scholarly journals Expression of endoplasmic-reticulum Ca2+-pump isoforms and of phospholamban in pig smooth-muscle tissues

1990 ◽  
Vol 271 (3) ◽  
pp. 649-653 ◽  
Author(s):  
J A Eggermont ◽  
F Wuytack ◽  
J Verbist ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Western Blot ◽  
Mrna Levels ◽  
Protection Assay ◽  
Western Blots ◽  
Specific Antibodies ◽  
Muscle Tissues ◽  
Low Levels

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.

scholarly journals MicroRNA-222 Promotes the Proliferation of Pulmonary Arterial Smooth Muscle Cells by Targeting P27 and TIMP3

2017 ◽  
Vol 43 (1) ◽  
pp. 282-292 ◽  
Author(s):  
Ying Xu ◽  
Yihua Bei ◽  
Shutong Shen ◽  
Jialiang Zhang ◽  
Yichao Lu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Western Blot ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Target Genes ◽  
Cell Number ◽  
Pulmonary Artery Hypertension ◽  
Luciferase Assay ◽  
Ki 67

Background/Aims: Aberrant vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of pulmonary artery hypertension (PAH). Dysregulated microRNAs (miRNAs, miRs) have been implicated in the progression of PAH. miR-222 has a pro-proliferation effect on VSMCs while it has an anti-proliferation effect on vascular endothelial cells (ECs). As the biological function of a single miRNA could be cell-type specific, the role of miR-222 in pulmonary artery smooth muscle cell (PASMC) proliferation is not clear and deserves to be explored. Methods: PASMCs were transfected with miR-222 mimic or inhibitor and PASMC proliferation was determined by Western blot for PCNA, Ki-67 and EdU staining, and cell number counting. The target genes of miR-222 including P27 and TIMP3 were determined by luciferase assay and Western blot. In addition, the functional rescue experiments were performed based on miR-222 inhibitor and siRNAs to target genes. Results: miR-222 mimic promoted PASMC proliferation while miR-222 inhibitor decreased that. TIMP3 was identified to be a direct target gene of miR-222 based on luciferase assay. Meanwhile, P27 and TIMP3 were up-regulated by miR-222 inhibitor and down-regulated by miR-222 mimic. Moreover, P27 siRNA and TIMP3 siRNA could both attenuate the anti-proliferation effect of miR-222 inhibitor in PASMCs, supporting that P27 and TIMP3 are at least partially responsible for the regulatory effect of miR-222 in PASMCs. Conclusion: miR-222 promotes PASMC proliferation at least partially through targeting P27 and TIMP3.

PGA1 and PGF2 alpha metabolism by pig pulmonary endothelium, smooth muscle, and fibroblasts

1979 ◽  
Vol 46 (2) ◽  
pp. 211-216 ◽  
Author(s):  
C. Ody ◽  
Y. Dieterle ◽  
I. Wand ◽  
H. Stalder ◽  
A. F. Junod
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Ethyl Acetate ◽  
Ethacrynic Acid ◽  
Inhibitory Effect ◽  
Cultured Fibroblasts ◽  
Pulmonary Endothelium ◽  
Muscle Tissues ◽  
Bromcresol Green

To determine the cellular site for uptake and degradation of circulating prostaglandins (PGs) by the lung, the metabolism of PGA1 and PGF2 alpha was studied in pig lung slices, smooth muscle preparations, and pulmonary valves, as well as in isolated and cultured endothelial cells and cultured fibroblasts. Formation of 15-keto metabolites of both PGA1 and PGF2 alpha by lung slices was confirmed. No evidence of PGF2 alpha degradation could be found in any of the remaining preparations. For PGA1, however, 15-hydroxyprostaglandin dehydrogenase activity was detected in the three smooth muscle preparations studied (trachea, aorta, pulmonary artery) and found to be similar to that measured in lung slices. But the inhibitory effect of diphloretin phosphate and bromcresol green was much more marked in smooth muscle tissues than in lung slices, which suggested that PGA1 metabolism by the lung was not due to smooth muscle cells. Endothelial cells, freshly isolated and cultured, originating from the pulmonary artery and from the aorta, formed a PGA1-glutathione adduct, poorly extractable in ethyl acetate. This reaction, also present in cultured fibroblasts, was inhibited by ethacrynic acid. The cellular site responsible for the pulmonary degradation of circulating PGs remains undetermined.

