Selected Contribution: Time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells

2001 ◽  
Vol 91 (2) ◽  
pp. 986-994 ◽  
Author(s):  
Ben Fabry ◽  
Geoffrey N. Maksym ◽  
Stephanie A. Shore ◽  
Paul E. Moore ◽  
Reynold A. Panettieri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Time Course ◽  
Detection System ◽  
Loss Modulus ◽  
Cell Stiffness ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Cells In Culture ◽  
Human Airway

We measured the time course and heterogeneity of responses to contractile and relaxing agonists in individual human airway smooth muscle (HASM) cells in culture. To this end, we developed a microrheometer based on magnetic twisting cytometry adapted with a novel optical detection system. Ferromagnetic beads (4.5 μm) coated with Arg-Gly-Asp peptide were bound to integrins on the cell surface. The beads were twisted in a sinusoidally varying magnetic field at 0.75 Hz. Oscillatory bead displacements were recorded using a phase-synchronized video camera. The storage modulus (cell stiffness; G′), loss modulus (friction; G"), and hysteresivity (η; ratio of G" to G′) could be determined with a time resolution of 1.3 s. Within 5 s after addition of histamine (100 μM), G′ increased by 2.2-fold, G" increased by 3.0-fold, and η increased transiently from 0.27 to 0.34. By 20 s, η decreased to 0.25, whereas G′ and G" remained above baseline. Comparable results were obtained with bradykinin (1 μM). These changes in G′, G", and η measured in cells were similar to but smaller than those reported for intact muscle strips. When we ablated baseline tone by adding the relaxing agonist dibutyryl cAMP (1 mM), G′ decreased within 5 min by 3.3-fold. With relaxing and contracting agonists, G′ could be manipulated through a contractile range of 7.3-fold. Cell populations exhibited a log-normal distribution of baseline stiffness (geometric SD = 2.8) and a heterogeneous response to both contractile and relaxing agonists, partly attributable to variability of baseline tone between cells. The total contractile range of the cells (from maximally relaxed to maximally stimulated), however, was independent of baseline stiffness. We conclude that HASM cells in culture exhibit a clear, although heterogeneous, response to contractile and relaxing agonists and express the essential mechanical features characteristic of the contractile response observed at the tissue level.

Prostanoids mediate IL-1β-induced β-adrenergic hyporesponsiveness in human airway smooth muscle cells

1998 ◽  
Vol 275 (3) ◽  
pp. L491-L501 ◽  
Author(s):  
Johanne D. Laporte ◽  
Paul E. Moore ◽  
Reynold A. Panettieri ◽  
Winfried Moeller ◽  
Joachim Heyder ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Cell Stiffness ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Heterologous Desensitization ◽  
Cox 2 ◽  
Inhibitor Of Protein Synthesis ◽  
Induced Changes

We have previously reported that pretreatment of cultured human airway smooth muscle (HASM) cells with interleukin-1β (IL-1β) results in decreased β-adrenergic responsiveness. The purpose of this study was to determine whether prostanoids released as a result of cyclooxygenase-2 (COX-2) induction by IL-1β contribute to this effect of the cytokine. Confluent serum-deprived HASM cells were studied in passages 4–7. IL-1β (20 ng/ml for 22 h) reduced the ability of the β-agonist isoproterenol (Iso) to decrease stiffness of HASM cells as measured by magnetic twisting cytometry. The effect of IL-1β on Iso-induced changes in cell stiffness was abolished by nonselective [indomethacin (Indo), 10−6 M] and selective (NS-398, 10−5 M) COX-2 inhibitors. Indo and NS-398 also inhibited both the increased basal cAMP and the decreases in Iso-stimulated cAMP production induced by IL-1β. IL-1β (20 ng/ml for 22 h) caused an increase in both basal (15-fold) and arachidonic acid (AA)-stimulated (10-fold) PGE2 release. Indo blocked basal and AA-stimulated PGE2 release in both control and IL-1β-treated cells. NS-398 also markedly reduced basal and AA-stimulated PGE2release in IL-1β-treated cells but had no significant effect on AA-stimulated PGE2 release in control cells. Western blot analysis confirmed the induction of COX-2 by IL-1β. Exogenously administered PGE2(10−7 M, 22 h) caused a significant reduction in the ability of Iso to decrease cell stiffness, mimicking the effects of IL-1β. Cycloheximide (10 μg/ml for 24 h), an inhibitor of protein synthesis, also abolished the effects of IL-1β on Iso-induced cell stiffness changes and cAMP formation. In summary, our results indicate that IL-1β significantly increases prostanoid release by HASM cells as a result of increased COX-2 expression. The prostanoids appear to contribute to β-adrenergic hyporesponsiveness, perhaps by heterologous desensitization of the β2 receptor.

