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Abstract: Background: Alterations of site‐specific gene expression profiles in disease‐relevant networks within the different layers of the intestinal wall may contribute to the onset and clinical course of gastrointestinal disorders. To date, no systematic analysis has assessed and compared sub‐regional gene expression patterns in all distinct layers of the gut using fresh frozen human samples. Our aim was to establish an optimized protocol for site‐specific RNA isolation in order to achieve maximum RNA quality and amount for subsequent gene expression analysis combining laser‐capture microdissection (LCM) with a probe‐based technology, the NanoString nCounter Analysis system. Methods: Four full‐thickness colon samples from patients who underwent surgery due to pathological conditions were processed and separated into epithelium, lamina propria, myenteric plexus, submucosa, and tunica muscularis by LCM. Site‐specific marker expression by nCounter technology was performed on total RNA from each sub‐region, respectively. Key Results: Collecting ~10 mm² (~100 000‐250 000 cells) of tissue from the epithelial layer, lamina propria, and myenteric plexus provided sufficient amounts of RNA of appropriate quality for subsequent analyses. In contrast, ~40 mm² (~250 000‐650 000 cells) of tissue were dissected from the less cell‐rich submucosal and tunica muscularis layer. nCounter analysis revealed a site‐specific expression pattern of marker genes in the different layers of the colonic wall which were highly correlating (r > .9). Conclusions and Inferences: LCM in combination with nCounter expression analysis enables site‐specific, sensitive, reliable detection, and quantification of mRNA from histologically heterogeneous tissues. [ABSTRACT FROM AUTHOR]

Background Impaired 5- HT3 receptor function is likely involved in the pathogenesis of functional gastrointestinal disorders ( FGID) and 5- HT3 receptor antagonists are effective treatments for chemotherapy-induced nausea and vomiting ( CINV) and irritable bowel syndrome ( IBS). The monoterpene alcohol menthol and the aporphine alkaloid boldine combat symptoms of gastrointestinal diseases; both interact with other members of the Cys-loop ligand-gated ion channel family and may therefore also act on 5- HT3 receptors. Methods The impact of boldine and menthol on human recombinant homomeric 5- HT3A- and heteromeric 5- HT3 AB receptors in HEK293 cells was determined by radioligand binding, a luminescence-based Ca2+ assay, and a membrane potential assay. 5- HT3 protein and m RNA expression was assessed in human colon tissue. Key Results Boldine and menthol inhibited the 5- HT-induced activation of 5- HT3 receptors in the low and middle micromolar range, respectively. Boldine was a competitive antagonist of both receptors being 6.5- to 10-fold more potent at 5- HT3A- vs 5- HT3 AB receptors. Menthol non-competitively and stereoselectively inhibited both receptors: In contrast to (+)-menthol, (−)-menthol was significantly more potent toward 5- HT3A- vs 5- HT3 AB receptors. We show co-expression of 5- HT3A and 5- HT3B subunits in the human gut epithelium, the lamina propria, the myenteric plexus, and the muscular cell layer. Conclusions & Inferences The demonstrated 5- HT3 inhibitory effects may be relevant for boldine's and menthol's alleviating properties on FGID and may encourage clinical studies with the compounds or the plant extracts for CINV and IBS treatment. The found receptor-discriminative properties make boldine and (−)-menthol to potentially useful tools for analyzing structural differences between these receptor subtypes. [ABSTRACT FROM AUTHOR]

Irritable bowel syndrome (IBS) is a gut-brain disorder in which symptoms are shaped by serotonin acting centrally and peripherally. The serotonin transporter gene SLC6A4 has been implicated in IBS pathophysiology, but the underlying genetic mechanisms remain unclear. We sequenced the alternative P2 promoter driving intestinal SLC6A4 expression and identified single nucleotide polymorphisms (SNPs) that were associated with IBS in a discovery sample. Identified SNPs built different haplotypes, and the tagging SNP rs2020938 seems to associate with constipation-predominant IBS (IBS-C) in females. rs2020938 validation was performed in 1978 additional IBS patients and 6,038 controls from eight countries. Meta-analysis on data from 2,175 IBS patients and 6,128 controls confirmed the association with female IBS-C. Expression analyses revealed that the P2 promoter drives SLC6A4 expression primarily in the small intestine. Gene reporter assays showed a functional impact of SNPs in the P2 region. In silico analysis of the polymorphic promoter indicated differential expression regulation. Further follow-up revealed that the major allele of the tagging SNP rs2020938 correlates with differential SLC6A4 expression in the jejunum and with stool consistency, indicating functional relevance. Our data consolidate rs2020938 as a functional SNP associated with IBS-C risk in females, underlining the relevance of SLC6A4 in IBS pathogenesis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)