Departments of 1 Physiology and 2 Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
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In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce Ca2+-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433-23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces Ca2+-independent contractions and/or Ca2+ sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of Ca2+ (pCa > 9), constitutively active glutathione-S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to ~40% of the maximal force generated by Ca2+ at pCa 4.4. In addition, GST-mPAK3 enhanced Ca2+ sensitivity of contraction by increasing force generation by 80% at intermediate Ca2+ concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK3K297R had no effect on force production. Using antibody against one of the PAK-phosphorylated sites (Ser657) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes Ca2+-independent contractions and produces Ca2+ sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation.
p21-activated kinase; monomeric G proteins; calcium sensitization; asthma; tonic smooth muscle
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INTRODUCTION |
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THE PRIMARY MECHANISM of smooth muscle activation is mediated by an increase in intracellular Ca2+, which initiates a cascade of mechanisms utilizing calmodulin, myosin light chain (MLC) kinase (MLCK), and phosphorylation of the regulatory MLC (MLC20) to cause cross-bridge cycling and contraction (20). However, the sensitivity of the contractile apparatus to Ca2+ can be modulated (18, 19, 21). A number of mechanisms for Ca2+ sensitization of smooth muscle implicate the involvement of two groups of proteins. Those in the first group modulate the steady-state level of MLC phosphorylation at constant intracellular Ca2+ concentration; these proteins include Rho-associated kinase (ROK) (22), the MLC phosphatase inhibitor CPI-17 (12), protein kinase C (10), calmodulin-dependent kinase II (25), integrin-associated kinase (31), and mitogen-activated protein kinase (MAPK) (24). The proteins in the second group, including caldesmon and calponin, modulate the actin-myosin interaction and actin-activated myosin ATPase activity (15).
The Rho family, which forms a subgroup of the superfamily of Ras small-molecular-weight GTPases (21 kDa), consists of Cdc42, Rac1, Rac2, RhoA, RhoB, and RhoC (9). These monomeric GTPases act as molecular switches, which alternate between the "on" GTP-binding state and the "off" GDP-binding state. The equilibrium of the two states is regulated by guanine nucleotide exchange factors, which promote the exchange of bound GDP for GTP, and GTPase-activating protein, which enhances GTPase activity of the Rho GTPases. ROK is a well-characterized downstream effector of RhoA (9, 22), and it is believed to play a major part in mediating RhoA-induced Ca2+-independent contractions and Ca2+ sensitization in tonic and phasic smooth muscle (20, 21). ROK inactivates MLC phosphatase by phosphorylating its myosin-binding subunit, resulting in an increase in the steady-state level of MLC20 phosphorylation at a constant Ca2+ concentration. There is strong evidence that other members of the Rho family of small GTPases are also involved in regulating the Ca2+ sensitivity of smooth muscle contraction. For example, Foster et al. (6) and Van Eyk and coworkers (28) reported that the Cdc42/Rac-activated p21 Ser/Thr kinase (PAK) induced a Ca2+-independent contraction in skinned guinea pig ileal smooth muscle with concomitant phosphorylation of caldesmon and desmin, suggesting that PAK may play a significant role in the modulation of Ca2+ sensitivity in smooth muscle contraction. At least four PAK isoforms, PAK1, PAK2, PAK3, and PAK4 (62-68 kDa), have been identified in mammalian tissues, and they share ~70% identity in overall amino acid sequences and >90% identity within the kinase domain (1). Although PAK1, PAK2, and PAK3 are enriched in the mammalian brain, PAK2 is ubiquitously expressed in mammalian tissues including brain, heart and cardiac muscle, kidney, liver, lung, spleen, and testes (26). Recently, PAK1, PAK2, and PAK3 have been identified in tracheal smooth muscle (5), and PAK2 and PAK3 have been identified in guinea pig taenia coli and rat aortic smooth muscle (28). There is no systematic study of functional diversity and/or redundancy of the PAK isoforms, although it is likely that they have similar substrate specificities on the basis of the high degree of homology in the amino acid sequences in their kinase domains (1).
Airway smooth muscle (ASM) exhibits Ca2+ sensitization to a range of agonists and antigen (3, 32, 33). Because abnormal contraction of airway and vascular smooth muscle may contribute to asthma and hypertension, respectively, the action of PAK in tonic smooth muscle is of considerable interest. In this study, we have examined whether PAK3 can produce Ca2+-independent contraction as well as Ca2+ sensitization of contraction of skinned tonic ASM and whether PAK phosphorylation of caldesmon plays a role in these processes.
