1 Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032; and 2 Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Actin is a major functional and structural cytoskeletal protein that mediates such diverse processes as motility, cytokinesis, contraction, and control of cell shape and polarity. While many extracellular signals are known to mediate actin filament polymerization, considerably less is known about signals that mediate depolymerization of the actin cytoskeleton. Human airway smooth muscle cells were briefly exposed to isoproterenol, forskolin, or the cAMP-dependent protein kinase A (PKA) agonist stimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphate (Sp-cAMPS). Actin polymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, forskolin, or Sp-cAMPS induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. The PKA inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) completely inhibited forskolin-stimulated depolymerization, whereas it only partially inhibited isoproterenol-induced depolymerization. The protein tyrosine kinase inhibitors genistein or tyrphostin A23 also partially inhibited isoproterenol-induced actin depolymerization. In contrast, the combination of Rp-cAMPS and either tyrosine kinase inhibitor had an additive effect at inhibiting isoproterenol-induced actin depolymerization. These results suggest that both PKA-dependent and -independent pathways mediate actin depolymerization in human airway smooth muscle cells.
protein kinase A
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INTRODUCTION |
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ACTIN IS A MAJOR FUNCTIONAL and structural cytoskeletal protein of all eukaryotic cells. It exists in cells in one of two forms: globular or G-actin, which is not polymerized, and filamentous or F-actin, which is the polymerized form. Regulation of actin filament dynamics is essential for cell motility, cytokinesis, muscle contraction, and control of cell shape and polarity. Actin polymerization is dynamically regulated in most cell types and can be initiated by many agonists that activate cell surface receptors. Such agonists include growth factors, neurotransmitters, hormones, extracellular matrix proteins, and chemoattractants. The signaling pathways involved converge on the Rho family of monomeric G proteins to induce actin polymerization through a family of proteins called Wiscott-Aldrich Syndrome proteins (24).
Considerable evidence has accumulated to implicate RhoA activation in
actin polymerization. The introduction of the C3 exoenzyme from
Clostridium botulinum into cells, which inactivates RhoA by
ADP-ribosylation, leads to a loss of actin stress fibers, followed by
cell rounding and loss of cell attachment (3, 11, 26). Conversely, microinjection of cells with constituitively activated RhoA
leads to a dramatic stimulation of actin polymerization
(23). Moreover, the signaling pathways leading from the
cell surface receptors to RhoA activation and actin polymerization have
been identified. Receptors that couple to Gq,
G
i-2, and G
12/13 have all been shown to
activate RhoA in a cell type-specific manner (12, 13, 27,
32).
Much less is known about the signaling pathways that mediate actin depolymerization. Many cell types undergo dramatic shape changes when exposed to agents that increase intracellular cAMP levels and cAMP-dependent protein kinase A (PKA) activity (1, 4, 6, 16, 18). This shape change is associated in some cells with decreases in cytoskeletal actin (6). Moreover, Dong and coworkers (4) have recently shown that phosphorylation of RhoA by PKA decreases the binding of RhoA to its downstream effector Rho kinase.
Until recently, most studies suggested that adenylyl cyclase was the
only downstream target of the -adrenergic
receptor/G
s complex. Stimulation of any of the
known
-adrenergic receptor subtypes was thought to result in
signaling to the heterotrimeric G protein, G
s, leading
to the activation of adenylyl cyclase, increased production of cAMP,
and subsequent activation of cAMP-dependent protein kinase A. Recent
studies, however, clearly indicate that G
s can signal
through alternate transduction pathways in such biological processes as
apoptosis and adipogenesis (9, 33, 35). Both of
these G
s-mediated events require activation of a protein
tyrosine kinase (9, 34).
Agonists that contract airway smooth muscle induce actin polymerization
in human airway smooth muscle cells via pathways involving the
heterotrimeric G proteins Gi-2 and G
q
signaling to RhoA (12, 13).
-Adrenergic agonists, other
agonists that couple to G
s, and agents that increase
intracellular cAMP levels all relax airway smooth muscle. The goal of
the present study was to determine whether
-adrenergic agonists
induced actin depolymerization in human airway smooth muscle, and, if
so, to identify the signaling pathway(s) involved.
