Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The current study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs) indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole-cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type whereas the NOS inhibitor L-NNA abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by L-NNA, but not MRS2500 suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since IRAG1 is expressed in ICC-IM it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. PDGFRᵯC+ cells but not ICC-IM expressed P2Y1R and SK3 suggesting that the purinergic pathway indirectly blocks whole-cell Ca2+ transients in Type II ICC-IM via PDGFRᵯC+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC and PDGFRᵯC+ cells via the SIP syncytium.

Mfge8 is expressed by pericytes in gastric antrum submucosa from patients with obesity.

The main function of the stomach is to digest ingested food. Gastric antrum muscular contractions mix ingested food with digestive enzymes and stomach acid and propel the chyme through the pyloric sphincter at a rate in which the small intestine can process the chyme for optimal nutrient absorption. Mfge8 binding to α8ß1 integrins helps regulate gastric emptying by reducing the force of antral smooth muscle contractions. The source of Mfge8 within gastric muscles is unclear. Since Mfge8 is a secreted protein, Mfge8 could be delivered via the circulation, or be locally secreted by cells within the muscle layers. In this study, we identify a source of Mfge8 within human gastric antrum muscles using spatial transcriptomic analysis. We show that Mfge8 is expressed in subpopulations of Mef2c+ perivascular cells within the submucosa layer of the gastric antrum. Mef2c is expressed in subpopulations of NG2+ and PDGFRB+ pericytes. Mfge8 is expressed in NG2+/Mef2c+ pericytes, but not in NG2+/Mef2c-, PDGFRB+/Mef2c-, or PDGFRB+/Mef2c+ pericytes. Mfge8 is absent from CD34+ endothelial cells but is expressed in a small population of perivascular ACTA2+ cells. We also show that α8 integrin is not expressed by interstitial cells of Cajal (ICC), supporting the findings that Mfge8 attenuates gastric antrum smooth muscle contractions by binding to α8ß1 integrins on enteric smooth muscle cells. These findings suggest a novel, supplementary mechanism of regulation of gastric antrum motility by cellular regulators of capillary blood flow, in addition to the regulation of gastric antrum motility by the enteric nervous system and the SMC, ICC, and PDGFRα+ cell (SIP) syncytium.

Unconventional Obturator Artery Nutrient Branch: Image of an Anatomical Variation.

Variations in vascular anatomy are of great concern to surgeons, as proper identification of aberrant arteries can reduce the risk of iatrogenic injury and improve patient outcomes. Several studies have highlighted the irregular branching pattern of pelvic arteries, with a recent focus on the obturator artery (OA). The OA has an inconstant origin from the internal iliac artery, external iliac artery, or inferior epigastric artery. Within the pelvis, the OA can give off muscular branches and nutrient vessels to the ilium and pubis. Though occasionally described in text, few resources employ images of human donors that depict branches arising from the OAs. Out of the 34 hemisected pelves studied, we identified 1 individual with a substantial nutrient vessel branching unilaterally from the OA. Herein, we present the first image of this unconventional nutrient artery. This vessel should be highlighted given that its size and course make it particularly vulnerable during intrapelvic surgeries such as pelvic lymph node dissection or in procedures requiring arterial embolization of the OA.

Insights on gastrointestinal motility through the use of optogenetic sensors and actuators.

The muscularis of the gastrointestinal (GI) tract consists of smooth muscle cells (SMCs) and various populations of interstitial cells of Cajal (ICC), platelet-derived growth factor receptor α+ (PDGFRα+ ) cells, as well as excitatory and inhibitory enteric motor nerves. SMCs, ICC and PDGFRα+ cells form an electrically coupled syncytium, which together with inputs from the enteric nervous system (ENS) regulates GI motility. Early studies evaluating Ca2+ signalling behaviours in the GI tract relied upon indiscriminate loading of tissues with Ca2+ dyes. These methods lacked the means to study activity in specific cells of interest without encountering contamination from other cells within the preparation. Development of mice expressing optogenetic sensors (green calmodulin fusion protein (GCaMP), red calmodulin fusion protein (RCaMP)) has allowed visualization of Ca2+ signalling behaviours in a cell specific manner. Additionally, availability of mice expressing optogenetic modulators (channelrhodopsins or halorhodospins) has allowed manipulation of specific signalling pathways using light. GCaMP-expressing animals have been used to characterize Ca2+ signalling behaviours of distinct classes of ICC and SMCs throughout the GI musculature. These findings illustrate how Ca2+ signalling in ICC is fundamental in GI muscles, contributing to tone in sphincters, pacemaker activity in rhythmic muscles and relaying enteric signals to SMCs. Animals that express channelrhodopsin in specific neuronal populations have been used to map neural circuitry and to examine post junctional neural effects on GI motility. Thus, optogenetic approaches provide a novel means to examine the contribution of specific cell types to the regulation of motility patterns within complex multi-cellular systems.

