Modelling Human Colonic Smooth Muscle Cell Electrophysiology.
Ontology highlight
ABSTRACT:
SUBMITTER: Yeoh JW
PROVIDER: S-EPMC6816779 | biostudies-literature | 2017 Apr
REPOSITORIES: biostudies-literature
Publications
Modelling Human Colonic Smooth Muscle Cell Electrophysiology.
Yeoh Jing Wui JW Corrias Alberto A Buist Martin L ML
Cellular and molecular bioengineering 20170206 2
The colon is a digestive organ that is subject to a wide range of motility disorders. However, our understanding of the etiology of these disorders is far from complete. In this study, a quantitative single cell model has been developed to describe the electrical behaviour of a human colonic smooth muscle cell (hCSMC). This model includes the pertinent ionic channels and intracellular calcium homoeostasis. These components are believed to contribute significantly to the electrical response of th ...[more]
Similar Datasets
Project description:Recently, a number of ion channel mutations have been identified in the smooth muscle cells of the human jejunum. Although these are potentially significant in understanding diseases that are currently of unknown etiology, no suitable computational cell model exists to evaluate the effects of such mutations. Here, therefore, a biophysically based single cell model of human jejunal smooth muscle electrophysiology is presented. The resulting cellular description is able to reproduce experimentally recorded slow wave activity and produces realistic responses to a number of perturbations, providing a solid platform on which the causes of intestinal myopathies can be investigated.
| S-EPMC3422293 | biostudies-literature
Project description:Mechanical stretch in obstruction induces expression of cyclooxygenase-2 (COX-2) in gut smooth muscle cells (SMCs). The stretch-induced COX-2 plays a critical role in motility dysfunction in obstructive bowel disorders (OBDs). The aims of the present study were to investigate the intracellular mechanism of mechanotranscription of COX-2 in colonic SMCs and to determine whether inhibition of mechanotranscription has therapeutic benefits in OBDs. Static stretch was mimicked in vitro in primary culture of rat colonic circular SMCs (RCCSMCs) and in colonic circular muscle strips. Partial obstruction was surgically induced with a silicon band in the distal colon of rats and COX-2-deficient mice. Static stretch of RCCSMCs significantly induced expression of COX-2 mRNA and protein and activated MAP kinases ERKs, p38, and JNKs. ERKs inhibitor PD98059, p38 inhibitor SB203580, and JNKs inhibitor SP600125 significantly blocked stretch-induced COX-2 expression. Pharmacological and molecular inhibition of stretch-activated ion channels (SACs) and integrins significantly suppressed stretch-induced expression of COX-2. SAC blockers inhibited stretch-activated ERKs, p38, and JNKs, but inhibition of integrins attenuated p38 activation only. In colonic circular muscle strips, stretch led to activation of MAPKs, induction of COX-2, and suppression of contractility. Inhibition of p38 with SB203580 blocked COX-2 expression and restored muscle contractility. Administration of SB203580 in vivo inhibited obstruction-induced COX-2 and improved motility function. Stretch-induced expression of COX-2 in RCCSMCs depends on mechanosensors, SACs, and integrins and an intracellular signaling mechanism involving MAPKs ERKs, p38, and JNKs. Inhibitors of the mechanotranscription pathway have therapeutic potentials for OBDs.
| S-EPMC3468553 | biostudies-literature
Project description:Background and purposeThe Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels.Experimental approachWe examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques.Key resultsThe Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTP?S-activated non-selective cation currents.Conclusions and implicationsThe Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus.
| S-EPMC3101624 | biostudies-literature
Project description:Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-?1. Molecular assays revealed higher level of ? smooth muscle actin (?-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the ?-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.
