Linden, David R.

Electrophysiology is the study of the electrical properties of biological tissues, which involves the movement of ions across cell membranes. The analysis of the movement of electrical charges through the body has a wide range of biomedical applications, such as diagnosing and planning treatment in cardiovascular, nervous systems, muscular, and gastrointestinal disorders. The dielectric properties of biological tissues change according to the water content in the tissue and are measured as permittivity and conductivity relative to the frequency of the electrical field. This principle has been applied in diagnostics and therapeutics using microwave energysuch as imaging and ablation, etc. This review article summarizes the potential use of measuring dielectric properties using microwave imaging and how it can augment electrophysiological studies in medicine.

Abstract Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5‐HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na + , K + , Ca 2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca 2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca 2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP‐1) resulted in a rapid increase in cytoplasmic Ca 2+ and a slower decrease in ER stores Ca 2+ , suggesting the involvement of intracellular Ca 2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca 2+ store receptors, a similar expression of IP 3 receptors, but a >30‐fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca 2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP 3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca 2+ responses and 5‐HT release. In tissue, pressure‐induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca 2+ stores to amplify mechanically induced Ca 2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca 2+ signalling, 5‐HT release and epithelial secretion. image Key points A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca 2+ ) with a longer duration than the stimulus, suggesting intracellular Ca 2+ signal amplification. In this study, we profiled the expression of intracellular Ca 2+ store receptors and found an enriched expression of the intracellular Ca 2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5‐HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca 2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca 2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

Irritable bowel syndrome is a globally prevalent disorder categorized into constipation, diarrhea or mixed (IBS‐C, IBD‐D, IBS‐M) based on the predominant stool form/frequency. While the gut microbiome is recognized as an important factor underlying the pathophysiology of IBS, the specific mechanisms remain an area of active investigation. In our recent longitudinal multi‐omics study in IBS patients, we found bacteria‐derived hypoxanthine was significantly decreased in stool from IBS‐C patients. The aim of this study was to determine if IBS‐C relevant molecular pathways are affected by hypoxanthine. We hypothesized that hypoxanthine acts on enterochromaffin (EC) cells to stimulate serotonin (5‐HT) release, where 5‐HT is an important determinant of gastrointestinal (GI) transit time. We used Ca 2+ imaging in QGP cells (EC model), T84 cells (colonocyte model), primary cells and organoids from NeuroD1‐cre;GCaMP5‐tdTomato mice [NeuroD1 is a marker for enteroendocrine cells (EECs) which express calcium indicator GCaMP in these mice] in the absence or presence of receptor antagonists, and in ex vivo colon preparations with intact innervation from E2aCre:GCaMP6s mice (GCaMP is expressed in neuronal and non‐neuronal cells), to test the effect of hypoxanthine. The physiologic relevance of hypoxanthine signaling was assessed by measuring whole gut transit time. We found hypoxanthine increased Ca 2+ influx in QGP cells [using Cal520 (qualitative) and Fura‐2AM (quantitative)], primary EECs and EECs within organoids (previous research showed that the majority of these EECs are EC cells), but not in T84 cells. Hypoxanthine‐evoked Ca 2+ response was absent in Ca 2+ ‐free medium (n=6‐8, t‐test, P <0.0001), inhibited by G αi/o ‐GPCR antagonist (pertussis toxin; n=8‐14, t‐test, P <0.01)), Transient Receptor Potential Cation Channel Subfamily C Member 4 (TRPC4) antagonist (ML204; n=14‐15, t‐test, P <0.0001) and TRPC4 siRNA (n=5‐21, t‐test, P <0.01), and was accompanied by increased 5‐HT release (measured using 5‐HT biosensor). In ex vivo experiments, luminal application of hypoxanthine activated a subset of epithelial cells (likely EC cells) followed by subsets of neurons (latency to response onset, n=4, Mann‐Whitney, P <0.0001), as well as movement of the imaging field, which indicates smooth muscle contraction/relaxation. Our in vitro findings were confirmed by in vivo experiments showing that germ‐free mice colonized with hypoxanthine‐producing bacteria had faster whole gut transit and higher stool 5‐HT level compared to hypoxanthine‐consuming bacteria (n=7‐8, t‐test, P <0.05). In summary, hypoxanthine increases Ca 2+ influx specifically in EC cells by activating a signaling cascade involving a G αi/o ‐GPCR and TRPC4 channels, which results in 5‐HT release. The delay in activation of myenteric neurons and motor activity suggests that EC cell 5‐HT release likely induces neurogenic motor patterns and may underlie the shorter GI transit time seen with increased bacterial hypoxanthine production. While the current empirically designed probiotics have proven ineffective in IBS, our findings will allow development of novel mechanism‐based probiotic therapies for IBS‐C using hypoxanthine‐producing bacteria.

