Gibbons, S J

The SCN5A-encoded voltage-gated mechanosensitive Na+channel NaV1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. NaV1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2–3% of patients have SCN5A missense mutations that alter NaV1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting NaV1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. NaV1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated NaV1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered NaV1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in NaV1.5 channels with abnormal voltage-dependent and mechanosensitive function.NEW & NOTEWORTHY The voltage-gated Na+channel NaV1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the NaV1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant NaV1.5 channels were loss of function in voltage dependence or mechanosensitivity.

New Findings What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl− homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl− homeostasis is regulated by anion‐permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl− (Cl−o) with the impermeant anions gluconate and isethionate. On reducing Cl−o, effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (Em). Restoration of Cl− hyperpolarized smooth muscle Em proportional to the change in Cl−o concentration. Replacement of 90% of Cl−o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration‐dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca2+ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl− and 0.13 mm free Ca2+, slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl−o was not observed in W/Wv mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl− conductance is low, whereas transmembrane Cl− movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle Em and that altering Cl− homeostasis in ICCs can alter smooth muscle Em.

Anoctamin1 (Ano1 and TMEM16A) is a calcium-activated chloride channel specifically expressed in the interstitial cells of Cajal (ICC) of the gastrointestinal tract muscularis propria. Ano1 is necessary for normal electrical slow waves and ICC proliferation. The full-length human Ano1 sequence includes an additional exon, exon “0,” at the NH2 terminus. Ano1 with exon 0 [Ano1(0)] had a lower EC50 for intracellular calcium ([Ca2+]i) and faster chloride current ( ICl) kinetics. The Ano1 alternative splice variant with segment “c” encoding exon 13 expresses on the first intracellular loop four additional amino acid residues, EAVK, which alter ICl at low [Ca2+]i. Exon 13 is expressed in 75–100% of Ano1 transcripts in most human tissues but only 25% in the human stomach. Our aim was to determine the effect of EAVK deletion on Ano1(0) ICl parameters. By voltage-clamp electrophysiology, we examined ICl in HEK293 cells transiently expressing Ano1(0) with or without the EAVK sequence [Ano1(0)ΔEAVK]. The EC50 values of activating and deactivating ICl for [Ca2+]i were 438 ± 7 and 493 ± 9 nM for Ano1(0) but higher for Ano1(0)ΔEAVK at 746 ± 47 and 761 ± 26 nM, respectively. Meanwhile, the EC50 values for the ratio of instantaneous to steady-state ICl were not different between variants. Congruently, the time constant of activation was slower for Ano1(0)ΔEAVK than Ano1(0) currents at intermediate [Ca2+]i. These results suggest that EAVK decreases the calcium sensitivity of Ano1(0) current activation and deactivation by slowing activation kinetics. Differential expression of EAVK in the human stomach may function as a switch to increase sensitivity to [Ca2+]i via faster gating of Ano1. NEW & NOTEWORTHY Calcium-activated chloride channel anoctamin1 (Ano1) is necessary for normal slow waves in the gastrointestinal interstitial cells of Cajal. Exon 0 encodes the NH2 terminus of full-length human Ano1 [Ano1(0)], while exon 13 encodes residues EAVK on its first intracellular loop. Splice variants lack EAVK more often in the stomach than other tissues. Ano1(0) without EAVK [Ano1(0)ΔEAVK] has reduced sensitivity for intracellular calcium, attributable to slower kinetics. Differential expression of EAVK may function as a calcium-sensitive switch in the human stomach.

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl–channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Flmice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+transients’ synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+transients.NEW & NOTEWORTHY The Ca2+-activated Cl−channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+transients in myenteric ICC but Ca2+transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.View this article's corresponding video summary at https://youtu.be/cyPtDP0KLY4 .

