Regulation Of Smooth Muscle Contraction Research Articles

Exploration and validation of ceRNA regulatory networks in colorectal cancer based on associations whole transcriptome sequencing

Colorectal cancer (CRC) is a wide-spread gastrointestinal cancer that is associated with augmented morbidity and mortality, and we do not yet have a deep understanding of its epidemiology and carcinogenicity. The transcriptome can reveal the complexity and heterogeneity of tumors and uncover new biomarkers or treatment options. In this study, we identified messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), round RNAs (circRNAs), and microRNAs (miRNAs) using whole-transcriptome sequencing and generated competing endogenous RNA (ceRNA) modulatory axes. We conducted whole transcriptome sequencing on 10 CRC and para-cancer (CRCP) samples and discovered 2465 differentially expressed (DE) mRNAs (DEmRNAs), 77 DE miRNAs (DEmiRNAs). 2852 DE lncRNAs (DElncRNAs) and 1477 DE circRNAs (DEcircRNAs). In addition, utilizing co-DE analysis, we generated the ceRNA axis. Subsequently, we employed the ceRNA axis to identify essential genes and corresponding associations with lncRNAs, circRNAs, and miRNAs in CRC. ceRNA regulatory network including mRNA-miRNA-lncRNA and mRNA-miRNA-circRNA. These modulatory axes potentially modulate the positive regulation of smooth muscle contraction, melanosome, plasma membrane, integral plasma membrane component and so on. Finally, the results of RNA sequencing (RNA-SEQ) were combined with the TCGA and GEO databases, and the DEGs strongly correlated with the TCGA-COAD overall survival (OS) as estimated by univariate cox and logarithmic rank analyses were cross-analyzed, and the co-upregulated DEGs were screened. Among the many DEs, KPNA2 was chosen for additional analysis. Using invitro experimentations, western blot, CCK8, EdU and other experiments were performed to verify the results. We found siRNA-based KPNA2 depletion reduces bladder cancer cells’ viability, migratory, and proliferative activities, which showed that the DEmRNA profiles were comparable to the sequencing information, confirming that the sequencing data were very reliable. These evidences highlight the ceRNA regulatory mechanisms in CRC and will aid future research into the molecular mechanisms behind colorectal cancer prevention and treatment.

Network Pharmacology, Integrated Bioinformatics, and Molecular Docking Reveals the Anti-Ovarian Cancer Molecular Mechanisms of Cinnamon (Cinnamomum cassia (L.) J. Presl)

Cinnamon ( Cinnamomum cassia (L.) J. Presl) is a popular natural spice with various pharmacological properties. This study was based on network pharmacology integrating bioinformatics and molecular docking to explore the potential molecular mechanisms of cinnamon in the treatment of ovarian cancer (OC). The chemical composition of cinnamon was collected from the TCMSP database to predict its targets and construct a “cinnamon active component target” network. OC-related genes were retrieved from Genecards and DisGeNET databases. The “disease-target” network was established, and the drug targets were mapped to the disease targets, and the key targets obtained from the mapping were subjected to DAVID analysis to construct a “component-target-pathway” network diagram. The active ingredients of cinnamon were molecularly docked to the core targets to predict the molecular mechanism of cinnamon in the treatment of ovarian cancer. From cinnamon, 105 chemical components were screened and de-duplicated to obtain 15 active components and 74 drug target proteins, and 26 common targets were obtained after mapping drug targets to disease targets. 368 entries were identified by GO enrichment analysis, mainly including biological progresses such as regulation of smooth muscle contraction and regulation of tube diameter, and molecular functions such as antioxidant activity, and peroxidase activity. The KEGG pathway enrichment analysis identified 4 signaling pathways, neuroactive ligand-receptor interaction, HIF-1 signaling pathway, regulation of lipolysis in adipocytes, and complement and coagulation cascades. Molecular docking analysis showed good affinity of these key targets with representative components of OC. There was a stable interaction between DIBP and ADRB2 and NR3C1. There is a stable interaction between oleic acid and C2K, EDN1, ERBB2, PLAU, PLG, PRSS3, PTGS1, PTGS2, SERPINE1 and SLC2A1. Cinnamon exerted its therapeutic effects on OC through multiple pathways and targets.