This study clearly demonstrates that -adrenergic agonists induce
actin depolymerization in human airway smooth muscle cells by a
cAMP-dependent signaling pathway. We also demonstrate for the first
time that isoproterenol induces actin depolymerization in these cells
by a cAMP-independent pathway that involves a tyrosine kinase as an intermediate.
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METHODS |
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Cell culture.
Primary cultures of human tracheal smooth muscle cells
(22) were maintained in Ham's F-12 medium containing 10%
fetal bovine serum at 37°C in an atmosphere of 5%
CO2-95% air. Immunoblot analysis of these cells identified
expression of -actin, myosin heavy chain, and desmin, confirming the
smooth muscle phenotype of the cells. The cells were plated on
eight-well microscope slides (Nunc Chambers, Naperville, IL) and grown
until almost confluent.
Fluorescence staining protocol. Fluorescence microscopy was performed by methods previously described in our laboratory with minor modifications (32). In brief, cells were fixed and agonist action was terminated by the addition of 3.7% (final concentration) fresh paraformaldehyde in PBS for 15 min. After three washes with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. Cells were next pretreated with blocking solution (1% BSA-0.1% Triton X-100 in PBS) for 15 min. Cells were simultaneously stained with FITC-labeled phalloidin (1 µg/ml) and Texas red-labeled DNase I (10 µg/ml) in 1% BSA in PBS to localize pools of F-actin and monomeric G-actin, respectively (15). The cell staining was performed for 20 min in a dark room at room temperature. The wells were washed twice with PBS, and a coverslip was mounted on the slide with the mounting medium Vectashield H-1000 to prevent rapid photobleaching. To ensure that fluorescent intensity measurements were obtained before significant photobleaching occurred, intensity measurements were obtained from the same image at 30-s intervals over a 3.5-min period with continuous exposure to ultraviolet (UV) excitation. Significant photobleaching of FITC-phalloidin occurred only after 2.5 min of exposure to continuous UV excitation, whereas significant photobleaching of Texas red DNase I occurred only after 3.5 min of exposure to continuous UV excitation. At 1 min, FITC-phalloidin fluorescence intensity was 95 ± 3.5% of the intensity obtained at time 0, and at 2.5 min, the fluorescence intensity was 73 ± 8.3% of the intensity obtained at time 0. At 1 min, Texas red DNase I fluorescence intensity was 96 ± 1.9% of the intensity obtained at time 0, and at 2.5 min, the fluorescence intensity was 88 ± 5.6% of the intensity obtained at time 0. Because all fluorescence intensity measurements are routinely obtained from all images within 15 s of exposure to UV excitation, photobleaching could not be a significant factor in our results. Incubation, fixation, and staining were always performed in parallel for all wells on a slide. Preliminary studies were performed omitting the quenching of the paraformaldehyde with NH4Cl, shortening of the washing periods, and washing cells with PBS instead of Triton X-100 in PBS or BSA, which yielded similar results.
Fluorescence microscopy. Actin pools were visualized with a fluorescence microscope (Olympus IX70; Tokyo, Japan), and the images were captured and stored using Metamorph software (Universal Imaging, West Chester, PA) on a personal computer. The fluorescence intensities of FITC-phalloidin and Texas red DNase I were calculated simultaneously from a view containing >15 cells. The excitation and emission wavelengths for FITC-phalloidin are 490 and 525 nm, respectively, whereas the excitation and emission wavelengths for Texas red DNase I are 596 and 615 nm, respectively. To standardize the fluorescence intensity measurements among experiments, the time of image capturing, the image intensity gain, the image enhancement, and the image black level in both channels were optimized before each experiment and kept constant throughout each experiment. Representative images were taken in triplicate from each well and were digitized (640 × 484 pixels) with a color resolution from 0- (minimum) to 255-bit (maximum) intensity. After the total and background intensity of FITC-phalloidin and Texas red DNase I were measured, background fluorescent intensity was subtracted from each image, and the F-actin:G-actin ratios were calculated. To control for day-to-day variations in staining intensity, untreated cells were always compared with treated cells on the same microscope slide because cells on the same slide undergo identical culture, fixation, permeabilization, staining, and microscopy conditions, allowing meaningful comparisons among samples.