| S-EPMC6188351 | biostudies-literature
Project description:Chronic intestinal pseudo-obstruction (CIPO) is a rare but life-threatening disease characterized by severe intestinal dysmotility. Histopathologic studies in CIPO patients have identified several different mechanisms that appear to be involved in the dysmotility, including defects in neurons, smooth muscle, or interstitial cells of Cajal. Currently there are few mouse models of the various forms of CIPO. We generated a mouse with a point mutation in the RNA recognition motif of the Nup35 gene, which encodes a component of the nuclear pore complex. Nup35 mutants developed a severe megacolon and exhibited a reduced lifespan. Histopathologic examination revealed a degenerative myopathy that developed after birth and specifically affected smooth muscle in the colon; smooth muscle in the small bowel and the bladder were not affected. Furthermore, no defects were found in enteric neurons or interstitial cells of Cajal. Nup35 mice are likely to be a valuable model for the subtype of CIPO characterized by degenerative myopathy. Our study also raises the possibility that Nup35 polymorphisms could contribute to some cases of CIPO.
| S-EPMC5012464 | biostudies-literature
Project description:Vascular smooth muscle cells (SMC) play a critical role in controlling blood pressure and blood distribution, as well as maintaining the structural integrity of the blood vessel. SMC also participate in physiological and pathological vascular remodeling due to their remarkable ability to dynamically modulate their phenotype. During the past decade, the development of in vivo fate mapping systems for unbiased identification and tracking of SMC and their progeny has led to major discoveries as well as the reevaluation of well-established concepts about the contribution of vascular SMC in major vascular diseases including atherosclerosis. Lineage tracing studies revealed that SMC undergoes multiple phenotypic transitions characterized by the expression of markers of alternative cell types (eg, macrophage-like and mesenchymal-stem cell-like) and populate injured or diseased vessels by oligoclonal expansion of a limited number of medial SMC. With the development of high-throughput transcriptomics and single-cell RNA sequencing (scRNAseq), the field is moving forward towards in-depth SMC phenotypic characterization. Herein, we review the major observations put forth by lineage and clonality tracing studies and the evidence in support for SMC phenotypic diversity in healthy and diseased vascular tissue. We will also discuss the opportunities and remaining challenges of combining lineage tracing and single-cell transcriptomics technologies, as well as studying the functional relevance of SMC phenotypic transitions and identifying the mechanisms controlling them.
| S-EPMC6986347 | biostudies-literature
Project description:The contractility of colonic smooth muscle is dysregulated due to immune/inflammatory responses in inflammatory bowel diseases. Inflammation in vitro induces up-regulation of regulator of G-protein signaling 4 (RGS4) expression in colonic smooth muscle cells.To characterize the immune/inflammatory responses and RGS4 expression pattern in colonic smooth muscle after induction of colitis.Colitis was induced in rabbits by intrarectal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Innate/adaptive immune response RT-qPCR array was performed using colonic circular muscle strips. At 1-9 weeks after colonic intramuscular microinjection of lentivirus, the distal and proximal colons were collected, and muscle strips and dispersed muscle cells were prepared from circular muscle layer. Expression levels of RGS4 and NF?B signaling components were determined by Western blot analysis. The biological consequences of RGS4 knockdown were assessed by measurement of muscle contraction and phospholipase C (PLC)-? activity in response to acetylcholine (ACh).Contraction in response to ACh was significantly inhibited in the inflamed colonic circular smooth muscle cells. RGS4, IL-1, IL-6, IL-8, CCL3, CD1D, and ITGB2 were significantly up-regulated, while IL-18, CXCR4, CD86, and C3 were significantly down-regulated in the inflamed muscle strips. RGS4 protein expression in the inflamed smooth muscles was dramatically increased. RGS4 stable knockdown in vivo augmented ACh-stimulated PLC-? activity and contraction in colonic smooth muscle cells.Inflamed smooth muscle exhibits up-regulation of IL-1-related signaling components, Th1 cytokines and RGS4, and inhibition of contraction. Stable knockdown of endogenous RGS4 in colonic smooth muscle increases PLC-? activity and contractile responses.