Distribution of neuromedin U (NMU)-like immunoreactivity in the goldfish brain, and effect of intracerebroventricular administration of NMU on emotional behavior in goldfish

Neuromedin U (NMU) is a multifunctional neuropeptide implicated in regulation of smooth muscle contraction in the circulatory and digestive systems, energy homeostasis and the stress response, but especially food intake in vertebrates. Recent studies have indicated the possible involvement of NMU in the regulation of psychomotor activity in rodents. We have identified four cDNAs encoding three putative NMU variants (NMU-21, −25 and −38) from the goldfish brain and intestine. Recently, we have also purified these NMUs and the truncated C-terminal form NMU-9 from these tissues, and demonstrated their anorexigenic action in goldfish. However, there is no information on the brain localization of NMU-like immunoreactivity and the psychophysiological roles of NMU in fish. Here, we investigated the brain distribution of NMU-like immunoreactivity and found that it was localized throughout the fore- and mid-brains. We subsequently examined the effect of intracerebroventricular (ICV) administration of NMU-21, which is abundant only in the brain on psychomotor activity in goldfish. As goldfish prefer the lower to the upper area of a tank, we developed an upper/lower area preference test in a tank for evaluating the psychomotor activity of goldfish using a personal tablet device without an automatic behavior-tracking device. ICV administration of NMU-21 at 10 pmol g-1 body weight (BW) prolonged the time spent in the upper area of the tank, and this action mimicked that of ICV administration of the central-type benzodiazepine receptor (CBR) agonist tofisopam at 100 pmol g-1 BW. These results suggest that NMU-21 potently induces anxiolytic-like action in the goldfish brain.

Bioinformatics and Experimental Analyses Reveal NFIC as an Upstream Transcriptional Regulator for Ischemic Cardiomyopathy.

Ischemic cardiomyopathy (ICM) caused by coronary artery disease always leads to myocardial infarction and heart failure. Identification of novel transcriptional regulators in ICM is an effective method to establish new diagnostic and therapeutic strategies. In this study, we used two RNA-seq datasets and one microarray dataset from different studies, including 25 ICM and 21 non-failing control (NF) samples of human left ventricle tissues for further analysis. In total, 208 differentially expressed genes (DEGs) were found by combining two RNA-seq datasets with batch effects removed. GO and KEGG analyses of DEGs indicated that the response to wounding, positive regulation of smooth muscle contraction, chromatin, PI3K-Akt signaling pathway, and transporters pathways are involved in ICM. Simple Enrichment Analysis found that NFIC-binding motifs are enriched in promoter regions of downregulated genes. The Gene Importance Calculator further proved that NFIC is vital. NFIC and its downstream genes were verified in the validating microarray dataset. Meanwhile, in rat cardiomyocyte cell line H9C2 cells, two genes (Tspan1 and Hopx) were confirmed, which decreased significantly along with knocking down Nfic expression. In conclusion, NFIC participates in the ICM process by regulating TSPAN1 and HOPX. NFIC and its downstream genes may be marker genes and potential diagnostic and therapeutic targets for ICM.

Relaxant effect of bioactive component compatibility of San-ao decoction on In vitro guinea pig airway smooth muscle: A dose-response relationship study

Background: Component compatibility is important to the modernization of traditional Chinese medicine. Studies have shown that San-ao decoction (SAD) can treat respiratory diseases by relaxing airway smooth muscle (ASM) and reducing airway hyper-responsiveness. However, whether its bioactive components and compatibility also present with similar relaxant effects remains unknown. This study aims to explore the potential relaxant property, dose-response relationship, and underlying mechanisms of the bioactive component compatibility in SAD. Methods: Network pharmacology was primarily used to identify the bioactive components of SAD and uncover its underlying mechanisms. ASM tension force measuring technique was utilized to verify the relaxant and dose-response effects on in vitro guinea pig ASM. Results: We postulated pseudoephedrine hydrochloride (PH), amygdalin (AM), and diammonium glycyrrhizate (DG) to be the bioactive components of SAD, which could effectively relax ASM in a dose-dependent manner on both acetylcholine-induced and spontaneous contraction. Both PH and AM could lead to DG dose–response curve shift. The regression equation of these three bioactive components was Y = −2.048 × X1 + 0.411 × X2 + 14.052 × X3 (X1, X2, X3 representing PH, AM, and DG, respectively). The underlying mechanisms of these components might be associated with the regulation of smooth muscle contraction. Conclusions: PH, AM, and DG are the bioactive components of SAD, which can relax ASM in a dose–response manner and exert a synergistic effect. Clinically, compatibility of these three bioactive components may serve as a new complementary and alternative treatment for respiratory diseases.

