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Ni Bhraonain EP, Turner JA, Hannigan KI, Sanders KM, Cobine CA. Immunohistochemical characterization of interstitial cells and their spatial relationship to motor neurons within the mouse esophagus. Cell Tissue Res 2025; 399:61-84. [PMID: 39607495 DOI: 10.1007/s00441-024-03929-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2024] [Accepted: 10/28/2024] [Indexed: 11/29/2024]
Abstract
Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract, yet their function in the esophagus remains unknown. The mouse esophagus has been described as primarily skeletal muscle; however, ICC have been identified in this region. This study characterizes the distribution of skeletal and smooth muscle cells (SMCs) and their spatial relationship to ICC, PDGFRα+ cells, and intramuscular motor neurons in the mouse esophagus. SMCs occupied approximately 30% of the distal esophagus, but their density declined in more proximal regions. Similarly, ANO1+ intramuscular ICC (ICC-IM) were distributed along the esophagus, with density decreasing proximally. While ICC-IM were closely associated with SMCs, they were also present in regions of skeletal muscle. Intramuscular, submucosal, and myenteric PDGFRα+ cells were densely distributed throughout the esophagus, yet only intramuscular PDGFRα+ cells in the lower esophageal sphincter (LES) and distal esophagus expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with intramuscular nNOS+, VIP+, VAChT+, and TH+ neurons and GFAP+ cells resembling intramuscular enteric glia. These findings suggest that ICC-IM and PDGFRα+ cells may have roles in regulating esophageal motility due to their close proximity to each other and to skeletal muscle and SMCs, although further functional studies are needed to explore their role in this region. The mixed muscular composition and presence of interstitial cells in the mouse distal esophagus is anatomically similar to the transitional zone found in the human esophagus, and therefore, motility studies in the mouse may be translatable to humans.
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Affiliation(s)
- Emer P Ni Bhraonain
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, 1664 N. Virginia St., MS 352, Reno, NV, 89557, USA
| | - Jack A Turner
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, 1664 N. Virginia St., MS 352, Reno, NV, 89557, USA
| | - Karen I Hannigan
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, 1664 N. Virginia St., MS 352, Reno, NV, 89557, USA
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, 1664 N. Virginia St., MS 352, Reno, NV, 89557, USA
| | - Caroline A Cobine
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, 1664 N. Virginia St., MS 352, Reno, NV, 89557, USA.
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2
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Hannigan KI, Ni Bhraonain EP, Gould TW, Keef KD, Cobine CA. Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter. Am J Physiol Gastrointest Liver Physiol 2024; 327:G382-G404. [PMID: 38860285 PMCID: PMC11427099 DOI: 10.1152/ajpgi.00309.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 05/30/2024] [Accepted: 06/03/2024] [Indexed: 06/12/2024]
Abstract
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 present 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 nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (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 inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ 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 PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).
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Affiliation(s)
- Karen I Hannigan
- Department of Physiology and Cell BiologyUniversity of NevadaRenoNevadaUnited States
| | - Emer P Ni Bhraonain
- Department of Physiology and Cell BiologyUniversity of NevadaRenoNevadaUnited States
| | - Thomas W Gould
- Department of Physiology and Cell BiologyUniversity of NevadaRenoNevadaUnited States
| | - Kathleen D Keef
- Department of Physiology and Cell BiologyUniversity of NevadaRenoNevadaUnited States
| | - Caroline A Cobine
- Department of Physiology and Cell BiologyUniversity of NevadaRenoNevadaUnited States
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Hwang SJ, Kim M, Jones A, Basma N, Baker SA, Sanders KM, Ward SM. Interstitial cells of the sip syncytium regulate basal membrane potential in murine gastric corpus. FASEB J 2024; 38:e23863. [PMID: 39143726 PMCID: PMC11587931 DOI: 10.1096/fj.202400982r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2024] [Revised: 07/11/2024] [Accepted: 07/23/2024] [Indexed: 08/16/2024]
Abstract
Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.
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Affiliation(s)
- Sung Jin Hwang
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
| | - MinKyung Kim
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
| | - Amanda Jones
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
| | - Naseer Basma
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
| | - Salah A Baker
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
| | - Kenton M Sanders
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
| | - Sean M Ward
- Department of Physiology & Cell Biology, Reno School of Medicine, University of Nevada, Reno, Nevada, USA
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Ni Bhraonain E, Turner J, Hannigan K, Sanders K, Cobine C. Immunohistochemical characterization of interstitial cells and their relationship to motor neurons within the mouse esophagus. RESEARCH SQUARE 2024:rs.3.rs-4474290. [PMID: 38947055 PMCID: PMC11213231 DOI: 10.21203/rs.3.rs-4474290/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/02/2024]
Abstract
Interstitial cells of Cajal (ICC) and PDGFRα+ cells regulate smooth muscle motility in the gastrointestinal (GI) tract. However, their role(s) in esophageal motility are still unclear. The mouse esophagus has traditionally been described as almost entirely skeletal muscle in nature though ICC have been identified along its entire length. The current study evaluated the distribution of skeletal and smooth muscle within the esophagus using a mouse selectively expressing eGFP in smooth muscle cells (SMCs). The relationship of SMCs to ICC and PDGFRα+ cells was also examined. SMCs declined in density in the oral direction however SMCs represented ~ 25% of the area in the distal esophagus suggesting a likeness to the transition zone observed in humans. ANO1+ intramuscular ICC (ICC-IM) were distributed along the length of the esophagus though like SMCs, declined proximally. ICC-IM were closely associated with SMCs but were also found in regions devoid of SMCs. Intramuscular and submucosal PDGFRα+ cells were densely distributed throughout the esophagus though only intramuscular PDGFRα+ cells within the LES and distal esophagus highly expressed SK3. ICC-IM and PDGFRα+ cells were closely associated with nNOS+, VIP+, VAChT+ and TH+ neurons throughout the LES and distal esophagus. GFAP+ cells resembling intramuscular enteric glia were observed within the muscle and were closely associated with ICC-IM and PDGFRα+ cells, occupying a similar location to c. These data suggest that the mouse esophagus is more similar to the human than thought previously and thus set the foundation for future functional and molecular studies using transgenic mice.
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Lu P, Lifshitz LM, Bellve K, ZhuGe R. TMEM16A in smooth muscle cells acts as a pacemaker channel in the internal anal sphincter. Commun Biol 2024; 7:151. [PMID: 38317010 PMCID: PMC10844222 DOI: 10.1038/s42003-024-05850-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Accepted: 01/23/2024] [Indexed: 02/07/2024] Open
Abstract
Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl- channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence.
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Affiliation(s)
- Ping Lu
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Lawrence M Lifshitz
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Karl Bellve
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Ronghua ZhuGe
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, USA.
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Veress B, Peruzzi N, Eckermann M, Frohn J, Salditt T, Bech M, Ohlsson B. Structure of the myenteric plexus in normal and diseased human ileum analyzed by X-ray virtual histology slices. World J Gastroenterol 2022; 28:3994-4006. [PMID: 36157532 PMCID: PMC9367237 DOI: 10.3748/wjg.v28.i29.3994] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Revised: 05/18/2022] [Accepted: 07/11/2022] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm.
AIM To examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS.
METHODS Six patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples.
RESULTS In the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings.
CONCLUSION The analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed.
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Affiliation(s)
- Bela Veress
- Department of Pathology, Skåne Universiity Hospital, Malmö 205 02, Sweden
| | - Niccolò Peruzzi
- Medical Radiation Physics, Department of Clinical Sciences, Lund University, Lund 221 00, Sweden
| | - Marina Eckermann
- Institute for X-Ray Physics, University of Göttingen, Göttingen 37077, Germany
- Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen 37077, Germany
- ESRF, The European Synchrotron, Grenoble 38043, France
| | - Jasper Frohn
- Institute for X-Ray Physics, University of Göttingen, Göttingen 37077, Germany
| | - Tim Salditt
- Institute for X-Ray Physics, University of Göttingen, Göttingen 37077, Germany
- Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen 37077, Germany
| | - Martin Bech
- Medical Radiation Physics, Department of Clinical Sciences, Lund University, Lund 221 00, Sweden
| | - Bodil Ohlsson
- Department of Internal Medicine, Skåne University Hospital, Lund University, Malmö S-205 02, Sweden
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Drumm BT, Cobine CA, Baker SA. Insights on gastrointestinal motility through the use of optogenetic sensors and actuators. J Physiol 2022; 600:3031-3052. [PMID: 35596741 DOI: 10.1113/jp281930] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 05/13/2022] [Indexed: 11/08/2022] Open
Abstract
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) regulate 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 (GCaMP, 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. Abstract Figure Legends Optogenetic activators and sensors can be used to investigate the complex multi-cellular nature of the gastrointestinal (GI tract). Optogenetic activators that are activated by light such as channelrhodopsins (ChR2), OptoXR and halorhodopsinss (HR) proteins can be genetically encoded into specific cell types. This can be used to directly activate or silence specific GI cells such as various classes of enteric neurons, smooth muscle cells (SMC) or interstitial cells, such as interstitial cells of Cajal (ICC). Optogenetic sensors that are activated by different wavelengths of light such as green calmodulin fusion protein (GCaMP) and red CaMP (RCaMP) make high resolution of sub-cellular Ca2+ signalling possible within intact tissues of specific cell types. These tools can provide unparalleled insight into mechanisms underlying GI motility and innervation. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Bernard T Drumm
- Smooth Muscle Research Centre, Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Co. Louth, Ireland.,Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Caroline A Cobine
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Salah A Baker
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
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Ji S, Traini C, Mischopoulou M, Gibbons SJ, Ligresti G, Faussone-Pellegrini MS, Sha L, Farrugia G, Vannucchi MG, Cipriani G. Muscularis macrophages establish cell-to-cell contacts with telocytes/PDGFRα-positive cells and smooth muscle cells in the human and mouse gastrointestinal tract. Neurogastroenterol Motil 2021; 33:e13993. [PMID: 33020982 PMCID: PMC7902307 DOI: 10.1111/nmo.13993] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 07/22/2020] [Accepted: 08/25/2020] [Indexed: 12/16/2022]
Abstract
BACKGROUND AND AIM Muscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine. METHODS The distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy. KEY RESULTS Immunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα-positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell-to-cell contacts with smooth muscle cells and TCs/PDGFRα-positive cells but not with ICCs and enteric neurons. CONCLUSION & INFERENCES This study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell-to-cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα-positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions.