Adenylyl cyclase assay.
To confirm that isoproterenol and forskolin increased cAMP levels in
these cells, adenylyl cyclase activity was measured as previously
described (7). Briefly, human airway smooth muscle cells
were grown in 24-well plates in Ham's F-12 media containing 10% FBS
at 37°C in a humidified atmosphere of 5% CO2-95% air. At confluence, cells were washed three times in warm PBS and
immediately lysed in 100 µl of lysis buffer (10 mM HEPES, pH 8.0, 2 mM EDTA, and 100 µM phenylmethylsulfonyl fluoride) for 45 min at
37°C. Adenylyl cyclase assays were performed for 10 min at 37°C in
a total volume of 150 µl composed of 100 µl of lysed cells and 50 µl of assay buffer; final concentrations were 0.5 mM
3-isobutyl-1-methylxanthine, 50 mM HEPES, pH 8.0, 50 mM NaCl, 0.4 mM
EGTA, 1 mM cAMP, 7 mM MgCl2, 0.1 mM ATP, 7 mM creatine
phosphate, 50 U/ml creatine phosphokinase, 0.1 mg/ml BSA, and 10 µCi/ml [-32P]ATP (specific activity 800 Ci/mmol)
without added effectors (control) or in the presence of 100 µM
isoproterenol or 10 µM forskolin. Preliminary experiments confirmed
the linearity of adenylyl cyclase activity at the protein
concentrations and incubation times used. The reactions were terminated
by addition of 150 µl of stop buffer {50 mM HEPES, pH 7.5, 2 mM
ATP, 0.5 mM cAMP, 2% SDS, and 1 µCi/ml [3H]cAMP
(specific activity 25 Ci/mmol)}. [
-32P]cAMP was
recovered by sequential column chromatography (28). Recovery rates of columns were 75-90%. Data were expressed as picomoles of cAMP per well per 10 min.
Materials. Isoproterenol, forskolin, and FITC-phalloidin were obtained from Sigma (St. Louis, MO). Texas red DNase I was obtained from Molecular Probes (Eugene, OR). Rp-cAMPS, Sp-cAMPS, and genistein were purchased from Calbiochem (La Jolla, CA). Vectashield H-1000 was obtained from Vector Laboratories (Burlingame, CA).
Statistical analysis of data. All data are presented as means ± SE. F-actin:G-actin ratios were compared with paired t-test or analysis of variance with repeated measures and Bonferroni posttest comparisons when appropriate with Instat software (Graph Pad, San Diego, CA). P < 0.05 was considered significant.
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RESULTS |
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Exposure of serum-deprived human airway smooth muscle cells to 10 µM forskolin or 100 µM isoproterenol for 5 min decreased the
FITC-phalloidin staining intensity of F-actin and increased the Texas
red DNase I staining of G-actin (Fig.
1A). This demonstrates that both 100 µM isoproterenol and 10 µM forskolin induced actin depolymerization in these cells, with isoproterenol inducing a significantly greater amount of actin depolymerization than forskolin in these cells (Fig. 1, A and B). The
F-actin:G-actin fluorescence ratio decreased from 2.8 ± 0.28 in
untreated cells to 1.7 ± 0.14 and 2.1 ± 0.23 in
isoproterenol- and forskolin-treated cells, respectively
(P < 0.001 for each agonist compared with the control group and P < 0.05 for isoproterenol- compared with
forskolin-treated cells; n = 11 experiments; Fig.
1B). To further investigate the dose dependence of
isoproterenol- and forskolin-induced actin depolymerization, additional
studies were performed. Isoproterenol (1-100 µM) dose
dependently decreased the F-actin:G-actin fluorescence ratio
(n = 6; Fig. 1C). Forskolin (3 and 10 µM)
significantly decreased F-actin:G-actin fluorescent ratio, whereas 1 µM forskolin was without effect (n = 5; Fig.
1D).
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Because isoproterenol was more effective than forskolin at inducing actin depolymerization in human airway smooth muscle cells, we questioned whether different signaling pathways were involved.
