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1
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Yang W, Liu R, Xu F. Glial cell line-derived neurotrophic factor improves impaired colonic motility in experimental colitis mice through connexin 43. World J Gastroenterol 2025; 31:100069. [DOI: 10.3748/wjg.v31.i8.100069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Revised: 12/06/2024] [Accepted: 12/25/2024] [Indexed: 01/23/2025] Open
Abstract
BACKGROUND Colonic motility dysfunction is a common symptom of ulcerative colitis (UC), significantly affecting patients’ quality of life. Evidence suggests that glial cell line-derived neurotrophic factor (GDNF) plays a role in restoring colonic function.
AIM To investigate whether GDNF enhances aberrant colonic motility in mice with experimental colitis via connexin 43 (Cx43).
METHODS An experimental colitis model was induced in male C57BL/6 mice using dextran sodium sulfate (DSS). The measurement of colonic transit time was conducted, and colon tissues were evaluated through transmission electron microscopy and hematoxylin and eosin staining. The mice were treated with exogenous GDNF and Gap 19, a selective Cx43 inhibitor. The Cx43 and GDNF levels were detected via immunofluorescence, immunohistochemistry, and real-time polymerase chain reaction. The levels of inflammatory markers, including interleukin-1β, tumor necrosis factor-α, interleukin-6, and C-reactive protein, were quantified using enzyme-linked immunosorbent assay.
RESULTS Experimental colitis was successfully induced using DSS, and the findings exhibited that the colonic transit time was significantly delayed in colitis mice relative to the UC group (P < 0.01). GDNF treatment improved colonic transit time and alleviated intestinal inflammation in DSS-induced colitis mice (P < 0.05). In the UC + Gap19 + GDNF group, colitis symptoms, colonic transit time, and inflammatory marker levels remained comparable to those in the UC group, indicating that the therapeutic effects of GDNF in UC mice were blocked by Gap 19.
CONCLUSION GDNF improves colonic motility in mice with experimental colitis through a partially Cx43-mediated mechanism. GDNF holds promise as a therapeutic option for improving colonic motility in patients with colitis.
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Affiliation(s)
- Wei Yang
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
| | - Rui Liu
- Medical School, Xiangyang Vocational and Technical College, Xiangyang 441021, Hubei Province, China
| | - Feng Xu
- Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China
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2
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Chen H, He M, Cao J, Zhang Y, Zhou Y, Yu Q, Wang A, Xuan J, Li T. Acupuncture and moxibustion intervention in functional dyspepsia: Gastric and duodenal regulation. Heliyon 2024; 10:e35696. [PMID: 39263151 PMCID: PMC11386019 DOI: 10.1016/j.heliyon.2024.e35696] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Revised: 07/31/2024] [Accepted: 08/01/2024] [Indexed: 09/13/2024] Open
Abstract
Functional dyspepsia (FD) is a brain-gut interaction disorder located in the stomach and duodenum, which has complex pathophysiological mechanisms, and there is no effective treatment for FD. Acupuncture and moxibustion have been proven to have definite and significant efficacy on FD. Focusing on the affected area and combined with the potential pathophysiology of FD, here we discuss the possible mechanisms of acupuncture and moxibustion in treating FD to guide future clinical and experimental research. We argue that the pathological causes of FD can be roughly divided into gastrointestinal dysfunction, duodenal low-grade inflammation, visceral hypersensitivity, and duodenal intestinal barrier and microbial imbalance. Correspondingly, the possible mechanisms of acupuncture and moxibustion in treating FD are elucidated from the perspective of how they improve gastric accommodation, regulate gastrointestinal motility, reduce gastric visceral sensitivity, regulate eosinophil-mast cell axis, inhibit low-grade inflammatory responses, and possibly regulate intestinal microbial homeostasis and duodenal barrier function through the microbiota-gut-brain axis. Although some evidence is still lacking, acupuncture remains a promising treatment for FD. In the future, it is necessary to conduct additional clinical and experimental research on acupuncture and moxibustion in treating FD to further explore their effects and mechanisms.
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Affiliation(s)
- Hongxiu Chen
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Min He
- Northeast Asian Institute of Traditional Chinese Medicine, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Jiazhen Cao
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Yifan Zhang
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Ying Zhou
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Qianhui Yu
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Anjie Wang
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
| | - Jing Xuan
- Affiliated Hospital of Changchun University of Traditional Chinese Medicine, No.1478, Gongnong Rd, Chaoyang District, 130021, Changchun, PR China
| | - Tie Li
- College of Acupuncture and Tuina, Changchun University of Chinese Medicine, No. 1035, Boshuo Rd, Jingyue Economic Development District, 130117, Changchun, PR China
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3
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Pourjamal N, Shirkoohi R, Rohani E, Hashemi M. The Expression Analysis of MEST1 and GJA1 Genes in Gastric Cancer in Association with Clinicopathological Characteristics. Int J Hematol Oncol Stem Cell Res 2024; 18:83-91. [PMID: 38680714 PMCID: PMC11055422 DOI: 10.18502/ijhoscr.v18i1.14747] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Accepted: 11/06/2023] [Indexed: 05/01/2024] Open
Abstract
Background: Gastric cancer is an invasive cancer, which is usually diagnosed in advanced stages. However, the markers affecting its progression, and invasion are of great importance in its diagnosis and treatment. The current research aimed to study the correlation of genes that contributed to epithelial-mesenchymal transition (EMT), Mest1, and GjA1, with some clinicopathological specifications in gastric cancer patients to better comprehend the functions of these genes in this tumor. Materials and Methods: RNA was extracted from the tumor, and normal tissues and cDNA were synthesized. Then, by designing specific primers for Gja1 and Mest1 genes, their expressions were studied by RT-PCR. The data was analyzed by GraphPad Prism 8 software. Results: Significant differences among the expressions of mentioned genes associated with clinicopathological variables of gastric cancer patients, including tumor size, grade, stage, metastasis, and lymphatic invasion were seen. Conclusion: The obtained data showed the important role of EMT-related genes, Gja1 and Mest1 in the clinical progression of the tumor. Further studies with larger sample sizes are required to confirm these genes as biomarker candidates for detecting gastric cancer.