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Affiliation(s)
- Sihan Ji
- Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota, USA,Department of Neuroendocrine Pharmacology, School of Pharmacy, China Medical University, Shenyang, Liaoning, China
| | - Chiara Traini
- Department of Experimental and Clinical Medicine, Research Unit of Histology and Embryology, University of Florence, Italy
| | | | - Simon J. Gibbons
- Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota, USA
| | | | | | - Lei Sha
- Department of Neuroendocrine Pharmacology, School of Pharmacy, China Medical University, Shenyang, Liaoning, China,Corresponding authors: Gianluca Cipriani, PhD, Mayo Clinic, Enteric NeuroScience Program, 200 First Street SW, Rochester, MN 55905, 507-210-6402, ; Maria Giuliana Vannucchi MD, PhD, Research Unit of Histology and Embryology, Dept of Experimental and Clinical Medicine, Viale G. Pieraccini,6, 50139 Florence, Italy, ; Lei Sha, MD, China Medical University, Department of Neuroendocrine Pharmacology, School of Pharmacy, Shenyang, Liaoning, China, 77 Pu He Road, Shenbei New District, Shenyang, Liaoning province, P. R. China,110122, 18900911003,
| | - Gianrico Farrugia
- Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota, USA
| | - Maria Giuliana Vannucchi
- Department of Experimental and Clinical Medicine, Research Unit of Histology and Embryology, University of Florence, Italy,Corresponding authors: Gianluca Cipriani, PhD, Mayo Clinic, Enteric NeuroScience Program, 200 First Street SW, Rochester, MN 55905, 507-210-6402, ; Maria Giuliana Vannucchi MD, PhD, Research Unit of Histology and Embryology, Dept of Experimental and Clinical Medicine, Viale G. Pieraccini,6, 50139 Florence, Italy, ; Lei Sha, MD, China Medical University, Department of Neuroendocrine Pharmacology, School of Pharmacy, Shenyang, Liaoning, China, 77 Pu He Road, Shenbei New District, Shenyang, Liaoning province, P. R. China,110122, 18900911003,
| | - Gianluca Cipriani
- Enteric NeuroScience Program, Mayo Clinic, Rochester, Minnesota, USA,Corresponding authors: Gianluca Cipriani, PhD, Mayo Clinic, Enteric NeuroScience Program, 200 First Street SW, Rochester, MN 55905, 507-210-6402, ; Maria Giuliana Vannucchi MD, PhD, Research Unit of Histology and Embryology, Dept of Experimental and Clinical Medicine, Viale G. Pieraccini,6, 50139 Florence, Italy, ; Lei Sha, MD, China Medical University, Department of Neuroendocrine Pharmacology, School of Pharmacy, Shenyang, Liaoning, China, 77 Pu He Road, Shenbei New District, Shenyang, Liaoning province, P. R. China,110122, 18900911003,
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Peruzzi N, Veress B, Dahlin LB, Salditt T, Andersson M, Eckermann M, Frohn J, Robisch AL, Bech M, Ohlsson B. 3D analysis of the myenteric plexus of the human bowel by X-ray phase-contrast tomography - a future method? Scand J Gastroenterol 2020; 55:1261-1267. [PMID: 32907418 DOI: 10.1080/00365521.2020.1815079] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
OBJECTIVES Light microscopical analysis in two dimensions, combined with immunohistochemistry, is presently the gold standard to describe the enteric nervous system (ENS). Our aim was to assess the usefulness of three-dimensional (3D) imaging by X-ray phase-contrast tomography in evaluating the ENS of the human bowel. MATERIAL AND METHODS Myenteric ganglia were identified in full-thickness biopsies of the ileum and colon by hematoxylin & eosin staining. A1-mm biopsy punch was taken from the paraffin blocks and placed into a Kapton® tube for subsequent tomographic investigation. The samples were scanned, without further preparation, using phase-contrast tomography at two different scales: overview scans (performed with laboratory setups), which allowed localization of the nervous tissue (∼1µm effective voxel size); and high-resolution scans (performed with a synchrotron endstation), which imaged localized regions of 320x320x320 µm3 (176 nm effective voxel size). RESULTS The contrast allowed us to follow the shape and the size changes of the ganglia, as well as to study their cellular components together with the cells and cellular projections of the periganglional space. Furthermore, it was possible to show the 3D network of the myenteric plexus and to quantify its volume within the samples. CONCLUSIONS Phase-contrast X-ray tomography can be applied for volume analyses of the human ENS and to study tissue components in unstained paraffin-embedded tissue biopsies. This technique could potentially be used to study disease mechanisms, and to compare healthy and diseased tissues in clinical research.
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Affiliation(s)
- Niccolò Peruzzi
- Division of Medical Radiation Physics, Department of Clinical Sciences, Lund University, Lund, Sweden
| | - Béla Veress
- Department of Pathology, Skåne University Hospital, Malmö, Sweden
| | - Lars B Dahlin
- Department of Translational Medicine - Hand Surgery, Lund University, Malmö, Sweden.,Department of Hand Surgery, Skåne University Hospital, Malmö, Sweden
| | - Tim Salditt
- Institute for X-Ray Physics, University of Göttingen, Göttingen, Germany.,Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany
| | - Mariam Andersson
- Department of Applied Mathematics and Computer Science, Technical University of Denmark, Lyngby, Denmark.,Danish Research Centre for Magnetic Resonance (DRCMR), Center for Functional and Diagnostic Imaging and Research, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark
| | - Marina Eckermann
- Institute for X-Ray Physics, University of Göttingen, Göttingen, Germany.,Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Germany
| | - Jasper Frohn
- Institute for X-Ray Physics, University of Göttingen, Göttingen, Germany
| | - Anna-Lena Robisch
- Institute for X-Ray Physics, University of Göttingen, Göttingen, Germany
| | - Martin Bech
- Division of Medical Radiation Physics, Department of Clinical Sciences, Lund University, Lund, Sweden
| | - Bodil Ohlsson
- Lund University, Skåne University Hospital, Department of Internal Medicine, Malmö, Sweden
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10
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Hannigan KI, Bossey AP, Foulkes HJL, Drumm BT, Baker SA, Ward SM, Sanders KM, Keef KD, Cobine CA. A novel intramuscular Interstitial Cell of Cajal is a candidate for generating pacemaker activity in the mouse internal anal sphincter. Sci Rep 2020; 10:10378. [PMID: 32587396 PMCID: PMC7316801 DOI: 10.1038/s41598-020-67142-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2019] [Accepted: 06/03/2020] [Indexed: 12/14/2022] Open
Abstract
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.
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Affiliation(s)
- Karen I Hannigan
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Aaron P Bossey
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Holly J L Foulkes
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Bernard T Drumm
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Salah A Baker
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Kathleen D Keef
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - Caroline A Cobine
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA.
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Drumm BT, Rembetski BE, Huynh K, Nizar A, Baker SA, Sanders KM. Excitatory cholinergic responses in mouse colon intramuscular interstitial cells of Cajal are due to enhanced Ca 2+ release via M 3 receptor activation. FASEB J 2020; 34:10073-10095. [PMID: 32539213 DOI: 10.1096/fj.202000672r] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2020] [Revised: 05/01/2020] [Accepted: 05/11/2020] [Indexed: 12/14/2022]
Abstract
Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca2+ release in ICC-IM activates Ano1, a Ca2+ -activated Cl- conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca2+ 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 (M2 , M3 ), and NK1 receptors were enriched in ICC-IM. NK1 receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca2+ transients. These effects were reversed by DAU 5884 (M3 receptor antagonist) but not AF-DX 116 (M2 receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca2+ transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca2+ stores with cyclopiazonic acid and reduced by inhibiting Ca2+ 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 M3 receptor activation. Enhancing ICC-IM Ca2+ release and Ano1 activation contributes to excitatory responses of colonic muscles.
