Microtubule assembly is regulated by externally applied strain in cultured smooth muscle cells

1998 ◽  
Vol 111 (22) ◽  
pp. 3379-3387 ◽  
Author(s):  
A.J. Putnam ◽  
J.J. Cunningham ◽  
R.G. Dennis ◽  
J.J. Linderman ◽  
D.J. Mooney
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Cellular Protein ◽  
Bovine Brain ◽  
Applied Strain ◽  
Microtubule Assembly ◽  
Mechanical Forces ◽  
Mechanical Stimuli

Mechanical forces clearly regulate the development and phenotype of a variety of tissues and cultured cells. However, it is not clear how mechanical information is transduced intracellularly to alter cellular function. Thermodynamic modeling predicts that mechanical forces influence microtubule assembly, and hence suggest microtubules as one potential cytoskeletal target for mechanical signals. In this study, the assembly of microtubules was analyzed in rat aortic smooth muscle cells cultured on silicon rubber substrates exposed to step increases in applied strain. Cytoskeletal and total cellular protein fractions were extracted from the cells following application of the external strain, and tubulin levels were quantified biochemically via a competitive ELISA and western blotting using bovine brain tubulin as a standard. In the first set of experiments, smooth muscle cells were subjected to a step-increase in strain and the distribution of tubulin between monomeric, polymeric, and total cellular pools was followed with time. Microtubule mass increased rapidly following application of the strain, with a statistically significant increase (P<0.05) in microtubule mass from 373+/−32 pg/cell (t=0) to 514+/−30 pg/cell (t=15 minutes). In parallel, the amount of soluble tubulin decreased approximately fivefold. The microtubule mass decreased after 1 hour to a value of 437+/−24 pg/cell. In the second set of experiments, smooth muscle cells were subjected to increasing doses of externally applied strain using a custom-built strain device. Monomeric, polymeric, and total tubulin fractions were extracted after 15 minutes of applied strain and quantified as for the earlier experiments. Microtubule mass increased with increasing strain while total cellular tubulin levels remained essentially constant at all strain levels. These findings are consistent with a thermodynamic model which predicts that microtubule assembly is promoted as a cell is stretched and compressional loads on the microtubules are presumably relieved. Furthermore, these data suggest microtubules are a potential target for translating changes in externally applied mechanical stimuli to alterations in cellular phenotype.

scholarly journals Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

1982 ◽  
Vol 92 (2) ◽  
pp. 462-470 ◽  
Author(s):  
E Schwartz ◽  
R S Bienkowski ◽  
B Coltoff-Schiller ◽  
S Goldfischer ◽  
O O Blumenfeld
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Time Course ◽  
Cultured Cells ◽  
Growth Parameters ◽  
Muscle Cells ◽  
Cellular Protein ◽  
Collagen Accumulation ◽  
The Matrix

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

Binding, degradation, and biological activity of insulin in vascular smooth muscle cells

1985 ◽  
Vol 249 (3) ◽  
pp. E292-E298
Author(s):  
N. Kaiser ◽  
A. Tur-Sinai ◽  
M. Hasin ◽  
E. Cerasi
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Insulin Binding ◽  
Maximal Response ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Igf I

The interaction of insulin with the vascular smooth muscle was studied using cultures derived from the bovine aortic arch. The cultured cells exhibited specific binding of 125I-insulin that was reversible and dependent on pH. Both insulin and insulinlike growth factor (IGF) I competed for 125I-insulin binding; IGF I, however, was less effective than insulin by at least an order of magnitude. Insulin binding was accompanied by internalization and degradation of the hormone in a temperature- and time-dependent manner. Chloroquine and other lysosomotropic agents elevated the internalized insulin and reduced its degradation. Pre-exposure of cell cultures to insulin resulted in downregulation of cell surface receptors. Insulin stimulated alpha-aminoisobutyric acid transport in confluent smooth muscle cells. The maximal response was observed at 100 ng/ml insulin with a half-maximal effect at 10 ng/ml. Sparse, serum-starved smooth muscle cells responded to insulin with a dose-dependent increase in [3H]-thymidine incorporation into DNA. Although the effect was already apparent at 1 ng/ml insulin, it reached near maximal level only at 10,000 ng/ml. IGF I also stimulated DNA synthesis in smooth muscle cells; however, at low concentrations insulin was more efficient in this respect. Human growth hormone was inactive. The data indicate the presence of specific receptors for insulin in bovine aortic smooth muscle cells. These receptors appear to mediate the metabolic activity as well as part of the mitogenic effect of insulin in these cells.

