A novel finding of anoctamin 5 expression in the rodent gastrointestinal tract.

Anoctamin 5 (Ano5) belongs to the anoctamin gene family and acts as a calcium-activated chloride channel (CaCC). A mutation in the Ano5 gene causes limb-girdle muscular dystrophy (LGMD) type 2L, the third most common LGMD in Northern and Central Europe. Defective sarcolemmal membrane repair has been reported in patients carrying this Ano5 mutant. It has also been noted that LGMD patients often suffer from nonspecific pharyngoesophageal motility disorders. One study reported that 8/19 patients carrying Ano5 nutations suffered from dysphagia, including the feeling that solid food items become lodged in the upper portion of the esophagus. Ano5 is widely distributed in bone, skeletal muscle, cardiac muscle, brain, heart, kidney and lung tissue, but no report has examined its expression in the gastrointestinal (GI) tract. In the present study, we investigated the distribution of Ano5 in the GI tracts of mice via reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunofluorescence analyses. The results indicated that Ano5 mRNA and protein are widely expressed in the esophagus, the stomach, the duodenum, the colon and the rectum but that Ano5 immunoreactivity was only detected in the mucosal layer, except for the muscular layer of the upper esophagus, which consists of skeletal muscle. In conclusion, our present results demonstrate for the first time the expression of Ano5 in the GI epithelium and in skeletal muscle in the esophagus. This novel finding facilitates clinical differential diagnosis and treatment. However, further investigation of the role of Ano5 in GI function is required.

Beta-adrenoceptors, but not dopamine receptors, mediate dopamine-induced ion transport in late distal colon of rats.

Dopamine, an important modulator in the gastrointestinal system, induces concentration-dependent transepithelial ion transport in the distal colon of the rat, as shown by a decrease in the short-circuit current, and acts in a segmentally dependent manner. However, the receptor(s) that mediates dopamine-induced ion transport is unknown. We have investigated the receptor mechanisms underlying dopamine-induced colonic ion transport by means of short-circuit current recording, real-time polymerase chain reaction, and Western blotting analysis, plus gene transfection and enzyme-linked immunosorbance assay. mRNA transcripts of adrenoceptors (alpha, beta) and dopaminergic receptors (D(1) and D(2)) were detected in the rat late distal colonic mucosa, with beta(2) displaying the highest expression. A similar result was found in human colorectal mucosa (equivalent of late distal colon in rat). Pretreatment with a beta(1)-adrenoceptor antagonist (CGP-20712A) and a beta(2)-adrenoceptor antagonist (ICI 118,551) inhibited the dopamine-induced short-circuit current response by 52.59% and 92.51%, respectively. However, neither dopamine D(1) receptor antagonist SCH-23390 nor dopamine D(2) receptor antagonist sulpiride blocked the effect of dopamine. Protein expression of both beta(1)- and beta(2)-adrenoceptors was found in the mucosa of rat distal colon and human sigmoid colon and rectum. Dopamine significantly increased intracellular cAMP levels in COS-7 cells transfected with beta(1)- or beta(2)-adrenoceptors. Thus, beta-adrenoceptors (mainly beta(2)-adrenoceptors), but not dopamine receptors, mediate dopamine-induced ion transport in the late distal colon of the rat. This extends our knowledge of the late distal colon (rats) or colorectum (human) and provides further experimental evidence that might aid the prevention, diagnosis, and clinical therapy of human colorectal diseases.

A dual role of 5-hydroxytryptamine receptor 3 in serotonin induced ion transport in rat distal colon.

5-hydroxytryptamine (5-HT)-evoked intestinal secretion can be divided into neural and non-neural pathway. Recently, 5-HT(3) receptor in neural pathway received much attention as a possible target in bowel diseases. The present study aims to investigate the effects of 5-HT(3) receptor in different enteric neural plexus (myenteric plexus and submucosal plexus) on rat colonic ion transport by using rat intact colon and mucosa/submucosa preparations. Ussing chamber and real-time PCR techniques were performed in our present study. Surprisingly, we found that in mucosa/submucosa preparations, 5-HT-induced DeltaI(SC) (change in short-circuit current) was not inhibited, but further increased by pretreatment with 5-HT(3) receptor antagonists, MDL72222 and Tropanyl-3, 5-dimethylbenzoate. And this response was significantly attenuated in the presence of tetrodotoxin (TTX). Conversely, in rat intact colon, 5-HT(3) receptor antagonists significantly inhibited 5-HT-induced DeltaI(SC). The results from real-time PCR proved the existence of 5-HT(3) receptor in muscularis externa and submucosa. Taken together, 5-HT(3) receptors possess a role of dual regulation on electrolyte secretion in rat distal colon, the neural stimulatory effect of 5-HT(3) receptor in myenteric plexus and the inhibitory effect of 5-HT(3) receptor in submucosal plexus.

Appearance of segmental discrepancy of anion transport in rat distal colon.

The present study investigated the segmental discrepancy of the rat distal colonic anion transport induced by luminal forskolin and the possible underlying mechanisms using short-circuit current recording technique and comparative quantity real-time PCR analysis. Forskolin-induced I(SC) in the segment next to lymph node (DC(1)) and the segment 4 cm away from lymph node (DC(4)) were 4.09+/-0.66 muA/cm(2) and 18.84+/-3.18 muA/cm(2) (n=13), respectively, which were blocked by luminal Cl(-) channel blocker, glybenclamide (1 mM) (n=5, p<0.01), as well as removal of extracellular Cl(-) and HCO(3)(-) in both DC(1) and DC(4) (n=5, p<0.001). Furthermore luminal pretreatment with K(+) blockers, TEA (5 mM) and glybenclamide (100 muM) didn't affect forskolin and bumetanide-enhanced I(SC). Reducing serosal Cl(-) concentration increased forskolin-induced I(SC) by 90% in DC(1) but decreased forskolin-induced I(SC) in DC(4) by 50%. Furthermore, pretreatment with serosal bumetanide, an inhibitor of Na(+)-K(+)-2Cl(-) cotransporter, enhanced forskolin-induced I(SC) by 87% in DC(1), from 4.09+/-0.66 muA/cm(2) to 7.65+/-0.53 muA/cm(2) (n=6, p<0.01), but inhibited forskolin-induced I(SC) by 50% in DC(4), from 29.19+/-4.51 muA/cm(2) to 15.06+/-4.10 muA/cm(2) (n=6, p<0.05). Pretreatment with luminal amiloride (10 muM), an inhibitor of ENaC, and serosal 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (200 muM), an inhibitor of NBC, significantly inhibited the forskolin-induced I(SC) in DC(1) (n=6, p<0.05), but not in DC(4). Real-time PCR analysis did not show the significant differences between the two segments in the expression amounts of CFTR and NKCC mRNAs, however the expression of NBC mRNA in DC(4) was significantly higher than that in DC(1). In conclusion, the rat distal colon manifests a segmental discrepancy in anion transport stimulated by luminal forskolin. HCO(3)(-) might be predominantly involved in the forskolin-induced anion secretion in DC(1), but Cl(-) might be the main component of the anion secretion in DC(4).