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Affiliation(s)
- Bernard T Drumm
- Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV, USA.,Department of Life & Health Science, Dundalk Institute of Technology, Dundalk, Ireland
| | - Benjamin E Rembetski
- Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV, USA
| | - Kaitlin Huynh
- Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV, USA
| | - Aqeel Nizar
- Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV, USA
| | - Salah A Baker
- Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV, USA
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, Reno School of Medicine, University of Nevada, Reno, NV, USA
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12
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Grainger N, Freeman RS, Shonnard CC, Drumm BT, Koh SD, Ward SM, Sanders KM. Identification and classification of interstitial cells in the mouse renal pelvis. J Physiol 2020; 598:3283-3307. [PMID: 32415739 DOI: 10.1113/jp278888] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2020] [Accepted: 04/29/2020] [Indexed: 12/13/2022] Open
Abstract
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.
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Affiliation(s)
- Nathan Grainger
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Ryan S Freeman
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Cameron C Shonnard
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Bernard T Drumm
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Sang Don Koh
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Sean M Ward
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Kenton M Sanders
- Department of Physiology & Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
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13
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Cobine CA, Hannigan KI, McMahon M, Bhraonain EPN, Baker SA, Keef KD. Rhythmic calcium transients in smooth muscle cells of the mouse internal anal sphincter. Neurogastroenterol Motil 2020; 32:e13746. [PMID: 31625250 PMCID: PMC7047590 DOI: 10.1111/nmo.13746] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2019] [Revised: 09/22/2019] [Accepted: 09/26/2019] [Indexed: 12/12/2022]
Abstract
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.
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Affiliation(s)
- Caroline A Cobine
- Corresponding Author: Caroline Cobine, Ph.D., Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, 1664 N. Virginia St., MS 352, Reno, NV 89557, USA, Phone: 1-775-682-8840, Fax: 1-775-784-6903,
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14
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Manousiouthakis E, Chen Y, Cairns DM, Pollard R, Gerlovin K, Dente MJ, Razavi Y, Kaplan DL. Bioengineered in vitro enteric nervous system. J Tissue Eng Regen Med 2019; 13:1712-1723. [PMID: 31278844 DOI: 10.1002/term.2926] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 06/06/2019] [Accepted: 06/19/2019] [Indexed: 12/13/2022]
Abstract
Bidirectional interactions between the human central nervous system and the gastrointestinal tract, via the enteric nervous system, are unmapped and central to many human conditions. There is a critical need to develop 3D human in vitro intestinal tissue models to emulate the intricate cell interactions of the human enteric nervous system within the gastrointestinal tract in order to better understand these complex interactions that cannot be studied utilizing in vivo animal models. In vitro systems, if sufficiently replicative of some in vivo conditions, may assist with the study of individual cell interactions. Here, we describe a 3D-innervated tissue model of the human intestine consisting of human-induced neural stem cells differentiated into relevant enteric nervous system neural cell types. Enterocyte-like (Caco-2) and goblet-like (HT29-MTX) cells are used to form the intestinal epithelial layer, and intestinal myofibroblasts are utilized to simulate the stromal layer. In vitro enteric nervous system cultures supported survival and function of the various cell types, with mucosal and neural transcription factors evident over 5 weeks. The human-induced neural stem cells migrated from the seeding location on the peripheral layer of the hollow scaffolds toward the luminal epithelial cells, prompted by the addition of neural growth factor. nNOS-expressing neurons and the substance P precursor gene TAC1 were expressed within the in vitro enteric nervous system to support the utility of the tissue model to recapitulate enteric nervous system phenotypes. This innervated tissue system offers a new tool to use to help in understanding neural circuits controlling the human intestine and associated communication networks.
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Affiliation(s)
| | - Ying Chen
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Dana M Cairns
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Rachel Pollard
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Kaia Gerlovin
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Michael J Dente
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - Yasmin Razavi
- Department of Biomedical Engineering, Tufts University, Medford, MA
| | - David L Kaplan
- Department of Biomedical Engineering, Tufts University, Medford, MA
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15
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Liang Y, Tarique I, Vistro WA, Liu Y, Wang Z, haseeb A, Gandahi NS, Iqbal A, Wang S, An T, Yang H, Chen Q, Yang P. Age-associated changes of the intrinsic nervous system in relation with interstitial cells in the pre-weaning goat rumen. Aging (Albany NY) 2019; 11:4641-4653. [PMID: 31305258 PMCID: PMC6660047 DOI: 10.18632/aging.102076] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Accepted: 07/01/2019] [Indexed: 05/04/2023]
Abstract
In this study, we investigated the neural changes and their relationships with interstitial cells (ICs) in the rumen of pre-weaning goats by transmission electron microscopy, western blot and immunofluorescence (antibody: general neuronal marker-Protein Gene Product (PGP9.5)/ IC marker-vimentin). The immunofluorescence results showed that PGP9.5-positive reaction was widely distributed in neuronal soma (NS) and nerve fibre (NF). The NSs were observed in the ganglia of the myenteric plexus (MP) but not in the submucosal plexus. The mean optical density (MOD) of the whole of PGP9.5-positive nerves and the protein expression level of PGP.5 in the rumen wall both decreased significantly with age. However an obvious increase MOD of PGP.5-positive NFs within the rumen epithelium were observed. In the MP, the nerves and ICs were interwoven to form two complex networks that gradually tightened with age. Furthermore, NSs and nerve trunks were surrounded by a ring-boundary layer consisting of several ICs that became physically closer with aging. Moreover, ICs were located nearby NFs within the ML, forming connections between ICs, smooth muscle cells and axons. This study describes the pattern of neural distribution and its association with ICs in the developing rumen which shed light on the postpartum development of ruminants.
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Affiliation(s)
- Yu Liang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Imran Tarique
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Waseem Ail Vistro
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Yifei Liu
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Ziyu Wang
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Abdul haseeb
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Noor Samad Gandahi
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Adeela Iqbal
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Siyi Wang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Tianci An
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Huan Yang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Qiusheng Chen
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
| | - Ping Yang
- MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
- College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
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Drumm BT, Hwang SJ, Baker SA, Ward SM, Sanders KM. Ca 2+ signalling behaviours of intramuscular interstitial cells of Cajal in the murine colon. J Physiol 2019; 597:3587-3617. [PMID: 31124144 DOI: 10.1113/jp278036] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Accepted: 05/23/2019] [Indexed: 12/13/2022] Open
Abstract
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 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.
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Affiliation(s)
- Bernard T Drumm
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
| | - Sung J Hwang
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
| | - Salah A Baker
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
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Generation of Spontaneous Tone by Gastrointestinal Sphincters. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019. [PMID: 31183822 DOI: 10.1007/978-981-13-5895-1_2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/05/2023]
Abstract
An important feature of the gastrointestinal (GI) muscularis externa is its ability to generate phasic contractile activity. However, in some GI regions, a more sustained contraction, referred to as "tone," also occurs. Sphincters are muscles oriented in an annular manner that raise intraluminal pressure, thereby reducing or blocking the movement of luminal contents from one compartment to another. Spontaneous tone generation is often a feature of these muscles. Four distinct smooth muscle sphincters are present in the GI tract: the lower esophageal sphincter (LES), the pyloric sphincter (PS), the ileocecal sphincter (ICS), and the internal anal sphincter (IAS). This chapter examines how tone generation contributes to the functional behavior of these sphincters. Historically, tone was attributed to contractile activity arising directly from the properties of the smooth muscle cells. However, there is increasing evidence that interstitial cells of Cajal (ICC) play a significant role in tone generation in GI muscles. Indeed, ICC are present in each of the sphincters listed above. In this chapter, we explore various mechanisms that may contribute to tone generation in sphincters including: (1) summation of asynchronous phasic activity, (2) partial tetanus, (3) window current, and (4) myofilament sensitization. Importantly, the first two mechanisms involve tone generation through summation of phasic events. Thus, the historical distinction between "phasic" versus "tonic" smooth muscles in the GI tract requires revision. As described in this chapter, it is clear that the unique functional role of each sphincter in the GI tract is accompanied by a unique combination of contractile mechanisms.
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18
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Keef KD, Cobine CA. Control of Motility in the Internal Anal Sphincter. J Neurogastroenterol Motil 2019; 25:189-204. [PMID: 30827084 PMCID: PMC6474703 DOI: 10.5056/jnm18172] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/25/2018] [Revised: 11/28/2018] [Accepted: 12/09/2018] [Indexed: 12/14/2022] Open
Abstract
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.
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Affiliation(s)
- Kathleen D Keef
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - Caroline A Cobine
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
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19
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Cobine CA, McKechnie M, Brookfield RJ, Hannigan KI, Keef KD. Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission. J Physiol 2018; 596:5319-5341. [PMID: 30198065 DOI: 10.1113/jp275437] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Accepted: 09/07/2018] [Indexed: 01/04/2023] Open
Abstract
KEY POINTS Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+ /VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were very similar. SK3 channel activation and inhibition caused faster/greater changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. ABSTRACT Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with a high gene sequence identity to humans. Electrical field stimulation produced nitric oxide synthase (NOS)-dependent contractile inhibition in both muscles whereas P2Y1-dependent purinergic NMT was restricted to rectum. An additional NOS-independent, α-chymotrypsin-sensitive component was identified in the IAS consistent with vasoactive intestinal peptide-ergic (VIPergic) NMT. Microelectrode recordings revealed slow NOS-dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1-dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+ /SK3+ cells were closely aligned with nNOS+ /VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2-14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was five times faster in rectum than in the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and the rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells.