Cystic ovary disease impairs transport speed, smooth muscle contraction, and epithelial ion transport in the bovine oviduct.

Cystic ovary disease (COD) is a common cause of bovine infertility but the impact of this disease on the oviduct is unknown. The aim of this study was to analyze the effects of COD on particle transport speed (PTS), ciliary beat frequency, myosalpinx contraction, and epithelial ion transport. Oviducts were obtained from cows affected by COD and compared with those of healthy, mid-diestrus cows. PTS and CBF were examined using live-cell imaging. Smooth muscle contraction and epithelial ion transport were investigated using organ baths and Ussing chambers. Our results showed that muscarinic receptors are involved in cholinergic signaling in the oviduct and that forskolin-induced cyclic AMP production is involved in active ion transport in the oviductal epithelium. Oviducts from cows with luteal cysts revealed significantly decreased PTS (p = 0.02). Further to that, in the oviducts of COD cows, the cholinergic regulation of smooth muscle contractions and active epithelial ion transport were significantly diminished (p < 0.0001). These results imply that in COD cows, oviductal transport is compromised by decreased fluid flow speed and reduced cholinergic regulation of smooth muscle contraction and ion transport. This knowledge contributes to a more comprehensive understanding of COD supporting the development of novel therapeutic concepts for infertility treatment.

The underlying mechanisms of FGF2 in carotid atherosclerotic plaque development revealed by bioinformatics analysis.

The purpose of this study was to explore the regulatory mechanisms of FGF2 in carotid atherosclerotic plaque development using bioinformatics analysis. Expression profiles of 32 atheroma plaque (AP) and 32 paired distant macroscopically intact (DMI) tissues samples in the GSE43292 dataset were downloaded from the Gene Expression Omnibus database. Following identification of differential expression genes (DEGs), correlation analysis of fibroblast growth factor 2 (FGF2) and DEGs was conducted. Subsequently, functional enrichment analysis and the protein-protein interaction network for FGF2 significantly correlated DEGs were constructed. Then, microRNAs (miRNAs) that regulated FGF2 and regulatory pairs of long noncoding RNA (lncRNA)-miRNA were predicted to construct the lncRNA-miRNA-FGF2 network. A total of 101 DEGs between AP and DMI samples were identified, and 31 DEGs were analyzed to have coexpression relationships with FGF2, including 23 positively correlated and 8 negatively correlated DEGs. VAV3 had the lowest r value among all FGF2 negatively correlated DEGs. FGF2 positively correlated DEGs were closely related to "regulation of smooth muscle contraction" (e.g., calponin 1 (CNN1)), while FGF2 negatively correlated DEGs were significantly associated with "platelet activation" (e.g., Vav guanine nucleotide exchange factor 3 (VAV3)). In addition, a total of 12 miRNAs that regulated FGF2 were predicted, and hsa-miR-15a-5p and hsa-miR-16-5p were highlighted in the lncRNA-miRNA-FGF2 regulatory network. CNN1 might cooperate with FGF2 to regulate smooth muscle contractility during CAP formation. VAV3 might cooperate with FGF2 to be responsible for the development of CAP through participating in platelet activation. Hsa-miR-15a-5p and hsa-miR-16-5p might participate in the development of CAP via regulating FGF2.

