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Gastrointestinal (GI) organs display spontaneous, non‐neurogenic electrical, and mechanical rhythmicity that underlies fundamental motility patterns, such as peristalsis and segmentation. Electrical rhythmicity (aka slow waves) results from pacemaker activity generated by interstitial cells of Cajal (ICC). ICC express a unique set of ionic conductances and Ca2+ handling mechanisms that generate and actively propagate slow waves. GI smooth muscle cells lack these conductances. Slow waves propagate actively within ICC networks and conduct electrotonically to smooth muscle cells via gap junctions. Slow waves depolarize smooth muscle cells and activate voltage‐dependent Ca2+ channels (predominantly CaV1.2), causing Ca2+ influx and excitation–contraction coupling. The main conductances responsible for pacemaker activity in ICC are ANO1, a Ca2+‐activated Cl− conductance, and CaV3.2. The pacemaker cycle, as currently understood, begins with spontaneous, localized Ca2+ release events in ICC that activate spontaneous transient inward currents due to activation of ANO1 channels. Depolarization activates CaV3.2 channels, causing the upstroke depolarization phase of slow waves. The upstroke is transient and followed by a long‐duration plateau phase that can last for several seconds. The plateau phase results from Ca2+‐induced Ca2+ release and a temporal cluster of localized Ca2+ transients in ICC that sustains activation of ANO1 channels and clamps membrane potential near the equilibrium potential for Cl− ions. The plateau phase ends, and repolarization occurs, when Ca2+ stores are depleted, Ca2+ release ceases and ANO1 channels deactivate. This review summarizes key mechanisms responsible for electrical rhythmicity in gastrointestinal organs. [ABSTRACT FROM AUTHOR]

Skeletal muscle's isometric contractile properties are one of the classic structure–function relationships in all of biology allowing for extrapolation of single fibre mechanical properties to whole muscle properties based on the muscle's optimal fibre length and physiological cross‐sectional area (PCSA). However, this relationship has only been validated in small animals and then extrapolated to human muscles, which are much larger in terms of length and PCSA. The present study aimed to measure directly the in situ properties and function of the human gracilis muscle to validate this relationship. We leveraged a unique surgical technique in which a human gracilis muscle is transferred from the thigh to the arm, restoring elbow flexion after brachial plexus injury. During this surgery, we directly measured subject specific gracilis muscle force–length relationship in situ and properties ex vivo. Each subject's optimal fibre length was calculated from their muscle's length‐tension properties. Each subject's PCSA was calculated from their muscle volume and optimal fibre length. From these experimental data, we established a human muscle fibre‐specific tension of 171 kPa. We also determined that average gracilis optimal fibre length is 12.9 cm. Using this subject‐specific fibre length, we observed an excellent fit between experimental and theorical active length‐tension curves. However, these fibre lengths were about half of the previously reported optimal fascicle lengths of 23 cm. Thus, the long gracilis muscle appears to be composed of relatively short fibres acting in parallel that may not have been appreciated based on traditional anatomical methods. Key points: Skeletal muscle's isometric contractile properties represent one of the classic structure–function relationships in all of biology and allow scaling single fibre mechanical properties to whole muscle properties based on the muscle's architecture.This physiological relationship has only been validated in small animals but is often extrapolated to human muscles, which are orders of magnitude larger.We leverage a unique surgical technique in which a human gracilis muscle is transplanted from the thigh to the arm to restore elbow flexion after brachial plexus injury, aiming to directly measure muscles properties in situ and test directly the architectural scaling predictions.Using these direct measurements, we establish human muscle fibre‐specific tension of ∼170 kPa.Furthermore, we show that the gracilis muscle actually functions as a muscle with relatively short fibres acting in parallel vs. long fibres as previously assumed based on traditional anatomical models. [ABSTRACT FROM AUTHOR]