Expression and epitopic conservation of calponin in different smooth muscles and during development

1996 ◽  
Vol 74 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Jian-Ping Jin ◽  
Michael P. Walsh ◽  
Mary E. Resek ◽  
Gail A. McMartin
Keyword(s):  
Smooth Muscle ◽  
Thin Filament ◽  
Smooth Muscles ◽  
Constitutive Expression ◽  
Young Chick ◽  
Adult Chicken ◽  
Muscle Tissues ◽  
Highly Sensitive ◽  
Cnbr Cleavage ◽  
Low Levels

Calponin is a thin filament associated protein found in smooth muscle as a potential modulator of contraction. Five mouse monoclonal antibodies (mAbs CP1, CP3, CP4, CP7, and CP8) were prepared against chicken gizzard α-calponin. The CP1 epitopic structure is conserved in smooth muscles across vertebrate phyla and is highly sensitive to CNBr cleavage in contrast with the chicken-specific CP4 and the avian–mammalian-specific CP8 epitopes that are resistant to CNBr fragmentation. Using this panel of mAbs against multiple epitopes, only α-calponin was detected in adult chicken smooth muscles and throughout development of the gizzard. Western blotting showed that the calponin content varied among different smooth muscle tissues and correlated with that of h-caldesmon. In contrast with the constitutive expression of calponin in phasic smooth muscle of the digestive tract, very low levels of calponin were detected in adult avian tracheas and no calponin expression was detected in embryonic and young chick tracheas. These results provide information on the structural conservation of calponins and suggest a relationship between calponin expression and smooth muscle functional states.Key words: smooth muscle calponin, caldesmon, expression, development, chicken trachea.

Constrictor-induced translocation of NFAT3 in human and rat pulmonary artery smooth muscle

2005 ◽  
Vol 289 (6) ◽  
pp. L1061-L1074 ◽  
Author(s):  
Asma Yaghi ◽  
Stephen M. Sims
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Rho Kinase ◽  
Pulmonary Vasculature ◽  
Resting Cells ◽  
Maximal Effect ◽  
Activated T Cells ◽  
Western Blots ◽  
Transient Rise ◽  
Low Amplitude

The transcription factor nuclear factor of activated T cells (NFAT) resides in the cytoplasm in resting cells and upon stimulation is dephosphorylated, translocates to the nucleus, and becomes transcriptionally active. NFAT is commonly activated by stimulation of receptors coupled to Ca2+ mobilization; however, little is known about the regulation of NFAT in pulmonary vascular smooth muscle. The aim of this study was to investigate regulation of NFAT in human and rat intralobar pulmonary artery by two constrictors: phenylephrine (PE) and 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 metabolite formed endogenously in lungs. Immunostaining of smooth muscle cells revealed cytoplasmic localization of NFAT in untreated cells, and PE or 20-HETE induced translocation to the nucleus, with maximal effect at 30 min. Cyclosporin A and FK-506 (both 1 μM) inhibited NFAT translocation, indicating involvement of calcineurin. Moreover, the Rho-kinase blocker Y-27632 prevented translocation. Translocation of NFAT was confirmed by Western blots, with NFAT3 the prominent isoform in pulmonary artery. Constrictors caused calcineurin-sensitive translocation of NFAT to nuclei in intact arteries, demonstrating regulation in native tissue. To investigate a role for Ca2+, cells were loaded with fura-2. Whereas PE caused an acute transient rise of [Ca2+]i, 20-HETE caused a prolonged low amplitude rise of [Ca2+]i. The involvement of Rho-kinase in PE- and 20-HETE-induced NFAT3 translocation in pulmonary artery suggests a level of control not previously recognized in smooth muscle. Constrictors of the pulmonary vasculature not only cause acute responses but also activate NFAT, which may alter gene expression in pulmonary health and disease.

scholarly journals Hypoxia Up-Regulates Hypoxia-Inducible Factor-1α Transcription by Involving Phosphatidylinositol 3-Kinase and Nuclear Factor κB in Pulmonary Artery Smooth Muscle Cells

2007 ◽  
Vol 18 (12) ◽  
pp. 4691-4697 ◽  
Author(s):  
Rachida S. BelAiba ◽  
Steve Bonello ◽  
Christian Zähringer ◽  
Stefanie Schmidt ◽  
John Hess ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Promoter Activity ◽  
Hypoxia Inducible Factor ◽  
Nuclear Factor Κb ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Pulmonary Artery Smooth Muscle

