Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models

Cephalalgia ◽  
2021 ◽  
pp. 033310242110535
Author(s):  
Sarah L Christensen ◽  
Rikke H Rasmussen ◽  
Sanne La Cour ◽  
Charlotte Ernstsen ◽  
Thomas F Hansen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Mouse Models ◽  
Ex Vivo ◽  
Target Tissue ◽  
Loss Of Function ◽  
Dilatory Response ◽  
Channel Opening ◽  
Nociceptive Responses ◽  
Channel Expression ◽  
Mrna And Protein Expression

Background Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. Methods In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. Results Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. Conclusion Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

scholarly journals Binding of EBP50 to Nox organizing subunit p47phox is pivotal to cellular reactive species generation and altered vascular phenotype

2016 ◽  
Vol 113 (36) ◽  
pp. E5308-E5317 ◽  
Author(s):  
Imad Al Ghouleh ◽  
Daniel N. Meijles ◽  
Stephanie Mutchler ◽  
Qiangmin Zhang ◽  
Sanghamitra Sahoo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Proximity Ligation Assay ◽  
Loss Of Function ◽  
Pdz Domains ◽  
Resistance Arteries ◽  
Functional Studies ◽  
Proximity Ligation ◽  
Critical Requirement ◽  
Gain Loss

Despite numerous reports implicating NADPH oxidases (Nox) in the pathogenesis of many diseases, precise regulation of this family of professional reactive oxygen species (ROS) producers remains unclear. A unique member of this family, Nox1 oxidase, functions as either a canonical or hybrid system using Nox organizing subunit 1 (NoxO1) or p47phox, respectively, the latter of which is functional in vascular smooth muscle cells (VSMC). In this manuscript, we identify critical requirement of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50; aka NHERF1) for Nox1 activation and downstream responses. Superoxide (O2•−) production induced by angiotensin II (AngII) was absent in mouse EBP50 KO VSMC vs. WT. Moreover, ex vivo incubation of aortas with AngII showed a significant increase in O2•− in WT but not EBP50 or Nox1 nulls. Similarly, lipopolysaccharide (LPS)-induced oxidative stress was attenuated in femoral arteries from EBP50 KO vs. WT. In silico analyses confirmed by confocal microscopy, immunoprecipitation, proximity ligation assay, FRET, and gain-/loss-of-function mutagenesis revealed binding of EBP50, via its PDZ domains, to a specific motif in p47phox. Functional studies revealed AngII-induced hypertrophy was absent in EBP50 KOs, and in VSMC overexpressing EBP50, Nox1 gene silencing abolished VSMC hypertrophy. Finally, ex vivo measurement of lumen diameter in mouse resistance arteries exhibited attenuated AngII-induced vasoconstriction in EBP50 KO vs. WT. Taken together, our data identify EBP50 as a previously unidentified regulator of Nox1 and support that it promotes Nox1 activity by binding p47phox. This interaction is pivotal for agonist-induced smooth muscle ROS, hypertrophy, and vasoconstriction and has implications for ROS-mediated physiological and pathophysiological processes.

scholarly journals G6PD activity contributes to the regulation of histone acetylation and gene expression in smooth muscle cells and to the pathogenesis of vascular diseases

2021 ◽  
Author(s):  
Vidhi Dhagia ◽  
Atsushi Kitagawa ◽  
Christina Jacob ◽  
Connie Zheng ◽  
Angelo D'Alessandro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Serum Response Factor ◽  
Ex Vivo ◽  
Primary Cultures ◽  
Vascular Diseases ◽  
G6pd Activity ◽  
Metabolic Reprogramming ◽  
Stem Cell Marker ◽  
Loss Of Function

We aimed to determine: 1) the mechanism(s) that enable glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)‑driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs; and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PDS188F) in rats promoted transcription of SMC‑restricted genes. G6PD knockdown or inhibition decreased (48.5%) HDAC activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control SD rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PDS188F rats a high-fat diet for 4 months increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PDS188F rats. Our findings demonstrate G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS.

scholarly journals The Potential Involvement of an ATP-Dependent Potassium Channel-Opening Mechanism in the Smooth Muscle Relaxant Properties of Tamarix dioica Roxb.

