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Background & Aims Interstitial cells of Cajal (ICCs) and pancreatic β cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve microRNA (miRNA)-mediated post-transcriptional gene silencing in KIT+ cells. Methods We performed miRNA-sequencing analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs antidiabetic and prokinetic medicines. Results miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+ cells or depletion of KIT+ cells in mice leads to degeneration of β cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Kruppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 small interfering RNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescue the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and gastrointestinal motility compared with common antidiabetic and prokinetic medications. Conclusions miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

High fidelity segmentation of both macro and microvascular structure of the retina plays a pivotal role in determining degenerative retinal diseases, yet it is a difficult problem. Due to successive resolution loss in the encoding phase combined with the inability to recover this lost information in the decoding phase, autoencoding based segmentation approaches are limited in their ability to extract retinal microvascular structure. We propose RV-GAN, a new multi-scale generative architecture for accurate retinal vessel segmentation to alleviate this. The proposed architecture uses two generators and two multi-scale autoencoding discriminators for better microvessel localization and segmentation. In order to avoid the loss of fidelity suffered by traditional GAN-based segmentation systems, we introduce a novel weighted feature matching loss. This new loss incorporates and prioritizes features from the discriminator’s decoder over the encoder. Doing so combined with the fact that the discriminator’s decoder attempts to determine real or fake images at the pixel level better preserves macro and microvascular structure. By combining reconstruction and weighted feature matching loss, the proposed architecture achieves an area under the curve (AUC) of 0.9887, 0.9914, and 0.9887 in pixel-wise segmentation of retinal vasculature from three publicly available datasets, namely DRIVE, CHASE-DB1, and STARE, respectively. Additionally, RV-GAN outperforms other architectures in two additional relevant metrics, mean intersection-over-union (Mean-IOU) and structural similarity measure (SSIM).

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contributing to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+signaling in ICC-SM and the relationship between ICC-SM Ca2+transients and activation of SMCs using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ∼15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+entry through voltage-dependent Ca2+conductances, L- and T-type Ca2+channels. Removal of Ca2+from the external solution abolished CTCs. Ca2+release mechanisms reduced the duration and amplitude of Ca2+transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+firing patterns and drive smooth muscle activity and overall colonic contractions.SynopsisHow Ca2+signaling in colonic submucosal pacemaker cells couples to smooth muscle responses is unknown. This study shows how ICC modulate colonic motility via complex Ca2+signaling and defines Ca2+transients’ sources using optogenetic techniques.

High-resolution Ca(2+) imaging to study cellular Ca(2+) behaviors has led to the creation of large datasets with a profound need for standardized and accurate analysis. To analyze these datasets, spatio-temporal maps (STMaps) that allow for 2D visualization of Ca(2+) signals as a function of time and space are often used. Methods of STMap analysis rely on a highly arduous process of user defined segmentation and event-based data retrieval. These methods are often time consuming, lack accuracy, and extremely variable between users. We designed a novel automated machine-learning based plugin for the analysis of Ca(2+) STMaps (STMapAuto). The plugin includes optimized tools for Ca(2+) signal preprocessing, automated segmentation, and automated extraction of key Ca(2+) event information such as: duration, spatial spread, frequency, propagation angle, and intensity in a variety of cell types including the Interstitial cells of Cajal (ICC). The plugin is fully implemented in Fiji and able to accurately detect and expeditiously quantify Ca(2+) transient parameters from ICC. The plugin’s speed of analysis of large-datasets was 197-fold faster than the commonly used single pixel-line method of analysis. The automated machine-learning based plugin described dramatically reduces opportunities for user error and provides a consistent method to allow high-throughput analysis of STMap datasets.

In this issue of Science Signaling, Thakore et al. report that the Ca(2+) permeable channel TRPML1 closely associates with ryanodine receptors to induce Ca(2+) sparks in native arterial myocytes. Functional studies revealed a key role for TRPML1 channels in regulation of arterial myocyte contractility and blood pressure.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

Patients with diabetes often develop gastrointestinal motor problems, including gastroparesis. Previous studies have suggested this gastric motor disorder was a consequence of an enteric neuropathy. Disruptions in interstitial cells of Cajal (ICC) have also been reported. A thorough examination of functional changes in gastric motor activity during diabetes has not yet been performed. We comprehensively examined the gastric antrums of Lepob mice using functional, morphological, and molecular techniques to determine the pathophysiological consequences in this type 2 diabetic animal model. Video analysis and isometric force measurements revealed higher frequency and less robust antral contractions in Lepob mice compared with controls. Electrical pacemaker activity was reduced in amplitude and increased in frequency. Populations of enteric neurons, ICC, and platelet-derived growth factor receptor α+ cells were unchanged. Analysis of components of the prostaglandin pathway revealed upregulation of multiple enzymes and receptors. Prostaglandin-endoperoxide synthase-2 inhibition increased slow wave amplitudes and reduced frequency of diabetic antrums. In conclusion, gastric pacemaker and contractile activity is disordered in type 2 diabetic mice, and this appears to be a consequence of excessive prostaglandin signaling. Inhibition of prostaglandin synthesis may provide a novel treatment for diabetic gastric motility disorders.

Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca(2+) release in ICC-IM activates Ano1, a Ca(2+)-activated Cl(−) conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca(2+) imaging of colonic ICC-IM in situ, using mice expressing GCaMP6f in ICC to evaluate ICC-IM responses to excitatory neurotransmission. Expression of muscarinic type 2, 3 (M(2), M(3)) and NK(1) receptors were enriched in ICC-IM. NK(1) receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca(2+) transients. These effects were reversed by DAU 5884 (M(3) receptor antagonist) but not AF-DX 116 (M(2) receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca(2+) transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca(2+) stores with cyclopiazonic acid and reduced by inhibiting Ca(2+) influx via Orai channels. Contractions induced by EFS were reduced by an Ano1 channel antagonist, abolished by DAU 5884 and unaffected by AF-DX 116. Colonic ICC-IM receive excitatory inputs from cholinergic neurons via M(3) receptor activation. Enhancing ICC-IM Ca(2+) release and Ano1 activation contributes to excitatory responses of colonic muscles.