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Affiliation(s)
- Nooshin Pourjamal
- Department of Genetics, Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran
| | - Reza Shirkoohi
- Cancer Biology Research Center, Cancer Research Institute, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran
| | - Elham Rohani
- Department of Genetics, Islamic Azad University, Tehran Medical Sciences Branch, Tehran, Iran
| | - Mehrdad Hashemi
- Farhikhtegan Medical Convergence Sciences Research Center, Farhikhtegan Hospital Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
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4
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Pavic B, Ogorevc M, Boric K, Vukovic D, Saraga-Babic M, Mardesic S. Connexin 37, 40, 43 and Pannexin 1 Expression in the Gastric Mucosa of Patients with Systemic Sclerosis. Biomedicines 2023; 11:2487. [PMID: 37760928 PMCID: PMC10525958 DOI: 10.3390/biomedicines11092487] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 08/30/2023] [Accepted: 09/06/2023] [Indexed: 09/29/2023] Open
Abstract
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Although its pathogenesis is not fully understood, connexins (Cxs) and pannexins (Panx) could be involved in the process of fibrosis. We analyzed the protein expression of Cx37, Cx40, Cx43, and Panx1 in the gastric mucosa of patients with SSc and healthy volunteers, using immunofluorescence staining. Protein levels of Cx37 were slightly increased, while the levels of Cx40 were significantly decreased in the lamina propria of the gastric mucosa of SSc patients compared to the controls. The changes were proportional to SSc severity, with the most prominent changes found in patients with severe diffuse cutaneous SSc. No differences in Cx43 or Panx1 levels were found between the analyzed groups of samples. The lack of changes in Cx43 expression, which has been previously associated with fibrosis, could be due to the weak expression of Cx43 in the gastric mucosa in general. Further studies on full-thickness gastric biopsies containing muscle layers and animal SSc models are needed to fully elucidate the role of Cxs and Panxs in SSc-associated fibrosis.
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Affiliation(s)
- Berna Pavic
- Renal Unit, University Hospital of Split, Šoltanska 1, 21000 Split, Croatia;
| | - Marin Ogorevc
- Department of Anatomy, Histology and Embryology, University of Split School of Medicine, Šoltanska 2, 21000 Split, Croatia; (M.O.); (M.S.-B.)
| | - Katarina Boric
- Department of Internal Medicine, University Hospital of Split, Šoltanska 1, 21000 Split, Croatia;
| | - Dubravka Vukovic
- Department of Dermatovenerology, University Hospital of Split, Šoltanska 1, 21000 Split, Croatia;
| | - Mirna Saraga-Babic
- Department of Anatomy, Histology and Embryology, University of Split School of Medicine, Šoltanska 2, 21000 Split, Croatia; (M.O.); (M.S.-B.)
| | - Snjezana Mardesic
- Department of Anatomy, Histology and Embryology, University of Split School of Medicine, Šoltanska 2, 21000 Split, Croatia; (M.O.); (M.S.-B.)
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5
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Mendes CE, Palombit K, Alves Pereira TT, Riceti Magalhães HI, Ferreira Caetano MA, Castelucci P. Effects of probenecid and brilliant blue G on rat enteric glial cells following intestinal ischemia and reperfusion. Acta Histochem 2023; 125:151985. [PMID: 36495673 DOI: 10.1016/j.acthis.2022.151985] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2022] [Accepted: 11/25/2022] [Indexed: 12/12/2022]
Abstract
The P2X7 receptor participates in several intracellular events and acts with the pannexin-1 channel. This study examined the effects of probenecid (PB) and brilliant blue G (BBG), which are antagonists of the pannexin-1 channel and P2X7 receptor, respectively, on rat ileum enteric glial cells after on ischemia and reperfusion. The ileal vessels were occluded for 45 min with nontraumatic vascular tweezers, and reperfusion was performed for periods of 24 h and 14 and 28 days. After ischemia (IR groups), the animals were treated with BBG (BG group) or PB (PB group). The double-labeling results demonstrated the following: the P2X7 receptor was present in enteric glial cells (S100β) and enteric neurons positive for HuC/D; enteric glial cells exhibited different phenotypes; some enteric glial cells were immunoreactive to only S100β or GFAP; and the pannexin-1 channel was present in enteric glial cells (GFAP). Density (in cells/cm2) analyses showed that the IR group exhibited a decrease in the number of cells immunoreactive for the P2X7 receptor, pannexin-1, and HuC/D and that treatment with BBG or PB resulted in the recovery of the numbers of these cells. The number of glial cells (S100β and GFAP) was higher in the IR group, and the treatments decreased the number of these cells to the normal value. However, the PB group did not exhibit recovery of S100β-positive glia. The cell profile area (μm2) of S100β-positive enteric glial cells decreased to the normal value after BBG treatment, whereas no recovery was observed in the PB group. The ileum contractile activity was decreased in the IR group and returned to baseline in the BG and PB groups. BBG and PB can effectively induce the recovery of neurons and glia cells and are thus potential therapeutic agents in the treatment of gastrointestinal tract diseases.
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Affiliation(s)
| | - Kelly Palombit
- Department of Morphology, University Federal of Piaui, Brazil
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6
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Lukowicz-Bedford RM, Farnsworth DR, Miller AC. Connexinplexity: the spatial and temporal expression of connexin genes during vertebrate organogenesis. G3 (BETHESDA, MD.) 2022; 12:jkac062. [PMID: 35325106 PMCID: PMC9073686 DOI: 10.1093/g3journal/jkac062] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Accepted: 02/24/2022] [Indexed: 11/28/2022]
Abstract
Animal development requires coordinated communication between cells. The Connexin family of proteins is a major contributor to intercellular communication in vertebrates by forming gap junction channels that facilitate the movement of ions, small molecules, and metabolites between cells. Additionally, individual hemichannels can provide a conduit to the extracellular space for paracrine and autocrine signaling. Connexin-mediated communication is widely used in epithelial, neural, and vascular development and homeostasis, and most tissues likely use this form of communication. In fact, Connexin disruptions are of major clinical significance contributing to disorders developing from all major germ layers. Despite the fact that Connexins serve as an essential mode of cellular communication, the temporal and cell-type-specific expression patterns of connexin genes remain unknown in vertebrates. A major challenge is the large and complex connexin gene family. To overcome this barrier, we determined the expression of all connexins in zebrafish using single-cell RNA-sequencing of entire animals across several stages of organogenesis. Our analysis of expression patterns has revealed that few connexins are broadly expressed, but rather, most are expressed in tissue- or cell-type-specific patterns. Additionally, most tissues possess a unique combinatorial signature of connexin expression with dynamic temporal changes across the organism, tissue, and cell. Our analysis has identified new patterns for well-known connexins and assigned spatial and temporal expression to genes with no-existing information. We provide a field guide relating zebrafish and human connexin genes as a critical step toward understanding how Connexins contribute to cellular communication and development throughout vertebrate organogenesis.