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 722 ◽  
Author(s):  
Syeda Imtiaz ◽  
Ambreen Aleem ◽  
Fatima Saqib ◽  
Alexe Ormenisan ◽  
Andrea Elena Neculau ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Potassium Channel ◽  
Gallic Acid ◽  
Castor Oil ◽  
Katp Channel ◽  
Ex Vivo ◽  
Phytochemical Study ◽  
Channel Opening ◽  
Cardiovascular Systems

Background: Tamarix dioica is traditionally used to manage various disorders related to smooth muscle in the gastrointestinal, respiratory, and cardiovascular systems. This study was planned to establish a pharmacological basis for the uses of Tamarix dioica in certain medical conditions related to the digestive, respiratory, and cardiovascular systems, and to explore the underlying mechanisms. Methods: A phytochemical study was performed by preliminary methods, followed by HPLC-DAD and spectrometric methods. In vivo evaluation of a crude hydromethanolic extract of T. dioica (TdCr) was done with a castor-oil-provoked diarrheal model in rats to determine its antidiarrheal effect. Ex vivo experiments were done by using isolated tissues to determine the effects on smooth and cardiac muscles and explore the possible mechanisms. Results: TdCr tested positive for flavonoids, saponins, phenols, and tannins as methanolic solvable constituents in a preliminary study. The maximum quantity of gallic acid equivalent (GAE), phenolic, and quercetin equivalent (QE) flavonoid content found was 146 ± 0.001 μg GAE/mg extract and 36.17 ± 2.35 μg QE/mg extract. Quantification based on HPLC-DAD (reverse phase) exposed the presence of rutin at the highest concentration, followed by catechin, gallic acid, myricetin, kaempferol, and apigenin in TdCr. In vivo experiments showed the significant antidiarrheal effect of TdCr (100, 200, and 400 mg/kg) in the diarrheal (castor-oil-provoked) model. Ex vivo experiments revealed spasmolytic, bronchodilatory, and vasorelaxant activities as well as partial cardiac depressant activity, which may be potentiated by a potassium channel opener mechanism, similar to that of cromakalim. The potassium channel (KATP channel)-opening activity was further confirmed by repeating the experiments in glibenclamide-pretreated tissues. Conclusions: In vivo and ex vivo studies of T. dioica provided evidence of the antidiarrheal, spasmolytic, bronchodilator, vasorelaxant, and partial cardiodepressant properties facilitated through the opening of the KATP channel.

scholarly journals Reduced Biaxial Contractility in the Descending Thoracic Aorta of Fibulin-5 Deficient Mice

2016 ◽  
Vol 138 (5) ◽  
Author(s):  
S.-I. Murtada ◽  
J. Ferruzzi ◽  
H. Yanagisawa ◽  
J. D. Humphrey
Keyword(s):  
Smooth Muscle ◽  
Mouse Models ◽  
Thoracic Aorta ◽  
Ex Vivo ◽  
Aortic Aneurysms ◽  
Passive State ◽  
Elastic Fibers ◽  
Smooth Muscle Contractility ◽  
Cell Contractility

The precise role of smooth muscle cell contractility in elastic arteries remains unclear, but accumulating evidence suggests that smooth muscle dysfunction plays an important role in the development of thoracic aortic aneurysms and dissections (TAADs). Given the increasing availability of mouse models of these conditions, there is a special opportunity to study roles of contractility ex vivo in intact vessels subjected to different mechanical loads. In parallel, of course, there is a similar need to study smooth muscle contractility in models that do not predispose to TAADs, particularly in cases where disease might be expected. Multiple mouse models having compromised glycoproteins that normally associate with elastin to form medial elastic fibers present with TAADs, yet those with fibulin-5 deficiency do not. In this paper, we show that deletion of the fibulin-5 gene results in a significantly diminished contractility of the thoracic aorta in response to potassium loading despite otherwise preserved characteristic active behaviors, including axial force generation and rates of contraction and relaxation. Interestingly, this diminished response manifests around an altered passive state that is defined primarily by a reduced in vivo axial stretch. Given this significant coupling between passive and active properties, a lack of significant changes in passive material stiffness may help to offset the diminished contractility and thereby protect the wall from detrimental mechanosensing and its sequelae.