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METHODS |
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All experimental procedures were approved by the Queen's University Animal Care Committee and conform to the guidelines of the Canadian Council on Animal Care. Smooth muscle from the trachea of freshly killed (pentobarbital anesthesia followed by saturated KCl, administered intravenously) dogs, pigs, rats, and mice was dissected free of cartilage. Airway epithelium of rodents was denuded by rubbing the lumen; epithelium and connective tissue of nonrodents were removed by dissection.
Solutions and drugs.
The tissue lysis buffer used to homogenize intact ASM had the following
composition: 20 mM Tris (pH 7.4), 0.1 mM phenylmethylsulfonyl fluoride,
5 µg/ml leupeptin, 5 µg/ml pepstatin, and 3 mM NaN3. For detection of caldesmon phosphorylation, 50 mM NaF and 2 mM Na3VO4 were added to the lysis buffer. Laemmli
sample buffer concentrate consisted of 60 mM Tris (pH 6.8), 2% SDS,
25% glycerol, 0.02% bromphenol, and 5% -mercaptoethanol. The
Western blot transfer buffer contained 2.5 mM Tris, 19 mM glycine, and
20% methanol.
Detection of PAK in ASM.
ASM was homogenized in tissue lysis buffer using a French press and
centrifuged to remove cell particles. Total protein content of the
clarified cell extract was measured (Bio-Rad detergent-compatible assay), and the samples were diluted 2:1 with Laemmli sample buffer concentrate. Samples were subjected to SDS-PAGE (10% acrylamide) at
equal total protein loads (by changing the volume loaded between 5 and
20 µl). Proteins were transferred to nitrocellulose membranes by
Western blot (1 h 15 min at 100 V and 250 mA) in Tris-glycine buffer.
Western blots were blocked overnight in 10% skim milk or 1% bovine
serum albumin (BSA) and probed with rabbit anti-PAK1 (-PAK N-20,
Santa Cruz Biotechnology, skim milk blocked) or goat anti-PAK3 (
-PAK
N-19, Santa Cruz Biotechnology, BSA blocked) antibodies diluted 1:500.
The blots were washed and incubated with anti-rabbit or anti-goat
secondary antibody linked to horseradish peroxidase diluted to
1:10,000. Enhanced chemiluminescence kits (Amersham Pharma) were used
for detection. Specific binding of the primary antibody was assessed by
adding the peptide used to generate the primary antibody (
-PAK N-20
or
-PAK C-19 blocking peptide, Santa Cruz Biotechnology) to the
primary incubation (1:50 dilution, i.e., 10-fold excess over the
primary antibody).
Detection of caldesmon phosphorylation. Phosphorylation of caldesmon at Ser657 was identified using phosphocaldesmon-specific antibodies. The antibodies were generated in rabbits against the synthetic phosphopeptide EGVNIKS(p) MWEKGC (a sequence based on Ser657 of chicken gizzard h-caldesmon) coupled to a carrier protein, keyhole limpet hemocyanin. Serum was first passed through an affinity column for the unphosphorylated peptide to remove antibodies directed against the unphosphorylated epitopes. The effluent was then passed through a second column coupled to the phosphorylated peptide. Specificity of the antibody for phosphorylated Ser657 (pSer657) caldesmon was determined by Western blot using purified chicken gizzard h-caldesmon and an aliquot of this caldesmon that was phosphorylated by GST-mPAK3. Chicken gizzard caldesmon was purified and phosphorylated by mPAK3 as described previously (6). Western blots of intact canine trachealis were probed with pSer657 caldesmon antibody (diluted 1:300 in 1% BSA). Western blots were performed as described above.
Preparation of Triton-skinned ASM.
Dog trachealis muscle was harvested from mongrel dogs as described
above. Short (~2-cm-long) sections of trachealis were incubated in
sucrose-potassium solution at 4°C twice for 30 min each to deplete
the tissue of Ca2+ and relax the smooth muscle
(23). Smooth muscle was skinned in sucrose-potassium
solution supplemented with Triton X-100 (1%) and DTE (0.5 mM) for
24 h at 4°C (23). After it was skinned, the
trachealis was washed for 1 h at 4°C in glycerol-relaxing storage solution to remove Triton X-100. Skinned trachealis was stored
at 20°C in glycerol-relaxing solution for up to 8 wk
(23).