Mechanotransduction in gastrointestinal smooth muscle cells: Role of mechanosensitive ion channels

2021 ◽  
Author(s):  
Vikram Joshi ◽  
Peter R Strege ◽  
Gianrico Farrugia ◽  
Arthur Beyder
Keyword(s):  
Smooth Muscle ◽  
Ion Channels ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Tissue Level ◽  
Cytoskeletal Proteins ◽  
Cell Junction ◽  
Mechanical Stimuli ◽  
Junction Molecules

Mechanosensation, the ability to properly sense mechanical stimuli and transduce them into physiologic responses, is an essential determinant of gastrointestinal (GI) function. Abnormalities in this process result in highly prevalent GI functional and motility disorders. In the GI tract, several cell types sense mechanical forces and transduce them into electrical signals, which elicit specific cellular responses. Some mechanosensitive cells like sensory neurons act as specialized mechanosensitive cells that detect forces and transduce signals into tissue-level physiologic reactions. Non-specialized mechanosensitive cells like smooth muscle cells (SMCs) adjust their function in response to forces. Mechanosensitive cells utilize various mechanoreceptors and mechanotransducers. Mechanoreceptors detect and convert force into electrical and biochemical signals, and mechanotransducers amplify and direct mechanoreceptor responses. Mechanoreceptors and mechanotransducers include ion channels, specialized cytoskeletal proteins, cell junction molecules, and G-protein coupled receptors. SMCs are particularly important due to their role as final effectors for motor function. Myogenic reflex-the ability of smooth muscle to contract in response to stretch rapidly-is a critical smooth muscle function. Such rapid mechanotransduction responses rely on mechano-gated and -sensitive ion channels, which alter their ion pores' opening in response to force, allowing fast electrical and Ca2+ responses. Though GI SMCs express a variety of such ion channels, their identities remain unknown. Recent advancements in electrophysiological, genetic, in vivo imaging, and multi-omic technologies broaden our understanding of how SMC mechano-gated and -sensitive ion channels regulate GI functions. This review discusses GI SMC mechanosensitivity's current developments with a particular emphasis on mechano-gated and -sensitive ion channels.

ETA and ETB receptors on single smooth muscle cells cooperate in mediating guinea pig tracheal contraction

1994 ◽  
Vol 266 (2) ◽  
pp. L113-L124 ◽  
Author(s):  
T. Inui ◽  
A. F. James ◽  
Y. Fujitani ◽  
M. Takimoto ◽  
T. Okada ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Dissociation Constants ◽  
Muscle Tension ◽  
Muscle Cells ◽  
Endothelin A

We investigated the distribution of endothelin A (ETA) and ETB receptors in single smooth muscle cells and their contribution to ET-induced contractions of guinea pig trachea. ETA and ETB receptors were detected in smooth muscle membranes (maximum binding capacities of 810 and 360 fmol/mg protein and dissociation constants of 38 and 5.1 pM for 125I-labeled ET-1 and 125I-ET-3, respectively) and visualized autoradiographically in primary cultured cells. ET-1 and ET-3 evoked concentration-dependent increases in intracellular Ca2+ concentration and smooth muscle tension. The half-maximally effective concentrations of ET-1 and ET-3 at inducing contractions were 1.9 and 2.7 nM, respectively. The Ca2+ responses showed tachyphylaxis to both ETs after stimulation with ET-1, but only to ET-3 after stimulation with ET-3. Consecutive applications of ET-3 and ET-1 (10 nM each) classified the cells into ETA dominant (approximately 30%) responding to only ET-1, ETB dominant (approximately 20%) responding to only ET-3, and ETA- and ETB-possessing (approximately 50%) cells responding to both. The ETA antagonist, 10 microM BQ-123, attenuated ET-1-induced contractions but did not affect the ET-3-induced contractions. The results indicate that both receptors coexist in a major population of smooth muscle cells and cooperate in mediating ET-1-induced contractions.