Sex Differences in Gene‐Expression between Brain Arteries and Cortical Microvessels in Mice revealed by RNA‐Sequencing

Background Brain microvessels (MVs) may represent a more vulnerable segment of the cerebral circulation than large arteries during aging, diabetes, and/or minor strokes. Adverse changes in MVs have been implicated in the development of cognitive impairment, vascular dementia, and Alzheimer's disease. However, development of effective therapies has been hampered by lack of understanding of the underlying mechanisms that negatively affect MVs compared to large arteries. The purpose of this study was to identify potential mechanisms underlying the vulnerability of MVs compared to large arteries to aging and pathological conditions and the impact of sex using quantitative RNA sequencing (RNAseq). Methods Cerebral arteries (anterior and middle cerebral, basilar, circle of Willis) and cortical MVs (end arterioles, capillaries, venules) were isolated from young age-matched male and female C57BL6J mice. Arteries and MVs were then used for 3 vs. 3 quantitative RNAseq. RNASeq analyses were performed using the Illumina NextSeq 550 sequencing system. RNASeq expression analysis was performed using Kallisto (v0.46.0), and Sleuth (v0.30.0) with the mouse transcriptome index. Results For females, the RNAseq analysis identified 14,325 genes out of 51,661genes examined with significant (p < 0.05) differences between arteries and cortical MVs. The greatest differences (3 to 6 times) were seen in the expression of the following genes: Thrombospondin-2 (calcium ion binding, negative regulation of angiogenesis), SPARC-related modular calcium-binding gene 1, Megakaryocyte-associated tyrosine protein-kinase (ATP binding), Prostaglandin-H2 D-isomerase (fatty acid synthesis), Solute carrier family 7 member 2, SOGA family member 3, Gelsolin (aging and apoptotic process), potassium voltage-gated channel subunits, and glutamate receptor subunits (Gria, Grin). While most of the Gria, Grin and potassium channels genes were abundantly expressed in MVs, thrombospondins, prostaglandins and apoptosis-related genes were more highly expressed in large arteries. The RNAseq profiles comparing arteries to MVs for males were very different than for females (extracellular matrix binding, regulation of smooth muscle contraction and growth factors, mostly). Conclusion We found substantial differences in the gene expression of brain large arteries and cortical MVs in mice and these differences may account for increased vulnerability of smaller blood vessels to aging and stress. For example, increased expression of glutamatergic receptors in MVs might be associated with enhanced vulnerability to damage and dysfunction of this segment of the cerebral circulation due to stress. The finding that RNAseq profiles comparing large arteries and cortical MVs differ between males and females may provide the basis for sex-dependent differences in the vulnerability and severity of neurological disorders in humans.

Regulation of smooth muscle contraction by monomeric non-RhoA GTPases.

Smooth muscle contraction in the cardiovascular system, airways, prostate and lower urinary tract is involved in the pathophysiology of many diseases, including cardiovascular and obstructive lung disease plus lower urinary tract symptoms, which are associated with high prevalence of morbidity and mortality. This prominent clinical role of smooth muscle tone has led to the molecular mechanisms involved being subjected to extensive research. In general smooth muscle contraction is promoted by three major signalling pathways, including the monomeric GTPase RhoA pathway. However, emerging evidence suggests that monomeric GTPases other than RhoA may be involved in signal transduction in smooth muscle contraction, including Rac GTPases, cell division control protein 42 homologue, adenosine ribosylation factor 6, Ras, Rap1b and Rab GTPases. Here, we review these emerging functions of non‐RhoA GTPases in smooth muscle contraction, which has now become increasingly more evident and constitutes an emerging and innovative research area of high clinical relevance.

Rho-kinase (ROCK) Inhibitors - A Neuroprotective Therapeutic Paradigm with a Focus on Ocular Utility.

