Muscarinic acetylcholine receptor expression in aganglionic bowel

In Hirschsprung's disease (HD) there exists an overabundance of acetylcholi ne (ACh), which in turn stimulates excessive production of the enzyme acety lcholinesterase. Muscarinic ACh receptors (mAChRs) play an important role i n smooth-muscle contraction. Recent studies have indicated five different s ubtypes of mAChRs encoded by five different genes, mi to m5. The purpose of this study was to investigate the expression of each mAChR subtype in agan glionic (AG) colon to further understand the pathophysiology of HD. Entire colon resected at the time of pull-through operation for HD was obtained fr om 14 patients. Specimens obtained at autopsy from 8 age-matched patients w ithout gastrointestinal disease acted as controls. Frozen sections were use d for indirect immunohistochemistry as well as in-situ hybridization. Immun ohistochemistry was performed using specific antiserum against each mAChR s ubtype and in-situ hybridization was performed using specific oligonucleoti de probes against mi to m5 subtypes. Messenger RNA (mRNA) was extracted fro m normoganglionic (NG) and AG bowel of HD patients and normal control bowel . Reverse transcription-polymerase chain reaction was performed to evaluate mRNA levels of each mAChR subtype. To adjust the levels of mRNA expression , a housekeeping gene G3PDH, known to be expressed normally, was used as an internal control. Strong m2 and m3 immunoreactivity was observed in the mu cosal layer, smooth-muscle layers, and myenteric plexus of NC bowel, wherea s mi immunoreactivity was only detected in the mucosal layer. The most stri king finding was the abundance of m3-immunorcactive fibers in muscle layers of NG bowel while there was a total lack of m3 fibers in smooth-muscle of AG bowel. Intense mRNA signals encoding m2 and m3 and to a lesser degree mi were detected in NG bowel, and these signals were weak in AC bowel. Immuno reactivity and mRNA expression of m4 and m5 was not detected in NG or AG bo wel. The lack of m3-immunoreactive fibers in the smooth-muscle layers of AG bowel and decreased m2 and m3 mRNA expression in AG bowel may be responsib le for the motility dysfunction in the aganglionic segment.