Gibbons, Simon J.

Background SCN5A ‐encoded Na V 1.5 is a mechanosensitive voltage‐gated sodium channel expressed in human gastrointestinal smooth muscle cells. In the gastrointestinal tract, alterations in Na V 1.5 current density by naturally occurring mutations or by pharmaceutical block of channel function are associated with constipation. In the heart loss of Na V 1.5 current is associated with cardiac contractility disorders such as dilated cardiomyopathy. Ion channel expression level is regulated by miRNAs, and Na V 1.5 is known to be regulated by miRNA in heart. However , it is not known if miRNAs regulate Na V 1.5 expression in GI smooth muscle. Aim To identify miRNAs that regulate Na V 1.5 expression in gastrointestinal smooth muscle in health and disease. Methods A qPCR array was used to screen for differential expression of 372 of the most common miRNAs in the miRBase database to test the differential expression of miRNA between control tissue and tissues from patients with slow transit constipation. qRT‐PCR was done to determine expression levels of SCN5A mRNA. A cell line (HuJej) was derived from human jejunum smooth muscle cells by immortalization using the SV40 large T antigen. Whole cell voltage clamp experiments were done by standard patch clamp techniques. Results SCN5A mRNA expression was reduced by 66±0.08% in patients with slow transit constipation (57.5±1.8 years) compared to age matched controls (53±1.7 years). qPCR array screening showed that 12 miRNAs were significantly upregulated in colonic muscularis externa from patients with STC compared to controls. Let‐7f miRNA was increased 6.46‐fold in slow transit constipation compared to controls and was the only miRNA with strong negative correlation to SCN5A mRNA expression in the same tissues. HuJej cells express Na V 1.5; therefore, we investigated the effect of let‐7f on Na V 1.5 currents by transfection with a stable miRNA mimic. 48 hours after transfection with let‐7f mimic, Na V 1.5 currents were significantly decreased compared to untransfected cells or to cells transfected with non‐targeting miRNA (let‐7f, −0.6±0.1 pA/pF versus untransfected, −3.9±0.6 pA/pF or non‐targeting, −2.3±0.6 pA/pF). Summary and Conclusions SCN5A mRNA is decreased in slow transit constipation. Expression levels of let‐7f and Na V 1.5 RNA are negatively correlated in human colonic smooth muscle. miRNA let‐7f regulates Na V 1.5 current density. Regulation of Na V 1.5 current density may have relevance in health and disease. Support or Funding Information This work was supported by grant DK52766.