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Affiliation(s)
- C A Cobine
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - M McKechnie
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - R J Brookfield
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - K I Hannigan
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
| | - K D Keef
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, USA
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Fidalgo S, Patel BA, Ranson RN, Saffrey MJ, Yeoman MS. Changes in murine anorectum signaling across the life course. Neurogastroenterol Motil 2018; 30:e13426. [PMID: 30062757 PMCID: PMC6175477 DOI: 10.1111/nmo.13426] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2018] [Accepted: 06/18/2018] [Indexed: 12/28/2022]
Abstract
BACKGROUND Increasing age is associated with an increase in the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to these conditions is not fully understood. This study examined the effects of increasing age on the function of the murine anorectum. METHODS The effects of increasing age on cholinergic, nitrergic, and purinergic signaling pathways in the murine anorectum were examined using classical organ bath assays to examine tissue function and electrochemical sensing to determine age-related changes in nitric oxide and acetylcholine release. KEY RESULTS Nitrergic relaxation increased between 3 and 6 months, peaked at 12 months and declined in the 18 and 24 months groups. These changes were in part explained by an age-related decrease in nitric oxide (NO) release. Cholinergic signaling was maintained with age by an increase in acetylcholine (ACh) release and a compensatory decrease in cholinesterase activity. Age-related changes in purinergic relaxation were qualitatively similar to nitrergic relaxation although the relaxations were much smaller. Increasing age did not alter the response of the anorectum smooth muscle to exogenously applied ACh, ATP, sodium nitroprusside or KCl. Similarly, there was no change in basal tension developed by the anorectum. CONCLUSIONS AND INFERENCES The decrease in nitrergic signaling with increasing age may contribute to the age-related fecal impaction and constipation previously described in this model by partially obstructing defecation.
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Affiliation(s)
- S. Fidalgo
- School of Pharmacy and Biomolecular ScienceCentre for Stress and Age‐Related DiseaseUniversity of BrightonBrightonUK
| | - B. A. Patel
- School of Pharmacy and Biomolecular ScienceCentre for Stress and Age‐Related DiseaseUniversity of BrightonBrightonUK
| | - R. N. Ranson
- Department of Applied SciencesFaculty of Health and Life SciencesNorthumbria UniversityNewcastle upon TyneUK
| | - M. J. Saffrey
- School of Life, Health and Chemical SciencesThe Open UniversityMilton KeynesUK
| | - M. S. Yeoman
- School of Pharmacy and Biomolecular ScienceCentre for Stress and Age‐Related DiseaseUniversity of BrightonBrightonUK
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21
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Gamage PPKM, Patel BA, Yeoman MS, Ranson RN, Saffrey MJ. Interstitial cell network volume is reduced in the terminal bowel of ageing mice. J Cell Mol Med 2018; 22:5160-5164. [PMID: 30047236 PMCID: PMC6156346 DOI: 10.1111/jcmm.13794] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2018] [Accepted: 05/18/2018] [Indexed: 12/15/2022] Open
Abstract
Ageing is associated with impaired neuromuscular function of the terminal gastrointestinal (GI) tract, which can result in chronic constipation, faecal impaction and incontinence. Interstitial cells of cajal (ICC) play an important role in regulation of intestinal smooth muscle contraction. However, changes in ICC volume with age in the terminal GI tract (the anal canal including the anal sphincter region and rectum) have not been studied. Here, the distribution, morphology and network volume of ICC in the terminal GI tract of 3‐ to 4‐month‐old and 26‐ to 28‐month‐old C57BL/6 mice were investigated. ICC were identified by immunofluorescence labelling of wholemount preparations with an antibody against c‐Kit. ICC network volume was measured by software‐based 3D volume rendering of confocal Z stacks. A significant reduction in ICC network volume per unit volume of muscle was measured in aged animals. No age‐associated change in ICC morphology was detected. The thickness of the circular muscle layer of the anal sphincter region and rectum increased with age, while that in the distal colon decreased. These results suggest that ageing is associated with a reduction in the network volume of ICC in the terminal GI tract, which may influence the normal function of these regions.
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Affiliation(s)
| | - Bhavik A Patel
- School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK.,Centre for Stress and Age-Related Disease, University of Brighton, Brighton, UK
| | - Mark S Yeoman
- School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK.,Centre for Stress and Age-Related Disease, University of Brighton, Brighton, UK
| | - Rachel N Ranson
- Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, UK
| | - M Jill Saffrey
- School of Life, Health and Chemical Sciences, Open University, Milton Keynes, UK.,Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, UK
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22
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Cobine CA, Hannah EE, Zhu MH, Lyle HE, Rock JR, Sanders KM, Ward SM, Keef KD. ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter. J Physiol 2017; 595:2021-2041. [PMID: 28054347 DOI: 10.1113/jp273618] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Accepted: 12/16/2016] [Indexed: 12/13/2022] Open
Abstract
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.
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Affiliation(s)
- C A Cobine
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - E E Hannah
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - M H Zhu
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - H E Lyle
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - J R Rock
- Department of Anatomy, UCSF School of Medicine, San Francisco, CA, 94143, USA
| | - K M Sanders
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - S M Ward
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
| | - K D Keef
- Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA
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23
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O’Donnell AM, Coyle D, Puri P. Deficiency of platelet-derived growth factor receptor-α-positive cells in Hirschsprung's disease colon. World J Gastroenterol 2016; 22:3335-3340. [PMID: 27022215 PMCID: PMC4806191 DOI: 10.3748/wjg.v22.i12.3335] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Revised: 11/19/2015] [Accepted: 12/21/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate whether the expression of platelet-derived growth factor receptor-α-positive (PDGFRα+)-cells is altered in Hirschsprung’s disease (HD).
METHODS: HD tissue specimens (n = 10) were collected at the time of pull-through surgery, while colonic control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 10). Immunolabelling of PDGFRα+-cells was visualized using confocal microscopy to assess the distribution of these cells, while Western blot analysis was undertaken to quantify PDGFRα protein expression.
RESULTS: Confocal microscopy revealed PDGFRα+-cells within the mucosa, myenteric plexus and smooth muscle in normal controls, with a marked reduction in PDGFRα+-cells in the HD specimens. Western blotting revealed high levels of PDGFRα protein expression in normal controls, while there was a striking decrease in PDGFRα protein expression in the HD colon.
CONCLUSION: These findings suggest that the altered distribution of PDGFRα+-cells in both the aganglionic and ganglionic HD bowel may contribute to the motility dysfunction in HD.
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24
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Wang GD, Wang XY, Liu S, Xia Y, Zou F, Qu M, Needleman BJ, Mikami DJ, Wood JD. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum. Am J Physiol Gastrointest Liver Physiol 2015; 308:G955-63. [PMID: 25813057 PMCID: PMC4451321 DOI: 10.1152/ajpgi.00430.2014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/02/2014] [Accepted: 03/18/2015] [Indexed: 01/31/2023]
Abstract
Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions.
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Affiliation(s)
- Guo-Du Wang
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio;
| | - Xi-Yu Wang
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio;
| | - Sumei Liu
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio;
| | - Yun Xia
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio; ,2Department of Anesthesiology, College of Medicine, The Ohio State University, Columbus, Ohio; and
| | - Fei Zou
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio;
| | - Meihua Qu
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio;
| | - Bradley J. Needleman
- 3Department of Surgery, College of Medicine, The Ohio State University, Columbus, Ohio
| | - Dean J. Mikami
- 3Department of Surgery, College of Medicine, The Ohio State University, Columbus, Ohio
| | - Jackie D. Wood
- 1Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio;
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25
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Peri LE, Koh BH, Ward GK, Bayguinov Y, Hwang SJ, Gould TW, Mullan CJ, Sanders KM, Ward SM. A novel class of interstitial cells in the mouse and monkey female reproductive tracts. Biol Reprod 2015; 92:102. [PMID: 25788664 DOI: 10.1095/biolreprod.114.124388] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2014] [Accepted: 03/12/2015] [Indexed: 01/14/2023] Open
Abstract
Growing evidence suggests important roles for specialized platelet-derived growth factor receptor alpha-positive (PDGFRalpha(+)) cells in regulating the behaviors of visceral smooth muscle organs. Examination of the female reproductive tracts of mice and monkeys showed that PDGFRalpha(+) cells form extensive networks in ovary, oviduct, and uterus. PDGFRalpha(+) cells were located in discrete locations within these organs, and their distribution and density were similar in rodents and primates. PDGFRalpha(+) cells were distinct from smooth muscle cells and interstitial cells of Cajal (ICC). This was demonstrated with immunohistochemical techniques and by performing molecular expression studies on PDGFRalpha(+) cells from mice with enhanced green fluorescent protein driven off of the endogenous promoter for Pdgfralpha. Significant differences in gene expression were found in PDGFRalpha(+) cells from ovary, oviduct, and uterus. Differences in gene expression were also detected in cells from different tissue regions within the same organ (e.g., uterine myometrium vs. endometrium). PDGFRalpha(+) cells are unlikely to provide pacemaker activity because they lack significant expression of key pacemaker genes found in ICC (Kit and Ano1). Gja1 encoding connexin 43 was expressed at relatively high levels in PDGFRalpha(+) cells (except in the ovary), suggesting these cells can form gap junctions to one another and neighboring smooth muscle cells. PDGFRalpha(+) cells also expressed the early response transcription factor and proto-oncogene Fos, particularly in the ovary. These data demonstrate extensive distribution of PDGFRalpha(+) cells throughout the female reproductive tract. These cells are a heterogeneous population of cells that are likely to contribute to different aspects of physiological regulation in the various anatomical niches they occupy.