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell‐specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC‐IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high‐speed video‐imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca2+ and post‐junctional electrical responses to neural stimulation. ICC‐IM were highly active, generating intracellular Ca2+‐transients that occurred stochastically, from multiple independent sites in single ICC‐IM. Ca2+‐transients were not entrained in single ICC‐IM or between neighbouring ICC‐IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca2+‐transients in ICC‐IM. This inhibitory response was often preceded by a summation of Ca2+‐transients that led to a global rise in Ca2+. Individual ICC‐IM responded to nerve stimulation by a global rise in Ca2+ followed by inhibition of Ca2+‐transients. The inhibition of Ca2+‐transients was blocked by the nitric oxide synthase antagonist l‐NNA. The global rise in intracellular Ca2+ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video‐imaging revealed that the rise in Ca2+ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca2+‐transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC‐IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. Key points: The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC‐IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs.Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC‐IM were examined. ICC‐IM were highly active, generating stochastic intracellular Ca2+‐transients.Stimulation of enteric motor nerves abolished Ca2+‐transients in ICC‐IM. This inhibitory response was preceded by a global rise in intracellular Ca2+. Individual ICC‐IM responded to nerve stimulation with a rise in Ca2+ followed by inhibition of Ca2+‐transients.Inhibition of Ca2+‐transients was blocked by the nitric oxide synthase antagonist l‐NNA. The global rise in Ca2+ was inhibited by the muscarinic antagonist atropine.Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca2+ and inhibition of Ca2+‐transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials.The data presented support the premise of serial innervation of ICC‐IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. [ABSTRACT FROM AUTHOR]

The lower oesophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Cav1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC‐IM), through activation of Ca2+‐activated Cl− channels (ANO1), set membrane potentials of SMCs favourable for activation of Cav1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC‐IM are activated by Ca2+ transients, but no studies have examined Ca2+ dynamics in ICC‐IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC‐IM and SMCs. These studies revealed that Ano1 and its gene product, ANO1, are expressed in c‐Kit+ cells (ICC‐IM) in mouse and monkey LES clasp muscles. Ca2+ signalling was imaged in situ, using mice expressing GCaMP6f specifically in ICC (Kit‐KI‐GCaMP6f). ICC‐IM exhibited spontaneous Ca2+ transients from multiple firing sites. Ca2+ transients were abolished by cyclopiazonic acid or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca2+ transients depended on Ca2+ influx and store reloading, as Ca2+ transient frequency was reduced in Ca2+ free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced ∼80% either by Ani9, an ANO1 antagonist or by the Cav1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca2+ activates ANO1 channels in ICC‐IM in the LES. Coupling of ICC‐IM to SMCs drives depolarisation, activation of Cav1.2 channels, Ca2+ entry and contractile tone. Key points: The lower oesophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the oesophagus. LES smooth muscle cells (SMCs) display depolarised membrane potentials facilitating activation of L‐type Ca2+ channels.Interstitial cells of Cajal (ICC) express Ca2+‐activated Cl− channels encoded by Ano1 in mouse and monkey LES. Ca2+ signalling in ICC activates ANO1 currents in ICC.ICC displayed spontaneous Ca2+ transients in mice from multiple firing sites in each cell and no entrainment of Ca2+ firing between sites or between cells.Inhibition of ANO1 channels with a specific antagonist caused hyperpolarisation of mouse LES and inhibition of tone in monkey and mouse LES muscles.Our data suggest a novel mechanism for LES tone in which Ca2+ transient activation of ANO1 channels in ICC generates depolarising inward currents that conduct to SMCs to activate L‐type Ca2+ currents, Ca2+ entry and contractile tone. [ABSTRACT FROM AUTHOR]