Background Microbial conversion of dietary substrate to bioactive molecules represents a potential regulatory mechanism by which gut microbes can alter intestinal physiology. Tryptamine, a monoamine structurally similar to serotonin (5‐HT), can be generated via decarboxylation of tryptophan by the gut bacteria but its physiological effect is not known. Aim To determine the effect of bacterial metabolite tryptamine on colonic secretion. Methods We mounted proximal colon mucosa‐submucosa preparations from germ free (GF), humanized (HM; ex‐GF colonized with human gut bacteria for 4–5 weeks), conventionally raised WT and 5‐HT4 receptor knockout (5‐HT4R −/− ) male mice in the Ussing chamber. The change in short circuit current (ΔIsc) was determined in response to application of increasing concentrations of tryptamine (3μM – 3mM) on the submucosal (basolateral) side either alone or in the presence of 5‐HT3R or 5‐HT4R antagonist, ondansetron (100nM) and GR‐113808 (30nM) respectively, with and without TTX (500nM). Colonoids (3‐dimensional epithelial organoids derived from colon) from GF and HM were used for swelling assay and ELISA based cAMP measurement. Results Tryptamine application significantly increased Isc in both GF and HM mice (n=6, P < 0.05, two‐way ANOVA with Bonferroni post‐hoc). Tryptamine mediated ΔIsc response was not significantly affected by amiloride, an inhibitor of epithelial sodium channels and Na + /H + exchangers, which are primarily responsible for absorption but was significantly decreased by 5‐Nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; anion blocker), a calcium‐sensitive chloride channel and sodium bicarbonate exchanger inhibitor responsible for anionic secretion in both GF and HM mice (n=4, P < 0.05, two‐way ANOVA with Bonferroni post‐hoc). Tryptamine evoked ΔIsc was not inhibited by 5‐HT3R (n=4, P > 0.05) but completely blocked by 5‐HT4R antagonist and was absent in 5HT4R knock out mice suggesting that the effect of tryptamine is mediated via 5HT4R. The increase in Isc in response to tryptamine and its inhibition by 5‐HT4R antagonist was also seen in presence of TTX (neuronal blocker; n=4, one‐way ANOVA , P < 0.05) suggesting that the epithelial 5‐HT4R alone can mediate the effect of tryptamine. Application of tryptamine (1mM) for 30 minutes led to a significant increase in the surface area of fluorescein labelled colonoids (n=5, one‐way ANOVA , P < 0.05), similar to forskolin, when compared to vehicle treated and 5HT4R antagonist pre‐treated GF and HM colonoids. Colonoids incubated with tryptamine (1mM) for 60 minutes had significantly higher cAMP levels (normalized to forskolin) compared to vehicle treated GF and HM colonoids, confirming that tryptamine activates 5‐HT4 GPCR (n=3, student's t‐test, P < 0.05). Conclusion Tryptamine, a tryptophan‐derived microbial metabolite, increases colonic secretion via 5HT4 GPCR activation. The identification of a bacterial metabolite activating a host GPCR to alter GI function represents an important advance in the field, and provides a distinct example of small molecule mediated host‐microbe interaction. Engineered probiotics producing tryptamine could be used as therapeutics in diseases associated with constipation. Support or Funding Information NIH DK111850, DK114007 (PCK) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

