scholarly journals Role of p47phox in regulating Cdc42GAP, vimentin, and contraction in smooth muscle cells

2009 ◽  
Vol 297 (6) ◽  
pp. C1424-C1433 ◽  
Author(s):  
Qing-Fen Li ◽  
Dale D. Tang
Keyword(s):  
Hydrogen Peroxide ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Contraction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Regulatory Subunit ◽  
Ros Generation ◽  
Vitro Assay

Cdc42GAP (GTPase activating protein) has been shown to regulate smooth muscle contraction as well as cell motility, adhesion, proliferation, and apoptosis. We have recently shown that Cdc42GAP activity is suppressed in smooth muscle cells during contractile activation, which is reversed by inhibitors of reactive oxygen species (ROS). Because p47phox, a regulatory subunit of NAD(P)H oxidase, has been implicated in smooth muscle signaling, we determined whether this subunit modulates Cdc42GAP activity in response to contractile stimulation. Transfection of smooth muscle cells with plasmids encoding short hairpin RNA (shRNA) against p47phox, but not plasmids for luciferase shRNA, inhibited the expression of p47phox. ROS production and the suppression of Cdc42GAP activity in response to stimulation with 5-hydroxytryptamine (5-HT) were attenuated in cells producing p47phox shRNA compared with cells producing luciferase shRNA. In contrast, the addition of hydrogen peroxide to p47phox-deficient cells suppressed the activity of Cdc42GAP. Furthermore, exposure to hydrogen peroxide led to a decrease in Cdc42GAP activity in an in vitro assay. Cdc42 activation, p21-activated kinase 1 (PAK1) phosphorylation at Thr-423 (an indication of PAK activation), and vimentin phosphorylation at Ser-56 in response to 5-HT activation were also attenuated in smooth muscle cells producing shRNA against p47phox. The knockdown of p47phox inhibited smooth muscle contraction during stimulation with 5-HT but not hydrogen peroxide. These results suggest that the p47phox subunit of NAD(P)H oxidase may mediate the agonist-induced GAP suppression by controlling ROS generation in smooth muscle cells during agonist stimulation. p47phox-regulated GAP affects smooth muscle contraction likely through the Cdc42/PAK1/vimentin pathway.

Agonist-induced association of tropomyosin with protein kinase Cα in colonic smooth muscle

2005 ◽  
Vol 288 (2) ◽  
pp. G268-G276 ◽  
Author(s):  
Sita Somara ◽  
Haiyan Pang ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Regulatory Protein ◽  
Direct Interaction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Particulate Fraction ◽  
Calcium Dependent

Smooth muscle contraction regulated by myosin light chain phosphorylation is also regulated at the thin-filament level. Tropomyosin, a thin-filament regulatory protein, regulates contraction by modulating actin-myosin interactions. Present investigation shows that acetylcholine induces PKC-mediated and calcium-dependent phosphorylation of tropomyosin in colonic smooth muscle cells. Our data also shows that acetylcholine induces a significant and sustained increase in PKC-mediated association of tropomyosin with PKCα in the particulate fraction of colonic smooth muscle cells. Immunoblotting studies revealed that in colonic smooth muscle cells, there is no significant change in the amount of tropomyosin or actin in particulate fraction in response to acetylcholine, indicating that the increased association of tropomyosin with PKCα in the particulate fraction may be due to acetylcholine-induced translocation of PKCα to the particulate fraction. To investigate whether the association of PKCα with tropomyosin was due to a direct interaction, we performed in vitro direct binding assay. Tropomyosin cDNA amplified from colonic smooth muscle mRNA was expressed as GST-tropomyosin fusion protein. In vitro binding experiments using GST-tropomyosin and recombinant PKCα indicated direct interaction of tropomyosin with PKCα. PKC-mediated phosphorylation of tropomyosin and direct interaction of PKCα with tropomyosin suggest that tropomyosin could be a substrate for PKC. Phosphorylation of tropomyosin may aid in holding the slided tropomyosin away from myosin binding sites on actin, resulting in actomyosin interaction and sustained contraction.

scholarly journals Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKCα

2004 ◽  
Vol 286 (4) ◽  
pp. G635-G644 ◽  
Author(s):  
Suresh B. Patil ◽  
Mercy D. Pawar ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Contraction ◽  
Membrane Fraction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Cell Length ◽  
Sustained Contraction ◽  
Hsp 27

Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 ± 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 ± .9% decreases in cell length ( n = 4)]. Furthermore, in 3D cells, translocation of both PKCα and of RhoA was greatly enhanced and resulted in a greater association of PKCα-RhoA in the membrane fraction. In 3G transfected cells, PKCα and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKCα-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKCα and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKCα-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.

