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Manjarres Z, Calvo M, Pacheco R. Regulation of Pain Perception by Microbiota in Parkinson Disease. Pharmacol Rev 2023; 76:7-36. [PMID: 37863655 DOI: 10.1124/pharmrev.122.000674] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2022] [Revised: 10/03/2023] [Accepted: 10/10/2023] [Indexed: 10/22/2023] Open
Abstract
Pain perception involves current stimulation in peripheral nociceptive nerves and the subsequent stimulation of postsynaptic excitatory neurons in the spinal cord. Importantly, in chronic pain, the neural activity of both peripheral nociceptors and postsynaptic neurons in the central nervous system is influenced by several inflammatory mediators produced by the immune system. Growing evidence has indicated that the commensal microbiota plays an active role in regulating pain perception by either acting directly on nociceptors or indirectly through the modulation of the inflammatory activity on immune cells. This symbiotic relationship is mediated by soluble bacterial mediators or intrinsic structural components of bacteria that act on eukaryotic cells, including neurons, microglia, astrocytes, macrophages, T cells, enterochromaffin cells, and enteric glial cells. The molecular mechanisms involve bacterial molecules that act directly on neurons, affecting their excitability, or indirectly on non-neuronal cells, inducing changes in the production of proinflammatory or anti-inflammatory mediators. Importantly, Parkinson disease, a neurodegenerative and inflammatory disorder that affects mainly the dopaminergic neurons implicated in the control of voluntary movements, involves not only a motor decline but also nonmotor symptomatology, including chronic pain. Of note, several recent studies have shown that Parkinson disease involves a dysbiosis in the composition of the gut microbiota. In this review, we first summarize, integrate, and classify the molecular mechanisms implicated in the microbiota-mediated regulation of chronic pain. Second, we analyze the changes on the commensal microbiota associated to Parkinson disease and propose how these changes affect the development of chronic pain in this pathology. SIGNIFICANCE STATEMENT: The microbiota regulates chronic pain through the action of bacterial signals into two main locations: the peripheral nociceptors and the postsynaptic excitatory neurons in the spinal cord. The dysbiosis associated to Parkinson disease reveals increased representation of commensals that potentially exacerbate chronic pain and reduced levels of bacteria with beneficial effects on pain. This review encourages further research to better understand the signals involved in bacteria-bacteria and bacteria-host communication to get the clues for the development of probiotics with therapeutic potential.
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Affiliation(s)
- Zulmary Manjarres
- Laboratorio de Neuroinmunología, Centro Científico y Tecnológico de Excelencia Ciencia & Vida, Fundación Ciencia & Vida, Santiago, Chile (Z.M., R.P.); Facultad de Ciencias Biológicas (Z.M., M.C.) and División de Anestesiología, Escuela de Medicina (M.C.), Pontificia Universidad Católica de Chile, Santiago, Chile; Millennium Nucleus for the Study of Pain, Santiago, Chile (Z.M., M.C.); and Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago, Chile (R.P.)
| | - Margarita Calvo
- Laboratorio de Neuroinmunología, Centro Científico y Tecnológico de Excelencia Ciencia & Vida, Fundación Ciencia & Vida, Santiago, Chile (Z.M., R.P.); Facultad de Ciencias Biológicas (Z.M., M.C.) and División de Anestesiología, Escuela de Medicina (M.C.), Pontificia Universidad Católica de Chile, Santiago, Chile; Millennium Nucleus for the Study of Pain, Santiago, Chile (Z.M., M.C.); and Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago, Chile (R.P.)
| | - Rodrigo Pacheco
- Laboratorio de Neuroinmunología, Centro Científico y Tecnológico de Excelencia Ciencia & Vida, Fundación Ciencia & Vida, Santiago, Chile (Z.M., R.P.); Facultad de Ciencias Biológicas (Z.M., M.C.) and División de Anestesiología, Escuela de Medicina (M.C.), Pontificia Universidad Católica de Chile, Santiago, Chile; Millennium Nucleus for the Study of Pain, Santiago, Chile (Z.M., M.C.); and Facultad de Medicina y Ciencia, Universidad San Sebastián, Santiago, Chile (R.P.)
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Seguella L, Gulbransen BD. Enteric glial biology, intercellular signalling and roles in gastrointestinal disease. Nat Rev Gastroenterol Hepatol 2021; 18:571-587. [PMID: 33731961 PMCID: PMC8324524 DOI: 10.1038/s41575-021-00423-7] [Citation(s) in RCA: 179] [Impact Index Per Article: 44.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 01/28/2021] [Indexed: 02/07/2023]
Abstract
One of the most transformative developments in neurogastroenterology is the realization that many functions normally attributed to enteric neurons involve interactions with enteric glial cells: a large population of peripheral neuroglia associated with enteric neurons throughout the gastrointestinal tract. The notion that glial cells function solely as passive support cells has been refuted by compelling evidence that demonstrates that enteric glia are important homeostatic cells of the intestine. Active signalling mechanisms between enteric glia and neurons modulate gastrointestinal reflexes and, in certain circumstances, function to drive neuroinflammatory processes that lead to long-term dysfunction. Bidirectional communication between enteric glia and immune cells contributes to gastrointestinal immune homeostasis, and crosstalk between enteric glia and cancer stem cells regulates tumorigenesis. These neuromodulatory and immunomodulatory roles place enteric glia in a unique position to regulate diverse gastrointestinal disease processes. In this Review, we discuss current concepts regarding enteric glial development, heterogeneity and functional roles in gastrointestinal pathophysiology and pathophysiology, with a focus on interactions with neurons and immune cells. We also present a working model to differentiate glial states based on normal function and disease-induced dysfunctions.