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

1997 ◽  
Vol 273 (4) ◽  
pp. C1250-C1258 ◽  
Author(s):  
Ashok K. Grover ◽  
Sue E. Samson
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Contractile Function ◽  
Ischemia Reperfusion ◽  
Muscle Cells ◽  
Intact Cells ◽  
Serca Pump

We examined the effects of peroxide on the sarco(endo)plasmic reticulum Ca2+ (SERCA) pump in pig coronary artery endothelium and smooth muscle at three organizational levels: Ca2+ transport in permeabilized cells, cytosolic Ca2+ concentration in intact cells, and contractile function of artery rings. We monitored the ATP-dependent, azide-insensitive, oxalate-stimulated45Ca2+uptake by saponin-permeabilized cultured cells. Low concentrations of peroxide inhibited the uptake less effectively in endothelium than in smooth muscle whether we added the peroxide directly to the Ca2+ uptake solution or treated intact cells with peroxide and washed them before the permeabilization. An acylphosphate formation assay confirmed the greater resistance of the SERCA pump in endothelial cells than in smooth muscle cells. Pretreating smooth muscle cells with 300 μM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic Ca2+ concentration in a Ca2+-free solution, but it did not affect the endothelial cells. Peroxide pretreatment inhibited the CPA-induced contraction in deendothelialized arteries with a 50% inhibitory concentration of 97 ± 13 μM, but up to 500 μM peroxide did not affect the endothelium-dependent, CPA-induced relaxation. Similarly, 500 μM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced, endothelium-dependent relaxation by only 40 ± 13%. The greater resistance of the endothelium to reactive oxygen may be important during ischemia-reperfusion or in the postinfection immune response.

Effect of hypertensive rat plasma on ion transport of cultured vascular smooth muscle

1986 ◽  
Vol 251 (5) ◽  
pp. H984-H990
Author(s):  
W. W. Magargal ◽  
H. W. Overbeck
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Sodium Intake ◽  
Muscle Cells ◽  
Rat Plasma ◽  
Sodium Content ◽  
Chronic Hypertension ◽  
Plasma Factors

We layered fresh, unprocessed plasma from healthy rats with early (less than or equal to 7 days) or benign, chronic (greater than 3 wk) one-kidney, one-clip hypertension and from paired one-kidney normotensive control rats over confluent primary-cultured rat aortic smooth muscle cells. Plasma from all rats increased cellular ouabain-sensitive 86Rb+ uptake and sodium content and decreased ouabain-insensitive 86Rb+ uptake compared with uptakes and content in the presence of balanced salt solution (P less than 0.01). Cells incubated in the presence of plasma from rats with early (P less than 0.02) or chronic hypertension (P less than 0.01) had significantly reduced ouabain-sensitive 86Rb+ uptake when compared with cells incubated in normotensive plasma, but their intracellular Na+ contents were not lower. We no longer detected this uptake difference when chronic hypertensives drank 0.9% NaCl instead of water. Plasma from hypertensive rats also altered ouabain-insensitive 86Rb+ uptake by the cultured cells. These findings of this new, reproducible, and specific assay system support the hypothesis that plasma factors inhibit the membrane sodium-potassium pump in vascular smooth muscle cells in this form of hypertension. The abnormality occurs in both early and chronic stages, but may not be related to sodium intake. The data also provide evidence for plasma factors in hypertension altering membrane K+ permeability.

Vascular smooth muscle cells' response to mechanical stimuli

2003 ◽  
Vol 2003.7 (0) ◽  
pp. 75-76
Author(s):  
Shigeru TADA ◽  
Tomoyuki HATAKEYAMA ◽  
Ken OKAZAKI
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Mechanical Stimuli

scholarly journals Mechanical stretch is a highly selective regulator of gene expression in human bladder smooth muscle cells

2004 ◽  
Vol 20 (1) ◽  
pp. 36-44 ◽  
Author(s):  
Rosalyn M. Adam ◽  
Samuel H. Eaton ◽  
Carlos Estrada ◽  
Ashish Nimgaonkar ◽  
Shu-Ching Shih ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Smooth Muscle Cells ◽  
Ex Vivo ◽  
Muscle Cells ◽  
Mechanical Stimuli ◽  
Bladder Smooth Muscle ◽  
Human Bladder ◽  
Cox 2