Key points: β‐Nicotinamide adenine dinucleotide (β‐NAD) is a key inhibitory neurotransmitter in the colon.The neuroeffector junction in the gut consists of enteric motor neurons and SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet‐derived growth factor receptor α (PDGFRα+ cells).Measuring metabolism of 1,N6‐etheno‐NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFRα+ cells with HPLC‐FLD, we report that (1) in tissues, eNAD is degraded to eADP‐ribose, eAMP and e‐adenosine (eADO) by CD38, ENPP1 and NT5E, (2) with SMCs and PDGFRα+ cells, eNAD is metabolized to eADO by ENPP1 and NT5E, (3) eNAD is not metabolized by ICC, (4) NT5E is expressed chiefly by SMCs and moderately by PDGFRα+ cells, (5) SIP cells are not the primary location of CD38.These data argue that the duration and strength of purinergic neurotransmission can be modulated by targeting multiple enzymes with specialized cellular distribution in the colon. Prior studies suggest that β‐nicotinamide adenine dinucleotide (β‐NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of β‐NAD at the neuroeffector junction (NEJ) is likely to be necessary for terminating inhibitory neurotransmission and may also produce bioactive metabolites. The enteric NEJ consists of enteric neurons and postjunctional cells of the SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet‐derived growth factor receptor α (PDGFRα+ cells). We examined possible specialized functions of the NEJ in β‐NAD metabolism by determining the degradation of 1,N6‐etheno‐NAD (eNAD) in colonic tunica muscularis of wild‐type, Cd38−/−, Nt5e−/−, Enpp1−/− and Cd38−/−/Nt5e−/− mice and in SIP cells from mice expressing cell‐specific fluorescent reporters purified by fluorescence activated cell sorting (FACS). We measured eNAD and its metabolites eADP‐ribose (eADPR), eAMP and e‐adenosine (eADO) from tissues and sorted SIP cells using liquid chromatography. eNAD exposed to colonic muscularis of wild‐type mice produced eADPR, eAMP and eADO. CD38 mediated the conversion of eNAD to eADPR, whereas ENPP1 mediated degradation of eNAD and eADPR to eAMP. NT5E (aka CD73) was the primary enzyme forming eADO from eAMP. PDGFRα+ cells and SMCs were involved in production of eADO from eNAD, and ICC were not involved in extracellular metabolism of eNAD. CD38 mediated the eNAD metabolism in whole tissues, but CD38 did not appear to be functionally expressed by SMCs or ICC. NT5E was expressed in SMCs > PDGFRα+ cells. Our data show that extracellular metabolism of β‐NAD in the colon is mediated by multiple enzymes with cell‐specific expression. Key points: β‐Nicotinamide adenine dinucleotide (β‐NAD) is a key inhibitory neurotransmitter in the colon.The neuroeffector junction in the gut consists of enteric motor neurons and SIP syncytium, including smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet‐derived growth factor receptor α (PDGFRα+ cells).Measuring metabolism of 1,N6‐etheno‐NAD (eNAD) in colonic tunica muscularis and in SMCs, ICC and PDGFRα+ cells with HPLC‐FLD, we report that (1) in tissues, eNAD is degraded to eADP‐ribose, eAMP and e‐adenosine (eADO) by CD38, ENPP1 and NT5E, (2) with SMCs and PDGFRα+ cells, eNAD is metabolized to eADO by ENPP1 and NT5E, (3) eNAD is not metabolized by ICC, (4) NT5E is expressed chiefly by SMCs and moderately by PDGFRα+ cells, (5) SIP cells are not the primary location of CD38.These data argue that the duration and strength of purinergic neurotransmission can be modulated by targeting multiple enzymes with specialized cellular distribution in the colon. [ABSTRACT FROM AUTHOR]

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. 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. 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 FROM AUTHOR]

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. 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. 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 FROM AUTHOR]