Noninvasive breath tests for gastric emptying are important techniques for understanding the changes in gastric motility that occur in disease or in response to drugs. Mice are often used as an animal model; however, the gamma variate model currently used for data analysis does not always fit the data appropriately. The aim of this study was to determine appropriate mathematical models to better fit mouse gastric emptying data including when two peaks are present in the gastric emptying curve. We fitted 175 gastric emptying data sets with two standard models (gamma variate and power exponential), with a gamma variate model that includes stretched exponential and with a proposed two-component model. The appropriateness of the fit was assessed by the Akaike Information Criterion. We found that extension of the gamma variate model to include a stretched exponential improves the fit, which allows for a better estimation of T1/2and Tlag. When two distinct peaks in gastric emptying are present, a two-component model is required for the most appropriate fit. We conclude that use of a stretched exponential gamma variate model and when appropriate a two-component model will result in a better estimate of physiologically relevant parameters when analyzing mouse gastric emptying data.

AbstractBackgroundOtilonium bromide (OB) is used as a spasmolytic drug in the treatment of the functional bowel disorder irritable bowel syndrome. Although its acute effects on colonic relaxation are well‐characterized, little is known about the effects of chronic administration ofOBon enteric neurons, neuromuscular transmission, and interstitial cells of Cajal (ICC), key regulators of the gut function.MethodsAdult Sprague–Dawley rats were treated withOBin drinking water at a dose of 2 mg/kg for 30 days. The colons ofOB‐treated and age‐matched control rats were studied by confocal immunohistochemistry to detect immunoreactivity (IR) in myenteric plexus neurons for nitrergic and tachykininergic markers, and also by microelectrode electrophysiology.Key ResultsUsing immunohistochemistry, chronicOBadministration did not change total neuron number, assessed by anti‐HuIR, but resulted in a significant increase inNK1 receptor positive neurons, a decrease in neuronal nitric oxide synthase expressing neurons, and a reduction in volume of substance P in nerve fibers in the myenteric plexus. ChronicOBadministration potentiated inhibitory and excitatory junction potentials evoked by repetitive electrical field stimulation. The various types of colonicICC, detected by KitIR, were not altered nor were slow waves or smooth muscle membrane potential.Conclusions & InferencesChronic treatment withOBcaused significant changes in the nitrergic and tachykinergic components of the myenteric plexus and in both inhibitory and excitatory neurotransmission in the rat colon.

The binding of Steel factor (SF) to c-kit initiates a signaling pathway essential for development and maintenance of interstitial cells of Cajal (ICC). Soluble and membrane-bound isoforms of SF are expressed in the gastrointestinal tract, but the role for either isoform in supporting ICC development is unknown. The aim of this study was to determine the role of SF in supporting ICC in culture. ICC were cultured from dissociated mouse jejunum and grown with fibroblast cell lines that produced either soluble, membrane-bound or membrane-restricted SF. ICC were identified and counted by c-kit immunoreactivity. The number of c-kit immunoreactive cells was greater in the coculture system compared with cultures grown without SF-producing fibroblasts. All forms of SF-producing fibroblasts increased ICC number in culture but physical separation of the fibroblasts from the c-kit immunoreactive cells, the addition of exogenous SF to the culture medium, or fibroblast-conditioned media did not. These results are consistent with the hypothesis that the membrane-bound form of SF preferentially contributes to expression of c-kit-positive ICC under cell culture conditions.