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Affiliation(s)
| | - Dylan R Farnsworth
- Institute of Neuroscience, Department of Biology, University of Oregon, Eugene, OR 97403, USA
| | - Adam C Miller
- Institute of Neuroscience, Department of Biology, University of Oregon, Eugene, OR 97403, USA
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7
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Sun Z, Xu C, Chen Y, Liu D, Wu P, Gao Q. Characterization of Pannexin1, Connexin32, and Connexin43 in Spotted Sea Bass ( Lateolabrax maculatus): They Are Important Neuro-Related Immune Response Genes Involved in Inflammation-Induced ATP Release. Front Immunol 2022; 13:870679. [PMID: 35514966 PMCID: PMC9062032 DOI: 10.3389/fimmu.2022.870679] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Accepted: 03/21/2022] [Indexed: 11/13/2022] Open
Abstract
Many immunological diseases can be treated by regulating neurobehavior, in which extracellular ATP is a vital member of endogenous danger-associated molecular pattern signaling molecule that plays a crucial part in innate neuro-related immunity. It is actively released through pannexin (Panx) and connexin (Cx) hemichannels from activated or stressed cells during inflammation, injury, or apoptosis. In addition to participating in ATP release, Panxs and Cxs also have crucial immune functions. In this study, pannexin1, three connexin32 isoforms and connexin43 were identified and characterized in spotted sea bass (Lateolabrax maculatus), which were named LmPanx1, LmCx32.2, LmCx32.3, LmCx32.7, and LmCx43. Their similar topological structures were discovered by sequence analysis: a relatively unconserved C-terminal region and four highly conserved transmembrane (TM) domains, and so on. Each extracellular (ECL) region of Panx1 has two conserved cysteine residues. Unlike Panx1, each ECL region of Cx32 and Cx43 contains three conserved cysteine residues, forming two conserved motifs: CX6CX3C motif in ECL1 and CX4CX5C motif in ECL2. Furthermore, Panx1 and Cx43 share similar genomic organization and synteny with their counterparts in selected vertebrates. Cx32 and CX43 were located in the same locus in fish, but diverged into two loci from amphibian. Moreover, despite varying expression levels, the identified genes were constitutively expressed in all examined tissues. All genes were upregulated by PAMP [lipopolysaccharide and poly(I:C)] stimulation or bacterial infection in vivo and in vitro, but they were downregulated in the brain at 6 or 12 h after stimulation. Especially, the three LmCx32 isoforms and LmCx43 were upregulated by ATP stimulation in primary head kidney leukocytes; however, downregulation of LmCx32.3 and LmCx43 expression were noted at 12 h. Conversely, ATP treatment inhibited the expression of LmPanx1. Importantly, we showed that the spotted sea bass Panx1, Cx43, and Cx32 were localized on the cellular membrane and involved in inflammation-induced ATP release. Taken together, our results demonstrated that Panx1, Cx32, and Cx43 are important neuro-related immune response genes involved in inflammation-induced ATP release.
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Affiliation(s)
- Zhaosheng Sun
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China
- International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China
- National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China
| | - Chong Xu
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China
- International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China
- National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China
| | - Yuxi Chen
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China
- International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China
- National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China
| | - Danjie Liu
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China
- International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China
- National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China
| | - Ping Wu
- College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Qian Gao
- Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China
- International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China
- National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China
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8
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Katturajan R, Evan Prince S. A role of connexin 43 on the drug-induced liver, kidney, and gastrointestinal tract toxicity with associated signaling pathways. Life Sci 2021; 280:119629. [PMID: 34004253 DOI: 10.1016/j.lfs.2021.119629] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Revised: 05/04/2021] [Accepted: 05/11/2021] [Indexed: 12/25/2022]
Abstract
Drug-induced organ toxicity/injury, especially in the liver, kidney, and gastrointestinal tract, is a systematic disorder that causes oxidative stress formation and inflammation resulting in cell death and organ failure. Current therapies target reactive oxygen species (ROS) scavenging and inhibit inflammatory factors in organ injury to restore the functions and temporary relief. Organ cell function and tissue homeostasis are maintained through gap junction intercellular communication, regulating connexin hemichannels. Mis-regulation of such connexin, especially connexin (Cx) 43, affects a comprehensive process, including cell differentiation, inflammation, and cell death. Aim to describe knowledge about the importance of connexin role and insights therapeutic targeting. Cx43 misregulation has been implicated in recent decades in various diseases. Moreover, in recent years there is increasing evidence that Cx43 is involved in the toxicity process, including hepatic, renal, and gastrointestinal disorders. Cx43 has the potential to initiate the immune system to cause cell death, which has been activated in the acceleration of apoptosis, necroptosis, and autophagy signaling pathway. So far, therapies targeting Cx43 have been under inspection and are subjected to clinical trial phases. This review elucidates the role of Cx43 in drug-induced vital organ injury, and recent reports compromise its function in the major signaling pathways.
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Affiliation(s)
- Ramkumar Katturajan
- Department of Biomedical Sciences, School of Biosciences and Technology, VIT, Vellore, Tamil Nadu, India.
| | - Sabina Evan Prince
- Department of Biomedical Sciences, School of Biosciences and Technology, VIT, Vellore, Tamil Nadu, India.