Key points: Colonic intramuscular interstitial cells of Cajal (ICC‐IM) exhibit spontaneous Ca2+ transients manifesting as stochastic events from multiple firing sites with propagating Ca2+ waves occasionally observed.Firing of Ca2+ transients in ICC‐IM is not coordinated with adjacent ICC‐IM in a field of view or even with events from other firing sites within a single cell.Ca2+ transients, through activation of Ano1 channels and generation of inward current, cause net depolarization of colonic muscles.Ca2+ transients in ICC‐IM rely on Ca2+ release from the endoplasmic reticulum via IP3 receptors, spatial amplification from RyRs and ongoing refilling of ER via the sarcoplasmic/endoplasmic‐reticulum‐Ca2+‐ATPase.ICC‐IM are sustained by voltage‐independent Ca2+ influx via store‐operated Ca2+ entry.Some of the properties of Ca2+ in ICC‐IM in the colon are similar to the behaviour of ICC located in the deep muscular plexus region of the small intestine, suggesting there are functional similarities between these classes of ICC. A component of the SIP syncytium that regulates smooth muscle excitability in the colon is the intramuscular class of interstitial cells of Cajal (ICC‐IM). All classes of ICC (including ICC‐IM) express Ca2+‐activated Cl− channels, encoded by Ano1, and rely upon this conductance for physiological functions. Thus, Ca2+ handling in ICC is fundamental to colonic motility. We examined Ca2+ handling mechanisms in ICC‐IM of murine proximal colon expressing GCaMP6f in ICC. Several Ca2+ firing sites were detected in each cell. While individual sites displayed rhythmic Ca2+ events, the overall pattern of Ca2+ transients was stochastic. No correlation was found between discrete Ca2+ firing sites in the same cell or in adjacent cells. Ca2+ transients in some cells initiated Ca2+ waves that spread along the cell at ∼100 µm s−1. Ca2+ transients were caused by release from intracellular stores, but depended strongly on store‐operated Ca2+ entry mechanisms. ICC Ca2+ transient firing regulated the resting membrane potential of colonic tissues as a specific Ano1 antagonist hyperpolarized colonic muscles by ∼10 mV. Ca2+ transient firing was independent of membrane potential and not affected by blockade of L‐ or T‐type Ca2+ channels. Mechanisms regulating Ca2+ transients in the proximal colon displayed both similarities to and differences from the intramuscular type of ICC in the small intestine. Similarities and differences in Ca2+ release patterns might determine how ICC respond to neurotransmission in these two regions of the gastrointestinal tract. Key points: Colonic intramuscular interstitial cells of Cajal (ICC‐IM) exhibit spontaneous Ca2+ transients manifesting as stochastic events from multiple firing sites with propagating Ca2+ waves occasionally observed.Firing of Ca2+ transients in ICC‐IM is not coordinated with adjacent ICC‐IM in a field of view or even with events from other firing sites within a single cell.Ca2+ transients, through activation of Ano1 channels and generation of inward current, cause net depolarization of colonic muscles.Ca2+ transients in ICC‐IM rely on Ca2+ release from the endoplasmic reticulum via IP3 receptors, spatial amplification from RyRs and ongoing refilling of ER via the sarcoplasmic/endoplasmic‐reticulum‐Ca2+‐ATPase.ICC‐IM are sustained by voltage‐independent Ca2+ influx via store‐operated Ca2+ entry.Some of the properties of Ca2+ in ICC‐IM in the colon are similar to the behaviour of ICC located in the deep muscular plexus region of the small intestine, suggesting there are functional similarities between these classes of ICC. [ABSTRACT FROM AUTHOR]

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. Interstitial cells of Cajal (ICC) generate slow waves and transduce neurotransmitter signals in the gastrointestinal (GI) tract, facilitating normal motility patterns. ICC express a Ca2+‐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 (KitCreERT2/+;Ano1tm2jrr/+) in GI muscles. The recombination efficiency of KitCreERT 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. 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 FROM AUTHOR]

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. 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. 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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 μ m) and to ATP (10 μ m). Ca2+ waves were inhibited by the nitric oxide donor DEA NONOate (10 μ m). 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 μ m) and xestospongin C (1 μ m) reduced Ca2+ waves. Ca2+ waves were insensitive to L‐type Ca2+ channel modulators nifedipine (1 μ m), nicardipine (1 μ m), isradipine (1 μ m) and FPL 64176 (1 μ m), and were unaffected by the T‐type Ca2+ channel antagonists NNC‐550396 (1 μ m) and TTA‐A2 (1 μ m). Ca2+ waves were reduced by the store operated Ca2+ entry blocker SKF 96365 (10 μ m) and by an Orai antagonist, GSK‐7975A (1 μ m). 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. [ABSTRACT FROM AUTHOR]

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 μ m) and ML‐133 (10 μ m). 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. [ABSTRACT FROM AUTHOR]

Key points We have developed novel techniques for paired, direct, real-time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability., Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel., The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth., Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin., Abstract The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real-time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17-3.02 μm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 μm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time-dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin. [ABSTRACT FROM AUTHOR]