Ca2+ signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower oesophageal sphincters.

The lower oesophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Cav 1.2 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 Cav 1.2 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 Cav 1.2 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 Cav 1.2 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.

Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon.

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.

A novel intramuscular Interstitial Cell of Cajal is a candidate for generating pacemaker activity in the mouse internal anal sphincter.

The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca2+ transients in mice expressing a genetically-encoded Ca2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.

Rhythmic calcium transients in smooth muscle cells of the mouse internal anal sphincter.

BACKGROUND: The internal anal sphincter (IAS) exhibits slow waves (SWs) and tone that are dependent upon L-type Ca2+ channels (CavL ) suggesting that phasic events (ie, SWs) play a fundamental role in tone generation. The present study further examined phasic activity in the IAS by measuring the spatiotemporal properties of Ca2+ transients (CTs) in IAS smooth muscle cells (SMCs). METHODS: Ca2+ transients were recorded with spinning disk confocal microscopy from the IAS of SM-GCaMP mice. Muscles were pinned submucosal surface up at two different lengths. Drugs were applied by inclusion in the superfusate. KEY RESULTS: Ca2+ transients displayed ongoing rhythmic firings at both lengths and were abolished by nifedipine and the KATP channel activator pinacidil indicating their dependence upon CavL . Like SWs, CTs were greatest in frequency (average 70.6 cpm) and amplitude at the distal extremity and conducted proximally. Removal of the distal IAS reduced but did not abolish CTs. The time constant for clearing cytoplasmic Ca2+ averaged 0.46 seconds and basal Ca2+ levels were significantly elevated. CONCLUSIONS & INFERENCES: The similarities in spatiotemporal and pharmacological properties of CTs and SWs suggest that SW gives rise to CTs while muscle stretch is not required. Elevated relative basal Ca2+ in the IAS is likely due to the inability of cells to clear or sequester Ca2+ between rapid frequency voltage-dependent Ca2+ entry events, that is, conditions that will lead to tone development. The conduction of CTs from distal to proximal IAS will lead to orally directed contractions and likely contribute to the maintenance of fecal continence.

Control of Motility in the Internal Anal Sphincter.

The internal anal sphincter (IAS) plays an important role in the maintenance of fecal continence since it generates tone and is responsible for > 70% of resting anal pressure. During normal defecation the IAS relaxes. Historically, tone generation in gastrointestinal muscles was attributed to mechanisms arising directly from smooth muscle cells, ie, myogenic activity. However, slow waves are now known to play a fundamental role in regulating gastrointestinal motility and these electrical events are generated by the interstitial cells of Cajal. Recently, interstitial cells of Cajal, as well as slow waves, have also been identified in the IAS making them viable candidates for tone generation. In this review we discuss four different mechanisms that likely contribute to tone generation in the IAS. Three of these involve membrane potential, L-type Ca2+ channels and electromechanical coupling (ie, summation of asynchronous phasic activity, partial tetanus, and window current), whereas the fourth involves the regulation of myofilament Ca2+ sensitivity. Contractile activity in the IAS is also modulated by sympathetic motor neurons that significantly increase tone and anal pressure, as well as inhibitory motor neurons (particularly nitrergic and vasoactive intestinal peptidergic) that abolish contraction and assist with normal defecation. Alterations in IAS motility are associated with disorders such as fecal incontinence and anal fissures that significantly decrease the quality of life. Understanding in greater detail how tone is regulated in the IAS is important for developing more effective treatment strategies for these debilitating defecation disorders.