NBCe1 is an electrogenic Na + bicarbonate cotransporter expressed as three isoforms. NBCe1A is mainly found in the kidney (100% activity), NBCe1B is a general isoform (~25% activity), and NBCe1C is found in the nervous system (20% activity). This project was to characterize mice with loss of NBCe1 in the kidney and urogenital tract and to test isoform activity ± IRBIT. Previous work has shown that NBCe1‐B is activated when IRBIT is present and interacts with the N‐terminus (Nt). Even though NBCe1C shares this Nt, the effects of IRBIT on human NBCe1C are unknown. Interstitial Cells of Cajal (ICC)pacemaker cells in the gastrointestinal tract ( c‐kit + ) have NBCe1C, IRBIT and require bicarbonate for normal gut activity. Developmentally similar ICC cells are found in the urogenital tract; however, NBCe1C locations within the kidney and urogenital tract are unknown. Using c‐kit ‐copGFP and NBCe1A‐knockout mice, kidneys, ureters, prostate, bladder, and urethra were dissected and analyzed via direct imaging, sectioning, and immunofluorescence. Oocytes injected with NBCe1C ± IRBIT were voltage clamped to determine the cotransporter's activity. “Strings” of c‐kit + ‐cells were found in the copGFP & nbce1A kidneys. In nbce1 (+/−) ‐(Het) mice (Figure), prostate ducts appeared enlarged, and in nbce1A − / − kidney, c‐kit protein was broadly expressed, showing cluster colocalization with NBCe1C. NBCe1C is not obviously expressed in WT or copGFP mice. Electrophysiology data showed that IRBIT activates NBCe1C by ~10‐fold. As c‐kit is also a marker of stem cells, NBCe1C/c‐kit association and c‐kit increase in nbce1A − / − mice, could suggest that NBCe1A loss elicits kidney repair itself (proliferation) and causes expression of other NBCe1‐isoforms. Support or Funding Information R25‐DK101405, R01‐DK057061, U54‐DK100227 (Mayo) and U54‐DK104310 (UW‐Madison) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Electrical slow wave activity, generated by interstitial cells of Cajal (ICC), is essential for efficient gastrointestinal (GI) transit. Electrophysiology of slow waves has been extensively studied; inclusion of bicarbonate (HCO 3 − ) in extracellular solutions is required for recording normal slow wave activity. HCO 3 − transport contributes to regulation of intracellular pH, which has multiple consequences for normal cellular function. Previous transcriptional profiling found that the Na + / HCO 3 − cotransporter (NBCe1/Slc4a4) is enriched in pacemaker myenteric ICC (ICC‐MY) compared to non pacemaker ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. Our aim was to determine the distribution and identity of Slc4a4/NBCe1 variants in ICC in the GI tract. NBCe1 immunoreactivity (IR) was detected by fluorescence labeling of parafomaldehyde‐fixed cross sections and whole mounts of adult mouse small intestine, colon and stomach using primary antibodies directed against a 329 amino acid sequence in the N terminus of the NBCe1 protein. Labeling of Kit receptor tyrosine kinase with a goat anti‐Kit polyclonal antibody was used to detect ICC. Slc4a4 knockout (KO) mice were bred from heterozygous founders. Slc4a4 fl/fl mice were bred with ETV1 CreERT2/− mice to allow targeted, conditional KO of Slc4a4 in ICC in response to tamoxifen treatment (130 mg/Kg). mRNA transcripts for Slc4a4 in Kit expressing cells were isolated from Kit‐ribotag mice, which were generated by breeding Kit creERT2 with Rpl22 tm1.1Psam mice. The abundance of specific Slc4a4 transcripts was measured by quantitative real time PCR (qRT‐PCR). Green fluorescent protein (GFP)‐labeled ICC were dissociated from the small intestine of neonatal Kit copGFP mice and RNA expression levels were determined using the Single Cell‐to‐CT™ qRT‐PCR Kit from Ambion. NBCe1‐IR was absent in all cells in Slc4a4‐KO tissues but co‐localized with Kit in ICC‐MY, but not in ICC‐DMP, in small intestine of wild type mice. In the proximal and distal colon, pacemaker submucosal ICC (ICC‐SMP) but not intramuscular ICC (ICC‐IM) or ICC‐MY showed NBCe1‐IR. In the corpus and antrum of the stomach NBCe1‐IR was detected in ICC‐MY but not in ICC‐IM. NBCe1‐IR did not co‐localize with Kit in the gastric fundus. mRNA for variant C, but not variants A or B of Slc4a4, was detected in small intestinal ICC‐MY. Reduced NBCe1‐IR was restricted to small intestinal ICC‐MY of Slc4a4 conditional KO mice. NBCe1 is enriched in subtypes of ICC responsible for pacemaker activity in all regions of the GI tract indicating a role for this transporter in slow wave generation. The presence of the C variant of Slc4a4 in ICC‐MY suggests dynamic regulation of NBCe1 activity in ICC by the I P 3 R b inding protein released with i nositol 1, 4, 5‐ t risphosphate (IRBIT). Support or Funding Information Supported by NIH R01 DK57061. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Ano1 is a Ca 2+ activated Cl − channel expressed in several proliferating cell types including various epithelia, gastrointestinal stromal tumors, interstitial cells of Cajal (ICC) and CFPAC‐1 cells. The aim of this study was to determine if Ano1 regulates proliferation. ICC from Ano1 (−/−) and Ano1 (+/+) mice were cultured with Cl − channel inhibitors or in low Cl − media. CFPAC‐1 cells grown in low Cl − media were pulsed with EdU for 1 h. Incorporation was measured by flow cytometry. For immunoblots on mouse jejunum, antibodies to pRb, phospho(Ser773)‐pRb and GAPDH were used. Fewer proliferating ICC were detected in Ano1 (−/−) cultures (15 ± 3.5% Ki67 + ICC vs 25 ± 4% in Ano1 (+/+) , mean ± SEM, n = 4, p < 0.05). Cl − channel inhibitors DIDS (10 μM), niflumate (10 μM) and tamoxifen (1 μM) decreased ICC proliferation by 53, 53 and 59%, respectively ( p < 0.05). This effect was reduced in cultures from Ano1 (−/−) mice (29% and 19 % decrease, 22% increase, p > 0.05). Fewer proliferating ICC and CFPAC‐1 cells were detected in low Cl − media (% EdU + ICC: 120 mM; 19.8 ± 5.3, 12 mM; 8.5 ± 3.2, n = 4, and % EdU + CFPAC‐1: 120 mM; 29.5, 12 mM; 14.3 p < 0.05). Proportionally less Ser773‐phosphorylated pRb was detected in jejunum from Ano1 (−/−) mice (Ano1 (+/+) ; 5.98 ± 0.78, Ano1 (−/−) ; 3.60 ± 0.49, mean ± SEM, n = 7, p < 0.05). In conclusion, Ano1 regulates proliferation possibly by promoting the G 1 /S transition of the cell cycle. Support: DK57061 and DK68055.

