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Background Cystic fibrosis transmembrane conductance regulator (CFTR) was recently found in the enteric nervous system, where its role is unclear. We aimed to identify which enteric neuronal structures express CFTR, whether CFTR modulates enteric neurotransmission and if altered CFTR expression is associated with slow transit constipation (STC). Methods Immunofluorescence double labeling was performed to localize CFTR with various neuronal and glial cell markers in the human colon. The immunoreactivity (IR) of CFTR and choline acetyltransferase (ChAT) on myenteric plexus of control and STC colon was quantitatively analyzed. In control colonic muscle strips, electrical field stimulation (EFS) evoked contractile responses and the release of acetylcholine (ACh) was measured in the presence of the CFTR channel inhibitor, CFTR(inh)-172. Results CFTR-IR was densely localized to myenteric ganglia, where it was co-localized with neuronal markers HuC/D and beta-tubulin, and glial marker S-100 but little with glial fibrillary acidic protein. Vesicular ACh transport was almost exclusively co-localized with CFTR, but neurons expressing nitric oxide synthase were CFTR negative. Significant reductions of CFTR-IR (P amp;lt; 0.01) and ChAT-IR (P amp;lt; 0.05) were observed on myenteric ganglia of STC compared to control. Pre-treatment of colonic muscle strips with CFTR(inh)-172 (10 mu M) significantly inhibited EFS-evoked contractile responses (P amp;lt; 0.01) and ACh release (P amp;lt; 0.05). Conclusions Co-localization of CFTR-IR with cholinergic markers, inhibition of EFS-induced colonic muscle contractility and ACh release by CFTR(inh)-172 suggest that CFTR modulates enteric cholinergic neurotransmission. The downregulation of CFTR and ChAT in myenteric ganglia of STC correlated with the impaired contractile responses to EFS., Funding Agencies|National Health and Medical Research Council of AustraliaNational Health and Medical Research Council of Australia [APP1048885]; UNSW; Shanghai Jiao Tong University

ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize CALHM1 and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. Gene expression by PCR and immunohistochemistry were performed in the porcine bladder. CALHM1 and pannexin-1 mediated ATP release in response to hypotonic solution (0.45% NaCl) induced stretch and extracellular Ca2+ depletion ([Ca2+]0) was measured in isolated urothelial, suburothelial and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial and detrusor muscle layers with a highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor,10Panx1, and by CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a non-selective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca2+]0 caused a marked but transient elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca2+]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells was insignificant. 10Panx1 showed no significant inhibition of [Ca2+]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1 that are densely expressed in the porcine bladder function as ATP release channels in response to bladder distension. Modulation of extracellular Ca2+ may also regulate ATP release in the porcine bladder through voltage gated CALHM1 ion channels.

ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize CALHM1 and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. Gene expression by PCR and immunohistochemistry were performed in the porcine bladder. CALHM1 and pannexin-1 mediated ATP release in response to hypotonic solution (0.45% NaCl) induced stretch and extracellular Ca2+ depletion ([Ca2+]0) was measured in isolated urothelial, suburothelial and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial and detrusor muscle layers with a highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor,10Panx1, and by CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a non-selective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca2+]0 caused a marked but transient elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca2+]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells was insignificant. 10Panx1 showed no significant inhibition of [Ca2+]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1 that are densely expressed in the porcine bladder function as ATP release channels in response to bladder distension. Modulation of extracellular Ca2+ may also regulate ATP release in the porcine bladder through voltage gated CALHM1 ion channels.