Insulin acutely increases agonist-induced airway smooth muscle contraction in human and rat

2021 ◽  
Author(s):  
Becky J. Proskocil ◽  
Gina N. Calco ◽  
Zhenying Nie
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Contraction ◽  
Intracellular Calcium ◽  
Mrna Expression ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Rat Tracheal Smooth Muscle ◽  
Isolated Rat

Obesity increases incidence and severity of asthma but the molecular mechanisms are not completely understood. Hyperinsulinemia potentiates vagally induced bronchoconstriction in obese rats. Since bronchoconstriction results from airway smooth muscle contraction, we tested whether insulin changed agonist-induced airway smooth muscle contraction. Obesity prone and resistant rats were fed a low-fat diet for 5 weeks and treated with insulin (Lantus, 3 units/rat s.c.) 16 h before vagally induced bronchoconstriction was measured. Ex vivo, contractile responses to methacholine were measured in isolated rat tracheal rings and human airway smooth muscle strips before and after incubation (0.5 - 2 h) with 100 nM insulin or 13.1 nM insulin like growth factor-1 (IGF-1). M2 and M3 muscarinic receptor mRNA expression was quantified by qRT-PCR and changes in intracellular calcium were measured in response to methacholine or serotonin in isolated rat tracheal smooth muscle cells treated with 1 µM insulin. Insulin, administered to animals 16 h prior, potentiated vagally induced bronchoconstriction in both obese prone and resistant rats. Insulin, not IGF-1, significantly increased methacholine-induced contraction of rat and human isolated airway smooth muscle. In cultured rat tracheal smooth muscle cells, insulin significantly increased M2, not M3, mRNA expression and enhanced methacholine- and serotonin-induced increase in intracellular calcium. Insulin alone did not cause an immediate increase in intracellular calcium. Thus, insulin, acutely potentiated agonist-induced increase in intracellular calcium and airway smooth muscle contraction. These findings may explain why obese individuals with hyperinsulinemia are prone to airway hyperreactivity and give insights into future targets for asthma treatment.

scholarly journals 1-O-alkyl-2-acetyl-sn-glycerol: a platelet-activating factor metabolite with biological activity in vascular smooth muscle cells.

1989 ◽  
Vol 1 (1) ◽  
pp. 13-25 ◽  
Author(s):  
L L Stoll ◽  
P H Figard ◽  
N R Yerram ◽  
M A Yorek ◽  
A A Spector
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Platelet Activating Factor ◽  
Ether Lipid ◽  
Muscle Cells ◽  
Biologically Active ◽  
Vitro Assay

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent vasoactive ether lipid produced by activated blood cells and endothelial cells. Vascular smooth muscle cells partially convert exogenous PAF to 1-O-alkyl-2-acetyl-sn-glycerol (AAG), a biologically active diacylglycerol analogue. AAG is formed rapidly (less than 15 s) after exposure of the smooth muscle cells and does not appear to be a substrate for diacylglycerol kinase in these cells. Although most of the compound is metabolized to 1-O-alkyl-sn-glycerol, a small quantity remains as AAG for greater than or equal to 6 h. AAG inhibits phorbol ester binding, and it is as effective an activator of protein kinase C as diolein in an in vitro assay. Furthermore, AAG and PAF produce the same pattern of effects on smooth muscle cell proliferation. These observations suggest that at least some of the actions of PAF in vascular smooth muscle may be mediated through the formation of AAG, a stable, bioactive metabolite that appears to function as a diacylglycerol analogue.

Direct association and translocation of PKC-α with calponin

2004 ◽  
Vol 286 (6) ◽  
pp. G954-G963 ◽  
Author(s):  
Suresh B. Patil ◽  
Mercy D. Pawar ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Western Blot ◽  
Smooth Muscle Cells ◽  
Direct Interaction ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Direct Association ◽  
Translocated Proteins

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-α and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-α. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-α in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-α, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351–972 corresponding to amino acids 92–229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX -KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-α in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-α, indicating a direct association of GST-calponin with PKC-α. These data suggest that there is a direct association of translocated calponin and PKC-α in the membrane and a role for the complex calponin-PKC-α-HSP27, in contraction of colonic smooth muscle cells.

Diacylglycerol metabolism in isolated aortic smooth muscle cells

1989 ◽  
Vol 256 (1) ◽  
pp. C11-C17 ◽  
Author(s):  
D. L. Severson ◽  
M. Hee-Cheong
Keyword(s):  
Smooth Muscle ◽  
Phosphatidic Acid ◽  
Smooth Muscle Cells ◽  
Total Phospholipid ◽  
Muscle Cells ◽  
Vitro Assay ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Pathway Formation