AbstractBackgroundNormal gastrointestinal motility depends on electrical slow‐wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3− in extracellular solutions used to record slow waves indicates a role for HCO3− transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+/HCO3− cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC‐MY) that generate slow waves. This study aimed to determine how extracellular HCO3− concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects.MethodsImmunohistochemistry and sharp electrode electrical recordings were used.Key ResultsThe NBCe1 immunoreactivity was localized to ICC‐MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3‐ from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow‐wave amplitude and frequency. In 100 mM HCO3−, the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+, an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3‐. There were no additional changes to electrical activity when HCO3‐ was removed from Li+ containing solutions. The Na+/HCO3− cotransport inhibitor, S‐0859 (30µM) significantly reduced the effect of removing HCO3− on electrical activity.Conclusions & InferencesThese studies demonstrate a major role for Na+/HCO3− cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.

In this study, we show that the electrogenic Na+/HCO3− cotransporter, NBCe1/Slc4a4, is expressed in subtypes of interstitial cells of Cajal (ICCs) responsible for electrical slow wave generation throughout the mouse gastrointestinal tract and is absent in other types of ICCs. The transcripts of Slc4a4 expressed in mouse ICCs and human gastrointestinal smooth muscle are the regulated isoforms. This indicates a key role for HCO3- transport in generation of gastrointestinal motility patterns.

AbstractBackground and AimMuscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine.MethodsThe distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy.Key ResultsImmunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα‐positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell‐to‐cell contacts with smooth muscle cells and TCs/PDGFRα‐positive cells but not with ICCs and enteric neurons.Conclusion & InferencesThis study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell‐to‐cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα‐positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.

AbstractBackgroundGastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading‐edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies.MethodWe have developed an easily achievable, labor‐saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time‐lapse video for offline analysis.Key ResultWe describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10‐15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach.Conclusion & InferencesA novel automated, inexpensive, and easily assembled whole gut transit setup is labor‐saving and allows minimal disruption to animal behavior compared with the conventional approach.

AbstractBackgroundThe gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut‐brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum.MethodsAdvillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin‐expressing enteric neurons using confocal microscopy, calcium imaging, and whole‐cell patch‐clamp electrophysiology.Key ResultsAdvillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene‐related peptide (CGRP), but not nNOS.Conclusions and InferencesAdvillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A , the gene that encodes the Na + channel Na V 1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na + current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na + channel Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.