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9
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Ma Y, Yang X, Chatterjee V, Wu MH, Yuan SY. The Gut-Lung Axis in Systemic Inflammation. Role of Mesenteric Lymph as a Conduit. Am J Respir Cell Mol Biol 2021; 64:19-28. [PMID: 32877613 DOI: 10.1165/rcmb.2020-0196tr] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Emerging evidence shows that after injury or infection, the mesenteric lymph acts as a conduit for gut-derived toxic factors to enter the blood circulation, causing systemic inflammation and acute lung injury. Neither the cellular and molecular identity of lymph factors nor their mechanisms of action have been well understood and thus have become a timely topic of investigation. This review will first provide a summary of background knowledge on gut barrier and mesenteric lymphatics, followed by a discussion focusing on the current understanding of potential injurious factors in the lymph and their mechanistic contributions to lung injury. We also examine lymph factors with antiinflammatory properties as well as the bidirectional nature of the gut-lung axis in inflammation.
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Affiliation(s)
- Yonggang Ma
- Department of Molecular Pharmacology and Physiology, and
| | - Xiaoyuan Yang
- Department of Molecular Pharmacology and Physiology, and
| | | | - Mack H Wu
- Department of Surgery, University of South Florida Morsani College of Medicine, Tampa, Florida
| | - Sarah Y Yuan
- Department of Molecular Pharmacology and Physiology, and.,Department of Surgery, University of South Florida Morsani College of Medicine, Tampa, Florida
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10
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Yu HZ, Fu MH, Ji XP, E-Ni RG. Progress in research of gastrointestinal motility regulation. Shijie Huaren Xiaohua Zazhi 2020; 28:1183-1191. [DOI: 10.11569/wcjd.v28.i23.1183] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Gastrointestinal motility is an important part of the physiological function of the digestive tract, and its dysfunction is one of the key factors that cause different gastrointestinal motility disorders. These diseases seriously affect patients' normal life. With the development of scientific research and technology, well-designed research studies have been conducted on the regulatory mechanisms of gastrointestinal motility, which mainly include the regulation of gastrointestinal hormones, intestinal microflora, neurotransmitters, brain-gut peptides, interstitial cells of Cajal, and gastrointestinal electrical activities. In addition, current studies have proved that bitter taste receptors have certain regulatory effects on gastrointestinal motility. This paper primarily discusses the relevant pathways controlling gastrointestinal motility.
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Affiliation(s)
- Hong-Zhen Yu
- School of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China
| | - Ming-Hai Fu
- School of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China
| | - Xiao-Ping Ji
- School of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China
| | - Rong-Gui E-Ni
- School of Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia Autonomous Region, China
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11
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Li S, Wang N, Zhang T, Feng Y, Wang L, Sun J. Characterization of three connexin32 genes and their role in inflammation-induced ATP release in the Japanese flounder Paralichthys olivaceus. FISH & SHELLFISH IMMUNOLOGY 2020; 106:181-189. [PMID: 32768708 DOI: 10.1016/j.fsi.2020.07.066] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/05/2020] [Revised: 07/13/2020] [Accepted: 07/31/2020] [Indexed: 06/11/2023]
Abstract
Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish.
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Affiliation(s)
- Shuo Li
- Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China.
| | - Nan Wang
- Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China
| | - Tongtong Zhang
- Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China
| | - Yu Feng
- Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China
| | - Liyan Wang
- Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China
| | - Jinsheng Sun
- Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin, 300387, China.
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12
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Sun T, Li D, Hu S, Huang L, Sun H, Yang S, Wu B, Ji F, Zhou D. Aging-dependent decrease in the numbers of enteric neurons, interstitial cells of Cajal and expression of connexin43 in various regions of gastrointestinal tract. Aging (Albany NY) 2019; 10:3851-3865. [PMID: 30530917 PMCID: PMC6326649 DOI: 10.18632/aging.101677] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2018] [Accepted: 11/18/2018] [Indexed: 12/20/2022]
Abstract
Aging is a significant risk factor for gastrointestinal dysmotility, but aging-associated differences between different organs and the exact time to start degenerating have remained obscure. Here we evaluated alterations of interstitial cells of Cajal, enteric neurons and connexin43 expression in the stomach, jejunum and colon in 2-, 12-, 16-, 20- and 24-month-old mice, as well as in aged human colon. Interstitial cells of Cajal, cholinergic and nitrergic neurons within the whole digestive tract were reduced over time, but their loss first appeared in stomach, then in intestine, helping to understand that gastric function was first impaired during aging. The decrease of connexin43 expression occurred before interstitial cells of Cajal and neurons loss, suggesting that connexin43 might be the major target influenced during senescence. Furthermore, changes in expressions of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin-1β, interleukin-6) and apoptosis-related proteins (B-cell lymphoma-2, caspase-3) which indicated “inflammaging”, might contribute to the loss of enteric neurons and interstitial cells of Cajal in aged gastrointestinal tract. Our results provide possible therapeutic time window for beneficial intervention for geriatric patients with gastrointestinal motility disorders.
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Affiliation(s)
- Tingyi Sun
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.,Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing, 100069, China.,Cancer Institute of Capital Medical University, Beijing, 100069, China
| | - Dandan Li
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China
| | - Shilong Hu
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China
| | - Li Huang
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China
| | - Haimei Sun
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.,Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing, 100069, China.,Cancer Institute of Capital Medical University, Beijing, 100069, China
| | - Shu Yang
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.,Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing, 100069, China.,Cancer Institute of Capital Medical University, Beijing, 100069, China
| | - Bo Wu
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.,Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing, 100069, China.,Cancer Institute of Capital Medical University, Beijing, 100069, China
| | - Fengqing Ji
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.,Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing, 100069, China.,Cancer Institute of Capital Medical University, Beijing, 100069, China
| | - Deshan Zhou
- Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China.,Beijing Key Laboratory of Cancer Invasion and Metastasis Research, Beijing, 100069, China.,Cancer Institute of Capital Medical University, Beijing, 100069, China
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13
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Yang L, Dong C, Tian L, Ji X, Yang L, Li L. Gadolinium Chloride Restores the Function of the Gap Junctional Intercellular Communication between Hepatocytes in a Liver Injury. Int J Mol Sci 2019; 20:E3748. [PMID: 31370360 PMCID: PMC6695937 DOI: 10.3390/ijms20153748] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2019] [Revised: 07/24/2019] [Accepted: 07/30/2019] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Gadolinium chloride (GdCl3) has been reported to attenuate liver injury caused by a variety of toxicants. Gap junctional intercellular communication (GJIC) is thought to be essential in controlling liver homeostasis and pathology. Here we evaluate the effects of GdCl3 on functional GJIC and connexin expression in mouse models and primary hepatocytes. METHODS Mice were administered GdCl3 intraperitoneally the day before a carbon tetrachloride (CCl4) injection or bile duct ligation (BDL) operation. Primary hepatocytes were treated with CCl4 or lipopolysaccharides (LPS), with or without GdCl3. A scrape loading/dye transfer assay was performed to assess the GJIC function. The expression of connexins was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. RESULTS CCl4 treatment or the BDL operation led to the dysfunction of GJIC and a down-regulation of Cx32 and Cx26 in injured liver. GdCl3 administration restored GJIC function between hepatocytes by facilitating the transfer of fluorescent dye from one cell into adjacent cells via GJIC, and markedly prevented the decrease of Cx32 and Cx26 in injured liver. In primary hepatocytes, CCl4 or LPS treatment induced an obvious decline of Cx32 and Cx26, whereas GdCl3 pretreatment prevented the down-regulation of connexins. In vivo GdCl3 protected hepatocytes and attenuated the liver inflammation and fibrosis in liver injury mouse models. CONCLUSION GdCl3 administration protects functional GJIC between hepatocytes, and prevents the decrease of connexin proteins at mRNA and protein levels during liver injury, leading to the alleviation of chronic liver injury.