Heparin and PGE2 inhibit DNA synthesis in human airway smooth muscle cells in culture

1995 ◽  
Vol 269 (4) ◽  
pp. L514-L519 ◽  
Author(s):  
P. R. Johnson ◽  
C. L. Armour ◽  
D. Carey ◽  
J. L. Black
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Dna Synthesis ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Cells In Culture ◽  
Human Airway ◽  
Inhibition Of Dna Synthesis

An increase in the bulk of the airway smooth muscle is a characteristic of asthma. Much of the research investigating the mechanisms of this increase in muscle has focused on mediators that are mitogenic for smooth muscle, while relatively few studies have focused on mediators inhibiting mitogenesis. In this study we have examined the effects of two mediators proposed as regulators of smooth muscle proliferation, namely heparin and prostaglandin (PG) E2, on human airway smooth muscle cells in culture stimulated with 1, 2.5, 5, and 10% fetal bovine serum (FBS) and platelet-derived growth factor AB (PDGF), 50 ng/ml. PGE2 had a biphasic effect on DNA synthesis in the presence of 1% FBS, with 10(-6) M causing inhibition and 10(-7) M causing an increase in DNA synthesis. PGE2 caused inhibition of DNA synthesis in the presence of 2.5, 5, and 10% FBS. Heparin (10 and 100 U/ml) caused an inhibition of DNA synthesis induced by 1% FBS, while 100 U/ml inhibited DNA synthesis induced by 5 and 10% FBS. PGE2 (10(-8), 10(-7), and 10(-6) M) inhibited the DNA synthesis induced by PDGF, while heparin (1, 10, and 100 U/ml) had no effect. These results indicate that both PGE2 and heparin may have a role in the control of human airway smooth muscle cell growth.

Pharmacological activation changes stiffness of cultured human airway smooth muscle cells

1996 ◽  
Vol 271 (5) ◽  
pp. C1660-C1668 ◽  
Author(s):  
R. D. Hubmayr ◽  
S. A. Shore ◽  
J. J. Fredberg ◽  
E. Planus ◽  
R. A. Panettieri ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
High Density ◽  
Cell Stiffness ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Cyclic Monophosphate ◽  
Human Airway

Using magnetic twisting cytometry (MTC), we measured the cytoskeletal stiffness of adherent human airway smooth muscle (HASM) cells. We hypothesized that modulation of actin-myosin interactions by application of contractile agonists would induce changes in cytoskeletal stiffness. In cells plated on high-density collagen, bradykinin (10(-6) M) and histamine (10(-4) M) increased stiffness by 85 +/- 15 and 68 +/- 16%, respectively. Increases in cell stiffness were also consistently observed after acetylcholine, substance P, and KCl. The bronchodilator agonists isoproterenol, prostaglandin E2, forskolin, dibutryl adenosine 3', 5'-cyclic monophosphate, and 8-bromoguanosine 3', 5'-cyclic monophosphate each caused a dose-dependent decrease in cell stiffness in unstimulated as well as bradykinin-treated cells. HASM cells plated on high-density collagen were stiffer than cells plated on low-density collagen (126 +/- 16 vs. 43 +/- 3 dyn/cm2) and developed more pronounced increases in stiffness in response to bradykinin as well as more pronounced decreases in stiffness in response to isoproterenol. These results are consistent with the hypothesis that modulation of actin-myosin interactions by application of contractile agonists causes changes in cytoskeletal stiffness of HASM cells. MTC may be a valuable tool for evaluating the mechanisms of pharmacomechanical coupling in airway smooth muscle cells in culture.