INTRODUCTION Intestinal enterochromaffin (EC) cells are the specialized epithelial mechanosensors of the gastrointestinal (GI) epithelium and are functionally and developmentally like Merkel cells in the skin. EC cells release serotonin (5‐HT) in response to mechanical stimuli. Recently, we discovered that like Merkel cells, human and mouse EC cells express the mechanosensitive (MS) ion channel Piezo2. However, little is known about the functional properties of this channel in primary EC cells. AIM To determine the role of Piezo2 in primary EC cells. METHODS The enteroendocrine cell transcription factor NeuroD1 was used to mark the late stage of EC cell development and generate NeuroD1‐cre::GCaMP5/tdTomato mouse to identify EC cells and examine their Ca 2+ dynamics. We examined colocalization of tdTomato with 5‐HT and Piezo2 by immunohistochemistry. We established and used primary colon epithelium cultures for whole‐cell patch clamp and Ca 2+ imaging experiments and mechanically stimulated by membrane displacement with a piezoelectrically driven glass probe or shear flow. RESULTS Immunostaining showed no tdTomato labeling outside of the GI epithelium, and epithelial tdTomato cells were 5‐HT+ and Piezo2+. Mechanical stimulation of primary EC cells by membrane displacement produced inward MS currents (20.8±4.9 pA/pF, C m =3.5 pF, n=6) with fast activation and inactivation kinetics (τ inact 11.1±2.8 ms), steep mechanosensitivity (Boltzmann function fit with a mid‐point of 3.46±0.80 μm and slope 0.79±0.27 μm) and a linear current‐voltage relationship (slope 0.61±0.17 pA/mV, x‐intercept −4.26±1.34 mV, n=5). Shear flow (20 s) transiently increased intracellular calcium ((Ca 2+ ) i ) (2.15±0.5 ΔF/F0, upstroke 3–5 s, return to baseline in 60 s, n=12). MS currents were inhibited by the MS ion channel blocker Gd 3+ (20.8±4.9 pA/pF versus 5.3±1.9 pA/pF), Piezo channel blocker GsMTx‐4 (20.8±4.9 pA/pF versus 2.5±0.9 pA/pF) and Piezo2 siRNA (20.8±4.9 pA/pF versus 1.1±0.3 pA/pF), but not non‐targeted (NT) siRNA (20.8±4.9 pA/pF versus 16.0±5.2 pA/pF and) (n=4–6 for each condition, p <0.05 for all except NT siRNA). Similarly, the (Ca 2+ ) i responses to force were inhibited by Gd 3+ (2.28±0.7 ΔF/F0 to 0.09±0.05 ΔF/F0), GsMTx‐4 (2.0±0.6 ΔF/F0 to 0.3±0.1 ΔF/F0), Piezo2 siRNA (2.15±0.5 ΔF/F0 to 0.08±0.05 ΔF/F0), but not NT siRNA (2.15±0.5 ΔF/F0 to 2.7±0.3 ΔF/F0) (n=4–6, p <0.05 for all except NT siRNA). CONCLUSIONS In the mouse epithelium, Piezo2+ EC cells were specifically marked by NeuroD1. Primary colonic NeuroD1+ cells had inward and non‐rectifying MS currents that could be blocked by Gd 3+ , GsMTx‐4 and Piezo2 siRNA. Similarly, shear flow evoked changes in (Ca 2+ ) i that were also blocked by Piezo2 inhibitors and dependent on extracellular Ca 2+ . These results suggest that the MS ion channel Piezo2 acts as the primary EC cell mechanosensor, whose activation by force leads to MS ionic current and (Ca 2+ ) i increase that may mediate mechanically‐induced 5‐HT release. Support or Funding Information This work was supported by NIH K08 DK106456, AGA Research Scholar Award, Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567) and DK52766. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and use of a simple method for small interfering RNA (siRNA) transfection of normal HBEs (NHBEs) in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology or morphology of NHBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.

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.

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.

Abstract Preterm infants can develop airway hyperreactivity and impaired bronchodilation following supplemental O 2 (hyperoxia) in early life, making it important to understand mechanisms of hyperoxia effects. Endogenous hydrogen sulfide (H 2 S) has anti‐inflammatory and vasodilatory effects with oxidative stress. There is little understanding of H 2 S signaling in developing airways. We hypothesized that the endogenous H 2 S system is detrimentally influenced by O 2 and conversely H 2 S signaling pathways can be leveraged to attenuate deleterious effects of O 2 . Using human fetal airway smooth muscle (fASM) cells, we investigated baseline expression of endogenous H 2 S machinery, and effects of exogenous H 2 S donors NaHS and GYY4137 in the context of moderate hyperoxia, with intracellular calcium regulation as a readout of contractility. Biochemical pathways for endogenous H 2 S generation and catabolism are present in fASM, and are differentially sensitive to O 2 toward overall reduction in H 2 S levels. H 2 S donors have downstream effects of reducing [Ca 2+ ] i responses to bronchoconstrictor agonist via blunted plasma membrane Ca 2+ influx: effects blocked by O 2 . However, such detrimental O 2 effects are targetable by exogenous H 2 S donors such as NaHS and GYY4137. These data provide novel information regarding the potential for H 2 S to act as a bronchodilator in developing airways in the context of oxygen exposure.