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Affiliation(s)
- Lauren E Peri
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Byoung H Koh
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Grace K Ward
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Yulia Bayguinov
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sung Jin Hwang
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Thomas W Gould
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Catrina J Mullan
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Kenton M Sanders
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sean M Ward
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada
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26
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Baker SA, Hennig GW, Ward SM, Sanders KM. Temporal sequence of activation of cells involved in purinergic neurotransmission in the colon. J Physiol 2015; 593:1945-63. [PMID: 25627983 DOI: 10.1113/jphysiol.2014.287599] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2014] [Accepted: 01/21/2015] [Indexed: 12/27/2022] Open
Abstract
KEY POINTS Platelet derived growth factor receptor α (PDGFRα(+) ) cells in colonic muscles are innervated by enteric inhibitory motor neurons. PDGFRα(+) cells generate Ca(2+) transients in response to exogenous purines and these responses were blocked by MRS-2500. Stimulation of enteric neurons, with cholinergic and nitrergic components blocked, evoked Ca(2+) transients in PDGFRα(+) and smooth muscle cells (SMCs). Responses to nerve stimulation were abolished by MRS-2500 and not observed in muscles with genetic deactivation of P2Y1 receptors. Ca(2+) transients evoked by nerve stimulation in PDGFRα(+) cells showed the same temporal characteristics as electrophysiological responses. PDGFRα(+) cells express gap junction genes, and drugs that inhibit gap junctions blocked neural responses in SMCs, but not in nerve processes or PDGFRα(+) cells. PDGFRα(+) cells are directly innervated by inhibitory motor neurons and purinergic responses are conducted to SMCs via gap junctions. ABSTRACT Interstitial cells, known as platelet derived growth factor receptor α (PDGFRα(+) ) cells, are closely associated with varicosities of enteric motor neurons and suggested to mediate purinergic hyperpolarization responses in smooth muscles of the gastrointestinal tract (GI), but this concept has not been demonstrated directly in intact muscles. We used confocal microscopy to monitor Ca(2+) transients in neurons and post-junctional cells of the murine colon evoked by exogenous purines or electrical field stimulation (EFS) of enteric neurons. EFS (1-20 Hz) caused Ca(2+) transients in enteric motor nerve processes and then in PDGFRα(+) cells shortly after the onset of stimulation (latency from EFS was 280 ms at 10 Hz). Responses in smooth muscle cells (SMCs) were typically a small decrease in Ca(2+) fluorescence just after the initiation of Ca(2+) transients in PDGFRα(+) cells. Upon cessation of EFS, several fast Ca(2+) transients were noted in SMCs (rebound excitation). Strong correlation was noted in the temporal characteristics of Ca(2+) transients evoked in PDGFRα(+) cells by EFS and inhibitory junction potentials (IJPs) recorded with intracellular microelectrodes. Ca(2+) transients and IJPs elicited by EFS were blocked by MRS-2500, a P2Y1 antagonist, and absent in P2ry1((-/-)) mice. PDGFRα(+) cells expressed gap junction genes, and gap junction uncouplers, 18β-glycyrrhetinic acid (18β-GA) and octanol blocked Ca(2+) transients in SMCs but not in neurons or PDGFRα(+) cells. IJPs recorded from SMCs were also blocked. These findings demonstrate direct innervation of PDGFRα(+) cells by motor neurons. PDGFRα(+) cells are primary targets for purinergic neurotransmitter(s) in enteric inhibitory neurotransmission. Hyperpolarization responses are conducted to SMCs via gap junctions.
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Affiliation(s)
- Salah A Baker
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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27
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Ranson RN, Saffrey MJ. Neurogenic mechanisms in bladder and bowel ageing. Biogerontology 2015; 16:265-84. [PMID: 25666896 PMCID: PMC4361768 DOI: 10.1007/s10522-015-9554-3] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2014] [Accepted: 01/28/2015] [Indexed: 01/18/2023]
Abstract
The prevalence of both urinary and faecal incontinence, and also chronic constipation, increases with ageing and these conditions have a major impact on the quality of life of the elderly. Management of bladder and bowel dysfunction in the elderly is currently far from ideal and also carries a significant financial burden. Understanding how these changes occur is thus a major priority in biogerontology. The functions of the bladder and terminal bowel are regulated by complex neuronal networks. In particular neurons of the spinal cord and peripheral ganglia play a key role in regulating micturition and defaecation reflexes as well as promoting continence. In this review we discuss the evidence for ageing-induced neuronal dysfunction that might predispose to neurogenic forms of incontinence in the elderly.
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Affiliation(s)
- Richard N Ranson
- Department of Applied Sciences (Biomedical Sciences), Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST, UK,
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Effect of da-cheng-qi decoction on the repair of the injured enteric nerve-interstitial cells of cajal-smooth muscle cells network in multiple organ dysfunction syndrome. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2014; 2014:596723. [PMID: 25477993 PMCID: PMC4247919 DOI: 10.1155/2014/596723] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/27/2014] [Revised: 10/18/2014] [Accepted: 10/21/2014] [Indexed: 01/22/2023]
Abstract
Wistar rats were randomly divided into control group, multiple organ dysfunction syndrome (MODS) group, and Da-Cheng-Qi decoction (DCQD) group. The network of enteric nerves-interstitial cells of Cajal- (ICC-) smooth muscle cells (SMC) in small intestine was observed using confocal laser scanning microscopy and transmission electron microscopy. The results showed that the numbers of cholinergic/nitriergic nerves, and the deep muscular plexus of ICC (ICC-DMP) and connexin43 (Cx43) in small intestine with MODS were significantly decreased. The network integrity of enteric nerves-ICC-SMC was disrupted. The ultrastructures of ICC-DMP, enteric nerves, and SMC were severely damaged. After treatment with DCQD, the damages were repaired and the network integrity of enteric nerves ICC-SMC was significantly recovered. In conclusion, the pathogenesis of gastrointestinal motility dysfunction in MODS in part may be due to the damages to enteric nerves-ICC-SMC network and gap junctions. The therapeutic mechanism of DCQD in part may be that it could repair the damages and maintain the integrity of enteric nerves ICC-SMC network.
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Cobine CA, Sotherton AG, Peri LE, Sanders KM, Ward SM, Keef KD. Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells. Am J Physiol Gastrointest Liver Physiol 2014; 307:G1057-72. [PMID: 25301187 PMCID: PMC4254957 DOI: 10.1152/ajpgi.00331.2014] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα(+) cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrα(egfp/+), Kit(copGFP/+), and smMHC(Cre-egfp) mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα(+) cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα(+) cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα(+) cells. The functional role of cGKI was investigated in cGKI(-/-) mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI(-/-) mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI(+/+) and cGKI(-/-) mice although there was a small reduction in the cGKI(-/-) mouse. N(ω)-nitro-l-arginine (l-NNA) abolished responses during the first 20-30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI(-/-) mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway.
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Affiliation(s)
- C. A. Cobine
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - A. G. Sotherton
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - L. E. Peri
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - K. M. Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - S. M. Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - K. D. Keef
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
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Kito Y, Kurahashi M, Mitsui R, Ward SM, Sanders KM. Spontaneous transient hyperpolarizations in the rabbit small intestine. J Physiol 2014; 592:4733-45. [PMID: 25217377 DOI: 10.1113/jphysiol.2014.276337] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Four types of electrical activity were recorded and related to cell structure by intracellular recording and dye injection into impaled cells in muscles of rabbit small intestine. The specific cell types from which recordings were made were longitudinal smooth muscle cells (LSMCs), circular smooth muscle cells (CSMCs), interstitial cells of Cajal distributed in the myenteric region (ICC-MY) and fibroblast-like cells (FLCs). Slow waves (slow wavesSMC) were recorded from LSMCs and CSMCs. Slow waves (slow wavesICC) were of greatest amplitude (>50 mV) and highest maximum rate of rise (>10 V s(-1)) in ICC-MY. The dominant activity in FLCs was spontaneous transient hyperpolarizations (STHs), with maximum amplitudes above 30 mV. STHs were often superimposed upon small amplitude slow waves (slow wavesFLC). STHs displayed a cyclical pattern of discharge irrespective of background slow wave activity. STHs were inhibited by MRS2500 (3 μm), a P2Y1 antagonist, and abolished by apamin (0.3 μm), a blocker of small conductance Ca(2+)-activated K(+) channels. Small amplitude STHs (<15 mV) were detected in smooth muscle layers, whereas STHs were not resolved in cells identified as ICC-MY. Electrical field stimulation evoked purinergic inhibitory junction potentials (IJPs) in CSMCs. Purinergic IJPs were not recorded from ICC-MY. These results suggest that FLCs may regulate smooth muscle excitability in the rabbit small intestine via generation of rhythmic apamin-sensitive STHs. Stimulation of P2Y1 receptors modulates the amplitudes of STHs. Our results also suggest that purinergic inhibitory motor neurons regulate the motility of the rabbit small intestine by causing IJPs in FLCs that conduct to CSMCs.