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Affiliation(s)
- Luisa Seguella
- Department of Physiology and Pharmacology "V. Erspamer", Sapienza University of Rome, Rome, Italy
- Department of Physiology, Neuroscience Program, Michigan State University, East Lansing, MI, USA
| | - Brian D Gulbransen
- Department of Physiology, Neuroscience Program, Michigan State University, East Lansing, MI, USA.
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Grubišić V, McClain JL, Fried DE, Grants I, Rajasekhar P, Csizmadia E, Ajijola OA, Watson RE, Poole DP, Robson SC, Christofi FL, Gulbransen BD. Enteric Glia Modulate Macrophage Phenotype and Visceral Sensitivity following Inflammation. Cell Rep 2020; 32:108100. [PMID: 32905782 PMCID: PMC7518300 DOI: 10.1016/j.celrep.2020.108100] [Citation(s) in RCA: 118] [Impact Index Per Article: 23.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2019] [Revised: 07/02/2020] [Accepted: 08/11/2020] [Indexed: 12/20/2022] Open
Abstract
Mechanisms resulting in abdominal pain include altered neuro-immune interactions in the gastrointestinal tract, but the signaling processes that link immune activation with visceral hypersensitivity are unresolved. We hypothesized that enteric glia link the neural and immune systems of the gut and that communication between enteric glia and immune cells modulates the development of visceral hypersensitivity. To this end, we manipulated a major mechanism of glial intercellular communication that requires connexin-43 and assessed the effects on acute and chronic inflammation, visceral hypersensitivity, and immune responses. Deleting connexin-43 in glia protected against the development of visceral hypersensitivity following chronic colitis. Mechanistically, the protective effects of glial manipulation were mediated by disrupting the glial-mediated activation of macrophages through the macrophage colony-stimulating factor. Collectively, our data identified enteric glia as a critical link between gastrointestinal neural and immune systems that could be harnessed by therapies to ameliorate abdominal pain.
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Affiliation(s)
- Vladimir Grubišić
- Department of Physiology and Neuroscience Program, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA
| | - Jonathon L McClain
- Department of Physiology and Neuroscience Program, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA
| | - David E Fried
- Department of Physiology and Neuroscience Program, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA
| | - Iveta Grants
- Department of Anesthesiology, The Wexner Medical Center, The Ohio State University, 420 West 12th Avenue, Room 216, Columbus, OH 43210, USA
| | - Pradeep Rajasekhar
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Melbourne, VIC, Australia
| | - Eva Csizmadia
- Division of Gastroenterology, Department of Medicine and of Anesthesia, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA
| | - Olujimi A Ajijola
- Cardiac Arrhythmia Center, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA, USA
| | - Ralph E Watson
- Department of Medicine, Michigan State University, East Lansing, MI 48824, USA
| | - Daniel P Poole
- Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia; ARC Centre of Excellence in Convergent Bio-Nano Science & Technology, Melbourne, VIC, Australia
| | - Simon C Robson
- Division of Gastroenterology, Department of Medicine and of Anesthesia, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA
| | - Fievos L Christofi
- Department of Anesthesiology, The Wexner Medical Center, The Ohio State University, 420 West 12th Avenue, Room 216, Columbus, OH 43210, USA
| | - Brian D Gulbransen
- Department of Physiology and Neuroscience Program, Michigan State University, 567 Wilson Road, East Lansing, MI 48824, USA.
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Boesmans W, Hao MM, Fung C, Li Z, Van den Haute C, Tack J, Pachnis V, Vanden Berghe P. Structurally defined signaling in neuro-glia units in the enteric nervous system. Glia 2019; 67:1167-1178. [PMID: 30730592 PMCID: PMC6593736 DOI: 10.1002/glia.23596] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2018] [Revised: 01/11/2019] [Accepted: 01/11/2019] [Indexed: 12/21/2022]
Abstract
Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron-to-glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high-resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron-to-glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro-glia units as an operational cellular assembly in the enteric nervous system.
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Affiliation(s)
- Werend Boesmans
- Laboratory for Enteric NeuroScience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium.,Department of Pathology, GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands.,Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium
| | - Marlene M Hao
- Laboratory for Enteric NeuroScience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium.,Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, Australia
| | - Candice Fung
- Laboratory for Enteric NeuroScience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium
| | - Zhiling Li
- Laboratory for Enteric NeuroScience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium
| | - Chris Van den Haute
- Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, University of Leuven, Leuven, Belgium.,Leuven Viral Vector Core, University of Leuven, Leuven, Belgium
| | - Jan Tack
- Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium
| | - Vassilis Pachnis
- Development and Homeostasis of the Nervous System Laboratory, The Francis Crick Institute, London, United Kingdom
| | - Pieter Vanden Berghe
- Laboratory for Enteric NeuroScience (LENS), Translational Research Center for Gastrointestinal Disorders (TARGID), University of Leuven, Leuven, Belgium
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Morales-Soto W, Gulbransen BD. Enteric Glia: A New Player in Abdominal Pain. Cell Mol Gastroenterol Hepatol 2018; 7:433-445. [PMID: 30739868 PMCID: PMC6369218 DOI: 10.1016/j.jcmgh.2018.11.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Revised: 11/16/2018] [Accepted: 11/19/2018] [Indexed: 12/14/2022]
Abstract
Chronic abdominal pain is the most common gastrointestinal issue and contributes to the pathophysiology of functional bowel disorders and inflammatory bowel disease. Current theories suggest that neuronal plasticity and broad alterations along the brain-gut axis contribute to the development of chronic abdominal pain, but the specific mechanisms involved in chronic abdominal pain remain incompletely understood. Accumulating evidence implicates glial cells in the development and maintenance of chronic pain. Astrocytes and microglia in the central nervous system and satellite glia in dorsal root ganglia contribute to chronic pain states through reactive gliosis, the modification of glial networks, and the synthesis and release of neuromodulators. In addition, new data suggest that enteric glia, a unique type of peripheral glia found within the enteric nervous system, have the potential to modify visceral perception through interactions with neurons and immune cells. Understanding these emerging roles of enteric glia is important to fully understand the mechanisms that drive chronic pain and to identify novel therapeutic targets. In this review, we discuss enteric glial cell signaling mechanisms that have the potential to influence chronic abdominal pain.