Key points Interstitial cells of Cajal at the level of the deep muscular plexus (ICC-DMP) in the small intestine generate spontaneous Ca2+ transients that consist of localized Ca2+ events and limited propagating Ca2+ waves., Ca2+ transients in ICC-DMP display variable characteristics: from discrete, highly localized Ca2+ transients to regionalized Ca2+ waves with variable rates of occurrence, amplitude, duration and spatial spread., Ca2+ transients fired stochastically, with no cellular or multicellular rhythmic activity being observed. No correlation was found between the firing sites in adjacent cells., Ca2+ transients in ICC-DMP are suppressed by the ongoing release of inhibitory neurotransmitter(s)., Functional intracellular Ca2+ stores are essential for spontaneous Ca2+ transients, and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump is necessary for maintenance of spontaneity., Ca2+ release mechanisms involve both ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs). Release from these channels is interdependent., ICC express transcripts of multiple RyRs and InsP3Rs, with Itpr1 and Ryr2 subtypes displaying the highest expression., Abstract 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 Ca2+-activated Cl− channels; thus, Ca2+ signalling is central to the behaviours of these cells. Confocal imaging was used to characterize the nature and mechanisms of Ca2+ transients in ICC-DMP within intact jejunal muscles expressing a genetically encoded Ca2+ indicator (GCaMP3) selectively in ICC. ICC-DMP displayed spontaneous Ca2+ transients that ranged from discrete, localized events to waves that propagated over variable distances. The occurrence of Ca2+ transients was highly variable, and it was determined that firing was stochastic in nature. Ca2+ transients were tabulated in multiple cells within fields of view, and no correlation was found between the events in adjacent cells. TTX (1 μ m) significantly increased the occurrence of Ca2+ transients, suggesting that ICC-DMP contributes to the tonic inhibition conveyed by ongoing activity of inhibitory motor neurons. Ca2+ transients were minimally affected after 12 min in Ca2+ free solution, indicating these events do not depend immediately upon Ca2+ influx. However, inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump and blockers of inositol triphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels blocked ICC Ca2+ transients. These data suggest an interdependence between RyR and InsP3R in the generation of Ca2+ transients. Itpr1 and Ryr2 were the dominant transcripts expressed by ICC. These findings provide the first high-resolution recording of the subcellular Ca2+ dynamics that control the behaviour of ICC-DMP in situ. [ABSTRACT FROM AUTHOR]

Key points Platelet-derived growth factor receptor-α-positive (PDGFRα+) interstitial cells in detrusor muscles may participate in post-junctional responses to neurotransmitters., PDGFRα+ interstitial cells express purinergic receptors (P2Y) and small conductance Ca2+-activated K+ channels (mainly SK3). ATP elicited large amplitude outward currents and hyperpolarization in PDGFRα+ cells. SK channel blockers and a P2Y1 receptor antagonist blocked responses to ATP., ATP elicited only minor responses in PDGFRα+ cells of P2ry1−/− mice. ATP elicited transient inward currents in smooth muscle cells and purinergic receptor (P2X) agonists had no effect on PDGFRα+ cells., A specific P2Y1 receptor blocker decreased electrical field stimulation-induced relaxation., Our findings provide an explanation for the purinergic relaxation of detrusor muscles and describe a novel mechanism for inhibitory regulation of bladder muscles that may control detrusor excitability during the filling phase., Abstract Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction could be due to activation of inward currents in smooth muscle cells, but the mechanism of purinergic relaxation has not been determined. We recently reported a new class of interstitial cells in detrusor muscles and showed that these cells could be identified with antibodies against platelet-derived growth factor receptor-α (PDGFRα+ cells). The current density of small conductance Ca2+-activated K+ (SK) channels in these cells is far higher (∼100 times) than in smooth muscle cells. Thus, we examined purinergic receptor (P2Y) mediated SK channel activation as a mechanism for purinergic relaxation. P2Y receptors (mainly P2ry1 gene) were highly expressed in PDGFRα+ cells. Under voltage clamp conditions, ATP activated large outward currents in PDGFRα+ cells that were inhibited by blockers of SK channels. ATP also induced significant hyperpolarization under current clamp conditions. A P2Y1 agonist, MRS2365, mimicked the effects of ATP, and a P2Y1 antagonist, MRS2500, inhibited ATP-activated SK currents. Responses to ATP were largely abolished in PDGFRα+ cells of P2ry1−/− mice, and no response was elicited by MRS2365 in these cells. A P2X receptor agonist had no effect on PDGFRα+ cells but, like ATP, activated transient inward currents in smooth muscle cells (SMCs). A P2Y1 antagonist decreased nerve-evoked relaxation. These data suggest that purines activate SK currents via mainly P2Y1 receptors in PDGFRα+ cells. Our findings provide an explanation for purinergic relaxation in detrusor muscles and show that there are no discrete inhibitory nerve fibres. A dual receptive field for purines provides the basis for inhibitory neural regulation of excitability. [ABSTRACT FROM AUTHOR]