Glaucoma is a progressive optic neuropathy causing visual impairment and Retinal Ganglionic Cells (RGCs) death gradually posing a need for neuroprotective strategies to minimize the loss of RGCs and visual field. It is recognized as a multifactorial disease, Intraocular Pressure (IOP) being the foremost risk factor. ROCK inhibitors have been probed for various possible indications, such as myocardial ischemia, hypertension, kidney diseases. Their role in neuroprotection and neuronal regeneration has been suggested to be of value in the treatment of neurological diseases, like spinal-cord injury, Alzheimer's disease and multiple sclerosis but recently Rho-associated Kinase inhibitors have been recognized as potential antiglaucoma agents. Rho-Kinase is a serine/threonine kinase with a kinase domain which is constitutively active and is involved in the regulation of smooth muscle contraction and stress fibre formation. Two isoforms of Rho-Kinase, ROCK-I (ROCK β) and ROCK-II (ROCK α) have been identified. ROCK II plays a pathophysiological role in glaucoma and hence the inhibitors of ROCK may be beneficial to ameliorate the vision loss. These inhibitors decrease the intraocular pressure in the glaucomatous eye by increasing the aqueous humour outflow through the trabecular meshwork pathway. They also act as anti-scarring agents and hence prevent post-operative scarring after the glaucoma filtration surgery. Their major role involves axon regeneration by increasing the optic nerve blood flow which may be useful in treating the damaged optic neurons. These drugs act directly on the neurons in the central visual pathway, interrupting the RGC apoptosis and therefore serve as a novel pharmacological approach for glaucoma neuroprotection. Based on the results of high-throughput screening, several Rho kinase inhibitors have been designed and developed comprising of diverse scaffolds exhibiting Rho kinase inhibitory activity from micromolar to subnanomolar ranges. This diversity in the scaffolds with inhibitory potential against the kinase and their SAR development will be intricated in the present review. Ripasudil is the only Rho kinase inhibitor marketed to date for the treatment of glaucoma. Another ROCK inhibitor AR-13324 has recently passed the clinical trials whereas AMA0076, K115, PG324, Y39983 and RKI-983 are still under trials. In view of this, a detailed and updated account of ROCK II inhibitors as the next generation therapeutic agents for glaucoma will be discussed in this review.

HuR (Human Antigen R) Regulates the Contraction of Vascular Smooth Muscle and Maintains Blood Pressure.

HuR (human antigen R)-an RNA-binding protein-is involved in regulating mRNA stability by binding adenylate-uridylate-rich elements. This study explores the role of HuR in the regulation of smooth muscle contraction and blood pressure. Approach and Results: Vascular HuRSMKO (smooth muscle-specific HuR knockout) mice were generated by crossbreeding HuRflox/flox mice with α-SMA (α-smooth muscle actin)-Cre mice. As compared with CTR (control) mice, HuRSMKO mice showed hypertension and cardiac hypertrophy. HuR levels were decreased in aortas from hypertensive patients and SHRs (spontaneously hypertensive rats), and overexpression of HuR could lower the blood pressure of SHRs. Contractile response to vasoconstrictors was increased in mesenteric artery segments isolated from HuRSMKO mice. The functional abnormalities in HuRSMKO mice were attributed to decreased mRNA and protein levels of RGS (regulator of G-protein signaling) protein(s) RGS2, RGS4, and RGS5, which resulted in increased intracellular calcium increase. Consistently, the degree of intracellular calcium ion increase in HuR-deficient smooth muscle cells was reduced by overexpression of RGS2, RGS4, or RGS5. Finally, administration of RGS2 and RGS5 reversed the elevated blood pressure in HuRSMKO mice. Our findings indicate that HuR regulates vascular smooth muscle contraction and maintains blood pressure by modulating RGS expression.

Transcriptional Suppression of CPI-17 Gene Expression in Vascular Smooth Muscle Cells by Tumor Necrosis Factor, Krüppel-Like Factor 4, and Sp1 Is Associated with Lipopolysaccharide-Induced Vascular Hypocontractility, Hypotension, and Mortality.

Vasodilatory shock in sepsis is caused by the failure of the vasculature to respond to vasopressors, which results in hypotension, multiorgan failure, and ultimately patient death. Recently, it was reported that CPI-17, a key player in the regulation of smooth muscle contraction, was downregulated by lipopolysaccharide (LPS) in mesenteric arteries concordant with vascular hypocontractilty. While Sp1 has been shown to activate CPI-17 transcription, it is unknown whether Sp1 is involved in LPS-induced smooth muscle CPI-17 downregulation. Here we report that tumor necrosis factor (TNF) was critical for LPS-induced smooth muscle CPI-17 downregulation. Mechanistically, we identified two GC boxes as a key TNF response element in the CPI-17 promoter and demonstrated that KLF4 was upregulated by TNF, competed with Sp1 for the binding to the GC boxes in the CPI-17 promoter, and repressed CPI-17 transcription through histone deacetylases (HDACs). Moreover, genetic deletion of TNF or pharmacological inhibition of HDACs protected mice from LPS-induced smooth muscle CPI-17 downregulation, vascular hypocontractility, hypotension, and mortality. In summary, these data provide a novel mechanism of the transcriptional control of CPI-17 in vascular smooth muscle cells under inflammatory conditions and suggest a new potential therapeutic strategy for the treatment of vasodilatory shock in sepsis.