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Affiliation(s)
| | - Brian D. Gulbransen
- Correspondence Address correspondence to: Brian D. Gulbransen, PhD, Neuroscience Program and Department of Physiology, Michigan State University, 567 Wilson Road, East Lansing, Michigan 48824. fax: (517) 355-5125.
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Delvalle NM, Fried DE, Rivera-Lopez G, Gaudette L, Gulbransen BD. Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility. Am J Physiol Gastrointest Liver Physiol 2018; 315:G473-G483. [PMID: 29927320 PMCID: PMC6230698 DOI: 10.1152/ajpgi.00155.2018] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.
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Affiliation(s)
| | - David E. Fried
- 2Department of Physiology, Michigan State University, East Lansing, Michigan
| | | | - Luke Gaudette
- 1Neuroscience Program, Michigan State University, East Lansing, Michigan
| | - Brian D. Gulbransen
- 1Neuroscience Program, Michigan State University, East Lansing, Michigan,2Department of Physiology, Michigan State University, East Lansing, Michigan
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Ha HS, Lee SE, Lee HS, Kim GH, Yoon CJ, Han JS, Lee JY, Sohn UD. The signaling of protease-activated receptor-2 activating peptide-induced contraction in cat esophageal smooth muscle cells. Arch Pharm Res 2017; 40:1443-1454. [PMID: 29098568 DOI: 10.1007/s12272-017-0975-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2017] [Accepted: 10/19/2017] [Indexed: 11/26/2022]
Abstract
Protease-activated receptors (PARs) are a family of G protein-coupled receptors with a unique activation mechanism involving proteolytic cleavage of the extracellular N-terminal domain of the receptor. PAR2 has a contractile effect on esophageal smooth muscle. We investigate the signaling pathways of the PAR2-activating peptide (PAR2-AP) induced contraction in cat esophageal smooth muscle cells. The length of freshly isolated smooth muscle cells and permeabilized cells from feline esophagus were measured by scanning micrometry, and by confirming molecular basis via western blot analysis. The responses to PAR2-AP were initial and sustained contractions, depending on time. The maximum contraction of the initial phase occurred at 60 s. The PAR2-AP-induced contraction was mediated by Gαi1, Gαi3, and Gαq protein activation, leading to phospholipase-c (PLC) and myosin light chain kinase (MLCK) activation. 20 kDa myosin light chain (MLC20) was phosphorylated by PAR2-AP. Rho kinase-2 (ROCK-2), an activator of 17 kDa C-kinase potentiated Protein phosphatase-1 Inhibitor (CPI-17), was increased by PAR2 receptor activation. In conclusion, PAR2-AP produced an initial contraction mediated by Gαi1, Gαi3, and Gαq protein activation, resulting in PLC and MLCK activation. The sustained contraction by PAR2-AP was mediated by the Rho/Rho kinase-dependent pathway.
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Affiliation(s)
- Hyun Su Ha
- College of Pharmacy, Chung-Ang University, Seoul, 156 -756, Republic of Korea
| | - Se Eun Lee
- College of Pharmacy, Chung-Ang University, Seoul, 156 -756, Republic of Korea
| | - Hyun Seok Lee
- College of Pharmacy, Chung-Ang University, Seoul, 156 -756, Republic of Korea
| | - Gil Hyung Kim
- College of Pharmacy, Chung-Ang University, Seoul, 156 -756, Republic of Korea
| | - Chan Jong Yoon
- College of Pharmacy, Chung-Ang University, Seoul, 156 -756, Republic of Korea
| | - Jong Soo Han
- College of Pharmacy, Chung-Ang University, Seoul, 156 -756, Republic of Korea
| | - Ji-Yun Lee
- Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul, 156-756, Republic of Korea.
| | - Uy Dong Sohn
- Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul, 156-756, Republic of Korea.