Interstitial cells of Cajal (ICC) are pacemaker cells that generate electrical slow waves in gastrointestinal (GI) smooth muscles. Slow waves organize basic motor patterns, such as peristalsis and segmentation in the GI tract. Slow waves depend upon activation of Ca2+-activated Cl- channels (CaCC) encoded by Ano1. Slow waves consist of an upstroke depolarization and a sustained plateau potential that is the main factor leading to excitation-contraction coupling. The plateau phase can last for seconds in some regions of the GI tract. How elevated Ca2+ is maintained throughout the duration of slow waves, which is necessary for sustained activation of CaCC, is unknown. Modeling has suggested a role for Na+/Ca2+ exchanger (NCX) in regulating CaCC currents in ICC, so we tested this idea on murine intestinal ICC. ICC of small and large intestine express NCX isoforms. NCX3 is closely associated with ANO1 in ICC, as shown by immunoprecipitation and proximity ligation assays (PLA). KB-R7943, an inhibitor of NCX, increased CaCC current in ICC, suggesting that NCX, acting in Ca2+ exit mode, helps to regulate basal [Ca2+] i in these cells. Shifting NCX into Ca2+ entry mode by replacing extracellular Na+ with Li+ increased spontaneous transient inward currents (STICs), due to activation of CaCC. Stepping ICC from -80 to -40 mV activated slow wave currents that were reduced in amplitude and duration by NCX inhibitors, KB-R7943 and SN-6, and enhanced by increasing the NCX driving force. SN-6 reduced the duration of clustered Ca2+ transients that underlie the activation of CaCC and the plateau phase of slow waves. Our results suggest that NCX participates in slow waves as modeling has predicted. Dynamic changes in membrane potential and ionic gradients during slow waves appear to flip the directionality of NCX, facilitating removal of Ca2+ during the inter-slow wave interval and providing Ca2+ for sustained activation of ANO1 during the slow wave plateau phase.

Key points Platelet-derived growth factor receptor-α (PDGFRα) is a novel biomarker along with smooth myosin heavy chain for the pacemaker cells (previously termed 'atypical' smooth muscle cells) in the murine and cynomolgus monkey pelvis-kidney junction. PDGFRα+ cells present in adventitial and urothelial layers of murine renal pelvis do not express smooth muscle myosin heavy chain (smMHC) but are in close apposition to nerve fibres. Most c-Kit+ cells in the renal pelvis are mast cells. Mast cells (CD117+ /CD45+ ) are more abundant in the proximal renal pelvis and pelvis-kidney junction regions whereas c-Kit+ interstitial cells (CD117+ /CD45- ) are found predominantly in the distal renal pelvis and ureteropelvic junction. PDGFRα+ cells are distinct from c-Kit+ interstitial cells. A subset of PDGFRα+ cells express the Ca2+ -activated Cl- channel, anoctamin-1, across the entire renal pelvis. Spontaneous Ca2+ transients were observed in c-Kit+ interstitial cells, smMHC+ PDGFRα cells and smMHC- PDGFRα cells using mice expressing genetically encoded Ca2+ sensors. Abstract Rhythmic contractions of the renal pelvis transport urine from the kidneys into the ureter. Specialized pacemaker cells, termed atypical smooth muscle cells (ASMCs), are thought to drive the peristaltic contractions of typical smooth muscle cells (TSMCs) in the renal pelvis. Interstitial cells (ICs) in close proximity to ASMCs and TSMCs have been described, but the role of these cells is poorly understood. The presence and distributions of platelet-derived growth factor receptor-α+ (PDGFRα+ ) ICs in the pelvis-kidney junction (PKJ) and distal renal pelvis were evaluated. We found PDGFRα+ ICs in the adventitial layers of the pelvis, the muscle layer of the PKJ and the adventitia of the distal pelvis. PDGFRα+ ICs were distinct from c-Kit+ ICs in the renal pelvis. c-Kit+ ICs are a minor population of ICs in murine renal pelvis. The majority of c-Kit+ cells were mast cells. PDGFRα+ cells in the PKJ co-expressed smooth muscle myosin heavy chain (smMHC) and several other smooth muscle gene transcripts, indicating these cells are ASMCs, and PDGFRα is a novel biomarker for ASMCs. PDGFRα+ cells also express Ano1, which encodes a Ca2+ -activated Cl- conductance that serves as a primary pacemaker conductance in ICs of the GI tract. Spontaneous Ca2+ transients were observed in c-Kit+ ICs, smMHC+ PDGFRα cells and smMHC- PDGFRα cells using genetically encoded Ca2+ sensors. A reporter strain of mice with enhanced green fluorescent protein driven by the endogenous promotor for Pdgfra was shown to be a powerful new tool for isolating and characterizing the phenotype and functions of these cells in the renal pelvis.

Transcriptome data revealed α1 adrenoceptors (ARs) expression in platelet-derived growth factor receptor α+ cells (PDGFRα+ cells) in murine colonic musculature. The role of PDGFRα+ cells in sympathetic neural regulation of murine colonic motility was investigated. Norepinephrine (NE), via α1A ARs, activated a small conductance Ca2+ -activated K+ (SK) conductance, evoked outward currents and hyperpolarized PDGFRα+ cells (the α1A AR-SK channel signal pathway). α1 AR agonists increased intracellular Ca2+ transients in PDGFRα+ cells and inhibited spontaneous phasic contractions (SPCs) of colonic muscle through activation of a SK conductance. Sympathetic nerve stimulation inhibited both contractions of distal colon and propulsive contractions represented by the colonic migrating motor complexes (CMMCs) via the α1A AR-SK channel signal pathway. Postsynaptic signaling through α1A ARs in PDGFRα+ cells is a novel mechanism that conveys part of stress responses in the colon. PDGFRα+ cells appear to be a primary effector of sympathetic neural regulation of murine colonic motility.