The oxygen sensitive α-subunit of the hypoxia-inducible factor-1 (HIF-1) is a major trigger of the cellular response to hypoxia. Although the posttranslational regulation of HIF-1α by hypoxia is well known, its transcriptional regulation by hypoxia is still under debate. We, therefore, investigated the regulation of HIF-1α mRNA in response to hypoxia in pulmonary artery smooth muscle cells. Hypoxia rapidly enhanced HIF-1α mRNA levels and HIF-1α promoter activity. Furthermore, inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT but not extracellular signal-regulated kinase 1/2 pathway blocked the hypoxia-dependent induction of HIF-1α mRNA and HIF-1α promoter activity, suggesting involvement of a PI3K/AKT-regulated transcription factor. Interestingly, hypoxia also induced nuclear factor-κB (NFκB) nuclear translocation and activity. In line, expression of the NFκB subunits p50 and p65 enhanced HIF-1α mRNA levels, whereas blocking of NFκB by an inhibitor of nuclear factor-κB attenuated HIF-1α mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NFκB site within the HIF-1α promoter, and mutation of this site abolished induction by hypoxia. In line, gel shift analysis and chromatin immunoprecipitation confirmed binding of p50 and p65 NFκB subunits to the HIF-1α promoter under hypoxia. Together, these findings provide a novel mechanism in which hypoxia induces HIF-1α mRNA expression via the PI3K/AKT pathway and activation of NFκB.

scholarly journals Evidence for two isoforms of the endoplasmic-reticulum Ca2+ pump in pig smooth muscle

1989 ◽  
Vol 260 (3) ◽  
pp. 757-761 ◽  
Author(s):  
J A Eggermont ◽  
F Wuytack ◽  
S De Jaegere ◽  
L Nelles ◽  
R Casteels
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Cdna Clones ◽  
Gene Transcript ◽  
Muscle Tissues ◽  
Class 1 ◽  
Terminal Amino ◽  
Slow Twitch ◽  
Primary Gene

cDNA clones coding for the endoplasmic reticulum Ca2+-transport ATPase have been cloned from a pig smooth-muscle cDNA library. The transcripts can be divided into two classes which differ in their 3′ ends due to alternative splicing of the primary gene transcript. The class 1 cDNA encodes a protein of 997 amino acids (Mr 110,000). The class 2 protein (1042 amino acids; Mr 115,000) is completely identical to the class 1 protein except that the four C-terminal amino acids of the class 1 protein are replaced in the class 2 protein with a tail of 49 amino acids. Comparison of these sequences with other Ca2+ pump sequences reveals that the class 1 isoform corresponds to the sarcoplasmic reticulum Ca2+ pump of slow-twitch skeletal/cardiac muscle, whereas the class 2 protein corresponds to a Ca2+ pump recently detected in non-muscle tissues.

scholarly journals Transforming growth factor-β downregulates sGC subunit expression in pulmonary artery smooth muscle cells via MEK and ERK signaling

2019 ◽  
Vol 316 (1) ◽  
pp. L20-L34 ◽  
Author(s):  
Lili Du ◽  
Jesse D. Roberts
Keyword(s):  
Smooth Muscle ◽  
Lung Injury ◽  
Pulmonary Artery ◽  
Protein Expression ◽  
Erk Signaling ◽  
Type I ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Pulmonary Artery Smooth Muscle ◽  
Erk Activity

TGFβ activation during newborn lung injury decreases the expression of pulmonary artery smooth muscle cell (PASMC)-soluble guanylate cyclase (sGC), a critical mediator of nitric oxide signaling. Using a rat PASMC line (CS54 cells), we determined how TGFβ downregulates sGC expression. We found that TGFβ decreases sGC expression through stimulating its type I receptor; TGFβ type I receptor (TGFβR1) inhibitors prevented TGFβ-1-mediated decrease in sGCα1 subunit mRNA levels in the cells. However, TGFβR1-Smad mechanisms do not regulate sGC; effective knockdown of Smad2 and Smad3 expression and function did not protect sGCα1 mRNA levels during TGFβ-1 exposure. A targeted small-molecule kinase inhibitor screen suggested that MEK signaling regulates sGC expression in TGFβ-stimulated PASMC. TGFβ activates PASMC MEK/ERK signaling; CS54 cell treatment with TGFβ-1 increased MEK and ERK phosphorylation in a biphasic, time- and dose-dependent manner. Moreover, MEK/ERK activity appears to be required for TGFβ-mediated sGC expression inhibition in PASMC; MEK and ERK inhibitors protected sGCα1 mRNA expression in TGFβ-1-treated CS54 cells. Nuclear ERK activity is sufficient for sGC regulation; heterologous expression of a nucleus-retained, constitutively active ERK2-MEK1 fusion protein decreased CS54 cell sGCα1 mRNA levels. The in vivo relevance of this TGFβ-MEK/ERK-sGC downregulation pathway is suggested by the detection of ERK activation and sGCα1 protein expression downregulation in TGFβ-associated mouse pup hyperoxic lung injury, and the determination that ERK decreases sGCα1 protein expression in TGFβ-1-treated primary PASMC obtained from mouse pups. These studies identify MEK/ERK signaling as an important pathway by which TGFβ regulates sGC expression in PASMC.