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Boesmans W, Martens MA, Weltens N, Hao MM, Tack J, Cirillo C, Vanden Berghe P. Imaging neuron-glia interactions in the enteric nervous system. Front Cell Neurosci 2013; 7:183. [PMID: 24155689 PMCID: PMC3801083 DOI: 10.3389/fncel.2013.00183] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2013] [Accepted: 10/01/2013] [Indexed: 12/13/2022] Open
Abstract
The enteric nervous system (ENS) is a network of neurons and glia within the wall of the gastrointestinal tract that is able to control many aspects of digestive function independently from the central nervous system. Enteric glial cells share several features with astrocytes and are closely associated with enteric neurons and their processes both within enteric ganglia, and along interconnecting fiber bundles. Similar to other parts of the nervous system, there is communication between enteric neurons and glia; enteric glial cells can detect neuronal activity and have the machinery to intermediate neurotransmission. However, due to the close contact between these two cell types and the particular characteristics of the gut wall, the recording of enteric glial cell activity in live imaging experiments, especially in the context of their interaction with neurons, is not straightforward. Most studies have used calcium imaging approaches to examine enteric glial cell activity but in many cases, it is difficult to distinguish whether observed transients arise from glial cells, or neuronal processes or varicosities in their vicinity. In this technical report, we describe a number of approaches to unravel the complex neuron-glia crosstalk in the ENS, focusing on the challenges and possibilities of live microscopic imaging in both animal models and human tissue samples.
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Affiliation(s)
- Werend Boesmans
- Laboratory for Enteric NeuroScience (LENS), Translational Research Center for GastroIntestinal Disorders, University of Leuven , Leuven, Belgium ; Translational Research Center for GastroIntestinal Disorders (TARGID), Department of Clinical and Experimental Medicine, University of Leuven , Leuven, Belgium
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Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2) in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA. PLoS One 2013; 8:e66743. [PMID: 23825105 PMCID: PMC3688948 DOI: 10.1371/journal.pone.0066743] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2013] [Accepted: 05/10/2013] [Indexed: 11/18/2022] Open
Abstract
We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser188.
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Effects of protease-activated receptors (PARs) on intracellular calcium dynamics of acinar cells in rat lacrimal glands. Histochem Cell Biol 2013; 140:463-76. [PMID: 23463389 DOI: 10.1007/s00418-013-1082-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/04/2013] [Indexed: 10/27/2022]
Abstract
Protease-activated receptors (PARs) represent a novel class of seven transmembrane domain G-protein coupled receptors, which are activated by proteolytic cleavage. PARs are present in a variety of cells and have been prominently implicated in the regulation of a number of vital functions. Here, lacrimal gland acinar cell responses to PAR activation were examined, with special reference to intracellular Ca(2+) concentration ([Ca(2+)]i) dynamics. In the present study, detection of acinar cell mRNA specific to known PAR subtypes was determined by reverse transcriptase polymerase chain reaction. Only PAR2 mRNA was detected in acinar cells of lacrimal glands. Both trypsin and a PAR2-activating peptide (PAR2-AP), SLIGRL-NH2, induced an increase in [Ca(2+)]i in acinar cells. The removal of extracellular Ca(2+) and the use of Ca(2+) channel blockers did not inhibit PAR2-AP-induced [Ca(2+)]i increases. Furthermore, U73122 and xestospongin C failed to inhibit PAR2-induced increases in [Ca(2+)]i. The origin of the calcium influx observed after activated PAR2-induced Ca(2+) release from intracellular Ca(2+) stores was also evaluated. The NO donor, GEA 3162, mimicked the effects of PAR2 in activating non-capacitative calcium entry (NCCE). However, both calyculin A (100 nM) and a low concentration of Gd(3+) (5 μM) did not completely block the PAR2-AP-induced increase in [Ca(2+)]i. These findings indicated that PAR2 activation resulted primarily in Ca(2+) mobilization from intracellular Ca(2+) stores and that PAR2-mediated [Ca(2+)]i changes were mainly independent of IP3. RT-PCR indicated that TRPC 1, 3 and 6, which play a role in CCE and NCCE, are expressed in acinar cells. We suggest that PAR2-AP differentially regulates both NCCE and CCE, predominantly NCCE. Finally, our results suggested that PAR2 may function as a key receptor in calcium-related cell homeostasis under pathophysiological conditions such as tissue injury or inflammation.
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Boesmans W, Cirillo C, Van den Abbeel V, Van den Haute C, Depoortere I, Tack J, Vanden Berghe P. Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells. Neurogastroenterol Motil 2013; 25:e151-60. [PMID: 23279281 DOI: 10.1111/nmo.12065] [Citation(s) in RCA: 60] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
BACKGROUND The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly. METHODS We studied the effect of acetylcholine (ACh), serotonin (5-HT), and adenosine triphosphate (ATP) on intracellular Ca(2+) signaling using aequorin-expressing and Fluo-4 AM-loaded CRL-2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c-Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT-PCR. KEY RESULTS Apart from ATP, also ACh and 5-HT induced a dose-dependent increase in intracellular Ca(2+) concentration in CRL-2690 cells. Similarly, these neurotransmitters also evoked Ca(2+) transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5-HT induced early gene expression in CRL-2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters. CONCLUSIONS & INFERENCES We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca(2+). On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.