Key points Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca(2+) from intracellular stores and activation of Ca(2+)-activated Cl(−) channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca(2+) handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na(+)/Ca(2+) exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca(2+) imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca(2+) transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca(2+) indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na(+)/Ca(2+) exchange inhibitor, CGP-37157 inhibited Ca(2+) transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca(2+) transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca(2+) (Ca(V)3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced Ca(V)3.2 currents. CCCP blocked Ca(V)3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca(2+) signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.

Key points Contraction of urethral smooth muscle cells (USMCs) contributes to urinary continence. Ca2+ signalling in USMCs was investigated in intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs were spontaneously active in situ, firing intracellular Ca2+ waves that were asynchronous at different sites within cells and between adjacent cells. Spontaneous Ca2+ waves in USMCs were myogenic but enhanced by adrenergic or purinergic agonists and decreased by nitric oxide. Ca2+ waves arose from inositol trisphosphate type 1 receptors and ryanodine receptors, and Ca2+ influx by store-operated calcium entry was required to maintain Ca2+ release events. Ca2+ release and development of Ca2+ waves appear to be the primary source of Ca2+ for excitation-contraction coupling in the mouse urethra, and no evidence was found that voltage-dependent Ca2+ entry via L-type or T-type channels was required for responses to α adrenergic responses. Abstract Urethral smooth muscle cells (USMCs) generate myogenic tone and contribute to urinary continence. Currently, little is known about Ca2+ signalling in USMCs in situ, and therefore little is known about the source(s) of Ca2+ required for excitation-contraction coupling. We characterized Ca2+ signalling in USMCs within intact urethral muscles using a genetically encoded Ca2+ sensor, GCaMP3, expressed selectively in USMCs. USMCs fired spontaneous intracellular Ca2+ waves that did not propagate cell-to-cell across muscle bundles. Ca2+ waves increased dramatically in response to the α1 adrenoceptor agonist phenylephrine (10 μm) and to ATP (10 μm). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 μm). Ca2+ influx and release from sarcoplasmic reticulum stores contributed to Ca2+ waves, as Ca2+ free bathing solution and blocking the sarcoplasmic Ca2+ -ATPase abolished activity. Intracellular Ca2+ release involved cooperation between ryanadine receptors and inositol trisphosphate receptors, as tetracaine and ryanodine (100 μm) and xestospongin C (1 μm) reduced Ca2+ waves. Ca2+ waves were insensitive to L-type Ca2+ channel modulators nifedipine (1 μm), nicardipine (1 μm), isradipine (1 μm) and FPL 64176 (1 μm), and were unaffected by the T-type Ca2+ channel antagonists NNC-550396 (1 μm) and TTA-A2 (1 μm). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10 μm) and by an Orai antagonist, GSK-7975A (1 μm). The latter also reduced urethral contractions induced by phenylephrine, suggesting that Orai can function effectively as a receptor-operated channel. In conclusion, Ca2+ waves in mouse USMCs are a source of Ca2+ for excitation-contraction coupling in urethral muscles.

KEY POINTS Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post-junctional responses are controversial. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC-IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC-IM. Conductances activated by cholinergic stimulation in isolated ICC-IM and SMCs were determined. A CaCC was activated by carbachol in ICC-IM and a non-selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild-type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC-IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC-IM and not SMCs. ABSTRACT Enteric motor neurotransmission is essential for normal gastrointestinal (GI) motility. Controversy exists regarding the cells and ionic conductance(s) that mediate post-junctional neuroeffector responses to motor neurotransmitters. Isolated intramuscular ICC (ICC-IM) and smooth muscle cells (SMCs) from murine fundus muscles were used to determine the conductances activated by carbachol (CCh) in each cell type. The calcium-activated chloride conductance (CaCC), anoctamin-1 (Ano1) is expressed by ICC-IM but not resolved in SMCs, and CCh activated a Cl- conductance in ICC-IM and a non-selective cation conductance in SMCs. We also studied responses to nerve stimulation using electrical-field stimulation (EFS) of intact fundus muscles from wild-type and Ano1 knockout mice. EFS activated excitatory junction potentials (EJPs) in wild-type mice, although EJPs were absent in mice with congenital deactivation of Ano1 and greatly reduced in animals in which the CaCC-Ano1 was knocked down using Cre/loxP technology. Contractions to cholinergic nerve stimulation were also greatly reduced in Ano1 knockouts. SMCs cells also have receptors and ion channels activated by muscarinic agonists. Blocking acetylcholine esterase with neostigmine revealed a slow depolarization that developed after EJPs in wild-type mice. This depolarization was still apparent in mice with genetic deactivation of Ano1. Pharmacological blockers of Ano1 also inhibited EJPs and contractile responses to muscarinic stimulation in fundus muscles. The data of the present study are consistent with the hypothesis that ACh released from motor nerves binds muscarinic receptors on ICC-IM with preference and activates Ano1. If metabolism of acetylcholine is inhibited, ACh overflows and binds to extrajunctional receptors on SMCs, eliciting a slower depolarization response.

