Cholinergic Modulation of Epithelial Integrity in the Proximal Colon of Pigs

Abstract

Background: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by ‘non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. Methods: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. Results: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µ<smlcap>M</smlcap>) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. Conclusion: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

“…Extended Life Span of PoCo83-3 Achieved by SV40 TAg Transduction As previously described, primary isolated pCECs generally entered a state of growth arrest after only a few passages of subcultivation [Petto et al, 2011;Lesko et al, 2013], a presumed consequence of cellular senescence [Campisi and Di d'Adda Fagagna, 2007]. In this study, pCECs were successfully transduced using a retroviral vector as confirmed by the stable expression of the SV40 TAg at the mRNA and protein levels ( Fig.…”

“…Furthermore, some work groups used a combination of SV40 TAg and hTERT to generate immortalized cell lines [Matsumura et al, 2004;Desmarets et al, 2013;Olyslaegers et al, 2013]. In previous studies using primary pCECs [Petto et al, 2011;Lesko et al, 2013], these cells seemed to be quite sensitive when subjected to cell culture, usually maintaining active proliferation for only a few passages (according to our own unpublished observations). Regarding the sensitivity of the cells, the use of SV40 provides some advantages.…”

“…As shown by Lesko et al [2013], firstpassage pCECs exhibited TEER values of approximately 1,000 Ω × cm 2 on day 6 after seeding, using ThinCert cellculture inserts. In contrast to the more heterogeneous primary and first-passage cultures of pCEC, which may have consisted of different epithelial cell types and a minor mesenchymal cell population [Petto et al, 2011;Lesko et al, 2013], substantially higher TEER values were expected for the clonal cell line PoCo83-3. We showed that PoCo83-3 cells at P20 and P30 exhibited nearly iden- Kaiser et ( Fig.…”

“…The primary cell isolate obtained from enzymatic digestions of colonic tissue consisted of a heterogeneous culture of epithelial cells and a minor mesenchymal cell population, as previously described [Petto et al, 2011;Lesko et al, 2013]. After 1 subcultivation and a subse- 273 quent retroviral transduction, the transduced cell line, referred to as PoCo83, exhibited a short-term growth depression.…”

“…TEER measurement is a widely used method for monitoring monolayer confluence, cell viability, and epithelial barrier integrity, and also for the functional assessment of TJ dynamics in cell culture models [Srinivasan et al, 2015]. For a comparison with studies that use primary isolated pCECs [Petto et al, 2011;Lesko et al, 2013], we used collagen-coated ThinCert cell-culture inserts as the standard setup for our experiments. We verified the monolayer formation of PoCo83-3 cells grown on ThinCert cell-culture inserts by examining paraffin sections, indicating that this type of membrane support is suitable for investigations concerning barrier function ( Fig.…”