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Affiliation(s)
- W Boesmans
- Laboratory of Enteric NeuroScience, KU Leuven, Leuven, Belgium
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Gulbransen BD, Sharkey KA. Novel functional roles for enteric glia in the gastrointestinal tract. Nat Rev Gastroenterol Hepatol 2012; 9:625-32. [PMID: 22890111 DOI: 10.1038/nrgastro.2012.138] [Citation(s) in RCA: 286] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Enteric glia are a unique class of peripheral glial cells within the gastrointestinal tract. Major populations of enteric glia are found in enteric ganglia in the myenteric and submucosal plexuses of the enteric nervous system (ENS); these cells are also found outside of the ENS, within the circular muscle and in the lamina propria of the mucosa. These different populations of cells probably represent unique classes of glial cells with differing functions. In the past few years, enteric glia have been found to be involved in almost every gut function including motility, mucosal secretion and host defence. Subepithelial glia seem to have a trophic and supporting relationship with intestinal epithelial cells, but the necessity of these roles in the maintenance of normal epithelial functions remains to be shown. Likewise, glia within enteric ganglia are activated by synaptic stimulation, suggesting an active role in synaptic transmission, but the precise role of glial activation in normal enteric network activity is unclear. Excitingly, enteric glia can also give rise to new neurons, but seemingly only under limited circumstances. In this Review, we discuss the current body of evidence supporting functional roles of enteric glia and identify key gaps in our understanding of the physiology of these unique cells.
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Affiliation(s)
- Brian D Gulbransen
- Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Drive North West Calgary, AB T2N 4N1, Canada
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Kugler EM, Mazzuoli G, Demir IE, Ceyhan GO, Zeller F, Schemann M. Activity of protease-activated receptors in primary cultured human myenteric neurons. Front Neurosci 2012; 6:133. [PMID: 22988431 PMCID: PMC3439632 DOI: 10.3389/fnins.2012.00133] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2012] [Accepted: 08/26/2012] [Indexed: 12/19/2022] Open
Abstract
Activity of the four known protease-activated receptors (PARs) has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (APs). Application of the PAR1-AP (TFLLR) or PAR4-AP (GYPGQV) evoked spike discharge in 79 or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs) of PAR1/PAR2 in 51%, PAR1/PAR4 in 43%, and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.
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Affiliation(s)
- Eva M Kugler
- Human Biology, Technische Universität München Freising, Germany
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Mueller K, Michel K, Krueger D, Demir IE, Ceyhan GO, Zeller F, Kreis ME, Schemann M. Activity of protease-activated receptors in the human submucous plexus. Gastroenterology 2011; 141:2088-2097.e1. [PMID: 21875497 DOI: 10.1053/j.gastro.2011.08.034] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2011] [Revised: 07/25/2011] [Accepted: 08/19/2011] [Indexed: 02/08/2023]
Abstract
BACKGROUND & AIMS Protease-activated receptors (PARs) are expressed in the enteric nervous system. Excessive release of proteases has been reported in functional and inflammatory bowel diseases. Studies in several animal models indicate the involvement of neural PARs. We studied the actions of different PAR-activating peptides (AP) in the human submucous plexus and performed comparative studies in guinea pig submucous neurons. METHODS We used voltage- and calcium-sensitive dye recordings to study the effects of PAR1-AP, PAR2-AP, PAR4-AP, the PAR1 activator thrombin, and the PAR2 activator tryptase on neurons and glia in human and guinea pig submucous plexus. Human preparations were derived from surgical resections. Levels of mucosal secretion evoked by PAR-APs were measured in Ussing chambers. RESULTS PAR1-AP and thrombin evoked a prominent spike discharge and intracellular Ca(2+) concentration ([Ca](i)) transients in most human submucous neurons and glia. PAR2-AP, tryptase, and PAR4-AP caused significantly weaker responses in a minor population. In contrast, PAR2-AP evoked much stronger responses in enteric neurons and glia of guinea pigs than did PAR1-AP or PAR4-AP. PAR1-AP, but not PAR2-AP or PAR4-AP, evoked a nerve-mediated secretion in human epithelium. The PAR1 antagonist SCH79797 inhibited the PAR1-AP, and thrombin evoked responses on neurons, glia, and epithelial secretion. In the submucous layer of human intestine, but not guinea pig intestine, PAR2-AP evoked [Ca](i) signals in CD68(+) macrophages. CONCLUSIONS In the human submucous plexus, PAR1, rather than PAR2 or PAR4, activates nerves and glia. These findings indicate that PAR1 should be the focus of future studies on neural PAR-mediated actions in the human intestine; PAR1 might be developed as a therapeutic target for gastrointestinal disorders associated with increased levels of proteases.
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Affiliation(s)
- Kerstin Mueller
- Human Biology, Technische Universität München, Freising, Germany
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WANG H, WU X, LI JY, CHAI BX, WANG J, MULHOLLAND MW, ZHANG W. Functional protease-activated receptors in the dorsal motor nucleus of the vagus. Neurogastroenterol Motil 2010; 22:431-8, e105. [PMID: 19719510 PMCID: PMC3052761 DOI: 10.1111/j.1365-2982.2009.01391.x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
BACKGROUND Protease-activated receptors (PARs), a family member of G-protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR-1 and PAR-2 in the dorsal motor nucleus of the vagus (DMV). METHODS DMNV neurons were isolated from neonatal rat brainstems using micro-dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca(2+)](i)) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining. KEY RESULT: Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca(2+)](i) expressed as DeltaF/F0 of 229 +/- 14% and 137 +/- 7%, respectively. Trypsin and PAR-2 agonist peptide activate PAR-2 with a maximum DeltaF/F0 change of 258 +/- 12% and 242 +/- 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 microm) decreased the maximal change in DeltaF/F0 induced by PAR-1 activation from 140 +/- 17% to 21 +/- 3%, while the PAR-2-mediated maximal change in DeltaF/F0 decreased from 185 +/- 21% to 19 +/- 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in DeltaF/F0 due to PAR-1 and PAR-2 activation by 72 +/- 13% and 71 +/- 20% respectively. PAR-1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR-1 and PAR-2 were detected in DMV tissues derived from IBD rats relative to control animals. CONCLUSIONS & INFERENCES Our results indicate that PAR-1 and PAR-2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3.