Key points Interstitial cells of Cajal (ICC) from murine colonic muscles express genes encoding inwardly rectifying K+ channels. Transcripts of Kcnj2 (Kir2.1), Kcnj4 (Kir2.3), Kcnj14 (Kir2.4), Kcnj5 (Kir3.4), Kcnj8 (Kir 6.1) and Kcnj11 (Kir6.2) were found in colonic ICC. A conductance with properties consistent with Kir2 channels was observed in ICC but not in smooth muscle cells (SMC). Despite expression of gene transcripts, G-protein gated K+ channel (Kir3) and KATP (Kir6) currents were not resolved in ICC. KATP is a conductance prominent in SMC. Kir2 antagonist caused depolarization of freshly dispersed ICC and colonic smooth muscles, suggesting that this conductance is active under resting conditions in colonic muscles. The conclusion of the present study is that ICC express the Ba2+ -sensitive, inwardly rectifying K+ conductance in colonic muscles. This conductance is most probably a result of heterotetramers of Kir2 gene products, with this regulating resting potentials and the excitability of colonic muscles. Abstract Membrane potentials of gastrointestinal muscles are important because voltage-dependent Ca2+ channels in smooth muscle cells (SMC) provide the Ca2+ that triggers contraction. Regulation of membrane potential is complicated because SMC are electrically coupled to interstitial cells of Cajal (ICC) and PDGFRα+ cells. Activation of conductances in any of these cells affects the excitability of the syncytium. We explored the role of inward rectifier K+ conductances in colonic ICC that might contribute to regulation of membrane potential. ICC expressed Kcnj2 (Kir2.1), Kcnj4 (Kir2.3), Kcnj14 (Kir2.4), Kcnj5 (Kir3.4), Kcnj8 (Kir 6.1) and Kcnj11 (Kir6.2). Voltage clamp experiments showed activation of inward current when extracellular K+ ([K+ ]o ) was increased. The current was inwardly rectifying and inhibited by Ba2+ (10 μm) and ML-133 (10 μm). A similar current was not available in SMC. The current activated in ICC by elevated [K+ ]o was not affected by Tertiapin-Q. Gβγ, when dialysed into cells, failed to activate a unique, Tertiapin-Q-sensitive conductance. Freshly dispersed ICC showed no evidence of functional KATP . Pinacidil failed to activate current and the inward current activated by elevated [K+ ]o was insensitive to glibenclamide. Under current clamp, ML-133 caused the depolarization of isolated ICC and also that of cells impaled with microelectrodes in intact muscle strips. These findings show that ICC, when isolated freshly from colonic muscles, expressed a Ba2+ -sensitive, inwardly rectifying K+ conductance. This conductance is most probably a result of the expression of multiple Kir2 family paralogues, and the inwardly rectifying conductance contributes to the regulation of resting potentials and excitability of colonic muscles.

Key points The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+-activated Cl− channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL, encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. Abstract The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+-activated Cl− channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Key points Rhythmic action potentials and intercellular Ca2+ waves are generated in smooth muscle cells of colonic longitudinal muscles (LSMC). Longitudinal muscle excitability is tuned by input from subserosal ICC (ICC-SS), a population of ICC with previously unknown function. ICC-SS express Ano1 channels and generate spontaneous Ca2+ transients in a stochastic manner. Release of Ca2+ and activation of Ano1 channels causes depolarization of ICC-SS and LSMC, leading to activation of L-type Ca2+ channels, action potentials, intercellular Ca2+ waves and contractions in LSMC. Nitrergic neural inputs regulate the Ca2+ events in ICC-SS. Pacemaker activity in longitudinal muscle is an emergent property as a result of integrated processes in ICC-SS and LSMC. Abstract Much is known about myogenic mechanisms in circular muscle (CM) in the gastrointestinal tract, although less is known about longitudinal muscle (LM). Two Ca2+ signalling behaviours occur in LM: localized intracellular waves not causing contractions and intercellular waves leading to excitation-contraction coupling. An Ano1 channel antagonist inhibited intercellular Ca2+ waves and LM contractions. Ano1 channels are expressed by interstitial cells of Cajal (ICC) but not by smooth muscle cells (SMCs). We investigated Ca2+ signalling in a novel population of ICC that lies along the subserosal surface of LM (ICC-SS) in mice expressing GCaMP6f in ICC. ICC-SS fired stochastic localized Ca2+ transients. Such events have been linked to activation of Ano1 channels in ICC. Ca2+ transients in ICC-SS occurred by release from stores most probably via inositol trisphosphate receptors. This activity relied on influx via store-operated Ca2+ entry and Orai channels. No voltage-dependent mechanism that synchronized Ca2+ transients in a single cell or between cells was found. Nitrergic agonists inhibited Ca2+ transients in ICC-SS, and stimulation of intrinsic nerves activated nitrergic responses in ICC-SS. Cessation of stimulation resulted in significant enhancement of Ca2+ transients compared to the pre-stimulus activity. No evidence of innervation by excitatory, cholinergic motor neurons was found. Our data suggest that ICC-SS contribute to regulation of LM motor activity. Spontaneous Ca2+ transients activate Ano1 channels in ICC-SS. Resulting depolarization conducts to SMCs, depolarizing membrane potential, activating L-type Ca2+ channels and initiating contraction. Rhythmic electrical and mechanical behaviours of LM are an emergent property of SMCs and ICC-SS.

Urethral smooth muscle (USM) generates tone to prevent urine leakage from the bladder during filling. USM tone has been thought to be a voltage-dependent process, relying on Ca

Carrying out clinical diagnosis of retinal vascular degeneration using Fluorescein Angiography (FA) is a time consuming process and can pose significant adverse effects on the patient. Angiography requires insertion of a dye that may cause severe adverse effects and can even be fatal. Currently, there are no non-invasive systems capable of generating Fluorescein Angiography images. However, retinal fundus photography is a non-invasive imaging technique that can be completed in a few seconds. In order to eliminate the need for FA, we propose a conditional generative adversarial network (GAN) to translate fundus images to FA images. The proposed GAN consists of a novel residual block capable of generating high quality FA images. These images are important tools in the differential diagnosis of retinal diseases without the need for invasive procedure with possible side effects. Our experiments show that the proposed architecture achieves a low FID score of 30.3 and outperforms other state-of-the-art generative networks. Furthermore, our proposed model achieves better qualitative results indistinguishable from real angiograms.