Mechanisms underlying TNF-α effects on agonist-mediated calcium homeostasis in human airway smooth muscle cells

1997 ◽  
Vol 273 (5) ◽  
pp. L1020-L1028 ◽  
Author(s):  
Yassine Amrani ◽  
Vera Krymskaya ◽  
Christopher Maki ◽  
Reynold A. Panettieri
Keyword(s):  
Protein Synthesis ◽  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Time Course ◽  
Airway Smooth Muscle Cells ◽  
Protein Synthesis Inhibitor ◽  
Potential Mechanism ◽  
Human Airway Smooth Muscle ◽  
Human Airway ◽  
Tnf Α

We have previously shown that tumor necrosis factor (TNF)-α, a cytokine involved in asthma, enhances Ca2+ responsiveness to bronchoconstrictor agents in cultured human airway smooth muscle (ASM) cells. In the present study, we investigated the potential mechanism(s) by which TNF-α modulates ASM cell responsiveness to such agents. In human ASM cells loaded with fura 2, TNF-α and interleukin (IL)-1β significantly enhanced thrombin- and bradykinin-evoked elevations of intracellular Ca2+. In TNF-α-treated cells, Ca2+responses to thrombin and bradykinin were 350 ± 14 and 573 ± 93 nM vs. 130 ± 17 and 247 ± 48 nM in nontreated cells, respectively ( P < 0.0001). In IL-1β-treated cells, the Ca2+response to bradykinin was 350 ± 21 vs. 127 ± 12 nM in nontreated cells ( P < 0.0001). The time course for TNF-α potentiation of agonist-induced Ca2+ responses requires a minimum of 6 h and was maximum after 12 h of incubation. In addition, cycloheximide, a protein synthesis inhibitor, completely blocked the potentiating effect of TNF-α on Ca2+ signals. We also found that TNF-α significantly enhanced increases in phosphoinositide (PI) accumulation induced by bradykinin. The percentage of change in PI accumulation over control was 115 ± 8 to 210 ± 15% in control cells vs. 128 ± 10 to 437 ± 92% in TNF-α-treated cells for 3 × 10−9 to 3 × 10−6 M bradykinin. The PI turnover to 10 mM NaF, a direct activator of G proteins, was also found to be enhanced by TNF-α. The percentage of change in PI accumulation over control increased from 280 ± 35% in control cells to 437 ± 92% in TNF-α-treated cells. Taken together, these results show that TNF-α can potently regulate G protein-mediated signal transduction in ASM cells by activating pathways dependent on protein synthesis. Our study demonstrates one potential mechanism underlying the enhanced Ca2+ response to bronchoconstrictor agents induced by cytokines in human ASM cells.

scholarly journals Spatial and temporal traction response in human airway smooth muscle cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1254-C1266 ◽  
Author(s):  
Iva Marija Tolić-Nørrelykke ◽  
James P. Butler ◽  
Jianxin Chen ◽  
Ning Wang
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Displacement Field ◽  
Time Course ◽  
Muscle Cells ◽  
Image Correlation ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Human Airway

Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

Anti-proliferative effect of Euphorbia stenoclada in human airway smooth muscle cells in culture

2007 ◽  
Vol 109 (1) ◽  
pp. 134-139 ◽  
Author(s):  
M. Chaabi ◽  
V. Freund-Michel ◽  
N. Frossard ◽  
A. Randriantsoa ◽  
R. Andriantsitohaina ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Airway Smooth Muscle Cells ◽  
Human Airway Smooth Muscle ◽  
Cells In Culture ◽  
Human Airway ◽  
Proliferative Effect

A human airway smooth muscle cell line that retains physiological responsiveness

1989 ◽  
Vol 256 (2) ◽  
pp. C329-C335 ◽  
Author(s):  
R. A. Panettieri ◽  
R. K. Murray ◽  
L. R. DePalo ◽  
P. A. Yadvish ◽  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
Airway Smooth Muscle ◽  
Cytosolic Calcium ◽  
Airway Smooth Muscle Cell ◽  
Airway Smooth Muscle Cells ◽  
Functional Coupling ◽  
Functional Responses ◽  
Human Airway Smooth Muscle ◽  
Human Airway

We report the development of a nontransformed line of human airway smooth muscle cells retaining smooth muscle-specific contractile protein expression and physiological responsiveness to agonists implicated in inflammatory airway diseases. Specific responses to histamine, leukotrienes, bradykinin, platelet-activating factor, substance P, and thromboxane analogues are demonstrated as well as functional coupling to beta-adrenergic receptors. The cell line was characterized using indirect immunofluorescence, as well as electrophoretic separation and immunoblot analysis of smooth muscle-specific actin. Functional responses were assessed by measurements of cytosolic calcium and stimulation of adenosine 3',5'-cyclic monophosphate production. The cells retain their responsiveness over many population doublings and should be a useful model to examine specific receptor-effector mechanisms, as well as the effects of neurohumoral agents on the regulation of airway smooth muscle growth and differentiation.

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