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Affiliation(s)
- H. WANG
- Department of Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China, Department of Surgery, University of Michigan, Ann Arbor, MI, USA
| | - X. WU
- Department of Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China, Department of Surgery, University of Michigan, Ann Arbor, MI, USA
| | - J.-Y. LI
- Department of Surgery, University of Michigan, Ann Arbor, MI, USA
| | - B.-X. CHAI
- Department of Surgery, University of Michigan, Ann Arbor, MI, USA
| | - J. WANG
- Department of Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - M. W. MULHOLLAND
- Department of Surgery, University of Michigan, Ann Arbor, MI, USA
| | - W. ZHANG
- Department of Surgery, University of Michigan, Ann Arbor, MI, USA
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Billich A, Urtz N, Reuschel R, Baumruker T. Sphingosine kinase 1 is essential for proteinase-activated receptor-1 signalling in epithelial and endothelial cells. Int J Biochem Cell Biol 2009; 41:1547-55. [PMID: 19162217 DOI: 10.1016/j.biocel.2009.01.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2008] [Revised: 12/24/2008] [Accepted: 01/05/2009] [Indexed: 01/07/2023]
Abstract
There is accumulating evidence that activation of sphingosine kinase 1 (SPHK1) is an important element in intracellular signalling cascades initiated by stimulation of multiple receptors, including certain growth factor, cytokine, and also G-protein coupled receptors. We here report that stimulation of the lung epithelial cell line A549 by thrombin leads to transient increase of SPHK1 activity and elevation of intracellular sphingosine-1-phosphate (S1P); abrogation of this stimulation by SPHK1-specific siRNA, pharmacological inhibition, or expression of a dominant-negative SPHK1 mutant blocks the response to thrombin, as measured by secretion of MCP-1, IL-6, IL-8, and PGE(2). Using selective stimulation of proteinase-activated receptors (PARs) a specific involvement of SPHK1 in the PAR-1 induced responses in A549 cell, including activation of NFkappaB, was evident, while PAR-2 and PAR-4 responses were independent of SPHK1. Moreover, PAR-1 or thrombin-induced cytokine production and adhesion factor expression of human umbilical vein endothelial cells was also seen to depend on SPHK1. Using dermal microvascular endothelial cells from SPHK1-deficient mice, we showed that absence of the enzyme abrogates MCP-1 production induced in these cells upon treatment with thrombin or PAR-1 activating peptide. We propose SPHK1 inhibition as a novel way to block PAR-1 mediated signalling, which could be useful in treatment of a number of diseases, in particular in atherosclerosis.
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Affiliation(s)
- Andreas Billich
- Novartis Institutes for BioMedical Research, Brunnerstrasse 59, Vienna, Austria.
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Bassotti G, Villanacci V, Fisogni S, Rossi E, Baronio P, Clerici C, Maurer CA, Cathomas G, Antonelli E. Enteric glial cells and their role in gastrointestinal motor abnormalities: introducing the neuro-gliopathies. World J Gastroenterol 2007; 13:4035-4041. [PMID: 17696219 PMCID: PMC4205302 DOI: 10.3748/wjg.v13.i30.4035] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/07/2007] [Revised: 05/03/2007] [Accepted: 05/12/2007] [Indexed: 02/06/2023] Open
Abstract
The role of enteric glial cells has somewhat changed from that of mere mechanical support elements, gluing together the various components of the enteric nervous system, to that of active participants in the complex interrelationships of the gut motor and inflammatory events. Due to their multiple functions, spanning from supporting elements in the myenteric plexuses to neurotransmitters, to neuronal homeostasis, to antigen presenting cells, this cell population has probably more intriguing abilities than previously thought. Recently, some evidence has been accumulating that shows how these cells may be involved in the pathophysiological aspects of some diseases. This review will deal with the properties of the enteric glial cells more strictly related to gastrointestinal motor function and the human pathological conditions in which these cells may play a role, suggesting the possibility of enteric neuro-gliopathies.
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Nasser Y, Keenan CM, Ma AC, McCafferty DM, Sharkey KA. Expression of a functional metabotropic glutamate receptor 5 on enteric glia is altered in states of inflammation. Glia 2007; 55:859-72. [PMID: 17405149 DOI: 10.1002/glia.20507] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The metabotropic glutamate receptor 5 (mGluR5) is expressed by astrocytes and its expression is modulated by inflammation. Enteric glia have many similarities to astrocytes and are the most numerous cell in the enteric nervous system (ENS). We investigated whether enteric glia express a functional mGluR5 and whether expression of this receptor was altered in colitis. In both enteric plexuses of the ileum and colon of guinea pigs and mice, we observed widespread glial mGluR5 expression. Incubation of isolated segments of the guinea pig ileum with the mGluR5 specific agonist RS-2-chloro-5-hydroxyphenylglycine (CHPG) caused a dose-dependent increase in the glial expression of c-Fos and the phosphorylated form of the extracellular signal-regulated kinase 1/2. Preincubation of tissues with the group I metabotropic glutamate receptor antagonist, S-4-carboxyphenylglycine, abolished the effects of CHPG. We examined mGluR5 expression in the guinea pig trinitrobenzene sulfonic acid and the IL-10 gene-deficient (IL-10(-/-)) mouse models of colitis. In guinea pigs, mGluR5 immunoreactivity became diffusely localized over the colonic myenteric ganglia, suggesting a change in receptor distribution. In contrast, glial mGluR5 expression was significantly reduced in the colonic myenteric plexus of IL-10(-/-) mice, as assessed with both real-time quantitative RT-PCR as well as immunohistochemistry and image analysis. These changes occurred without concomitant changes to enteric ganglia or glial fibrillary acidic protein expression in the IL-10(-/-) mouse. Our data suggest that enteric glia are a functional target of the glutamatergic neurotransmitter system in the ENS and that changes in mGluR5 expression may be of physiological significance during colitis.