Mechanism of persistent hyperalgesia in neuropathic pain caused by chronic constriction injury.

Transmembrane member 16A (TMEM16A) is involved in many physiological functions, such as epithelial secretion, sensory conduction, nociception, control of neuronal excitability, and regulation of smooth muscle contraction, and may be important in peripheral pain transmission. To explore the role of TMEM16A in the persistent hyperalgesia that results from chronic constriction injury-induced neuropathic pain, a rat model of the condition was established by ligating the left sciatic nerve. A TMEM16A selective antagonist (10 μg T16Ainh-A01) was intrathecally injected at L5-6. For measurement of thermal hyperalgesia, the drug was administered once at 14 days and thermal withdrawal latency was recorded with an analgesia meter. For measurement of other indexes, the drug was administered at 12 days, once every 6 hours, totally five times. The measurements were performed at 14 days. Western blot assay was conducted to analyze TMEM16A expression in the L4-6 dorsal root ganglion. Immunofluorescence staining was used to detect the immunoreactivity of TMEM16A in the L4-6 dorsal root ganglion on the injured side. Patch clamp was used to detect electrophysiological changes in the neurons in the L4-6 dorsal root ganglion. Our results demonstrated that thermal withdrawal latency was shortened in the model rats compared with control rats. Additionally, TMEM16A expression and the number of TMEM16A positive cells in the L4-6 dorsal root ganglion were higher in the model rats, which induced excitation of the neurons in the L4-6 dorsal root ganglion. These findings were inhibited by T16Ainh-A01 and confirm that TMEM16A plays a key role in persistent chronic constriction injury-induced hyperalgesia. Thus, inhibiting TMEM16A might be a novel pharmacological intervention for neuropathic pain. All experimental protocols were approved by the Animal Ethics Committee at the First Affiliated Hospital of Shihezi University School of Medicine, China (approval No. A2017-170-01) on February 27, 2017.

Regulation of gastric smooth muscle contraction via Ca2+-dependent and Ca2+-independent actin polymerization.

In gastrointestinal smooth muscle, acetylcholine induced muscle contraction is biphasic, initial peak followed by sustained contraction. Contraction is regulated by phosphorylation of 20 kDa myosin light chain (MLC) at Ser19, interaction of actin and myosin, and actin polymerization. The present study characterized the signaling mechanisms involved in actin polymerization during initial and sustained muscle contraction in response to muscarinic M3 receptor activation in gastric smooth muscle cells by targeting the effectors of initial (phospholipase C (PLC)-β/Ca2+ pathway) and sustained (RhoA/focal adhesion kinase (FAK)/Rho kinase pathway) contraction. The initial Ca2+ dependent contraction and actin polymerization is mediated by sequential activation of PLC-β1 via Gαq, IP3 formation, Ca2+ release and Ca2+ dependent phosphorylation of proline-rich-tyrosine kinase 2 (Pyk2) at Tyr402. The sustained Ca2+ independent contraction and actin polymerization is mediated by activation of RhoA, and phosphorylation of FAK at Tyr397. Both phosphorylation of Pyk2 and FAK leads to phosphorylation of paxillin at Tyr118 and association of phosphorylated paxillin with the GEF proteins p21-activated kinase (PAK) interacting exchange factor α, β (α and β PIX) and DOCK 180. These GEF proteins stimulate Cdc42 leading to the activation of nucleation promoting factor N-WASP (neuronal Wiskott-Aldrich syndrome protein), which interacts with actin related protein complex 2/3 (Arp2/3) to induce actin polymerization and muscle contraction. Acetylcholine induced muscle contraction is inhibited by actin polymerization inhibitors. Thus, our results suggest that a novel mechanism for the regulation of smooth muscle contraction is mediated by actin polymerization in gastrointestinal smooth muscle which is independent of MLC20 phosphorylation.