To investigate the role of protein kinase A in isoproterenol- and
forskolin-induced actin depolymerization, cells were pretreated with
the specific competitive antagonist of the action of cAMP on protein
kinase A, Rp-cAMPS, or its S isomer, Sp-cAMPS, which functions as a
cAMP agonist. Sp-cAMPS (100 µM) given for 30 min induced significant
actin depolymerization (Fig.
2A). The F-actin:G-actin fluorescence ratio decreased from 3.0 ± 0.27 to 2.3 ± 0.29 in Sp-cAMPS-treated cells (P = 0. 009;
n = 6 experiments; Fig. 2B). Sp-cAMPS (10 µM) given for 30 min had no significant effect on actin
polymerization or depolymerization (data not shown).
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In a separate series of studies, cells were pretreated with Rp-cAMPS
(100 µM) for 30 min before isoproterenol or forskolin exposure.
Rp-cAMPS, in the absence of any -adrenoceptor agonist, had no
significant effect on actin polymerization or depolymerization (Fig.
2A). The F-actin:G-actin fluorescence ratio averaged
3.4 ± 0.20 in the absence and 3.7 ± 0.28 in the presence of
Rp-cAMPS (P = 0.3011; n = 7 experiments; Fig. 2B). However, pretreatment with Rp-cAMPS
(100 µM) totally blocked forskolin-induced actin depolymerization
(Fig. 3A) and only partially
blocked isoproterenol-induced actin depolymerization (Fig.
4A). In the absence of
Rp-cAMPS pretreatment, forskolin exposure decreased the F-actin:G-actin
fluorescence ratio from a control value of 2.3 ± 0.41 to 1.9 ± 0.41 (P < 0.01), but in the presence of both
forskolin and Rp-cAMPS, the F-actin:G-actin ratio was 2.2 ± 0.38 (P > 0.05 compared with control; n = 5 experiments; Fig. 3B). In contrast, isoproterenol decreased
the F-actin:G-actin fluorescence ratio from 3.2 ± 0.30 to
1.8 ± 0.16 in the absence of Rp-cAMPS (P < 0.001) and to 2.3 ± 0.06 in the presence of Rp-cAMPS (P < 0.001; n = 5 experiments; Fig.
4B).
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In a separate series of studies, to determine whether protein tyrosine
kinases were involved in isoproterenol-induced actin depolymerization,
human airway smooth muscle cells were pretreated with 30 µM genistein
or 150 µM tyrphostin A23 for 30 min before agonist exposure in the
presence and absence of Rp-cAMPS. Tyrphostin A23, in the absence of
agonist, produced a small but significant decrease in the
F-actin:G-actin fluorescence ratio. The F-actin:G-actin ratio decreased
from 2.9 ± 0.08 in the untreated cells to 2.6 ± 0.11 in the
tyrphostin A23-pretreated cells (P = 0.01;
n = 7 experiments). Tyrphostin A23 and Rp-cAMPS
pretreatment each only partially inhibited the actin depolymerization
induced by isoproterenol. However, the combined pretreatment with
tyrphostin A23 and Rp-cAMPS totally inhibited the actin
depolymerization induced by isoproterenol (Fig.
5A). In the absence of
pretreatment, isoproterenol decreased the F-actin:G-actin fluorescence
ratio from 2.9 ± 0.08 to 1.9 ± 0.10 (P < 0.001). In cells pretreated with tyrphostin A23, isoproterenol decreased the F-actin:G-actin ratio to only 2.5 ± 0.07 (P < 0.001). In cells pretreated with Rp-cAMPS,
isoproterenol decreased the F-actin:G-actin fluorescence ratio to only
2.4 ± 0.05. In contrast, when cells were pretreated with both
tyrphostin A23 and Rp-cAMPS, the F-actin:G-actin ratio in cells
exposed to isoproterenol was 2.8 ± 0.11 (P > 0.05 compared with control; n = 7 experiments; Fig.
5B).