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Affiliation(s)
- Yoshihiko Kito
- Department of Pharmacology, Faculty of Medicine, Saga University, Nabeshima, Saga, 849-8501, Japan Department of Cell Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya, 467-8601, Japan
| | - Masaaki Kurahashi
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
| | - Retsu Mitsui
- Department of Cell Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya, 467-8601, Japan
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
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Jiménez M, Clavé P, Accarino A, Gallego D. Purinergic neuromuscular transmission in the gastrointestinal tract; functional basis for future clinical and pharmacological studies. Br J Pharmacol 2014; 171:4360-75. [PMID: 24910216 DOI: 10.1111/bph.12802] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2014] [Revised: 05/14/2014] [Accepted: 05/24/2014] [Indexed: 12/13/2022] Open
Abstract
Nerve-mediated relaxation is necessary for the correct accomplishment of gastrointestinal (GI) motility. In the GI tract, NO and a purine are probably released by the same inhibitory motor neuron as inhibitory co-transmitters. The P2Y1 receptor has been recently identified as the receptor responsible for purinergic smooth muscle hyperpolarization and relaxation in the human gut. This finding has been confirmed in P2Y1 -deficient mice where purinergic neurotransmission is absent and transit time impaired. However, the mechanisms responsible for nerve-mediated relaxation, including the identification of the purinergic neurotransmitter(s) itself, are still debatable. Possibly different mechanisms of nerve-mediated relaxation are present in the GI tract. Functional demonstration of purinergic neuromuscular transmission has not been correlated with structural studies. Labelling of purinergic neurons is still experimental and is not performed in routine pathology studies from human samples, even when possible neuromuscular impairment is suspected. Accordingly, the contribution of purinergic neurotransmission in neuromuscular diseases affecting GI motility is not known. In this review, we have focused on the physiological mechanisms responsible for nerve-mediated purinergic relaxation providing the functional basis for possible future clinical and pharmacological studies on GI motility targeting purine receptors.
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Affiliation(s)
- Marcel Jiménez
- Department of Cell Biology, Physiology and Immunology, Neurosciences Institute, Universitat Autònoma de Barcelona, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain
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Sanders KM, Ward SM, Koh SD. Interstitial cells: regulators of smooth muscle function. Physiol Rev 2014; 94:859-907. [PMID: 24987007 DOI: 10.1152/physrev.00037.2013] [Citation(s) in RCA: 347] [Impact Index Per Article: 31.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.
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Affiliation(s)
- Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
| | - Sang Don Koh
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada
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Blair PJ, Rhee PL, Sanders KM, Ward SM. The significance of interstitial cells in neurogastroenterology. J Neurogastroenterol Motil 2014; 20:294-317. [PMID: 24948131 PMCID: PMC4102150 DOI: 10.5056/jnm14060] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/21/2014] [Revised: 06/06/2014] [Accepted: 06/07/2014] [Indexed: 12/21/2022] Open
Abstract
Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRα(+)) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα(+) cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.
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Affiliation(s)
- Peter J Blair
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA; and
| | - Poong-Lyul Rhee
- Division of Gastroenterology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA; and
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA; and
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Blair PJ, Rhee PL, Sanders KM, Ward SM. The significance of interstitial cells in neurogastroenterology. J Neurogastroenterol Motil 2014. [PMID: 24948131 DOI: 10.5056/jnm140] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRα(+)) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα(+) cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.
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Affiliation(s)
- Peter J Blair
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA
| | - Poong-Lyul Rhee
- Division of Gastroenterology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Kenton M Sanders
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA
| | - Sean M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA
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Lies B, Groneberg D, Friebe A. Toward a better understanding of gastrointestinal nitrergic neuromuscular transmission. Neurogastroenterol Motil 2014; 26:901-12. [PMID: 24827638 DOI: 10.1111/nmo.12367] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 04/21/2014] [Indexed: 12/30/2022]
Abstract
BACKGROUND Nitric oxide (NO) is an important inhibitory neurotransmitter in the gastrointestinal (GI) tract. The majority of nitrergic effects are transduced by NO-sensitive guanylyl cyclase (NO-GC) as the receptor for NO, and, thus, mediated by cGMP-dependent mechanisms. Work carried out during the past years has demonstrated NO to be largely involved in GI smooth muscle relaxation and motility. However, detailed investigation of nitrergic signaling has turned out to be complicated as NO-GC was identified in several different GI cell types such as smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. With regards to nitrergic neurotransmission, special focus has been placed on the role of interstitial cells of Cajal using mutant mice with reduced populations of ICC. Recently, global and cell-specific knockout mice for enzymes participating in nitrergic signaling have been generated providing a suitable approach to further examine the role of NO-mediated signaling in GI smooth muscle. PURPOSE This review discusses the current knowledge on nitrergic mechanisms in gastrointestinal neuromuscular transmission with a focus on genetic models and outlines possible further investigations to gain better understanding on NO-mediated effects in the GI tract.
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Affiliation(s)
- B Lies
- Physiologisches Institut I, Universität Würzburg, Würzburg, Germany
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Hall KA, Ward SM, Cobine CA, Keef KD. Spatial organization and coordination of slow waves in the mouse anorectum. J Physiol 2014; 592:3813-29. [PMID: 24951622 DOI: 10.1113/jphysiol.2014.272542] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
The internal anal sphincter (IAS) develops tone and is important for maintaining a high anal pressure while tone in the rectum is less. The mechanisms responsible for tone generation in the IAS are still uncertain. The present study addressed this question by comparing the electrical properties and morphology of the mouse IAS and distal rectum. The amplitude of tone and the frequency of phasic contractions was greater in the IAS than in rectum while membrane potential (Em) was less negative in the IAS than in rectum. Slow waves (SWs) were of greatest amplitude and frequency at the distal end of the IAS, declining in the oral direction. Dual microelectrode recordings revealed that SWs were coordinated over a much greater distance in the circumferential direction than in the oral direction. The circular muscle layer of the IAS was divided into five to eight 'minibundles' separated by connective tissue septa whereas few septa were present in the rectum. The limited coordination of SWs in the oral direction suggests that the activity in adjacent minibundles is not coordinated. Intramuscular interstitial cells of Cajal and platelet-derived growth factor receptor alpha-positive cells were present in each minibundle suggesting a role for one or both of these cells in SW generation. In summary, three important properties distinguish the IAS from the distal rectum: (1) a more depolarized Em; (2) larger and higher frequency SWs; and (3) the multiunit configuration of the muscle. All of these characteristics may contribute to greater tone generation in the IAS than in the distal rectum.
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Affiliation(s)
- K A Hall
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
| | - S M Ward
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
| | - C A Cobine
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
| | - K D Keef
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Vannucchi MG, Traini C, Manetti M, Ibba-Manneschi L, Faussone-Pellegrini MS. Telocytes express PDGFRα in the human gastrointestinal tract. J Cell Mol Med 2014; 17:1099-108. [PMID: 24151977 PMCID: PMC4118169 DOI: 10.1111/jcmm.12134] [Citation(s) in RCA: 147] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2013] [Accepted: 08/09/2013] [Indexed: 12/25/2022] Open
Abstract
Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34-positive/c-kit-negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c-kit-positive/CD34-negative/platelet-derived growth factor receptor α (PDGFRα)-negative interstitial cells of Cajal (ICC) and the PDGFRα-positive/c-kit-negative fibroblast-like cells (FLC). As TC display the same features and locations of the PDGFRα-positive cells, we investigated whether TC and PDGFRα-positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c-kit and CD34/c-kit double immunolabelling was performed in full-thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34-positive. TC formed a three-dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c-kit-positive and CD34/PDGFRα-negative. In conclusion, in the human GI tract the TC are PDGFRα-positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region-specific roles.
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Affiliation(s)
- Maria-Giuliana Vannucchi
- Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Florence, Italy
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Abstract
BACKGROUND Interstitial cells of Cajal, expressing the proto-oncogene c-kit, have been shown to regulate the spontaneous activity of the gastrointestinal tract. They have been described in the human internal anal sphincter; however, their function is still unclear. OBJECTIVE We examined the effects of the c-kit tyrosine kinase inhibitor imatinib mesylate on sphincter strips to investigate the function of the interstitial cells. DESIGN This was a case series study. SETTIGS This was a single-center study conducted at the University of Oxford. PATIENTS Internal anal sphincter strips were collected from 10 patients undergoing abdominoperineal resection or proctectomy and mounted in organ bath. Responses to electrical field stimulation and chemical agents were monitored in the absence of drugs and after the administration of increasing doses of imatinib mesylate. Immunohistochemistry was performed to identify interstitial cells. MAIN OUTCOME MEASURES The role of the interstitial cells in the internal anal sphincter was assessed. RESULTS Imatinib mesylate significantly reduced the tone and the spontaneous activity of the strips. In the absence of drugs, the tone generated was 147.7 ± 33.0 mg/mg of tissue. Administration of ≥5 μM of imatinib mesylate caused a dose-dependent reduction in the tone. Strips exhibited spontaneous activity characterized by intermittent low-amplitude contractions superimposed on basal tone (135.6 ± 4.6 contractions in 10 minutes). Imatinib mesylate significantly reduced the number of contractions at concentration >5 μM. No differences were observed in the responses to electrical field stimulation, carbachol, or phenylephrine. Immunohistochemistry showed c-kit-positive cells. LIMITATIONS This study was limited by the relatively small number of patients enrolled and thus the difficulty of finding human tissue for laboratory studies. CONCLUSIONS Our results suggest that the interstitial cells modulate the tone and the spontaneous activity of the internal anal sphincter. This provides a foundation for new approaches to preclinical and clinical research. Moreover, these cells may represent a target for drugs inhibiting the c-kit receptor and provide a new approach for treating anorectal diseases.