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Affiliation(s)
- Le Yang
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China
| | - Chengbin Dong
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China
| | - Lei Tian
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China
| | - Xiaofang Ji
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China
| | - Lin Yang
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China
| | - Liying Li
- Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China.
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14
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Zhao X, Yu C, Zheng M, Sun J. Prognostic value of the mRNA expression of gap junction α members in patients with gastric cancer. Oncol Lett 2019; 18:1669-1678. [PMID: 31423234 PMCID: PMC6614678 DOI: 10.3892/ol.2019.10516] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2018] [Accepted: 05/02/2019] [Indexed: 12/15/2022] Open
Abstract
Gastric cancer remains one of the primary causes of cancer-associated death worldwide. The gap junction α (GJA) family has been demonstrated to be involved in the cellular proliferation and metastasis of gastric cancer. However, the prognostic value of GJA in gastric cancer is yet to be elucidated. In the present study, the overall survival (OS) of patients with gastric cancer and the mRNA expression of GJA family members, including GJA1, GJA3, GJA4, GJA10 and GJA12, were analyzed using 593 patients with gastric cancer from the Kaplan-Meier plotter database. High GJA1 and GJA10 mRNA expression levels were associated with a poorer patient outcome (P=0.0066 and P=0.015, respectively), whereas high mRNA expression levels of GJA4 and GJA12 were associated with longer survival times (P=0.0056 and P=0.0054, respectively). Furthermore, the values of specific prognostic indicators of different subtypes of gastric cancer, including human epidermal growth factor receptor 2 status, Lauren differentiation and tumor stage, were also analyzed. The findings of the present study suggested a potential role for GJA family members in gastric cancer, which warrants further investigation.
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Affiliation(s)
- Xuan Zhao
- Department of General Surgery, Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China
| | - Chaoran Yu
- Department of General Surgery, Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China
| | - Minhua Zheng
- Department of General Surgery, Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China
| | - Jing Sun
- Department of General Surgery, Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R. China
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15
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Wong J, Chopra J, Chiang LLW, Liu T, Ho J, Wu WKK, Tse G, Wong SH. The Role of Connexins in Gastrointestinal Diseases. J Mol Biol 2019; 431:643-652. [PMID: 30639409 DOI: 10.1016/j.jmb.2019.01.007] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2018] [Revised: 10/03/2018] [Accepted: 01/04/2019] [Indexed: 12/13/2022]
Abstract
Gap junctions are hexagonal arrays of protein molecules in the plasma membrane and were first described in Mauthner cell synapses of goldfish. They form pathways for coupling between cells, allowing passive, electrotonic spread of ions and also passage of larger molecules such as amino acids and nucleotides. They are expressed in both excitable and non-excitable tissues. Each gap junction is made of two connexons, which are hexameric proteins of the connexin subunit. In this review, the roles that connexins play in gastrointestinal motility, the mechanisms of altered connexin expression leading to inflammatory bowel disease, gastrointestinal infections, and gastrointestinal symptoms in autistic spectrum disorder are discussed in detail.
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Affiliation(s)
- Jeremy Wong
- Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, PR China
| | - Jasmine Chopra
- Faculty of Arts and Science, University of Toronto, Toronto, Canada
| | | | - Tong Liu
- Tianjin Key Laboratory of Ionic-Molecular Function of Cardiovascular disease, Department of Cardiology, Tianjin Institute of Cardiology, Second Hospital of Tianjin Medical University, Tianjin 300211, PR China
| | - Jeffery Ho
- Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, PR China; Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China
| | - William K K Wu
- Department of Anaesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, PR China; Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China
| | - Gary Tse
- Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China; Li Ka Shing Institute of Health Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, PR China.
| | - Sunny Hei Wong
- Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong, PR China; Li Ka Shing Institute of Health Sciences, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, PR China.
