Actin depolymerization via the beta -adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway

Carol A. Hirshman1, Defen Zhu1, Reynold A. Panettieri2, and Charles W. Emala1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

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 Galpha q, Galpha i-2, and Galpha 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 beta -adrenergic receptor/Galpha s complex. Stimulation of any of the known beta -adrenergic receptor subtypes was thought to result in signaling to the heterotrimeric G protein, Galpha 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 Galpha s can signal through alternate transduction pathways in such biological processes as apoptosis and adipogenesis (9, 33, 35). Both of these Galpha 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 Galpha i-2 and Galpha q signaling to RhoA (12, 13). beta -Adrenergic agonists, other agonists that couple to Galpha s, and agents that increase intracellular cAMP levels all relax airway smooth muscle. The goal of the present study was to determine whether beta -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 beta -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.


    METHODS
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INTRODUCTION
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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 alpha -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.

The cells were serum deprived for 24 h, after which they were exposed to no treatment, 1-100 µM isoproterenol, or 1-10 µM forskolin for 5 min. In separate studies, 100 µM isoproterenol or 10 µM forskolin was used in the presence or absence of 100 µM Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), which was administered 30 min before the agonists. In other studies, cells were exposed to no treatment or to stimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphate (Sp-cAMPS; 10 and 100 µM) for 30 min. In a fourth set of studies, we sought to determine whether protein tyrosine kinases played a role in isoproterenol- and forskolin-induced actin depolymerization. Cells were pretreated with 20 µM genistein, 150 µM tyrphostin A23, 100 µM Rp-cAMPS, or both Rp-cAMPS and either tyrphostin A23 or genistein for 30 min before agonist treatment. Rp-cAMPS and Sp-cAMPS were dissolved in water; genistein and tyrphostin were dissolved in DMSO such that the final concentration of DMSO was 0.05%. Preliminary studies in our laboratory have shown that this concentration of DMSO has no effect on actin polymerization or depolymerization in these cells.

Because activation of the cytoskeleton by nonspecific stimuli is a major concern, physical manipulation of the slides was kept to a minimum. Termination of agonist activation was achieved by the addition, directly to the media, of an equal volume of 7.4% fresh paraformaldehyde in PBS for 15 min.

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 [alpha -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)}. [alpha -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.


    RESULTS
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ABSTRACT
INTRODUCTION
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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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Fig. 1.   A: representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin and Texas red DNase I to illustrate filamentous (F)-actin (top row) and globular (G)-actin (bottom row) fibers. Cells treated with isoproterenol (100 µM) or forskolin (10 µM) for 5 min showed decreased F-actin staining compared with the untreated control cells. B: fluorescent staining ratios of F-actin:G-actin (F/G) in human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas red DNase I were measured in the same field in triplicate for each treatment. Isoproterenol and forskolin (n = 11 experiments) each decreased the ratio of F-actin:G-actin, indicating that actin depolymerization with isoproterenol has a significantly greater effect than forskolin. *P < 0.001 for each agonist compared with control; #P < 0.05 for isoproterenol compared with forskolin. C: fluorescence staining ratios of F-actin:G-actin in human airway smooth muscle cells exposed to isoproterenol (1 - 100 µM) for 5 min. Each dose of isoproterenol significantly decreased the ratio of F-actin:G-actin, indicating actin depolymerization. n = 6; *P < 0.01; **P < 0.001 compared with control; #P < 0.05 compared with 100 µM isoproterenol. D: fluorescence staining ratios of F-actin:G-actin in human airway smooth muscle cells exposed to forskolin (1 - 10 µM) for 5 min. Forskolin (3 and 10 µM) significantly decreased the ratio of F-actin:G-actin, indicating actin depolymerization. n = 5; *P < 0.05; **P < 0.01 compared with control; #P < 0.01 compared with 1 µM. Iso, isoproterenol; forsk, forskolin.