Inhibitory Neural Regulation of the Ca 2+ Transients in Intramuscular Interstitial Cells of Cajal in the Small Intestine.

Gastrointestinal motility is coordinated by enteric neurons. Both inhibitory and excitatory motor neurons innervate the syncytium consisting of smooth muscle cells (SMCs) interstitial cells of Cajal (ICC) and PDGFRα+ cells (SIP syncytium). Confocal imaging of mouse small intestines from animals expressing GCaMP3 in ICC were used to investigate inhibitory neural regulation of ICC in the deep muscular plexus (ICC-DMP). We hypothesized that Ca2+ signaling in ICC-DMP can be modulated by inhibitory enteric neural input. ICC-DMP lie in close proximity to the varicosities of motor neurons and generate ongoing Ca2+ transients that underlie activation of Ca2+-dependent Cl- channels and regulate the excitability of SMCs in the SIP syncytium. Electrical field stimulation (EFS) caused inhibition of Ca2+ for the first 2-3 s of stimulation, and then Ca2+ transients escaped from inhibition. The NO donor (DEA-NONOate) inhibited Ca2+ transients and Nω-Nitro-L-arginine (L-NNA) or a guanylate cyclase inhibitor (ODQ) blocked inhibition induced by EFS. Purinergic neurotransmission did not affect Ca2+ transients in ICC-DMP. Purinergic neurotransmission elicits hyperpolarization of the SIP syncytium by activation of K+ channels in PDGFRα+ cells. Generalized hyperpolarization of SIP cells by pinacidil (KATP agonist) or MRS2365 (P2Y1 agonist) also had no effect on Ca2+ transients in ICC-DMP. Peptidergic transmitter receptors (VIP and PACAP) are expressed in ICC and can modulate ICC-DMP Ca2+ transients. In summary Ca2+ transients in ICC-DMP are blocked by enteric inhibitory neurotransmission. ICC-DMP lack a voltage-dependent mechanism for regulating Ca2+ release, and this protects Ca2+ handling in ICC-DMP from membrane potential changes in other SIP cells.

Laboratory practical to study the differential innervation pathways of urinary tract smooth muscle.

In the mammalian lower urinary tract, there is a reciprocal relationship between the contractile state of the bladder and urethra. As the bladder fills with urine, it remains relaxed to accommodate increases in volume, while the urethra remains contracted to prevent leakage of urine from the bladder to the exterior. Disruptions to the normal contractile state of the bladder and urethra can lead to abnormal micturition patterns and urinary incontinence. While both the bladder and urethra are smooth-muscle organs, they are differentially contracted by input from cholinergic and sympathetic nerves, respectively. The laboratory practical described here provides an experiential approach to understanding the anatomy of the lower urinary tract. Several key factors in urinary tract physiology are outlined, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α1-adrenoceptors. This is achieved by measuring the force generated by bladder and urethra smooth muscle to demonstrate that acetylcholine contracts the smooth muscle of the bladder, whereas adrenergic agonists contract the urethral smooth muscle. An inhibition of these effects is also demonstrated by application of the muscarinic receptor antagonist atropine and the α1-adrenergic receptor blocker phentolamine. A list of suggested techniques and exam questions to evaluate student understanding on this topic is also provided.

Ca2+ signalling in mouse urethral smooth muscle in situ: role of Ca2+ stores and Ca2+ influx mechanisms.

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.

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter.

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα(+)), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα(+) cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα(+) cells and neuronal nitric-oxide-synthase-positive (nNOS(+)) nerve fibers in the IAS of wild-type (WT) and W/W ( v ) mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα(+) cells (PDGFRα(+)-IM). Other populations of PDGFRα(+) cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ( v ) IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα(+)-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ( v ) IAS. Minimum surface distance analysis revealed that nNOS(+) nerve fibers were closely aligned with both ICC-IM and PDGFRα(+)-IM. An even closer association was seen between ICC-IM and PDGFRα(+)-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα(+)-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery.

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor Gö-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or Gö-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.