Multiple cell types generate and organize the movements of the gut muscles required to process nutrients and wastes in the GI tract. Central to motility are smooth muscle cells (SMCs) that generate the forces necessary to propel luminal contents or restrict movements. SMCs are not capable of organ level cooperation without complex regulatory mechanisms. Unrestrained, SMCs are a detriment to propulsive activity because of their intrinsic but cell-to-cell uncoordinated excitability properties. SMCs are coupled electrically to interstitial cells forming a regulatory unit called the SIP syncytium. SIP is an acronym that describes a syncytium of electrical network cells formed by the SMCs, ICC (Interstitial Cells of Cajal) and PDGFR2 + (Platelet-derived growth factor receptor alpha). Interstitial cells generate electrical rhythmicity and synchronize the contractions of SMCs into phasic contractions. Interstitial cells also receive and transduce neural inputs. Superimposed upon the regulation provided by the SIP syncytium, the enteric nervous system senses the volume and composition of luminal contents through intrinsic primary sensory nerves (IPANs) and generates motility patterns at the whole organ level. These effects are further mediated through 5-hydroxy tryptamine (Serotonin), acetyl choline, vasoactive intestinal peptide, nitric oxide and others. Neuropathies of the enteric nervous system are associated with serious motility disorders.

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

Oviducts (also called fallopian tubes) are smooth muscle-lined tubular organs that at one end extend in a trumpet bell-like fashion to surround the ovary, and at the other connect to the uterus. Contractions of the oviduct smooth muscle (myosalpinx) and the wafting motion of the ciliated epithelium that lines these tubes facilitate bidirectional transport of gametes so that newly released ovum(s) are transported in one direction (pro-uterus) while spermatozoa are transported in the opposite direction (pro-ovary). These transport processes must be temporally coordinated so that the ovum and spermatozoa meet in the ampulla, the site of fertilization. Once fertilized, the early embryo begins another precisely timed journey towards the uterus for implantation. Myosalpinx contractions facilitate this journey too, while luminal secretions from secretory epithelial cells aid early embryo maturation.The previous paradigm was that oviduct transport processes were primarily controlled by fluid currents generated by the incessant beat of the ciliated epithelium towards the uterus. More recently, video imaging and spatiotemporal mapping have suggested a novel paradigm in which ovum/embryo transport is highly dependent upon phasic and propulsive contractions of the myosalpinx. A specialized population of pacemaker cells, termed oviduct interstitial cells of Cajal (ICC-OVI), generate the electrical activity that drives these contractions. The ionic mechanisms underlying this pacemaker activity are dependent upon the calcium-activated chloride conductance, Ano1.This chapter discusses the basis of oviduct pacemaker activity, its hormonal regulation, and the underlying mechanisms and repercussions when this activity becomes disrupted during inflammatory responses to bacterial infections, such as Chlamydia.

KEY POINTS: Electrical pacemaking in gastrointestinal muscles is generated by specialized interstitial cells of Cajal that produce the patterns of contractions required for peristalsis and segmentation in the gut. The calcium‐activated chloride conductance anoctamin‐1 (Ano1) has been shown to be responsible for the generation of pacemaker activity in GI muscles, but this conclusion is established from studies of juvenile animals in which effects of reduced Ano1 on gastric emptying and motor patterns could not be evaluated. Knocking down Ano1 expression using Cre/LoxP technology caused dramatic changes in in gastric motor activity, with disrupted slow waves, abnormal phasic contractions and delayed gastric emptying; modest changes were noted in the small intestine. Comparison of the effects of Ano1 antagonists on muscles from juvenile and adult small intestinal muscles suggests that conductances in addition to Ano1 may develop with age and contribute to pacemaker activity. ABSTRACT: Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca(2+)‐activated Cl(−) conductance (CaCC), and constitutive knockout of the channel protein anoctamin‐1 leads to loss of slow waves in gastric and intestinal muscles. These knockout experiments were performed on juvenile mice. However, additional experiments demonstrated significant differences in the sensitivity of gastric and intestinal muscles to antagonists of anoctamin‐1 channels. Furthermore, the significance of anoctamin‐1 and the electrical and mechanical behaviours facilitated by this conductance have not been evaluated on the motor behaviours of adult animals. Cre/loxP technology was used to generate cell‐specific knockdowns of anoctamin‐1 in ICC (Kit(CreERT2/+);Ano1(tm2jrr/+)) in GI muscles. The recombination efficiency of Kit(CreERT) was evaluated with an eGFP reporter, molecular techniques and immunohistochemistry. Electrical and contractile experiments were used to examine the consequences of anoctamin‐1 knockdown on pacemaker activity, mechanical responses, gastric motility patterns, gastric emptying and GI transit. Reduced anoctamin‐1 caused loss of gastric, but not intestinal slow waves. Irregular spike complexes developed in gastric muscles, leading to uncoordinated antral contractions, delayed gastric emptying and increased total GI transit time. Slow waves in intestinal muscles of juvenile mice were more sensitive to anoctamin‐1 antagonists than slow waves in adult muscles. The low susceptibility to anoctamin‐1 knockdown and weak efficacy of anoctamin‐1 antagonists in inhibiting slow waves in adult small intestinal muscles suggest that a conductance in addition to anoctamin‐1 may develop in small intestinal ICC with ageing and contribute to pacemaker activity.