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Affiliation(s)
- Yasmin Nasser
- Institute for Infection, Immunity and Inflammation, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada
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Lin T, Zhang W, Fan Y, Mulholland M. Interleukin-1β and Interleukin-6 Stimulate Matrix Metalloproteinase-9 Secretion in Cultured Myenteric Glia. J Surg Res 2007; 137:38-45. [PMID: 17109889 DOI: 10.1016/j.jss.2006.05.043] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2005] [Revised: 05/12/2006] [Accepted: 05/16/2006] [Indexed: 10/23/2022]
Abstract
Western blotting of culture media of myenteric glia stained positive for GFAP revealed increased secretion of matrix metalloproteinase-9 (MMP-9) in interleukin-1beta (IL-1beta) stimulated cells (10 ng/mL) versus control (142 +/- 19 versus 42 +/- 19, P < 0.05). Interleukin-6 (IL-6) stimulated cells also showed increased expression of MMP-9 (10 ng/mL) versus control (69 +/- 14 versus 4 +/- 2, P < 0.01). Control and cytokine-stimulated cells secreted MMP-2 constituitively. Gelatin zymography demonstrated that products were biologically active. Cytoplasmic staining for MMP-9 was detected in IL-1beta and IL-6-stimulated cells but was negligible in controls. Cultured myenteric glia are responsive to IL-1beta and IL-6 stimulation by secreting MMPs into the extracellular environment.
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Affiliation(s)
- Theodore Lin
- Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, USA
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20
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Grundy D, Al-Chaer ED, Aziz Q, Collins SM, Ke M, Taché Y, Wood JD. Fundamentals of neurogastroenterology: basic science. Gastroenterology 2006; 130:1391-411. [PMID: 16678554 DOI: 10.1053/j.gastro.2005.11.060] [Citation(s) in RCA: 180] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/17/2005] [Accepted: 11/03/2005] [Indexed: 02/06/2023]
Abstract
The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs.
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Affiliation(s)
- David Grundy
- Department of Biomedical Sciences, University of Sheffield, Sheffield, England
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21
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Abstract
The enteric nervous system is composed of both neurons and glia. Recent evidence indicates that enteric glia-which vastly outnumber enteric neurons-are actively involved in the control of gastrointestinal functions: they contain neurotransmitter precursors, have the machinery for uptake and degradation of neuroligands, and express neurotransmitter-receptors which makes them well suited as intermediaries in enteric neurotransmission and information processing in the ENS. Novel data further suggest that enteric glia have an important role in maintaining the integrity of the mucosal barrier of the gut. Finally, enteric glia may also serve as a link between the nervous and immune systems of the gut as indicated by their potential to synthesize cytokines, present antigen and respond to inflammatory insults. The role of enteric glia in human disease has not yet been systematically studied, but based on the available evidence it is predictable that enteric glia are involved in the etiopathogenesis of various pathological processes in the gut, particularly such with neuroinflammatory or neurodegenerative components.
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Affiliation(s)
- A Rühl
- Department of Human Biology, Technical University of Munich, Germany.
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22
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Park GH, Ryu JR, Shin CY, Choi MS, Han BH, Kim WK, Kim HC, Ko KH. Evidence that protease-activated receptor-2 mediates trypsin-induced reversal of stellation in cultured rat astrocytes. Neurosci Res 2005; 54:15-23. [PMID: 16256233 DOI: 10.1016/j.neures.2005.09.007] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2005] [Revised: 09/05/2005] [Accepted: 09/22/2005] [Indexed: 11/25/2022]
Abstract
Serine proteases such as thrombin and trypsin play a key role in the development and repair processes in the central nervous system. Molecular actions of serine proteases include multiple cellular events like activation of protease-activated receptors (PARs). PARs belong to a family of G protein-coupled receptors that can be stimulated through their proteolytic cleavage by ligands. PAR-2 has been implicated in neurodegenerative diseases including astrogliosis. Although recent studies have shown that low concentration of trypsin activates PAR-2, its role in morphological changes in primary astrocytes has not been studied. In the present study, we investigated the effects of PAR-2 in astrocyte stellation in rat primary astrocyte culture. Both trypsin (0.1-1 U/ml) and a PAR-2-activating peptide SLIGRL-NH2 (1-50 microM) significantly reversed the stellation induced by serum deprivation in rat astrocytes. Treatment of astrocytes with trypsin or SLIGRL-NH2 resulted in a transient rise of the intracellular Ca2+ level and trypsin-induced morphological changes were blocked by BAPTA, a Ca2+ chelator. In addition, a protein kinase C (PKC) inhibitor, bisindolylmaleimide significantly inhibited the trypsin-induced morphological changes, whereas activation of PKC by phorbol-12-myristate-13-acetate acted as trypsin. Taken together, these results suggest that activation of PAR-2 by trypsin caused reversal of stellation in cultured astrocytes, in part, via the mobilization of intracellular Ca2+ and activation of PKC.