Hyperactivation of RAP1 and JAK/STAT Signaling Pathways Contributes to Fibrosis during the Formation of Nasal Capsular Contraction

2021 ◽  
pp. 1-12
Author(s):  
Meng Wu ◽  
Ming Li ◽  
Hong-Ju Xie ◽  
Hong-Wei Liu
Keyword(s):  
Signaling Pathways ◽  
Type I Collagen ◽  
Ex Vivo ◽  
Capsular Contracture ◽  
Silicone Implant ◽  
Type I ◽  
Loss Of Function ◽  
Sequencing Data ◽  
Functional Changes ◽  
Stat Signaling

Silicone implant-based augmentation rhinoplasty or mammoplasty induces capsular contracture, which has been acknowledged as a process that develops an abnormal fibrotic capsule associated with the immune response to allogeneic materials. However, the signaling pathways leading to the nasal fibrosis remain poorly investigated. We aimed to explore the molecular mechanism underlying the pathogenesis of nasal capsular contracture, with a specific research interest in the signaling pathways involved in fibrotic development at the advanced stage of contracture. By examining our recently obtained RNA sequencing data and global gene expression profiling between grade II and grade IV nasal capsular tissues, we found that both the RAP1 and JAK/STAT signaling pathways were hyperactive in the contracted capsules. This was verified on quantitative real-time PCR which demonstrated upregulation of most of the representative component signatures in these pathways. Loss-of-function assays through siRNA-mediated Rap1 silencing and/or small molecule-directed inhibition of JAK/STAT pathway in ex vivo primary nasal fibroblasts caused a series of dramatic behavioral and functional changes, including decreased cell viability, increased apoptosis, reduced secretion of proinflammatory cytokines, and synthesis of type I collagen, compared to control cells, and indicating the essential role of the RAP1 and JAK/STAT signaling pathways in nasal capsular fibrosis. Our results sheds light on targeting downstream signaling pathways for the prevention and therapy of silicone implant-induced nasal capsular contracture.

scholarly journals Frequency and average gray-level information for thermal ablation status in ultrasound B-Mode sequences

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Jens Ziegle ◽  
Alfredo Illanes ◽  
Axel Boese ◽  
Michael Friebe
Keyword(s):  
Thermal Ablation ◽  
Ex Vivo ◽  
Cell Damage ◽  
Potential Temperature ◽  
Image Sequences ◽  
Target Tissue ◽  
Gray Level ◽  
Porcine Liver ◽  
Time Frequency ◽  
Average Gray Level

AbstractDuring thermal ablation in a target tissue the information about temperature is crucial for decision making of successful therapy. An observable temporal and spatial temperature propagation would give a visual feedback of irreversible cell damage of the target tissue. Potential temperature features in ultrasound (US) B-Mode image sequences during radiofrequency (RF) ablation in ex-vivo porcine liver were found and analysed. These features could help to detect the transition between reversible and irreversible damage of the ablated target tissue. Experimental RF ablations of ex-vivo porcine liver were imaged with US B-Mode imaging and image sequences were recorded. Temperature was simultaneously measured within the liver tissue around a bipolar RF needle electrode. In the B-Mode images, regions of interest (ROIs) around the centre of the measurement spots were analysed in post-processing using average gray-level (AVGL) compared against temperature. The pole of maximum energy level in the time-frequency domain of the AVGL changes was investigated in relation to the measured temperatures. Frequency shifts of the pole were observed which could be related to transitions between the states of tissue damage.

scholarly journals Combination of histochemical analyses and micro-MRI reveals regional changes of the murine cervix in preparation for labor

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Antara Chatterjee ◽  
Rojan Saghian ◽  
Anna Dorogin ◽  
Lindsay S. Cahill ◽  
John G. Sled ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Diffusion Tensor ◽  
Ex Vivo ◽  
Muscle Cells ◽  
Pregnant Mouse ◽  
Collagen Fibers ◽  
Muscular Layer ◽  
Tissue Organization ◽  
Picrosirius Red

AbstractThe cervix is responsible for maintaining pregnancy, and its timely remodeling is essential for the proper delivery of a baby. Cervical insufficiency, or “weakness”, may lead to preterm birth, which causes infant morbidities and mortalities worldwide. We used a mouse model of pregnancy and term labor, to examine the cervical structure by histology (Masson Trichome and Picrosirius Red staining), immunohistochemistry (Hyaluronic Acid Binding Protein/HABP), and ex-vivo MRI (T2-weighted and diffusion tensor imaging), focusing on two regions of the cervix (i.e., endocervix and ectocervix). Our results show that mouse endocervix has a higher proportion of smooth muscle cells and collagen fibers per area, with more compact tissue structure, than the ectocervix. With advanced gestation, endocervical changes, indicative of impending delivery, are manifested in fewer smooth muscle cells, expansion of the extracellular space, and lower presence of collagen fibers. MRI detected three distinctive zones in pregnant mouse endocervix: (1) inner collagenous layer, (2) middle circular muscular layer, and (3) outer longitudinal muscular layer. Diffusion MRI images detected changes in tissue organization as gestation progressed suggesting the potential application of this technique to non-invasively monitor cervical changes that precede the onset of labor in women at risk for preterm delivery.