PL-015 Aerobic exercise increases BKCa channel expression to enhance tracheal smooth muscle relaxation in a murine asthma model

Objective Increasing evidence has shown that moderate-intensity aerobic exercise training reduces airway hyperresponsiveness (AHR) in patients with asthma. However, the mechanisms underlying exercise-induced improvements in smooth muscle contractility have not been fully elucidated. Large-conductance Ca2+-activated K+ channels (BKCa) are expressed broadly on smooth muscle cells and play an important role in the regulation of smooth muscle contraction. We tested the hypothesis that exercise training increases the contribution of BKCa channel to tracheal smooth muscle relaxation in in ovalbumin (OVA)-challenged asthmatic rats.&#x0D; Methods Rats were sensitized/challenged with OVA or saline and exercised at a moderate intensity 5 times/week for 4 weeks. Tracheal smooth muscle contractility was tested. Membrane potential of primary cultured tracheal smooth muscle cells was measured. In addition, western immunoblotting was performed to study the expression levels of BKCa channel protein.&#x0D; Results The contraction of rat airway smooth muscle induced by carbachol was significantly increased with asthma and exercise training reversed this alteration. Application of BKCa channel agonist, NS1619, induced tracheal smooth muscle relaxation. NS1619-induced relaxation was decreased in asthmatic rats, however exercise training significantly increased NS1619-induced relaxation. In primary cultured smooth muscle cells, NS1619-induced membrane potential was reduced with asthma and this alteration was diminished after exercise training. Additionally, western blotting revealed that the protein expression of BKCa was reduced in asthmatic group and aerobic exercise significantly improved BKCa expression.&#x0D; Conclusions The present study reveals that aerobic exercise training increases BKCa expression on tracheal smooth muscle, which partly underlies the beneficial effect of exercise on improving airway smooth muscle relaxation in asthma.

ML-7 attenuates airway inflammation and remodeling via inhibiting the secretion of Th2 cytokines in mice model of asthma.

Previous studies have indicated that smooth muscle myosin light chain kinase (MLCK) has a prominent role in the regulation of smooth muscle contraction, which tends to be upregulated in asthma. In recent years, numerous studies have reported that MLCK is intimately connected with the immunoregulatory mechanism of T cells. The imbalance of T helper type 1 cells (Th1)/Th2 constitutes the immune-associated pathological basis of chronic asthma. Th2-associated cytokines, including interleukin-4, −5, −13, −25 and −33, are involved in airway inflammation, hyperresponsiveness and remodeling, which leads to a progressive decline in lung function. The purpose of the present study was to verify whether inhibition of bronchial MLCK attenuated the expression Th2-associated cytokines in asthmatic mice, including the above-mentioned ones. Female BALB/c mice were used to establish an ovalbumin (OVA)-induced model of asthma, of which one group was treated with the MLCK inhibitor (5-iodonaphthalene-1-sulfonyl) homopiperazine (ML-7). The inhibitor of MLCK, ML-7 attenuated airway inflammation and remodeling by reducing inflammatory cell infiltration and the secretion of Th2 cytokines in mice model of asthma, which may represent a promising therapeutic strategy for asthma.

Effects of newly synthetized isoquinoline derivatives on rat uterine contractility and ROCK II activity

Protein kinases have an important role in signal transduction in the cellular system via protein phosphorylation. RhoA activated Rho-kinases have a pivotal role in the regulation of smooth muscle contraction. ROCK I and ROCK II phosphorylate myosin-phosphatase and myosin-kinase, which induces contraction in the myometrium. Several studies have investigated the affinity of isoquinoline alkaloids (HA-1077, H1152P) to Rho-kinases, and these compounds notably inhibited the Ca2+-independent process.We measured the efficiency of 25 original, newly synthesized isoquinoline derivatives for the Rho-kinase activity using Rho-associated kinase activity assay and determined their effects on the non-pregnant, 20-day pregnant and parturient rat myometrial contraction in vitro.The IC50 values of 11 from among the 25 derivatives were significantly lower on the oxytocin-induced non-pregnant rat uterine contraction compared with Y-27632 and fasudil, although their maximal inhibitory effects were weaker than those of Y-27632 and fasudil. We measured the effects of 11 isoquinoline molecules with significant IC50 values on ROCK II activity. We found two isoquinolines out of 11 compounds (218 and 852) which decreased the active ROCK II level similarly as Y-27632. Then we found that 218 and 852 relaxed the 20th-day pregnant and parturient rat uterus with greater potency as compared with fasudil.The majority of the synthesized isoquinoline derivatives have uterus relaxant effects and two of them significantly suppress the Rho-kinase mediated myosin light chain phosphorylation. Our results may suggest that the isoquinoline structure has a promising prospect for the development of new and effective inhibitors of uterine contractions in preterm birth.

Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction.

A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions. Also, the knowledge is expected to facilitate devising innovative strategies that more precisely monitor and normalize organ functions by targeting individual smooth muscles. Evidence has established a current paradigm that the myosin light chain phosphatase (MLCP) is a master regulator of smooth muscle responsiveness to stimuli. Cellular MLCP activity is negatively and positively regulated in response to G-protein activation and cAMP/cGMP production, respectively, through the MYPT1 regulatory subunit and an endogenous inhibitor protein named CPI-17. In this article we review the outcomes from two decade of research on the CPI-17 signaling and discuss emerging paradoxes in the view of signaling pathways regulating smooth muscle functions through MLCP.

СОTRANSPORTER OF SODIUM, POTASSIUM AND CHLORIDE AS A REGULATOR OF VASCULAR TONE: ROLE IN THE PATHOGENESIS OF SYSTEMIC AND PULMONARY HYPERTENSION

This review summarizes the data on the implication of ubiquitous isoform of the carrier providing electroneutral symport of sodium, potassium and chloride (Na+, K+, 2Cl–-cotransport — NKCC1) in the regulation of smooth muscle contraction in the systemic and pulmonary circulation and its involvement in the pathogenesis of systemic (essential) and pulmonary hypertension with the special emphasis on the usage of NKCC1 as a novel antihypertensive target. In this connection we consider the data on the attenuation of the myogenic tone in the resistant vessels of the systemic circulation and afferent arteriole of the kidney as well as the action of inhaled loop diuretic on the contraction of the airway smooth muscle cells.

Analysis of phosphorylation of the myosin targeting subunit of smooth muscle myosin light chain phosphatase by Phos‐tag SDS‐PAGE

Phosphorylation of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase plays an important role in the regulation of smooth muscle contraction, and several sites of phosphorylation by different protein Ser/Thr kinases have been identified. Furthermore, in some instances, phosphorylation at specific sites affects phosphorylation at neighboring sites, with functional consequences. Characterization of the complex phosphorylation of MYPT1 in tissue samples at rest and in response to contractile and relaxant stimuli is, therefore, challenging. We have exploited Phos‐tag SDS‐PAGE in combination with western blotting using antibodies to MYPT1 to separate multiple phosphorylated MYPT1 species and quantify MYPT1 phosphorylation stoichiometry using purified, full‐length recombinant MYPT1 phosphorylated by Rho‐associated coiled‐coil kinase (ROCK) and cyclic AMP‐dependent protein kinase (PKA). This approach confirmed that phosphorylation of MYPT1 by ROCK occurs at Thr697 and Thr855, PKA phosphorylates these two sites and the neighboring Ser696 and Ser854, and prior phosphorylation at Thr697 and Thr855 by ROCK precludes phosphorylation at Ser696 and Ser854 by PKA. Furthermore, phosphorylation at Thr697 and Thr855 by ROCK exposes two other sites of phosphorylation by PKA. Treatment of Triton‐skinned rat caudal arterial smooth muscle strips with the membrane‐impermeant phosphatase inhibitor microcystin, or of intact tissue with the membrane‐permeant calyculin A, induced slow, sustained contractions that correlated with phosphorylation of MYPT1 at 7 to ≥10 sites. Phos‐tag SDS‐PAGE thus provides a suitable and convenient method for analysis of the complex, multi‐site MYPT1 phosphorylation events involved in the regulation of myosin light chain phosphatase activity and smooth muscle contraction.Support or Funding InformationFunded by the Canadian Institutes of Health Research (MOP‐111262)