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Affiliation(s)
- Gyu Hwan Park
- Department of Pharmacology, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, South Korea
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Fan Y, Zhang W, Mulholland M. Thrombin and PAR-1-AP increase proinflammatory cytokine expression in C6 cells. J Surg Res 2005; 129:196-201. [PMID: 16143343 DOI: 10.1016/j.jss.2005.07.041] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2005] [Revised: 07/01/2005] [Accepted: 07/29/2005] [Indexed: 11/24/2022]
Abstract
BACKGROUND In addition to a recognized role in the coagulation cascade, thrombin is known to have other functions via G protein-coupled receptors, including protease-activated receptor-1 (PAR-1). To investigate the relationship between PAR-1 activation and proinflammatory cytokine expression, we studied the responsiveness of C6 cells to thrombin and to the agonist PAP-1-activating peptide (PAR-1-AP). MATERIALS AND METHODS Cultured C6 rat glioma cells were stimulated with human alpha-thrombin or PAR-1-AP. To study mRNA expression changes, total RNA was isolated from the C6 cells, reverse transcribed, and amplified by real-time polymerase chain reaction. Three proinflammatory cytokines were studied: interleukin-6 (IL-6), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha). To measure cytokine release, cell-free supernatants were assayed using enzyme-linked immunosorbent assay (ELISA). RESULTS By quantitative real time reverse transcriptase polymerase chain reaction, thrombin (5 U/mL) exposure significantly increased mRNA expression of the proinflammatory cytokines: IL-6 (2.8 +/- 0.4, multiple of control), IL-1beta (4.8 +/- 1.6), and TNF-alpha (16.5 +/- 4.2). Effects on IL-6 mRNA expression were dose-dependent and matched by increments in IL-6 protein secretion. Effects of thrombin on IL-6 mRNA expression could be inhibited by hirudin. PAR-1-AP exposure also significantly increased mRNA expression of IL-6, IL-1beta and TNF-alpha. PAR-1 mRNA is expressed in C6 cells. CONCLUSION Both thrombin and its agonist, PAR-1-AP, significantly increased mRNA expression of pro-inflammatory cytokines in C6 glioma cells via PAR-1 activation.
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Affiliation(s)
- Yongyi Fan
- Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA
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Steinhoff M, Buddenkotte J, Shpacovitch V, Rattenholl A, Moormann C, Vergnolle N, Luger TA, Hollenberg MD. Proteinase-activated receptors: transducers of proteinase-mediated signaling in inflammation and immune response. Endocr Rev 2005; 26:1-43. [PMID: 15689571 DOI: 10.1210/er.2003-0025] [Citation(s) in RCA: 369] [Impact Index Per Article: 18.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Serine proteinases such as thrombin, mast cell tryptase, trypsin, or cathepsin G, for example, are highly active mediators with diverse biological activities. So far, proteinases have been considered to act primarily as degradative enzymes in the extracellular space. However, their biological actions in tissues and cells suggest important roles as a part of the body's hormonal communication system during inflammation and immune response. These effects can be attributed to the activation of a new subfamily of G protein-coupled receptors, termed proteinase-activated receptors (PARs). Four members of the PAR family have been cloned so far. Thus, certain proteinases act as signaling molecules that specifically regulate cells by activating PARs. After stimulation, PARs couple to various G proteins and activate signal transduction pathways resulting in the rapid transcription of genes that are involved in inflammation. For example, PARs are widely expressed by cells involved in immune responses and inflammation, regulate endothelial-leukocyte interactions, and modulate the secretion of inflammatory mediators or neuropeptides. Together, the PAR family necessitates a paradigm shift in thinking about hormone action, to include proteinases as key modulators of biological function. Novel compounds that can modulate PAR function may be potent candidates for the treatment of inflammatory or immune diseases.
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Affiliation(s)
- Martin Steinhoff
- Department of Dermatology and Boltzmann Institute for Immunobiology of the Skin, University of Münster, von-Esmarch-Strasse 58, 48149 Münster, Germany.
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Abstract
The enteric nervous system is composed of both enteric neurones and enteric glia. Enteric glial cells were first described by Dogiel and are now known to outnumber neurones approximately 4 : 1. In the past, these cells were assumed to subserve a largely supportive role; however, recent evidence indicates that enteric glial cells may play a more active role in the control of gut function. In transgenic mouse models, where enteric glial cells are selectively ablated, the loss of glia results in intestinal inflammation and disruption of the epithelial barrier. Enteric glia are activated specifically by inflammatory insults and may contribute actively to inflammatory pathology via antigen presentation and cytokine synthesis. Enteric glia also express receptors for neurotransmitters and so may serve as intermediaries in enteric neurotransmission. Thus, enteric glia may serve as a link between the nervous and immune systems of the gut and may also have an important role in maintaining the integrity of the mucosal barrier and in other aspects of intestinal homeostasis.
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Affiliation(s)
- A Rühl
- Department of Human Biology, Technical University Munich, Freising-Weihenstephan, Germany.
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Tjwa ETTL, Bradley JM, Keenan CM, Kroese ABA, Sharkey KA. Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon. Am J Physiol Gastrointest Liver Physiol 2003; 285:G1268-76. [PMID: 12881225 DOI: 10.1152/ajpgi.00073.2003] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.
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Affiliation(s)
- Eric T T L Tjwa
- Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada
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