scholarly journals NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 147
Author(s):  
Romuald Brice Babou Kammoe ◽  
Gilles Kauffenstein ◽  
Julie Pelletier ◽  
Bernard Robaye ◽  
Jean Sévigny
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Smooth Muscle Contraction ◽  
Receptor Activation ◽  
Nucleoside Triphosphate ◽  
Nucleotide Receptors ◽  
Nerve Terminals ◽  
Wild Type ◽  
Nucleoside Triphosphate Diphosphohydrolase ◽  
Bladder Contraction

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

scholarly journals Reduced biomechanical models for precision-cut lung-slice stretching experiments

2021 ◽  
Vol 82 (5) ◽  
Author(s):  
Hannah J. Pybus ◽  
Amanda L. Tatler ◽  
Lowell T. Edgar ◽  
Reuben D. O’Dea ◽  
Bindi S. Brook
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Ex Vivo ◽  
Finite Thickness ◽  
Biomechanical Model ◽  
Local Stress ◽  
Smooth Muscle Contraction ◽  
Biomechanical Models ◽  
Cytokine Activation ◽  
Asymptotic Reduction

AbstractPrecision-cut lung-slices (PCLS), in which viable airways embedded within lung parenchyma are stretched or induced to contract, are a widely used ex vivo assay to investigate bronchoconstriction and, more recently, mechanical activation of pro-remodelling cytokines in asthmatic airways. We develop a nonlinear fibre-reinforced biomechanical model accounting for smooth muscle contraction and extracellular matrix strain-stiffening. Through numerical simulation, we describe the stresses and contractile responses of an airway within a PCLS of finite thickness, exposing the importance of smooth muscle contraction on the local stress state within the airway. We then consider two simplifying limits of the model (a membrane representation and an asymptotic reduction in the thin-PCLS-limit), that permit analytical progress. Comparison against numerical solution of the full problem shows that the asymptotic reduction successfully captures the key elements of the full model behaviour. The more tractable reduced model that we develop is suitable to be employed in investigations to elucidate the time-dependent feedback mechanisms linking airway mechanics and cytokine activation in asthma.

scholarly journals Myorelaxant Effect of the Dysphania ambrosioides Essential Oil on Sus scrofa domesticus Coronary Artery and Its Toxicity in the Drosophila melanogaster Model

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 2041
Author(s):  
Luiz Jardelino de Lacerda Neto ◽  
Andreza Guedes Barbosa Ramos ◽  
Renata Evaristo Rodrigues da Silva ◽  
Luís Pereira-de-Morais ◽  
Fernanda Maria Silva ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Essential Oil ◽  
Coronary Arteries ◽  
Sus Scrofa ◽  
Ex Vivo ◽  
Alternative Methods ◽  
Major Constituent ◽  
Sus Scrofa Domesticus

Purpose: Alternative methods for the use of animals in research have gained increasing importance, due to assessments evaluating the real need for their use and the development of legislation that regulates the subject. The principle of the 3R’s (replacement, reduction and refinement) has been an important reference, such that in vitro, ex vivo and cord replacement methods have achieved a prominent place in research. Methods: Therefore, due to successful results from studies developed with these methods, the present study aimed to evaluate the myorelaxant effect of the Dysphania ambrosioides essential oil (EODa) using a Sus scrofa domesticus coronary artery model, and the toxicity of both the Dysphania ambrosioides essential oil and its major constituent, α-terpinene, against Drosophila melanogaster in toxicity and negative geotaxis assays. Results: The EODa relaxed the smooth muscle of swine coronary arteries precontracted with K+ and 5-HT in assays using Sus scrofa domesticus coronary arteries. The toxicity results presented LC50 values of 1.546 mg/mL and 2.282 mg/mL for the EODa and α-terpinene, respectively, thus showing the EODa and α-terpinene presented toxicity to these dipterans, with the EODa being more toxic. Conclusions: Moreover, the results reveal the possibility of using the EODa in vascular disease studies since it promoted the relaxation of the Sus scrofa domesticus coronary smooth muscle.

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