Spontaneous excitability and contractions of colonic smooth muscle cells (SMCs) are normally suppressed by inputs from inhibitory motor neurons, a behavior known as tonic inhibition. The post-junctional cell(s) mediating tonic inhibition have not been elucidated. We investigated the post-junctional cells mediating tonic inhibition in the proximal colon and whether tonic inhibition results from suppression of the activity of Ano1 channels, which are expressed exclusively in interstitial cells of Cajal (ICC). We found that tetrodotoxin (TTX), an inhibitor of nitric oxide (NO) synthesis, L-NNA, and an inhibitor of soluble guanylyl cyclase, ODQ, greatly enhanced colonic contractions. Ano1 antagonists, benzbromarone and Ani9 inhibited the effects of TTX, L-NNA and ODQ. Ano1 channels are activated by Ca2+ release from the endoplasmic reticulum (ER) in ICC, and blocking Ca2+ release with a SERCA inhibitor (thapsigargin) or a store-operated Ca2+ entry blocker (GSK 7975 A) reversed the effects of TTX, L-NNA and ODQ. Ca2+ imaging revealed that TTX, L-NNA and ODQ increased Ca2+ transient firing in colonic ICC. Our results suggest that tonic inhibition in the proximal colon occurs through suppression of Ca2+ release events in ICC. Suppression of Ca2+ release in ICC limits the open probability of Ano1 channels, reducing the excitability of electrically-coupled SMCs.

Pacemaker depolarization in interstitial cells of Cajal (ICCs) is believed to be induced by Ca(2+) transients and activation of anoctamin-1 (Ano1) channels in the plasma membrane. However, block of store-operated calcium entry (SOCE) or the Na-K-2Cl cotransporter (NKCC1) terminates pacemaker activity in ICC, indicating these transporters are involved in the initiation or maintenance of pacemaker activity. We hypothesized that SOCE contributes to pacemaker depolarization by maintaining [Ca(2+)] in the endoplasmic reticulum, which is the underlying source of Ca(2+) transients for activation of Ano1. NKCC1 maintains the Cl(−) gradient supporting the driving force for inward current mediated by Ano1. Currently mechanisms sustaining release of Ca(2+) and activation of Ano1 channels during the plateau phase of slow waves are unknown, but the reverse mode of the Na(+)/Ca(2+) exchange may contribute. We generated a mathematical model of pacemaker activity based on current empirical observations from ICC of mouse small intestine that incorporates functions of SOCE and NKCC1. This model reproduces experimental findings, suggesting roles for SOCE and Ano1 channels: blocking of either NKCC1 or SOCE in our model terminates pacemaker activity. Direct contribution of NKCC1 to pacemaker activity in a beat-to-beat manner is not predicted by our model. Instead, NKCC1 plays a maintenance role supporting the driving force for Cl(−) efflux. Incorporation of SOCE allows the model to drive pacemaker activity without a diastolic depolarization, as observed in cardiac pacemaking. Further biological experiments are necessary to validate and further refine the roles of NKCC1, Na(+)/Ca(2+) exchange, and Ano1 in the pacemaker mechanism of ICC.

Background & Aims Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine [5-HT]), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiologic roles of 5-HT deficiency in gastric motility in mice and humans. Methods We developed an inducible, EC cell–specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell–depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell–depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG). Results Tph1-tdTom mice showed EC cells that were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate. Conclusions The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiologic mechanism of 5-HT deficiency in IG.

Electrical slow waves in the small intestine are generated by pacemaker cells called interstitial cells of Cajal. Drumm et al. record clusters of Ca2+ transients in these cells that are entrained by voltage-dependent Ca2+ entry and which define the duration of the electrical slow waves., Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl− channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves.

Inhibitory motor neurons regulate several gastric motility patterns including receptive relaxation, gastric peristaltic motor patterns, and pyloric sphincter opening. Nitric oxide (NO) and purines have been identified as likely candidates that mediate inhibitory neural responses. However, the contribution from each neurotransmitter has received little attention in the distal stomach. The aims of this study were to identify the roles played by NO and purines in inhibitory motor responses in the antrums of mice and monkeys. By using wild-type mice and mutants with genetically deleted neural nitric oxide synthase ( Nos1 −/−) and P2Y1 receptors ( P2ry1 −/−) we examined the roles of NO and purines in postjunctional inhibitory responses in the distal stomach and compared these responses to those in primate stomach. Activation of inhibitory motor nerves using electrical field stimulation (EFS) produced frequency-dependent inhibitory junction potentials (IJPs) that produced muscle relaxations in both species. Stimulation of inhibitory nerves during slow waves terminated pacemaker events and associated contractions. In Nos1 −/− mice IJPs and relaxations persisted whereas in P2ry1 −/− mice IJPs were absent but relaxations persisted. In the gastric antrum of the non-human primate model Macaca fascicularis, similar NO and purine neural components contributed to inhibition of gastric motor activity. These data support a role of convergent inhibitory neural responses in the regulation of gastric motor activity across diverse species.