| S-EPMC4920730 | biostudies-literature
Project description:Regulator of G protein signaling 4 (RGS4) regulates the strength and duration of G protein signaling and plays an important role in smooth muscle contraction, cardiac development, and psychiatric disorders. Little is known about the posttranscriptional regulation of RGS4 expression. We cloned the full-length cDNA of rabbit RGS4, which contains a long 3'-untranslated region (UTR) with several AU-rich elements (AREs). We determined whether RGS4 mRNA stability is mediated by the RNA-binding protein human antigen R (HuR) and contributes to IL-1β-induced upregulation of RGS4 expression. We show that IL-1β treatment in colonic smooth muscle cells doubled the half-life of RGS4 mRNA. Addition of RGS4 3'-UTR to the downstream of Renilla luciferase reporter induced dramatic reduction in the enzyme activity and mRNA expression of luciferase, which was attenuated by the site-directed mutation of the two 3'-most ARE sites. IL-1β increased luciferase mRNA stability in a UTR-dependent manner. Knockdown of HuR significantly aggravated UTR-mediated instability of luciferase and inhibited IL-1β-induced upregulation of RGS4 mRNA. In addition, IL-1β increased cytosolic translocation and RGS4 mRNA binding of HuR. These findings suggest that 3'-most ARE sites within RGS4 3'-UTR are essential for the instability of RGS4 mRNA and IL-1β promotes the stability of RGS4 mRNA through HuR.
| S-EPMC3006324 | biostudies-literature
Project description:BackgroundAltered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca(2+) signalling contributes to the impaired contractility of colon circular smooth muscles.MethodsAcute colitis was induced in C57Bl/6 mice with dextran sulphate sodium (DSS) in the drinking water for 5 days.Key resultsSpontaneous and acetylcholine-evoked contractions, caffeine-evoked hyperpolarization, and SERCA2 and phospholamban expression were reduced compared with controls. Tetrodotoxin did not restore control levels of contractile activity. The amplitudes, but not the frequency, of intracellular Ca(2+) waves were increased compared with controls. Caffeine abolished intracellular Ca(2+) waves in control smooth muscle cells, but not in smooth muscle cells from DSS-treated mice. CaM kinase II activity and cytosolic levels of HDAC4 were increased, and I kappaB alpha levels were decreased in distal colon smooth muscles from DSS-treated mice.Conclusions & inferencesThese results suggest that disruptions in intracellular Ca(2+) mobilization due to down-regulation of SERCA2 and phospholamban expression lead to increased CaM kinase II activity and cytosolic HDAC4 that may contribute to the dysmotility of colonic smooth muscles in colitis by enhancing NF-kappaB activity.
| S-EPMC2806503 | biostudies-literature
Project description:Patients with inflammatory bowel disease who are in remission and those who developed irritable bowel syndrome after enteric infection continue to have symptoms of diarrhea or constipation in the absence of overt inflammation, indicating motility dysfunction. We investigated whether oxidative stress during inflammation impairs integrity of the promoter of Cacna1c, which encodes the pore-forming ?1C subunit of Ca(v)1.2b calcium channels.We used long-extension polymerase chain reaction to evaluate DNA integrity in tissues from distal colons of rats; trinitrobenzene sulfonic acid was used to induce inflammation.The H2O2 increased in the muscularis externa 1-7 days after inflammation was induced with trinitrobenzene sulfonic acid. The oxidative stress significantly impaired DNA integrity in 2 specific segments of the Cacna1c promoter: -506 to -260 and -2193 to -1542. The impairment peaked at day 3 and recovered partially by day 7 after induction of inflammation; expression of the products of Cacna1c followed a similar time course. Oxidative stress suppressed the expression of nuclear factor-erythroid-2-related factor 2 (Nrf2), an important regulator of anti-oxidant proteins. Intraperitoneal administration of sulforaphane significantly reversed the suppression of Nrf2, oxidative damage in the promoter of Cacna1c, and suppression of Cacna1c on day 7 of inflammation. The inflammation subsided completely by 56 days after inflammation was induced; however, impairment of DNA integrity, expression of Nrf2 and Cacna1c, and smooth muscle reactivity to acetylcholine remained suppressed at this time point.Oxidative stress during inflammation impairs the integrity of the promoter of Cacna1c; impairment persists partially after inflammation has subsided. Reduced transcription of Cacna1c contributes to smooth muscle dysfunction in the absence of inflammation.
| S-EPMC3186840 | biostudies-literature