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Baker SA, Hennig GW, Salter AK, Kurahashi M, Ward SM, Sanders KM. Distribution and Ca(2+) signalling of fibroblast-like (PDGFR(+)) cells in the murine gastric fundus. J Physiol 2013; 591:6193-208. [PMID: 24144881 DOI: 10.1113/jphysiol.2013.264747] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Platelet-derived growth factor receptor α positive (PDGFRα(+)) cells are suggested to mediate purinergic inputs in GI muscles, but the responsiveness of these cells to purines in situ has not been evaluated. We developed techniques to label and visualize PDGFRα(+) cells in murine gastric fundus, load cells with Ca(2+) indicators, and follow their activity via digital imaging. Immunolabelling demonstrated a high density of PDGFRα(+) cells in the fundus. Cells were isolated and purified by fluorescence-activated cell sorting (FACS) using endogenous expression of enhanced green fluorescent protein (eGFP) driven off the Pdgfra promoter. Quantitative PCR showed high levels of expression of purinergic P2Y1 receptors and SK3 K(+) channels in PDGFRα(+) cells. Ca(2+) imaging was used to characterize spontaneous Ca(2+) transients and responses to purines in PDGFRα(+) cells in situ. ATP, ADP, UTP and β-NAD elicited robust Ca(2+) transients in PDGFRα(+) cells. Ca(2+) transients were also elicited by the P2Y1-specific agonist (N)-methanocarba-2MeSADP (MRS-2365), and inhibited by MRS-2500, a P2Y1-specific antagonist. Responses to ADP, MRS-2365 and β-NAD were absent in PDGFRα(+) cells from P2ry1((-/-)) mice, but responses to ATP were retained. Purine-evoked Ca(2+) transients were mediated through Ca(2+) release mechanisms. Inhibitors of phospholipase C (U-73122), IP3 (2-APB), ryanodine receptors (Ryanodine) and SERCA pump (cyclopiazonic acid and thapsigargin) abolished Ca(2+) transients elicited by purines. This study provides a link between purine binding to P2Y1 receptors and activation of SK3 channels in PDGFRα(+) cells. Activation of Ca(2+) release is likely to be the signalling mechanism in PDGFRα(+) cells responsible for the transduction of purinergic enteric inhibitory input in gastric fundus muscles.
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Affiliation(s)
- Salah A Baker
- K. M. Sanders: Department of Physiology and Cell Biology, University of Nevada School of Medicine, MS 352, Reno, NV 89557, USA.
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Bombardi C, Grandis A, Gardini A, Sorteni C, Clavenzani P, Chiocchetti R. Expression of β2 adrenoceptors within enteric neurons of the horse ileum. Res Vet Sci 2013; 95:837-45. [PMID: 23941962 DOI: 10.1016/j.rvsc.2013.07.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2013] [Revised: 05/10/2013] [Accepted: 07/08/2013] [Indexed: 12/31/2022]
Abstract
The activity of the gastrointestinal tract is regulated through the activation of adrenergic receptors (ARs). Since data concerning the distribution of ARs in the horse intestine is virtually absent, we investigated the distribution of β2-AR in the horse ileum using double-immunofluorescence. The β2-AR-immunoreactivity (IR) was observed in most (95%) neurons located in submucosal plexus (SMP) and in few (8%) neurons of the myenteric plexus (MP). Tyrosine hydroxylase (TH)-IR fibers were observed close to neurons expressing β2-AR-IR. Since β2-AR is virtually expressed in most neurons located in the horse SMP and in a lower percentage of neurons in the MP, it is reasonable to retain that this adrenergic receptor could regulate the activity of both secretomotor neurons and motor neurons innervating muscle layers and blood vessels. The high density of TH-IR fibers near β2-AR-IR enteric neurons indicates that the excitability of these cells could be directly modulated by the sympathetic system.
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Affiliation(s)
- Cristiano Bombardi
- Department of Veterinary Medical Science, University of Bologna, 40064 Ozzano dell'Emilia, Bologna, Italy.
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Wang C, Houghton MJ, Gamage PPKM, Collins HE, Patel BA, Yeoman MS, Ranson RN, Saffrey MJ. Changes in the innervation of the mouse internal anal sphincter during aging. Neurogastroenterol Motil 2013; 25:e469-77. [PMID: 23634828 DOI: 10.1111/nmo.12144] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2013] [Accepted: 03/28/2013] [Indexed: 12/20/2022]
Abstract
BACKGROUND The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.
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Affiliation(s)
- C Wang
- Department of Life, Health and Chemical Sciences, The Open University, Milton Keynes, UK
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Hieda K, Cho KH, Arakawa T, Fujimiya M, Murakami G, Matsubara A. Nerves in the intersphincteric space of the human anal canal with special reference to their continuation to the enteric nerve plexus of the rectum. Clin Anat 2013; 26:843-54. [PMID: 23512701 DOI: 10.1002/ca.22227] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2012] [Revised: 12/10/2012] [Accepted: 12/20/2012] [Indexed: 11/09/2022]
Abstract
In the intersphincteric space of the anal canal, nerves are thought to "change" from autonomic to somatic at the level of the squamous-columnar epithelial junction of the anal canal. To compare the nerve configuration in the intersphincteric space with the configuration in adjacent areas of the human rectum, we immunohistochemically assessed tissue samples from 12 donated cadavers, using antibodies to S100, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH). Antibody to S100 revealed a clear difference in intramuscular nerve distribution patterns between the circular and longitudinal muscle layers of the most inferior part of the rectum, with the former having a plexus-like configuration, while the latter contained short, longitudinally running nerves. Most of the intramural ganglion cells in the anal canal were restricted to above the epithelial junction, but some were located just below that level. Near or at the level of the epithelial junction, the nerves along the rectal adventitia and Auerbach's nerve plexus joined to form intersphincteric nerves, with all these nerves containing both nNOS-positive parasympathetic and TH-positive sympathetic nerve fibers. Thus, it was histologically difficult to distinguish somatic intersphincteric nerves from the autonomic Auerbach's plexus. In the intersphincteric space, the autonomic nerve elements with intrapelvic courses seemed to "borrow" a nerve pathway in the peripheral branches of the pudendal nerve. Injury to the intersphincteric nerve during surgery may result in loss of innervation in the major part of the internal anal sphincter.
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Affiliation(s)
- Keisuke Hieda
- Department of Urology, Hiroshima University School of Medicine, Hiroshima, Japan
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Rumessen JJ, Vanderwinden JM, Hansen A, Horn T. Ultrastructure of Interstitial Cells in Subserosa of Human Colon. Cells Tissues Organs 2013; 197:322-32. [DOI: 10.1159/000346314] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/04/2012] [Indexed: 11/19/2022] Open
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Rusu MC, Didilescu AC, Stănescu R, Pop F, Mănoiu VM, Jianu AM, Vâlcu M. The mandibular ridge oral mucosa model of stromal influences on the endothelial tip cells: an immunohistochemical and TEM study. Anat Rec (Hoboken) 2012. [PMID: 23192856 DOI: 10.1002/ar.22630] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This study aimed to evaluate by immunohistochemistry and transmission electron microscopy (TEM) the morphological features of the oral mucosa endothelial tip cells (ETCs) and to determine the immune and ultrastructural patterns of the stromal nonimmune cells which could influence healing processes. Immune labeling was performed on bioptic samples obtained from six edentulous patients undergoing surgery for dental implants placement; three normal samples were collected from patients prior to the extraction of the third mandibular molar. The antibodies were tested for CD34, CD117(c-kit), platelet derived growth factor receptor-alpha (PDGFR-α), Mast Cell Tryptase, CD44, vimentin, CD45, CD105, alpha-smooth muscle actin, FGF2, Ki67. In light microscopy, while stromal cells (StrCs) of the reparatory and normal oral mucosa, with a fibroblastic appearance, were found positive for a CD34/CD44/CD45/CD105/PDGFR-α/vimentin immune phenotype, the CD117/c-kit labeling led to a positive stromal reaction only in the reparatory mucosa. In TEM, non-immune StrCs presenting particular ultrastructural features were identified as circulating fibrocytes (CFCs). Within the lamina propria CFCs were in close contact with ETCs. Long processes of the ETCs were moniliform, and hook-like collaterals were arising from the dilated segments, suggestive for a different stage migration. Maintenance and healing of oral mucosa are so supported by extensive processes of angiogenesis, guided by ETCs that, in turn, are influenced by the CFCs that populate the stromal compartment both in normal and reparatory states. Therefore, CFCs could be targeted by specific therapies, with pro- or anti-angiogenic purposes.
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Affiliation(s)
- Mugurel Constantin Rusu
- Division of Anatomy, Faculty of Dental Medicine, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania.
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Koh BH, Roy R, Hollywood MA, Thornbury KD, McHale NG, Sergeant GP, Hatton WJ, Ward SM, Sanders KM, Koh SD. Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells. J Cell Mol Med 2012; 16:691-700. [PMID: 22151424 PMCID: PMC3822840 DOI: 10.1111/j.1582-4934.2011.01506.x] [Citation(s) in RCA: 80] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit(+) interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet-derived growth factor receptor-α (PDGFR-α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR-α(+) cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet-derived growth factor receptor-α(+) cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.
