scholarly journals Microinjection of nonmuscle and smooth muscle caldesmon into fibroblasts and muscle cells.

1990 ◽  
Vol 111 (6) ◽  
pp. 2487-2498 ◽  
Author(s):  
Y Yamakita ◽  
S Yamashiro ◽  
F Matsumura
Keyword(s):  
Smooth Muscle ◽  
Molecular Mass ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Biochemical Properties ◽  
Stress Fibers ◽  
Relative Molecular Mass ◽  
Short Period ◽  
Nonmuscle Cells ◽  
Mass Form

Caldesmon is present in a high molecular mass form in smooth muscle and predominantly in a low molecular mass form in nonmuscle cells. Their biochemical properties are very similar. To examine whether these two forms of caldesmon behave differently in cultured cells, we microinjected fluorescently labeled smooth muscle and nonmuscle caldesmons into fibroblasts. Simultaneous injection of both caldesmons into the same cells has revealed that both high and low relative molecular mass caldesmons are quickly (within 10 min) and stably (over 3 d) incorporated into the same structures of microfilaments including stress fibers and membrane ruffles, suggesting that nonmuscle cells do not distinguish nonmuscle caldesmon from smooth muscle caldesmon. The effect of calmodulin on the incorporation of caldesmon has been examined by coinjection of caldesmon with calmodulin. We have found that calmodulin retards the incorporation of caldesmon into stress fibers for a short period (10 min) but not for a longer incubation (30 min). The behavior of caldesmon in developing muscle cells was also examined because we previously observed that caldesmon disappears during myogenesis (Yamashiro, S., R. Ishikawa, and F. Matsumura. 1988. Protoplasma Suppl. 2: 9-21). We have found that, in contrast to its stable incorporation into stress fibers of fibroblasts, caldesmon is unable to be incorporated into thin filament structure (I-band) of differentiated muscle.

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.

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.

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.

A 90-kDa surface antigen of immature smooth muscle cells is ICAM-1

1991 ◽  
Vol 261 (4) ◽  
pp. L21-L22
Author(s):  
O. Yu. Printseva ◽  
M. M. Peclo ◽  
A. V. Tjurmin ◽  
A. M. Gown
Keyword(s):  
Monoclonal Antibody ◽  
Smooth Muscle ◽  
Monoclonal Antibodies ◽  
Smooth Muscle Cells ◽  
Molecular Mass ◽  
Surface Antigen ◽  
Muscle Cells ◽  
Human Aorta ◽  
Long Term Culture

A monoclonal antibody, designated 10F3, that reacts with an antigen of molecular mass 90,000 Da has been developed by immunization of BALB/c mice with smooth muscle cells in long-term culture. The cells were originally isolated from fetal human aorta. The 10F3 was identified as an antibody that reacts with the ICAM-1 molecule. ICAM-1 is a mesenchymal antigen that is lost during differentiation of cells other than endothelium but is reexpressed by the intimal cells of vessels involved in atherogenesis. atherosclerosis; monoclonal antibodies

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.

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