The metabolism of the cell-permeable diacylglycerol (DG) analogue, 1,2-dioctanoyl-sn-glycerol (diC8), by isolated intact smooth muscle cells from rabbit aorta was determined. Radiolabeled diC8 was rapidly converted to monoacylglycerol, with smaller amounts of radioactivity recovered as free glycerol and in the total phospholipid fraction that included phosphatidic acid. The greater metabolism of diC8 by the lipase pathway (formation of monoacylglycerol and glycerol) as compared with the kinase pathway (formation of phosphatidic acid and other phospholipids) was observed consistently when experimental conditions were changed by varying the time of incubation (5-30 min), diC8 concentration (0.5-50 microM), and the content of smooth muscle cells in the incubation (0.5-5 X 10(5) cells). These results with the metabolism of exogenous diC8 by intact smooth muscle cells are consistent with in vitro determinations of DG kinase and lipase activities, where lipase activity (especially hydrolysis of the sn-1 position to yield the monoacylglycerol intermediate) was much greater than kinase activity measured with both soluble and particulate subcellular fractions from aortic smooth muscle cells. Both DG kinase and lipase activities were inhibited when 1-monoolein was added to the in vitro assay. Treatment of intact smooth muscle cells with 1-monoolein (200 microM), however, did not reduce the formation of monoacylglycerol from diC8.

scholarly journals Cdc42GAP, reactive oxygen species, and the vimentin network

2009 ◽  
Vol 297 (2) ◽  
pp. C299-C309 ◽  
Author(s):  
Qing-Fen Li ◽  
Amy M. Spinelli ◽  
Dale D. Tang
Keyword(s):  
Reactive Oxygen Species ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Reactive Oxygen ◽  
Muscle Cells ◽  
Oxygen Species ◽  
Wild Type ◽  
Vitro Assay ◽  
Contractile Activation

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.

scholarly journals Paraoxon Attenuates Vascular Smooth Muscle Contraction through Inhibiting Ca2+Influx in the Rabbit Thoracic Aorta

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Shouhong Zhou ◽  
Liying Liu ◽  
Xuhong Yang ◽  
Shujin Wu ◽  
Gengrong Chen
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Contraction ◽  
Thoracic Aorta ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Inhibitory Effect ◽  
Aortic Smooth Muscle ◽  
Vascular Smooth Muscle Contraction

We investigated the effect of paraoxon on vascular contractility using organ baths in thoracic aortic rings of rabbits and examined the effect of paraoxon on calcium homeostasis using a whole-cell patch-clamp technique in isolated aortic smooth muscle cells of rabbits. The findings show that administration of paraoxon (30 μM) attenuated thoracic aorta contraction induced by phenylephrine (1 μM) and/or a highK+environment (80 mM) in both the presence and absence of thoracic aortic endothelium. This inhibitory effect of paraoxon on vasoconstrictor-induced contraction was abolished in the absence of extracellularCa2+, or in the presence of theCa2+channel inhibitor, verapamil. But atropine had little effect on the inhibitory effect of paraoxon on phenylephrine-induced contraction. Paraoxon also attenuated vascular smooth muscle contraction induced by the cumulative addition of CaCl2and attenuated an increase of intracellularCa2+concentration induced byK+in vascular smooth muscle cells. Moreover, paraoxon (30 μM) inhibited significantly L-type calcium current in isolated aortic smooth muscle cells of rabbits. In conclusion, our results demonstrate that paraoxon attenuates vasoconstrictor-induced contraction through inhibitingCa2+influx in the rabbits thoracic aorta.

scholarly journals Direct association of calponin with specific domains of PKC-α

2008 ◽  
Vol 295 (6) ◽  
pp. G1246-G1254 ◽  
Author(s):  
Sita Somara ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Assay ◽  
Active Form ◽  
Muscle Cells ◽  
Smooth Muscle Contraction ◽  
Full Length ◽  
Direct Association

Calponin contributes to the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated Mg-ATPase activity of phosphorylated myosin. Previous studies have shown that the contractile agonist acetylcholine induced a direct association of translocated calponin and PKC-α in the membrane. In the present study, we have determined the domain of PKC-α involved in direct association with calponin. In vitro binding assay was carried out by incubating glutathione S-transferase-calponin aa 92-229 with His-tagged proteins of individual domains and different combinations of domains of PKC-α. Calponin was found to bind directly to the full-length PKC-α. Calponin bound to C2 and C4 domains but not to C1 and C3 domains of PKC-α. When incubated with proteins of different combination of domains, calponin bound to C2-C3, C3-C4, and C2-C3-C4 but not to C1-C2 or C1-C2-C3. To determine whether these in vitro bindings mimic the in vivo associations, and in vivo binding assay was performed by transfecting colonic smooth muscle cells with His-tagged proteins of individual domains and different combinations of domains of PKC-α. Coimmunoprecipitation of calponin with His-tagged truncated forms of PKC-α showed that C1-C2, C1-C2-C3, C2-C3, and C3-C4 did not associate with calponin. Calponin associated only with full-length PKC-α and with C2-C3-C4 in cells in the resting state, and this association increased upon stimulation with acetylcholine. These data suggest that calponin bound to fragments that may mimic the active form of PKC-α and that the functional association of PKC-α with calponin requires both C2 and C4 domains during contraction of colonic smooth muscle cells.

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