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16
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Vieira C, Ferreirinha F, Magalhães-Cardoso MT, Silva I, Marques P, Correia-de-Sá P. Post-inflammatory Ileitis Induces Non-neuronal Purinergic Signaling Adjustments of Cholinergic Neurotransmission in the Myenteric Plexus. Front Pharmacol 2017; 8:811. [PMID: 29167643 PMCID: PMC5682326 DOI: 10.3389/fphar.2017.00811] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2017] [Accepted: 10/26/2017] [Indexed: 12/11/2022] Open
Abstract
Uncoupling between ATP overflow and extracellular adenosine formation changes purinergic signaling in post-inflammatory ileitis. Adenosine neuromodulation deficits were ascribed to feed-forward inhibition of ecto-5′-nucleotidase/CD73 by high extracellular adenine nucleotides in the inflamed ileum. Here, we hypothesized that inflammation-induced changes in cellular density may also account to unbalance the release of purines and their influence on [3H]acetylcholine release from longitudinal muscle-myenteric plexus preparations of the ileum of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-treated rats. The population of S100β-positive glial cells increase, whereas Ano-1-positive interstitial cells of Cajal (ICCs) diminished, in the ileum 7-days after the inflammatory insult. In the absence of changes in the density of VAChT-positive cholinergic nerves detected by immunofluorescence confocal microscopy, the inflamed myenteric plexus released smaller amounts of [3H]acetylcholine which also became less sensitive to neuronal blockade by tetrodotoxin (1 μM). Instead, [3H]acetylcholine release was attenuated by sodium fluoroacetate (5 mM), carbenoxolone (10 μM) and A438079 (3 μM), which prevent activation of glial cells, pannexin-1 hemichannels and P2X7 receptors, respectively. Sodium fluoroacetate also decreased ATP overflow without significantly affecting the extracellular adenosine levels, thus indicating that surplus ATP release parallels reactive gliosis in post-inflammatory ileitis. Conversely, loss of ICCs may explain the lower amounts of adenosine detected in TNBS-treated preparations, since blockade of Cav3 (T-type) channels existing in ICCs with mibefradil (3 μM) or inhibition of the equilibrative nucleoside transporter 1 with dipyridamole (0.5 μM), both decreased extracellular adenosine. Data indicate that post-inflammatory ileitis operates a shift on purinergic neuromodulation reflecting the upregulation of ATP-releasing enteric glial cells and the depletion of ICCs accounting for decreased adenosine overflow via equilibrative nucleoside transporters.
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Affiliation(s)
- Cátia Vieira
- Laboratório de Farmacologia e Neurobiologia, Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Fátima Ferreirinha
- Laboratório de Farmacologia e Neurobiologia, Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Maria T Magalhães-Cardoso
- Laboratório de Farmacologia e Neurobiologia, Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Isabel Silva
- Laboratório de Farmacologia e Neurobiologia, Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Patrícia Marques
- Laboratório de Farmacologia e Neurobiologia, Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Paulo Correia-de-Sá
- Laboratório de Farmacologia e Neurobiologia, Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal
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17
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Willebrords J, Cogliati B, Pereira IVA, da Silva TC, Crespo Yanguas S, Maes M, Govoni VM, Lima A, Felisbino DA, Decrock E, Nogueira MS, de Castro IA, Leclercq I, Leybaert L, Rodrigues RM, Vinken M. Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice. Sci Rep 2017; 7:8268. [PMID: 28811572 PMCID: PMC5557827 DOI: 10.1038/s41598-017-08583-w] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2017] [Accepted: 07/27/2017] [Indexed: 12/26/2022] Open
Abstract
While gap junctions mediate intercellular communication and support liver homeostasis, connexin hemichannels are preferentially opened by pathological stimuli, including inflammation and oxidative stress. The latter are essential features of non-alcoholic steatohepatitis. In this study, it was investigated whether connexin32 and connexin43 hemichannels play a role in non-alcoholic steatohepatitis. Mice were fed a choline-deficient high-fat diet or normal diet for 8 weeks. Thereafter, TAT-Gap24 or TAT-Gap19, specific inhibitors of hemichannels composed of connexin32 and connexin43, respectively, were administered for 2 weeks. Subsequently, histopathological examination was carried out and various indicators of inflammation, liver damage and oxidative stress were tested. In addition, whole transcriptome microarray analysis of liver tissue was performed. Channel specificity of TAT-Gap24 and TAT-Gap19 was examined in vitro by fluorescence recovery after photobleaching analysis and measurement of extracellular release of adenosine triphosphate. TAT-Gap24 and TAT-Gap19 were shown to be hemichannel-specific in cultured primary hepatocytes. Diet-fed animals treated with TAT-Gap24 or TAT-Gap19 displayed decreased amounts of liver lipids and inflammatory markers, and augmented levels of superoxide dismutase, which was supported by the microarray results. These findings show the involvement of connexin32 and connexin43 hemichannels in non-alcoholic steatohepatitis and, simultaneously, suggest a role as potential drug targets in non-alcoholic steatohepatitis.
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Affiliation(s)
- Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-270, São Paulo, Brazil
| | - Isabel Veloso Alves Pereira
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-270, São Paulo, Brazil
| | - Tereza Cristina da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-270, São Paulo, Brazil
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Veronica Mollica Govoni
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-270, São Paulo, Brazil
| | - Andressa Lima
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-270, São Paulo, Brazil
| | - Daniele Aparecida Felisbino
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, 05508-270, São Paulo, Brazil
| | - Elke Decrock
- Department of Basic Medical Sciences, Physiology Group, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium
| | - Marina Sayuri Nogueira
- Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580, 05508-270, São Paulo, Brazil
| | - Inar Alves de Castro
- Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Prof. Lineu Prestes 580, 05508-270, São Paulo, Brazil
| | - Isabelle Leclercq
- Laboratory of hepatogastroenterology, Institut de Recherche Expérimentale et clinique, Université catholique de Louvain, Avenue Mounier 53, 1200, Brussels, Belgium
| | - Luc Leybaert
- Department of Basic Medical Sciences, Physiology Group, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium
| | - Robim Marcelino Rodrigues
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
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18
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Maes M, Crespo Yanguas S, Willebrords J, Weemhoff JL, da Silva TC, Decrock E, Lebofsky M, Pereira IVA, Leybaert L, Farhood A, Jaeschke H, Cogliati B, Vinken M. Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice. Toxicol Lett 2017; 278:30-37. [PMID: 28687253 PMCID: PMC5800489 DOI: 10.1016/j.toxlet.2017.07.007] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Revised: 06/27/2017] [Accepted: 07/01/2017] [Indexed: 02/07/2023]
Abstract
Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure.
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Affiliation(s)
- Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
| | - Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
| | - James L Weemhoff
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States.
| | - Tereza Cristina da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
| | - Elke Decrock
- Department of Basic Medical Sciences, Physiology Group, Ghent University, Ghent, Belgium.
| | - Margitta Lebofsky
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States.
| | - Isabel Veloso Alves Pereira
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
| | - Luc Leybaert
- Department of Basic Medical Sciences, Physiology Group, Ghent University, Ghent, Belgium.
| | - Anwar Farhood
- Department of Pathology, St. David's North Austin Medical Center, Austin, United States.
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States.
