In vitro assay of invasion using endothelial and smooth muscle cells

1982 ◽  
pp. 251-266
Author(s):  
P. A. Jones
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
In Vitro Assay ◽  
Vitro Assay

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.

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 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.

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.

Epicardial Cells of Human Adults Can Undergo an Epithelial-to-Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro

Stem Cells ◽  
2007 ◽  
Vol 25 (2) ◽  
pp. 271-278 ◽  
Author(s):  
John van Tuyn ◽  
Douwe E. Atsma ◽  
Elizabeth M. Winter ◽  
Ietje van der Velde-van Dijke ◽  
Daniel A. Pijnappels ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Epithelial To Mesenchymal Transition ◽  
Muscle Cells ◽  
Mesenchymal Transition ◽  
Epicardial Cells ◽  
Human Adults

Characterization and confocal imaging of calponin in gastrointestinal smooth muscle

1993 ◽  
Vol 265 (5) ◽  
pp. C1371-C1378 ◽  
Author(s):  
M. P. Walsh ◽  
J. D. Carmichael ◽  
G. J. Kargacin
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Thin Filament ◽  
Muscle Cells ◽  
Biochemical Properties ◽  
Confocal Imaging ◽  
Isolated Cells ◽  
Independent Manner

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.

Light and dark smooth muscle cells in estrogen-stimulated rat myometrium

1976 ◽  
Vol 54 (6) ◽  
pp. 822-833 ◽  
Author(s):  
R. E. Garfield ◽  
E. E. Daniel
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Atp Depletion ◽  
Metabolic Inhibitors ◽  
Volume Control ◽  
Control Mechanisms ◽  
Dark Cells ◽  
Light Cells

Smooth muscle cells of different densities to transmission of electrons (termed light and dark cells) were found in rat myometrium examined in the electron microscope following fixation by immersion in glutaraldehyde. Light cells accounted for about 4% of the total population of cells. No light cells were found in tissues fixed in situ by intraarterial perfusion with glutaraldehyde. In addition to staining differences, light cells were distinguished from most dark cells by differences in nuclear, mitochondrial, endoplasmic reticular, and surface structures. The relative number of light and dark cells after in vitro fixation was not changed in tissues relaxed with adrenaline or contracted with oxytocin. Mechanical injury resulted in increased numbers of light cells. Similarly, chemical injury with metabolic inhibitors resulted in ATP depletion, followed by increased numbers of light cells and gain in water content. We concluded that light cells were produced by mechanical or metabolic damage, leading to loss of volume control mechanisms, swelling, and leakage of protein. Light cells found after fixation in vitro in numerous prior studies represent cells damaged during isolation, and not a physiological variant among smooth muscle cells.

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