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In a separate series of experiments, genistein pretreatment also partially reversed the actin depolymerization induced by isoproterenol, and the combined pretreatment with genistein and Rp-cAMPS had a significantly greater inhibitory effect on isoproterenol-induced actin depolymerization than either genistein or Rp-cAMPS alone. In the absence of pretreatment, isoproterenol decreased the F-actin:G-actin fluorescence ratio from 3.3 ± 0.30 to 1.8 ± 0.16 (P < 0.001). In cells pretreated with genistein, isoproterenol decreased the F-actin:G-actin ratio to 2.4 ± 0.25 (P < 0.01). In cells pretreated with Rp-cAMPS, isoproterenol decreased the F-actin:G-actin ratio to 2.3 ± 0.22, and in cells pretreated with both genistein and Rp-cAMPS, isoproterenol decreased the F-actin:G-actin ratio to 2.8 ± 0.19 (P < 0.001; n = 6 experiments). In contrast, genistein pretreatment was without effect on forskolin-induced actin depolymerization. In cells exposed to forskolin, the F-actin:G-actin ratio was 2.3 ± 0.25 in the absence and 2.6 ± 0.28 in the presence of genistein (P > 0.05; n = 6 experiments).
To confirm that isoproterenol and forskolin are coupled to increased
cAMP in these cells, adenylyl cyclase activity was measured. Both 100 µM isoproterenol and 10 µM forskolin increased adenylyl cyclase
activity. Control adenylyl cyclase activity yielded 204 ± 61 picomoles of cAMP per well per 10 min. Activity increased 231 ± 44% and 707 ± 141% above control levels in the presence of
isoproterenol and forskolin, respectively (Fig.
6).
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DISCUSSION |
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This study demonstrates for the first time that isoproterenol induced actin depolymerization in human airway smooth muscle cells by two separate signaling pathways: a cAMP/PKA-dependent pathway and a cAMP-independent pathway involving a protein tyrosine kinase. Pretreatment of human airway smooth muscle cells with Rp-cAMPS only partially inhibited isoproterenol-induced actin depolymerization but totally inhibited forskolin-induced actin depolymerization. Pretreatment with the protein tyrosine kinase inhibitors genistein or tyrphostin A23 partially inhibited isoproterenol-induced actin depolymerization but was without effect on forskolin-induced actin depolymerization, whereas pretreatment with both Rp-cAMPS and a protein tyrosine kinase inhibitor had an additive effect and totally prevented isoproterenol-induced actin depolymerization in these cells.
To image and quantify actin depolymerization in these cells, we used dual fluorescence labeling with FITC-phalloidin and Texas red DNase I adapted from the method of Knowles and McCulloch (15). This technique has been used for more than ten years to quantify changes in stress fibers and reorganization of the actin cytoskeleton (8, 13, 21, 32). FITC-phalloidin specifically labels F-actin while Texas red DNase I specifically labels G-actin. Dual labeling techniques with the additional staining of the total protein content (8) or concurrent labeling of F-actin and G-actin pools (15, 25, 31) are widely used to correct for differences in cell size or number per image and to reduce their influence on fluorescence intensities. Because the staining patterns of F-actin and G-actin are thought to be spatially separate and distinct and since there is little interference due to differences in emission and absorption spectra (15), one can simultaneously observe the relative amount and configuration of the F-actin and the G-actin in the same cell. The total actin content measured by quantitative fluorescence has been shown to correspond closely to the DNase inhibition assay (25). Although variations in staining intensity do occur between individual experiments, the influence of variations in staining is diminished by the inclusion of treated groups and control groups on the same slide, which undergo identical culture, staining, and microscopy conditions. Thus the results obtained in this study could not be due to variations in staining intensity because untreated cells were always compared with treated cells on the same microscope slide. Although the fluorochromes used in this study (FITC and Texas red) undergo photobleaching at different rates (that could potentially effect calculated F-actin:G-actin fluorescent staining ratios), we have shown that our fluorescent intensity measurements are obtained before either fluorochrome exhibits significant photobleaching.
Isoproterenol increases adenylyl cyclase and cAMP in human airway
smooth muscle cells as in airway smooth muscle cells from other species
(7). Forskolin, which bypasses the -adrenergic receptor by activating adenylyl cyclases directly (30), is
even more effective than isoproterenol at increasing adenylyl cyclase activity at the concentrations used in this study in these cells. These
data agree with a previous study in cultured canine airway smooth
muscle cells (7) and studies in every other cell in which
cAMP or adenylyl cyclase has been measured. Thus our results demonstrating that isoproterenol was more effective than forskolin at
inducing actin depolymerization was unexpected.