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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Fig. 2.   A: representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Pretreatment for 30 min with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; 100 µM) did not alter F-actin staining of cells, whereas pretreatment with stimulatory diastereoisomer of adenosine 3',5'-cyclic monophosphate (Sp-cAMPS; 100 µM) decreased F-actin staining compared with the untreated control cells. B: fluorescent staining ratios of F-actin:G-actin in human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas red DNase I was measured in the same field in triplicate for each treatment. Rp-cAMPS (n = 7 experiments) had no significant effect on the F-actin:G-actin ratio. P = 0.3. Sp-cAMPS (100 µM; n = 6 experiments) decreased the F-actin:G-actin ratio compared with the untreated control (P = 0.009).

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 beta -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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Fig. 3.   A: representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Pretreatment for 30 min with Rp-cAMPS (100 µM) prevented the decrease in F-actin staining induced by forskolin (10 µM). B: fluorescent staining ratios of F-actin:G-actin in human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas red DNase I was measured in the same field in triplicate for each treatment. Rp-cAMPS (n = 5 experiments) completely blocked the decrease in the F-actin:G-actin ratio induced by forskolin. P < 0.01 forskolin compared with control; P > 0.05 forskolin + Rp-cAMPS compared with control.



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Fig. 4.   A: representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Pretreatment for 30 min with Rp-cAMPS (100 µM) in part prevented the decrease in F-actin staining induced by isoproterenol (100 µM). B: fluorescent staining ratios of F-actin:G-actin in human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas red DNase I was measured in the same field in triplicate for each treatment. Rp-cAMPS (n = 5 experiments) partly blocked the decrease in the F-actin:G-actin ratio induced by isoproterenol. P < 0.001 isoproterenol alone or isoproterenol + Rp-cAMPS compared with control.

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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Fig. 5.   A: representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin to illustrate F-actin fibers. Pretreatment with either tyrphostin A23 (A23; 150 µM) or Rp-cAMPS (Rp; 100 µM) for 30 min in part prevented the isoproterenol-induced decrease in F-actin staining, whereas pretreatment with both tyrphostin A23 and Rp-cAMPS totally prevented the decrease in F-actin staining induced by isoproterenol (100 µM). B: fluorescent staining ratios of F-actin:G-actin in human airway smooth muscle cells. Fluorescent intensity of F-actin staining by FITC-phalloidin and G-actin staining by Texas red DNase I was measured in the same field in triplicate for each treatment. Tyrphostin A23 or Rp-cAMPS each partially prevented the decrease in the F-actin:G-actin ratio induced by isoproterenol. P < 0.05 for either isoproterenol + tyrphostin A23 or isoproterenol + Rp-cAMPS compared with isoproterenol alone. The combined treatment of tyrphostin A23 and Rp-cAMPS completely blocked the decrease in the F-actin:G-actin ratio induced by isoproterenol. P > 0.05 for isoproterenol + Rp-cAMPS + tyrphostin A23 compared with control.

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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Fig. 6.   Adenylyl cyclase activity in cultured human airway smooth muscle cells. Isoproterenol (100 µM) or forskolin (10 µM) increased adenylyl cyclase activity above basal levels; n = 6; *P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -adrenergic agonists and Galpha s proteins that do not use the classical PKA pathway have been identified, and recent studies have identified protein tyrosine kinases in the pathways. Galpha 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 beta -adrenergic receptor-dependent regulation of cardiac Ca2+ channels (29). In S49 mouse lymphoma, beta -adrenergic receptor stimulation induces apoptosis though a pathway involving the Lck family of protein tyrosine kinases (9). In HEK-293 cells, beta -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, Galpha 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 beta -adrenergic receptors and Galpha 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 beta -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 beta -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 beta -adrenergic receptor/Galpha s proteins in these cells can signal through pathways separate from cAMP.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-62340 and HL-58519.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(5):C1468-C1476
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