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Affiliation(s)
- Byoung H Koh
- Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Grover M, Bernard CE, Pasricha PJ, Parkman HP, Abell TL, Nguyen LA, Snape W, Shen KR, Sarr M, Swain J, Kendrick M, Gibbons S, Ordog T, Farrugia G. Platelet-derived growth factor receptor α (PDGFRα)-expressing "fibroblast-like cells" in diabetic and idiopathic gastroparesis of humans. Neurogastroenterol Motil 2012; 24:844-52. [PMID: 22650155 PMCID: PMC3756591 DOI: 10.1111/j.1365-2982.2012.01944.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND Emerging evidence suggests that "fibroblast-like cells" (FLC) may play a role in the regulation of gastrointestinal (GI) motor function. FLC are ultrastructurally distinct from other interstitial cells, including interstitial cells of Cajal (ICC), and express small-conductance Ca(2+) -activated K(+) channels (SK3). In mice, platelet-derived growth factor receptor α (PDGFRα) antibody has also been shown to label FLC. The aims of this study were to determine the morphology and distribution of PDGFRα-immunoreactive (ir) FLC in human gastric muscle and to determine if FLC are altered in gastroparesis, where ICC are reduced. METHODS Full thickness gastric body biopsies from five healthy subjects, 10 diabetic, and 10 idiopathic gastroparesis patients were immunolabeled using SK3 and PDGFRα staining for FLC and Kit staining for ICC. Intramuscular FLC and ICC were quantified. KEY RESULTS Intramuscular PDGFRα-ir cells had slender cell bodies and long, thin processes and were more abundant in the longitudinal compared with the circular muscle. In the region of myenteric plexus, FLC had smaller, rounder cell bodies with 3-4 processes and formed networks, often around ganglia. All SK3-ir cell structures showed complete overlap with PDGFRα-ir. FLC were in close proximity to ICC, but their cell bodies did not overlap. No differences were seen in the distribution, morphology, or overall numbers of FLC in gastroparesis patients. CONCLUSIONS & INFERENCES In conclusion, PDGFRα identifies FLC in human gastric smooth muscle. FLC were not altered in distribution or overall numbers in gastroparesis. Additional studies are required to determine their role in human GI function.
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Affiliation(s)
| | | | | | | | | | | | - William Snape
- California Pacific Medical Center, San Francisco, CA
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Blair PJ, Bayguinov Y, Sanders KM, Ward SM. Relationship between enteric neurons and interstitial cells in the primate gastrointestinal tract. Neurogastroenterol Motil 2012; 24:e437-49. [PMID: 22805588 PMCID: PMC4854185 DOI: 10.1111/j.1365-2982.2012.01975.x] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Morphological studies have revealed a close anatomical relationship between enteric nerve terminals and intramuscular ICC (ICC-IM) which supports a role for ICC-IM as intermediaries in enteric motor neurotransmission. Recently, a second type of interstitial cell previously described as 'fibroblast-like' but can now be identified by platelet-derived growth factor receptor-α expression, has also been implicated in enteric neurotransmission in rodents. The present study was performed to determine if enteric nerve fibers form close anatomical relationships with ICC and PDGFRα(+) cells throughout the primate GI tract. METHODS Immunohistochemical experiments and confocal microscopy were performed to examine the relationship between excitatory and inhibitory motor neurons, ICC and PDGFRα(+) cells throughout the monkey GI tract. KEY RESULTS The pan neuronal marker. Protein gene product 9.5 (PGP9.5) was used to label all enteric neurons and substance-P (sub-P) and neuronal nitric oxide synthase (nNOS) to label excitatory and inhibitory neurons, respectively. Double labeling with Kit revealed that both classes of nerve fibers were closely apposed with ICC-IM in the stomach, small intestine and colon (taenia and inter-taenia regions), but not with ICC at the level of the myenteric plexus (ICC-MY). Varicose enteric nerve fibers were closely associated with ICC-IM for distances up to 250 μm. Both excitatory and inhibitory nerve fibers were also closely apposed to PDGFRα(+) cells throughout the primate GI tract. CONCLUSIONS & INFERENCES The close anatomical relationship between enteric nerve fibers and ICC-IM and PDGFRα(+) cells throughout the GI tract of the Cynomolgus monkey provides morphological evidence that these two classes of interstitial cells may provide a similar physiological function in primates as has been attributed in rodent animal models.
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Affiliation(s)
- P J Blair
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
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Blair PJ, Bayguinov Y, Sanders KM, Ward SM. Interstitial cells in the primate gastrointestinal tract. Cell Tissue Res 2012; 350:199-213. [PMID: 22864981 DOI: 10.1007/s00441-012-1468-7] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2012] [Accepted: 06/20/2012] [Indexed: 02/06/2023]
Abstract
Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine(+) mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.
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Affiliation(s)
- Peter J Blair
- Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, NV, 89557, USA
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Gallego D, Gil V, Martínez-Cutillas M, Mañé N, Martín MT, Jiménez M. Purinergic neuromuscular transmission is absent in the colon of P2Y(1) knocked out mice. J Physiol 2012; 590:1943-56. [PMID: 22371472 DOI: 10.1113/jphysiol.2011.224345] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Purinergic and nitrergic co-transmission is the dominant mechanism responsible for neural-mediated smooth muscle relaxation in the gastrointestinal tract. The aim of the present paper was to test whether or not P2Y(1) receptors are involved in purinergic neurotransmission using P2Y(1)(−/−) knock-out mice. Tension and microelectrode recordings were performed on colonic strips. In wild type (WT) animals, electrical field stimulation (EFS) caused an inhibitory junction potential (IJP) that consisted of a fast IJP (MRS2500 sensitive, 1 μm) followed by a sustained IJP (N(ω)-nitro-L-arginine (L-NNA) sensitive, 1 mm). The fast component of the IJP was absent in P2Y(1)(−/−) mice whereas the sustained IJP (L-NNA sensitive) was recorded. In WT animals, EFS-induced inhibition of spontaneous motility was blocked by the consecutive addition of L-NNA and MRS2500. In P2Y(1)(−/−) mice, EFS responses were completely blocked by L-NNA. In WT and P2Y(1)(−/−) animals, L-NNA induced a smooth muscle depolarization but ‘spontaneous' IJP (MRS2500 sensitive) could be recorded in WT but not in P2Y(1)(−/−) animals. Finally, in WT animals, 1 μm MRS2365 caused a smooth muscle hyperpolarization that was blocked by 1 μm MRS2500. In contrast, 1 μm MRS2365 did not modify smooth muscle resting membrane potential in P2Y(1)(−/−) mice. β-Nicotinamide adenine dinucleotide (β-NAD, 1 mm) partially mimicked the effect of MRS2365. We conclude that P2Y(1) receptors mediate purinergic neurotransmission in the gastrointestinal tract and β-NAD partially fulfils the criteria to participate in rodent purinergic neurotransmission. The P2Y(1)(−/−) mouse is a useful animal model to study the selective loss of purinergic neurotransmission.
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Affiliation(s)
- Diana Gallego
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) Instituto de Salud Carlos III, Barcelona, Spain
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Duffy AM, Cobine CA, Keef KD. Changes in neuromuscular transmission in the W/W(v) mouse internal anal sphincter. Neurogastroenterol Motil 2012; 24:e41-55. [PMID: 22074497 PMCID: PMC3245326 DOI: 10.1111/j.1365-2982.2011.01806.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Intramuscular interstitial cells of Cajal (ICC-IM) have been shown to participate in nitrergic neuromuscular transmission (NMT) in various regions of the gastrointestinal (GI) tract, but their role in the internal anal sphincter (IAS) is still uncertain. Contractile studies of the IAS in the W/W(v) mouse (a model in which ICC-IM numbers are markedly reduced) have reported that nitrergic NMT persists and that ICC-IM are not required. However, neither the changes in electrical events underlying NMT nor the contributions of other non-nitrergic neural pathways have been examined in this model. METHODS The role of ICC-IM in NMT was examined by recording the contractile and electrical events associated with electrical field stimulation (EFS) of motor neurons in the IAS of wildtype and W/W(v) mice. Nitrergic, purinergic, and cholinergic components were identified using inhibitors of these pathways. KEY RESULTS Under NANC conditions, purinergic and nitrergic pathways both contribute to EFS-induced inhibitory junction potentials (IJPs) and relaxation. Purinergic IJPs and relaxation were intact in the W/W(v) mouse IAS, whereas nitrergic IJPs were reduced by 50-60% while relaxation persisted. In the presence of L-NNA (NOS inhibitor) and MRS2500 (P2Y1 receptor antagonist), EFS gave rise to cholinergic depolarization and contractions that were abolished by atropine. Cholinergic depolarization was absent in the W/W(v) mouse IAS while contraction persisted. CONCLUSIONS & INFERENCES ICC-IM significantly contributes to the electrical events underlying nitrergic and cholinergic NMT, whereas contractile events persist in the absence of ICC-IM. The purinergic inhibitory neural pathway appears to be independent of ICC-IM.
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Affiliation(s)
| | | | - KD Keef
- Individual to whom correspondences should be addressed: Kathleen Keef, Ph.D., Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, , 1-775-784-4302
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