Application of mechanical stimuli has been shown to alter gene expression in bladder smooth muscle cells (SMC). To date, only a limited number of “stretch-responsive” genes in this cell type have been reported. We employed oligonucleotide arrays to identify stretch-sensitive genes in primary culture human bladder SMC subjected to repetitive mechanical stimulation for 4 h. Differential gene expression between stretched and nonstretched cells was assessed using Significance Analysis of Microarrays (SAM). Expression of 20 out of 11,731 expressed genes (∼0.17%) was altered >2-fold following stretch, with 19 genes induced and one gene (FGF-9) repressed. Using real-time RT-PCR, we tested independently the responsiveness of 15 genes to stretch and to platelet-derived growth factor-BB (PDGF-BB), another hypertrophic stimulus for bladder SMC. In response to both stimuli, expression of 13 genes increased, 1 gene (FGF-9) decreased, and 1 gene was unchanged. Six transcripts (HB-EGF, BMP-2, COX-2, LIF, PAR-2, and FGF-9) were evaluated using an ex vivo rat model of bladder distension. HB-EGF, BMP-2, COX-2, LIF, and PAR-2 increased with bladder stretch ex vivo, whereas FGF-9 decreased, consistent with expression changes observed in vitro. In silico analysis of microarray data using the FIRED algorithm identified c-jun, AP-1, ATF-2, and neurofibromin-1 (NF-1) as potential transcriptional mediators of stretch signals. Furthermore, the promoters of 9 of 13 stretch-responsive genes contained AP-1 binding sites. These observations identify stretch as a highly selective regulator of gene expression in bladder SMC. Moreover, they suggest that mechanical and growth factor signals converge on common transcriptional regulators that include members of the AP-1 family.

scholarly journals Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells

1987 ◽  
Vol 248 (1) ◽  
pp. 237-242 ◽  
Author(s):  
J P Slotte ◽  
E L Bierman
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Human Skin ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Arterial Smooth Muscle Cells

The spontaneous turnover of plasma-membrane sterols, as measured by their transfer to the endoplasmic reticulum, was measured in quiescent cultured human skin fibroblasts and monkey arterial smooth-muscle cells. The plasma-membrane sterol pool was pulse-labelled with trace amounts of either [3H]desmosterol or [3H]cholesterol. We then measured the enzymic conversion of [3H]desmosterol into [3H]cholesterol and of [3H]cholesterol into [3H]cholesteryl esters in intact cells. Depending on the probe used, markedly different transfer or conversion rates were found in these cells. In quiescent human skin fibroblasts, incubated in a serum-free medium, about 1.1% of the plasma-membrane [3H]desmosterol was converted into [3H]cholesterol/h, whereas in monkey arterial smooth-muscle cells the corresponding rate was 0.4%. Under similar experimental conditions, these cells esterified less than 0.02% (fibroblasts) and 0.12% (smooth-muscle cells) of the plasma-membrane [3H]cholesterol/h. The movement of sterols from the plasma membrane to the endoplasmic reticulum, as measured by the conversion of [3H]desmosterol into [3H]cholesterol was not blocked by colchicine, but was markedly enhanced by 3% (w/v) dimethyl sulphoxide. In all, these results indicate that plasma-membrane sterols of cultured cells are continuously transferred to the interior of the cell at a rate substantially higher than previously appreciated. This turnover of plasma-membrane sterol molecules took place even when there was no mass transfer of sterols into the cells.

Characterization of colonic circular smooth muscle cells in culture

1992 ◽  
Vol 263 (3) ◽  
pp. G365-G370 ◽  
Author(s):  
H. S. Ennes ◽  
J. A. McRoberts ◽  
P. E. Hyman ◽  
W. J. Snape
Keyword(s):  
Smooth Muscle ◽  
Adenylate Cyclase ◽  
Smooth Muscle Cells ◽  
Primary Culture ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Intracellular Camp ◽  
Isolated Cells ◽  
Vip Receptors ◽  
Circular Smooth Muscle

The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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