oxidase inhibitor, concentration-dependently caused an increase of the sum of 5-HT plus 5-HIAA accumulation in the presence of 5-HTP (10 mM), but these drugs did not significantly affect the 5-HT-metabolite in the colonic mucosa measured as the ratio of 5-HIAA /5-HT. Exogenously applied H2O2 (10 -100 m M) concentration-dependently inhibited the sum of 5-HT plus 5-HIAA accumulation. In contrast, neither superoxide dismutase (SOD, 100 -300 U / ml), superoxide anion scavenger, nor dimetyl sulfoxide (1 -5%, DMSO), a hydroxyl radical scavenger affected the sum of 5-HT plus 5-HIAA accumulation. Moreover, mucosa ROS generation was estimated using the chemiluminescence technique. SOD (100 -300 U /ml), catalase (3000 -6000 U/ ml) or DPI (10 -100 m M), concentration-dependently reduced luminolenhanced chemiluminescence signal from the colonic mucosa, while allopurinol (10 -100 mM), a xanthine oxidase inhibitor, did not affect the chemiluminescence signal. These results suggest that ROS is formed through an NADPH oxidase system in the guinea pig colonic mucosa, where it exerts a modulatory effect on mucosal 5-HT formation upon addition of 5-HTP. Thus, ROS formation from normal colonic mucosa could be considered to contribute to the control of 5-HT production in mucosa enterochromaffin cells.

We have compared the reactivity to carbachol and high potassium of circular smooth muscle isolated from segments of human colon which was freeze-stored in different preservative solutions for more than one month following surgical resection. Concentration-dependent contractions in response to carbachol were reduced in terms of both their sensitivity (pEC50) and reactivity (Emax), depending on the preservative solutions used. Similar reduction of reactivity to 100 mM KCl was also observed. The best responsiveness was shown when the tissue was freeze-stored in SFM101. It is concluded that the freeze-storage of surgically excised human colon in SFM101 or phosphate buffer solution for more than one month provided the best preservation of smooth muscle function for in vitro pharmacological examination.