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
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19
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Abstract
Being critical mediators of liver homeostasis, connexins and their channels are frequently involved in liver toxicity. In the current paper, specific attention is paid to actions of hepatotoxic drugs on these communicative structures. In a first part, an overview is provided on the structural, regulatory and functional properties of connexin-based channels in the liver. In the second part, documented effects of acetaminophen, hypolipidemic drugs, phenobarbital and methapyriline on connexin signaling are discussed. Furthermore, the relevance of this subject for the fields of clinical and in vitro toxicology is demonstrated. Relevance for patients: The role of connexin signaling in drug-induced hepatotoxicity may be of high clinical relevance, as it offers perspectives for the therapeutic treatment of such insults by interfering with connexin channel opening.
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Affiliation(s)
- Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
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20
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Willebrords J, Crespo Yanguas S, Maes M, Decrock E, Wang N, Leybaert L, Kwak BR, Green CR, Cogliati B, Vinken M. Connexins and their channels in inflammation. Crit Rev Biochem Mol Biol 2016; 51:413-439. [PMID: 27387655 PMCID: PMC5584657 DOI: 10.1080/10409238.2016.1204980] [Citation(s) in RCA: 90] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Inflammation may be caused by a variety of factors and is a hallmark of a plethora of acute and chronic diseases. The purpose of inflammation is to eliminate the initial cell injury trigger, to clear out dead cells from damaged tissue and to initiate tissue regeneration. Despite the wealth of knowledge regarding the involvement of cellular communication in inflammation, studies on the role of connexin-based channels in this process have only begun to emerge in the last few years. In this paper, a state-of-the-art overview of the effects of inflammation on connexin signaling is provided. Vice versa, the involvement of connexins and their channels in inflammation will be discussed by relying on studies that use a variety of experimental tools, such as genetically modified animals, small interfering RNA and connexin-based channel blockers. A better understanding of the importance of connexin signaling in inflammation may open up towards clinical perspectives.
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Affiliation(s)
- Joost Willebrords
- Department of In Vitro Toxicology and
Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels,
Belgium; Joost Willebrords: + Tel: 32 2 477 45 87, Michaël Maes: Tel: +32 2
477 45 87, Sara Crespo Yanguas: Tel: +32 2 477 45 87
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and
Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels,
Belgium; Joost Willebrords: + Tel: 32 2 477 45 87, Michaël Maes: Tel: +32 2
477 45 87, Sara Crespo Yanguas: Tel: +32 2 477 45 87
| | - Michaël Maes
- Department of In Vitro Toxicology and
Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels,
Belgium; Joost Willebrords: + Tel: 32 2 477 45 87, Michaël Maes: Tel: +32 2
477 45 87, Sara Crespo Yanguas: Tel: +32 2 477 45 87
| | - Elke Decrock
- Department of Basic Medical Sciences, Physiology Group, Ghent
University, De Pintelaan 185, 9000 Ghent, Belgium; Elke Decrock: Tel: +32 9 332 39
73, Nan Wang: Tel: +32 9 332 39 38, Luc Leybaert: Tel: +32 9 332 33 66
| | - Nan Wang
- Department of Basic Medical Sciences, Physiology Group, Ghent
University, De Pintelaan 185, 9000 Ghent, Belgium; Elke Decrock: Tel: +32 9 332 39
73, Nan Wang: Tel: +32 9 332 39 38, Luc Leybaert: Tel: +32 9 332 33 66
| | - Luc Leybaert
- Department of Basic Medical Sciences, Physiology Group, Ghent
University, De Pintelaan 185, 9000 Ghent, Belgium; Elke Decrock: Tel: +32 9 332 39
73, Nan Wang: Tel: +32 9 332 39 38, Luc Leybaert: Tel: +32 9 332 33 66
| | - Brenda R. Kwak
- Department of Pathology and Immunology and Division of Cardiology,
University of Geneva, Rue Michel-Servet 1, CH-1211 Geneva, Switzerland; Brenda R.
Kwak: Tel: +41 22 379 57 37
| | - Colin R. Green
- Department of Ophthalmology and New Zealand National Eye Centre,
University of Auckland, New Zealand; Colin R. Green: Tel: +64 9 923 61 35
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal
Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87,
05508-270 São Paulo, Brazil; Bruno Cogliati: Tel: +55 11 30 91 12 00
| | - Mathieu Vinken
- Department of In Vitro Toxicology and
Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels,
Belgium; Joost Willebrords: + Tel: 32 2 477 45 87, Michaël Maes: Tel: +32 2
477 45 87, Sara Crespo Yanguas: Tel: +32 2 477 45 87
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21
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Tse G, Lai ETH, Yeo JM, Tse V, Wong SH. Mechanisms of Electrical Activation and Conduction in the Gastrointestinal System: Lessons from Cardiac Electrophysiology. Front Physiol 2016; 7:182. [PMID: 27303305 PMCID: PMC4885840 DOI: 10.3389/fphys.2016.00182] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2016] [Accepted: 05/06/2016] [Indexed: 12/12/2022] Open
Abstract
The gastrointestinal (GI) tract is an electrically excitable organ system containing multiple cell types, which coordinate electrical activity propagating through this tract. Disruption in its normal electrophysiology is observed in a number of GI motility disorders. However, this is not well characterized and the field of GI electrophysiology is much less developed compared to the cardiac field. The aim of this article is to use the established knowledge of cardiac electrophysiology to shed light on the mechanisms of electrical activation and propagation along the GI tract, and how abnormalities in these processes lead to motility disorders and suggest better treatment options based on this improved understanding. In the first part of the article, the ionic contributions to the generation of GI slow wave and the cardiac action potential (AP) are reviewed. Propagation of these electrical signals can be described by the core conductor theory in both systems. However, specifically for the GI tract, the following unique properties are observed: changes in slow wave frequency along its length, periods of quiescence, synchronization in short distances and desynchronization over long distances. These are best described by a coupled oscillator theory. Other differences include the diminished role of gap junctions in mediating this conduction in the GI tract compared to the heart. The electrophysiology of conditions such as gastroesophageal reflux disease and gastroparesis, and functional problems such as irritable bowel syndrome are discussed in detail, with reference to ion channel abnormalities and potential therapeutic targets. A deeper understanding of the molecular basis and physiological mechanisms underlying GI motility disorders will enable the development of better diagnostic and therapeutic tools and the advancement of this field.