Cytokines decrease sGC in pulmonary artery smooth muscle cells via NO-dependent and NO-independent mechanisms

2001 ◽  
Vol 280 (2) ◽  
pp. L272-L278 ◽  
Author(s):  
Masao Takata ◽  
Galina Filippov ◽  
Heling Liu ◽  
Fumito Ichinose ◽  
Stefan Janssens ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
No Production ◽  
Mrna Levels ◽  
Protein Levels ◽  
Subunit Mrna ◽  
Tnf Α ◽  
Pulmonary Artery Smooth Muscle

Exposure of rat pulmonary artery smooth muscle cells (rPASMC) to cytokines leads to nitric oxide (NO) production by NO synthase 2 (NOS2). NO stimulates cGMP synthesis by soluble guanylate cyclase (sGC), a heterodimer composed of α1- and β1-subunits. Prolonged exposure of rPASMC to NO decreases sGC subunit mRNA and protein levels. The objective of this study was to determine whether levels of NO produced endogenously by NOS2 are sufficient to decrease sGC expression in rPASMC. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) increased NOS2 mRNA levels and decreased sGC subunit mRNA levels. Exposure of rPASMC to IL-1β and TNF-α for 24 h decreased sGC subunit protein levels and NO-stimulated sGC enzyme activity.l- N 6-(1-iminoethyl)lysine (NOS2 inhibitor) or 1 H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (sGC inhibitor) partially prevented the cytokine-mediated decrease in sGC subunit mRNA levels. However, cytokines also decreased sGC subunit mRNA levels in PASMC derived from NOS2-deficient mice. These results demonstrate that levels of NO and cGMP produced in cytokine-exposed PASMC are sufficient to decrease sGC subunit mRNA levels. In addition, cytokines can decrease sGC subunit mRNA levels via NO-independent mechanisms.

Chronic intrauterine pulmonary hypertension selectively modifies pulmonary artery smooth muscle cell gene expression

2006 ◽  
Vol 290 (3) ◽  
pp. L426-L433 ◽  
Author(s):  
Ernesto Resnik ◽  
Jean Herron ◽  
Maggie Keck ◽  
David Sukovich ◽  
Bradley Linden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Mrna Levels ◽  
Α Subunit ◽  
Pulmonary Vasodilation ◽  
Pulmonary Artery Smooth Muscle

Pulmonary artery smooth muscle cell (PASMC) relaxation at birth results from an increase in cytosolic cGMP, cGMP-dependent and kinase-mediated activation of the Ca2+-sensitive K+ channel (KCa), and closure of voltage-operated Ca2+ channels (VOCC). How chronic intrauterine pulmonary hypertension compromises perinatal pulmonary vasodilation remains unknown. We tested the hypothesis that chronic intrauterine pulmonary hypertension selectively modifies gene expression to mitigate perinatal pulmonary vasodilation mediated by the cGMP kinase-KCa-VOCC pathway. PASMC were isolated from late-gestation fetal lambs that had undergone either ligation of the ductus arteriosus (hypertensive) or sham operation (control) at 127 days of gestation and were maintained under either hypoxic (∼25 Torr) or normoxic (∼120 Torr) conditions in primary culture. We studied mRNA levels for cGMP kinase Iα (PKG-1α), the α-chain of VOCC (Cav1.2), and the α-subunit of the KCa channel. Compared with control PASMC, hypertensive PASMC had decreased VOCC, KCa, and PKG-1α expression. In response to sustained normoxia, expression of VOCC and KCa channel decreased and expression of PKG-1α increased. In contrast, sustained normoxia had no effect on PKG-1α levels and an attenuated effect on VOCC and KCa channel expression in hypertensive PASMC. Protein expression of PKG-1α was consistent with the mRNA data. We conclude that chronic intrauterine pulmonary hypertension decreases PKG expression and mitigates the genetic effects of sustained normoxia on pulmonary vasodilation, because gene expression remains compromised even after sustained exposure to normoxia.

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