The pacemaker function of interstitial cells of Cajal (ICC) is impaired during intestinal inflammation. The aim of this study is to clarify the pathophysiological mechanisms of ICC dysfunction during inflammatory condition by using intestinal cell clusters. Cell clusters were prepared from smooth muscle layer of murine jejunum and treated with interferon-gamma and lipopolysaccharide (IFN-γ+LPS) for 24h to induce inflammation. Pacemaker function of ICC was monitored by measuring cytosolic Ca(2+) oscillation in the presence of nifedipine. Treatment with IFN-γ+LPS impaired the pacemaker activity of ICC with increasing mRNA level of interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6 in cell clusters; however, treatment with these cytokines individually had little effect on pacemaker activity of ICC. Treatment with IFN-γ+LPS also induced the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and resident macrophages, but not in ICC. Pretreatment with NOS inhibitor, L-NAME or iNOS inhibitor, 1400W ameliorated IFN-γ+LPS-induced pacemaker dysfunction of ICC. Pretreatment with guanylate cyclase inhibitor, ODQ did not, but antioxidant, apocynin, to suppress NO-induced oxidative stress, significantly suppressed the impairment of ICC function induced by IFN-γ+LPS. Treatment with IFN-γ+LPS also decreased c-Kit-positive ICC, which was prevented by pretreatment with L-NAME. However, apoptotic ICC were not detected in IFN-γ+LPS-treated clusters, suggesting IFN-γ+LPS stimulation just changed the phenotype of ICC but not induced cell death. Moreover, ultrastructure of ICC was not disturbed by IFN-γ+LPS. In conclusion, ICC dysfunction during inflammation is induced by NO-induced oxidative stress rather than NO/cGMP signaling. NO-induced oxidative stress might be the main factor to induce phenotypic changes of ICC.

Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca(2+) transients that consist of localized Ca(2+) events and limited propagating Ca(2+) waves. Ca(2+) transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca(2+) transients to regionalized Ca(2+) waves with variable rates of occurrence, amplitude, duration and spatial spread. Ca(2+) transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells. Ca(2+) transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s). Functional intracellular Ca(2+) stores are essential for spontaneous Ca(2+) transients, and the sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump is necessary for maintenance of spontaneity. Ca(2+) release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3 Rs). Release from these channels is interdependent. ICC express transcripts of multiple RyRs and InsP3 Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression.Interstitial cells of Cajal in the deep muscular plexus of the small intestine (ICC-DMP) are closely associated with varicosities of enteric motor neurons and generate responses contributing to neural regulation of intestinal motility. Responses of ICC-DMP are mediated by activation of Ca(2+) -activated Cl(-) channels; thus, Ca(2+) signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca(2+) transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca(2+) indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca(2+) transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca(2+) transients was highly variable, and it was determined that firing was stochastic in nature. Ca(2+) transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 ?m) significantly increased the occurrence of Ca(2+) transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca(2+) transients were minimally affected after 12 min in Ca(2+) free solution, indicating these events do not depend immediately upon Ca(2+) influx. However, inhibitors of sarco/endoplasmic reticulum Ca(2+) -ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3 R) and ryanodine receptor (RyR) channels blocked ICC Ca(2+) transients. These data suggest an interdependence between RyR and InsP3 R in the generation of Ca(2+) transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca(2+) dynamics that control the behaviour of ICC-DMP in situ.

Background and Purpose High-throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second-generation. low MW inhibitors of the calcium-activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors. Experimental Approach Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose-dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves. Key Results The efficacy of second-generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh-A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh-A01 in both preparations. Conclusions and Implications We have identified several second-generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.

High-resolution Ca2+ imaging to study cellular Ca2+ behaviors has led to the creation of large datasets with a profound need for standardized and accurate analysis. To analyze these datasets, spatio-temporal maps (STMaps) that allow for 2D visualization of Ca2+ signals as a function of time and space are often used. Methods of STMap analysis rely on a highly arduous process of user defined segmentation and event-based data retrieval. These methods are often time consuming, lack accuracy, and are extremely variable between users. We designed a novel automated machine-learning based plugin for the analysis of Ca2+ STMaps (STMapAuto). The plugin includes optimized tools for Ca2+ signal preprocessing, automated segmentation, and automated extraction of key Ca2+ event information such as duration, spatial spread, frequency, propagation angle, and intensity in a variety of cell types including the Interstitial cells of Cajal (ICC). The plugin is fully implemented in Fiji and able to accurately detect and expeditiously quantify Ca2+ transient parameters from ICC. The plugin's speed of analysis of large-datasets was 197-fold faster than the commonly used single pixel-line method of analysis. The automated machine-learning based plugin described dramatically reduces opportunities for user error and provides a consistent method to allow high-throughput analysis of STMap datasets.

Background & Aims Colonic musculature contain smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and platelet-derived growth factor receptor α+ cells (PDGFRα+ cells), which are electrically coupled and operate together as the SIP syncytium. PDGFRα+ cells have enriched expression of small conductance Ca2+-activated K+ (SK) channels. Purinergic enteric neural input activates SK channels in PDGFRα+ cells, hyperpolarizes SMC, and inhibits colonic contractions. Recently we discovered that PDGFRα+ cells in mouse colon have enriched expression of α1A adrenoceptors (ARs), which coupled to activation of SK channels and inhibited colonic motility, and α1A ARs were principal targets for sympathetic regulation of colonic motility. Here we investigated whether PDGFRα+ cells in human colon express α1A ARs and share the roles as targets for sympathetic regulation of colonic motility. Methods Isometric tension recording, intracellular recording, and Ca2+ imaging were performed on muscles of the human colon. Responses to α1 ARs agonists or electric field stimulation with AR antagonists and neuroleptic reagents were studied. Results Exogenous or endogenous norepinephrine released from nerve fibers inhibited colonic contractions through binding to α1A ARs or enhanced colonic contractions by acting on α1D ARs. Inhibitory responses were blocked by apamin, an antagonist of SK channels. Phenylephrine, α1 AR agonists, or norepinephrine increased intracellular [Ca2+] in PDGFRα+ cells, but not in ICC, and hyperpolarized SMCs by binding to α1 ARs expressed by PDGFRα+ cells. Conclusions Human colonic contractions are inhibited by α1A ARs expressed in PDGFRα+ cells and activated by α1D ARs expressed in SMC., Graphical abstract

Transcriptome data on the quantitative numbers of transcriptional variants expressed in primary cells offer essential clues into specific cellular functions and biological processes. We have previously collected transcriptomes from primary smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and PDGFRα+ cells (fibroblast-like cells) isolated from murine jejunal and colonic smooth muscle and/or mucosal tissues as well as transcriptomes from the associated tissues (jejunal smooth muscle, colonic smooth muscle, and colonic mucosa). In this study, we have built the Smooth Muscle Transcriptome Browser (SMTB), https://med.unr.edu/physio/transcriptome, a web-based, graphical user interface that offers genetic references and expression profiles of all transcripts expressed at both the cellular (SMC, ICC, and PDGFRα+ cells) and tissue level (smooth muscle and mucosal tissue). This browser brings new insights into the cellular and biological functions of the cell types in gastrointestinal smooth muscle biology.

