Role of A2B receptors in the control of colonic cholinergic motility in the presence of bowel inflammation

Objective: Adenosine A2B receptors (A2BR) contribute to the physiological regulation of immune responses, intestinal secretive/absorptive functions and colonic motility. However, the involvement of these receptors in enteric motor alterations associated with bowel inflammation is unknown. This study evaluated A2BR expression in rat colon and investigated their role in the control of cholinergic motility in the presence of colitis. Methods: Colitis was induced by intrarectal 2, 4-dinitrobenzenesulfonic acid (DNBS) in Sprague-Dawley rats. After 6 days, A2BR expression and localization in the colonic neuromuscular layer were examined by RT-PCR and immunofluorescence. Colonic longitudinal muscle strips (LMS) were suspended in organ baths with Krebs solution, containing guanethidine, Nomega- propyl-L-arginine and antagonists of NK 1, 2 and 3 receptors. The effects of MRS 1754 (MRS, A2BR antagonist), NECA (A2BR agonist), dipyridamole (DIP, adenosine reuptake inhibitor) and adenosine deaminase (ADA) were assayed on atropine-sensitive cholinergic contractions evoked by electrical stimulation (ES; 0.5 ms, 28 V, 10 Hz), or by carbachol in the presence of tetrodotoxin. Results: RT-PCR revealed the presence of A2BR mRNA in normal colon, and showed that the expression pattern did not vary in inflamed tissues. Immunofluorescence displayed a predominant localization of A2BR in the longitudinal muscle layer and myenteric plexus, however, in the presence of colitis, A2BR expression was enhanced at muscular level, but reduced in myenteric ganglia. MRS enhanced ESinduced contractions in normal LMS (+37%), while being less effective in inflamed tissues (+13.2%). Under incubation with DIP plus ADA, to abate extracellular adenosine levels, NECA decreased the motor responses to ES in normal LMS (EC50 = 300 n mol L-1, Emax = -58%), and it was more effective in rats with colitis (EC50 = 275 n mol L-1, Emax = -83.8%). Carbachol-evoked contractions were enhanced by MRS in normal LMS (+27%), but not in inflamed rats, while they were inhibited by NECA in normal and, to a greater extent, in inflamed LMS (-34% and -52%, respectively). Conclusion: These results suggest that, under normal conditions, adenosine modulates the colonic cholinergic motility via activation of A2BR, mainly located on smooth muscle. In the presence of colitis, this inhibitory control is impaired, despite an up-regulated A2BR expression, probably resulting from a reduced endogenous adenosine availability.