We therefore used Rp-cAMPS and its S isomer Sp-cAMPS to further explore the role of cAMP in actin depolymerization in human airway smooth muscle cells. Rp-cAMPS acts as intracellular antagonists of cAMP by competing specifically with cAMP for binding sites on the regulatory subunits of PKA. Sp-cAMPS also competes for these sites but is an activator of PKA and thus acts as a cAMP agonist (10). Our data demonstrating that Sp-cAMPS induces actin depolymerization, in combination with data demonstrating that pretreatment with Rp-cAMPS inhibits both isoproterenol- and forskolin-induced actin depolymerization, clearly implicate cAMP in this signaling pathway. The present study in human airway smooth muscle cells agrees with studies in fibroblasts (18) and neutrophils (5), demonstrating actin microfilament disassembly by agonists that increase intracellular levels of cAMP. A possible pathway by which cAMP and its downstream effector PKA induce actin depolymerization is by RhoA inhibition. Dong and coworkers (4) have recently shown that PKA inactivates RhoA by specifically phosphorylating Ser-188 on the protein. This possibility is consistent with previous studies from our laboratory in these cells demonstrating that C3 exotoxin, which ADP ribosylates an asparagine residue at the codon 41 position on RhoA and inactivates the protein, also induces actin depolymerization in unstimulated human airway smooth muscle cells (32).
Other biological processes mediated by the -adrenergic agonists and
G
s proteins that do not use the classical PKA pathway have been identified, and recent studies have identified protein tyrosine kinases in the pathways. G
s directly activates
Ca2+-activated K+ (KCa) channels in
airway smooth muscle cells (17) and
dihydropyridine-sensitive Ca2+ channels in cardiac myocytes
(37). A recent study demonstrated that tyrosine
phosphatase inhibitors selectively antagonize
-adrenergic receptor-dependent regulation of cardiac Ca2+ channels
(29). In S49 mouse lymphoma,
-adrenergic receptor stimulation induces apoptosis though a pathway involving the
Lck family of protein tyrosine kinases (9). In HEK-293
cells,
-adrenergic receptor stimulation of mitogen-activated protein
kinases ERK1 and ERK2 (extracellular regulated kinases 1 and 2)
requires the activation of the Src family of protein tyrosine kinase
(19). In 3T3-L1 cells, G
s activation
inhibits adipogenesis through the protein tyrosine kinase Syc
(34). Thus phosphorylation of tyrosine residues by protein
tyrosine kinases represents another means of modulating target proteins.
In the present study, the effects of protein tyrosine kinase inhibition
and Rp-cAMPS were additive. This indicates that -adrenergic receptors and G
s proteins in human airway smooth cells
use both tyrosine kinase-dependent signaling pathways and the classic
PKA pathway to induce actin depolymerization. The identity of the precise protein tyrosine kinase and the signaling pathway by which phosphorylation of tyrosine residues modulate
-adrenergic-induced actin depolymerization are a subject for future investigations.
Several recent papers have documented the finding that cytochalasin or
latrunculin, agents that inhibit actin polymerization, either block or
significantly alter the contractile properties of smooth muscle
(2, 14, 20, 36). Thus it is likely that the actin
depolymerization induced by -adrenergic receptor activation in this
study plays a role in the regulation of airway smooth muscle tone.
In conclusion, this study demonstrates for the first time that
isoproterenol induces actin depolymerization in human airway smooth
muscle cells by two separate signaling pathways: a cAMP PKA-dependent
pathway and a cAMP-independent pathway involving a protein tyrosine
kinase. These observations indicate that the -adrenergic
receptor/G
s proteins in these cells can signal through pathways separate from cAMP.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-62340 and HL-58519.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. A. Hirshman, Dept. of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St., P&S Box 46, New York, NY 10032 (E-mail: cah63{at}columbia.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 February 2001; accepted in final form 10 July 2001.
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