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Affiliation(s)
- Gary Tse
- Li Ka Shing Faculty of Medicine, School of Biomedical Sciences, University of Hong KongHong Kong, China
| | - Eric Tsz Him Lai
- Li Ka Shing Faculty of Medicine, School of Biomedical Sciences, University of Hong KongHong Kong, China
| | - Jie Ming Yeo
- School of Medicine, Imperial College LondonLondon, UK
| | - Vivian Tse
- Department of Physiology, McGill UniversityMontreal, QC, Canada
| | - Sunny Hei Wong
- Department of Medicine and Therapeutics, Institute of Digestive Disease, LKS Institute of Health Sciences, Chinese University of Hong KongHong Kong, China
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22
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Maes M, McGill MR, da Silva TC, Abels C, Lebofsky M, Maria Monteiro de Araújo C, Tiburcio T, Veloso Alves Pereira I, Willebrords J, Crespo Yanguas S, Farhood A, Beschin A, Van Ginderachter JA, Zaidan Dagli ML, Jaeschke H, Cogliati B, Vinken M. Involvement of connexin43 in acetaminophen-induced liver injury. Biochim Biophys Acta Mol Basis Dis 2016; 1862:1111-21. [PMID: 26912412 DOI: 10.1016/j.bbadis.2016.02.007] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Revised: 02/06/2016] [Accepted: 02/17/2016] [Indexed: 01/17/2023]
Abstract
BACKGROUND AND AIMS Being goalkeepers of liver homeostasis, gap junctions are also involved in hepatotoxicity. However, their role in this process is ambiguous, as gap junctions can act as both targets and effectors of liver toxicity. This particularly holds true for drug-induced liver insults. In the present study, the involvement of connexin26, connexin32 and connexin43, the building blocks of liver gap junctions, was investigated in acetaminophen-induced hepatotoxicity. METHODS C57BL/6 mice were overdosed with 300mg/kg body weight acetaminophen followed by analysis of the expression and localization of connexins as well as monitoring of hepatic gap junction functionality. Furthermore, acetaminophen-induced liver injury was compared between mice genetically deficient in connexin43 and wild type littermates. Evaluation of the toxicological response was based on a set of clinically relevant parameters, including protein adduct formation, measurement of alanine aminotransferase activity, cytokines and glutathione. RESULTS It was found that gap junction communication deteriorates upon acetaminophen intoxication in wild type mice, which is associated with a switch in mRNA and protein production from connexin32 and connexin26 to connexin43. The upregulation of connexin43 expression is due, at least in part, to de novo production by hepatocytes. Connexin43-deficient animals tended to show increased liver cell death, inflammation and oxidative stress in comparison with wild type counterparts. CONCLUSION These results suggest that hepatic connexin43-based signaling may protect against acetaminophen-induced liver toxicity.
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Affiliation(s)
- Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Mitchell R McGill
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States
| | - Tereza Cristina da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Chloé Abels
- Myeloid Cell Immunology Lab, VIB Inflammation Research Center, Ghent, Belgium; Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Margitta Lebofsky
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States
| | | | - Taynã Tiburcio
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Isabel Veloso Alves Pereira
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Anwar Farhood
- Department of Pathology, St. David's North Austin Medical Center, Austin, United States
| | - Alain Beschin
- Myeloid Cell Immunology Lab, VIB Inflammation Research Center, Ghent, Belgium; Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Jo A Van Ginderachter
- Myeloid Cell Immunology Lab, VIB Inflammation Research Center, Ghent, Belgium; Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Maria Lucia Zaidan Dagli
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
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23
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Puebla C, Cisterna BA, Salas DP, Delgado-López F, Lampe PD, Sáez JC. Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism. Biochim Biophys Acta Mol Cell Biol Lipids 2016; 1861:439-48. [PMID: 26869446 DOI: 10.1016/j.bbalip.2016.02.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2015] [Revised: 01/25/2016] [Accepted: 02/06/2016] [Indexed: 02/08/2023]
Abstract
Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.
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Affiliation(s)
- Carlos Puebla
- Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
| | - Bruno A Cisterna
- Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile
| | - Daniela P Salas
- Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Fernando Delgado-López
- Laboratorios de Biomedicina, Departamento de Ciencias Preclínicas, Facultad de Medicina, Universidad Católica del Maule, Talca, Chile
| | - Paul D Lampe
- Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States
| | - Juan C Sáez
- Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile; Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile.
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24
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Willebrords J, Maes M, Yanguas SC, Cogliati B, Vinken M. Detection of Connexins in Liver Cells Using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis and Immunoblot Analysis. Methods Mol Biol 2016; 1437:37-53. [PMID: 27207285 DOI: 10.1007/978-1-4939-3664-9_3] [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] [Indexed: 12/21/2022]
Abstract
Since connexin expression is partly regulated at the protein level, immunoblot analysis represents a frequently addressed technique in the connexin research field. The present chapter describes the setup of an immunoblot procedure, including protein extraction and quantification from biological samples, gel electrophoresis, protein transfer, and immunoblotting, which is optimized for analysis of connexins in liver tissue. In essence, proteins are separated on a polyacrylamide gel using sodium dodecyl sulfate followed by transfer of proteins on a nitrocellulose membrane. The latter allows specific detection of connexins with antibodies combined with revelation through enhanced chemiluminescence.
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Affiliation(s)
- Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium.
| | - Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium
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25
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Maes M, Willebrords J, Crespo Yanguas S, Cogliati B, Vinken M. Analysis of Liver Connexin Expression Using Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction. Methods Mol Biol 2016; 1437:1-19. [PMID: 27207283 DOI: 10.1007/978-1-4939-3664-9_1] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Although connexin production is mainly regulated at the protein level, altered connexin gene expression has been identified as the underlying mechanism of several pathologies. When studying the latter, appropriate methods to quantify connexin RNA levels are required. The present chapter describes a well-established reverse transcription quantitative real-time polymerase chain reaction procedure optimized for analysis of hepatic connexins. The method includes RNA extraction and subsequent quantification, generation of complementary DNA, quantitative real-time polymerase chain reaction, and data analysis.
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Affiliation(s)
- Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium.
| | - Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, SP, Brazil
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarkbeeklaan 103, 1090, Jette, Brussel, Belgium
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