Ca(2+) imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca(2+) signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca(2+) signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca(2+) imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca(2+) signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca(2+)dyes or optogenetic Ca(2+) imaging, appropriate spatial and temporal analysis of the Ca(2+) signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca(2+) imaging recordings to facilitate more complete analysis and quantification of Ca(2+) signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca(2+) signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca(2+) signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Oviducts (also called fallopian tubes) are smooth muscle-lined tubular organs that at one end extend in a trumpet bell-like fashion to surround the ovary, and at the other connect to the uterus. Contractions of the oviduct smooth muscle (myosalpinx) and the wafting motion of the ciliated epithelium that lines these tubes facilitate bidirectional transport of gametes so that newly released ovum(s) are transported in one direction (pro-uterus) while spermatozoa are transported in the opposite direction (pro-ovary). These transport processes must be temporally coordinated so that the ovum and spermatozoa meet in the ampulla, the site of fertilization. Once fertilized, the early embryo begins another precisely timed journey towards the uterus for implantation. Myosalpinx contractions facilitate this journey too, while luminal secretions from secretory epithelial cells aid early embryo maturation.The previous paradigm was that oviduct transport processes were primarily controlled by fluid currents generated by the incessant beat of the ciliated epithelium towards the uterus. More recently, video imaging and spatiotemporal mapping have suggested a novel paradigm in which ovum/embryo transport is highly dependent upon phasic and propulsive contractions of the myosalpinx. A specialized population of pacemaker cells, termed oviduct interstitial cells of Cajal (ICC-OVI), generate the electrical activity that drives these contractions. The ionic mechanisms underlying this pacemaker activity are dependent upon the calcium-activated chloride conductance, Ano1.This chapter discusses the basis of oviduct pacemaker activity, its hormonal regulation, and the underlying mechanisms and repercussions when this activity becomes disrupted during inflammatory responses to bacterial infections, such as Chlamydia.

The gastrointestinal (GI) tract has multifold tasks of ingesting, processing, and assimilating nutrients and disposing of wastes at appropriate times. These tasks are facilitated by several stereotypical motor patterns that build upon the intrinsic rhythmicity of the smooth muscles that generate phasic contractions in many regions of the gut. Phasic contractions result from a cyclical depolarization/repolarization cycle, known as electrical slow waves, which result from intrinsic pacemaker activity. Interstitial cells of Cajal (ICC) are electrically coupled to smooth muscle cells (SMCs) and generate and propagate pacemaker activity and slow waves. The mechanism of slow waves is dependent upon specialized conductances expressed by pacemaker ICC. The primary conductances responsible for slow waves in mice are Ano1, Ca(2+)-activated Cl(−) channels (CaCCs), and Ca(V)3.2, T-type, voltage-dependent Ca(2+) channels. Release of Ca(2+) from intracellular stores in ICC appears to be the initiator of pacemaker depolarizations, activation of T-type current provides voltage-dependent Ca(2+) entry into ICC, as slow waves propagate through ICC networks, and Ca(2+)-induced Ca(2+) release and activation of Ano1 in ICC amplifies slow wave depolarizations. Slow waves conduct to coupled SMCs, and depolarization elicited by these events enhances the open-probability of L-type voltage-dependent Ca(2+) channels, promotes Ca(2+) entry, and initiates contraction. Phasic contractions timed by the occurrence of slow waves provide the basis for motility patterns such as gastric peristalsis and segmentation. This chapter discusses the properties of ICC and proposed mechanism of electrical rhythmicity in GI muscles.

Spontaneous excitability and contractions of colonic smooth muscle cells (SMCs) are normally suppressed by inputs from inhibitory motor neurons, a behavior known as tonic inhibition. The post-junctional cell(s) mediating tonic inhibition have not been elucidated. We investigated the post-junctional cells mediating tonic inhibition in the proximal colon and whether tonic inhibition results from suppression of the activity of Ano1 channels, which are expressed exclusively in interstitial cells of Cajal (ICC). We found that tetrodotoxin (TTX), an inhibitor of nitric oxide (NO) synthesis, L-NNA, and an inhibitor of soluble guanylyl cyclase, ODQ, greatly enhanced colonic contractions. Ano1 antagonists, benzbromarone and Ani9 inhibited the effects of TTX, L-NNA and ODQ. Ano1 channels are activated by Ca2+ release from the endoplasmic reticulum (ER) in ICC, and blocking Ca2+ release with a SERCA inhibitor (thapsigargin) or a store-operated Ca2+ entry blocker (GSK 7975 A) reversed the effects of TTX, L-NNA and ODQ. Ca2+ imaging revealed that TTX, L-NNA and ODQ increased Ca2+ transient firing in colonic ICC. Our results suggest that tonic inhibition in the proximal colon occurs through suppression of Ca2+ release events in ICC. Suppression of Ca2+ release in ICC limits the open probability of Ano1 channels, reducing the excitability of electrically-coupled SMCs.