Anoctamin‐1 (Ano1) is a widely expressed protein responsible for endogenous Ca 2+ ‐activated Cl 2 currents. Ano1 is overexpressed in cancer. Differential expression of transcriptional variants is also found in other diseases. However, the mechanisms underlying regulation of Ano1 are unknown. This study identifies the Ano1 promoter and defines a mechanism for regulating its expression. Next‐generation RNA sequencing (RNA‐seq) analysis in human gastric muscle found a new exon upstream of the reported exon 1 and identified a promoter proximal to this new exon. Reporter assays in human embryonic kidney 293 cells showed a 6.7 ± 2.1fold increase in activity over empty vector. Treatment with a known regulator of Ano1 expression, IL‐4, increased promoter activity by 1.6 ± 0.02‐fold over untreated cells. The promoter region contained putative binding sites for multiple transcription factors including signal transducer and activator of transcription 6 (STAT6), a downstream effector of IL‐4. Chromatin immunoprecipitation (ChIP) experiments on T84 cells, which endogenously express Ano1, showed a 2.1 ± 0.12‐fold increase in binding of STAT6 to P0 after IL‐4 treatment. These results were confirmed by mutagenesis, expression, and RNA interference techniques. This work allows deeper understanding of the regulation of Ano1 in physiology and as a potential therapeutic target in a variety of diseases—Mazzone, A., Gibbons, S. J., Bernard, C. E., Nowsheen, S., Middha, S., Almada, L. L., Ordog, T., Kendrick, M. L., Reid Lombardo, KM., Shen, K. R., Galietta, L. J. V., Fernandez‐Zapico, M. E., and Farrugia, G., Identification and characterization of a novel promoter for the human ANO1 gene regulated by the transcription factor signal transducer and activator of transcription 6 (STAT6). FASEB J. 29, 152–163 (2015). www.fasebj.org

Abstract Background Diabetic gastroparesis results in significant morbidity for patients and major economic burden for society. Treatment options for diabetic gastroparesis are currently directed at symptom control rather than the underlying disease and are limited. The pathophysiology of diabetic gastroparesis includes damage to intrinsic and extrinsic neurons, smooth muscle, and interstitial cells of C ajal ( ICC ). Oxidative damage in diabetes appears to be one of the primary insults involved in the pathogenesis of several complications of diabetes, including gastroparesis. Recent studies have highlighted the potential role of macrophages as key cellular elements in the pathogenesis of diabetic gastroparesis. Macrophages are important for both homeostasis and defense against a variety of pathogens. Heme oxygenase 1 ( HO 1), an enzyme expressed in a subset of macrophages has emerged as a major protective mechanism against oxidative stress. Activation of macrophages with high levels of HO 1 expression protects against development of delayed gastric emptying in animal models of diabetes, while activation of macrophages that do not express HO 1 are linked to neuromuscular cell injury. Targeting macrophages and HO 1 may therefore be a therapeutic option in diabetic gastroparesis. Purpose This report briefly reviews the pathophysiology of diabetic gastroparesis with a focus on oxidative damage and how activation and polarization of different subtypes of macrophages in the muscularis propria determines development of delay in gastric emptying or protects against its development.

AbstractBackgroundHeme oxygenase 1 (HO‐1) degrades heme and protects against oxidative stress. In vitro and animal models suggest that HO‐1 is beneficial in several diseases (e.g., postoperative ileus, gastroparesis, acute pancreatitis, and colitis). However, the only drugs (i.e., hemin and heme arginate) which pharmacologically upregulate HO‐1 in humans are expensive and can only be administered intravenously. Our aims were to compare the effects of placebo, aspirin, and simvastatin alone, and with α‐lipoic acid, on HO‐1 protein concentration and activity in humans.MethodsThis randomized, double‐blind, placebo‐controlled study compared the effects of three oral regimens administered for 7 days, i.e., placebo; aspirin (325 mg twice daily) and simvastatin (40 mg twice daily); aspirin, simvastatin, and the sodium salt of R‐ α‐lipoic acid (NaRLA, 600 mg three times daily) on markers of HO‐1 activation (i.e., plasma HO‐1 protein concentration and venous monocyte HO‐1 protein activity) in 18 healthy subjects (14 females). Markers of HO‐1 activation were evaluated at baseline, days 2, and 7.Key ResultsBaseline HO‐1 protein concentrations and activity were similar among the three groups. Compared to placebo, aspirin and simvastatin combined, or together with NaRLA did not affect HO‐1 protein concentration or activity at 2 or 7 days. HO‐1 protein concentrations and activity were correlated on day 7 (r = 0.75, p = 0.0004) but not at baseline and on day 2.Conclusions & InferencesAt therapeutic doses, aspirin, simvastatin, and α‐lipoic acid do not increase plasma HO‐1 protein concentration or venous monocyte HO‐1 activity in healthy humans.