Skinned ASM protocols. Thin fiber bundles (~200-300 µm) were mounted on an optical force transducer connected to a computerized data acquisition program (Codas). Resting tension was set at ~200 µN using a micromanipulator attached to one of the mounting posts. Fibers were equilibrated in Ca2+-free relaxing solution (pCa > 9) for 10 min and then exposed to maximal Ca2+ contracting solution (pCa 4.4) or a submaximal Ca2+ concentration (pCa 6.2), which produces approximately half-maximal force. After peak contraction, the fiber was relaxed in pCa > 9 solution and then placed in one of the following: pCa > 9 GST-mPAK3, pCa > 9 GST-mPAK3K297R, GST-mPAK3 and Ca2+ at the same pCa as the first Ca2+ challenge (6.2 or 4.4), or Ca2+ at the same pCa as the first Ca2+ challenge (6.2 or 4.4) without PAK (see Figs. 2-4). The final PAK concentration varied from 0.5 to 5 µg/ml. The free Ca2+ concentration was calculated from the total Ca2+ concentration and EGTA concentration using the WinMaxC computer program (version 1.92, Stanford University) (2). Corrections for ATP, magnesium, temperature, and pH were included in the calculation of free Ca2+ (2).
Production of GST-mPAK3 and GST-mPAK3K297R. Active (mPAK3) and kinase dead inactive (mPAK3K297R) PAK were obtained from recombinant sources as GST fusion proteins (28). Escherichia coli (jm110) containing a plasmid (pGEX-KG) encoding ampicillin resistance and GST-mPAK3 (constitutively active) or GST-mPAK3K297R (inactive) were cultured in LB broth. Harvested cells were homogenized by sonication in lysis buffer, and cell fragments were removed by ultracentrifugation. GST fusion PAK was collected by binding to glutathione-Sepharose using a batch preparation protocol and dialyzed against imidazole (10 mM, pH 6.7) to remove glutathione and salts. The extract was concentrated by the Centriprep (Amicon) system. As reported previously, GST-mPAK3 is susceptible to degradation, resulting in different protein concentrations and enzymatic activities for each preparation (28). PAK stock solutions had a protein concentration of ~0.5 mg/ml and were used within 7 days of preparation.
Bacteria were cultured in LB broth containing 10 g/l tryptone (N-Z-Case Plus), 5 g/l yeast extract, 5 g/l NaCl, 0.5 mM NaOH, and 100 mg/l ampicillin. The lysis buffer used in preparing recombinant PAK contained 50 mM NaCl, 20 mM Tris (pH 7.4), 5 mM EGTA, 3 mM NaN3, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM DTE, 20 µg/ml lyposol, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 1% Triton X-100.Statistics. The results of skinned fiber force measurements are reported as means ± SE, with n representing the number of fibers studied. At least three animals and two PAK preparations (where appropriate) were included in each group. Repeated challenges in the same fibers were compared by paired t-test, with P < 0.05 considered significant. The response to GST-mPAK3 in Ca2+-free solution was compared with zero force production using Student's t-test against a fixed value (zero). For Western blots, tissues from at least four different animals were studied.
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RESULTS |
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PAK immunoreactivity in ASM.
Western blot analysis (Fig.
1A) showed a band at ~68 kDa
corresponding to PAK1 in mouse, rat, pig, and dog trachealis samples probed with anti-PAK1 N-20 antibody, which is specific for the NH2-terminal 20-amino acid sequence of PAK1. No
immunoreactivity was observed when a 10-fold excess of the synthetic
peptide used to generate the PAK1 antibody was included in the primary
antibody incubation (data not shown). As reported by others, PAK1 is
highly enriched in rat brain. In porcine and canine ASM, a few bands of
low molecular weight were usually detected in the cell lysates, which
likely represent degradation products. Surprisingly, we failed to
detect PAK3 in dog (Fig. 1B) as well as mouse, pig, and rat
tracheal smooth muscles (data not shown) using different batches of
anti-PAK3 N-19 antibodies from a commercial supplier (see
METHODS) and our laboratory, even when large amounts of
lysate samples were loaded (up to 80 µg of protein load).
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Contraction of skinned ASM by GST-mPAK3 in the absence of
Ca2+.
A constitutively active GST-mPAK3 induces Ca2+-independent
contraction of Triton-skinned fibers of trachealis (Figs.
2 and 3). The canine skinned fibers contracted when bathed in the contracting buffer containing maximal Ca2+ (pCa 4.4) or when incubated
with Ca2+-free relaxing buffer (pCa > 9) containing
~1 µg/ml of constitutively active GST-mPAK3 (Fig. 2, A
and B). GST-mPAK3 produced a significant increase in force
over basal tension (n = 10 fibers from 7 dogs, P < 0.005 by fixed-value t-test) that was
~40% of the force produced by maximal Ca2+ at pCa 4.4 (Fig. 3). The response to maximal Ca2+ had a rise time of
40 ± 5 min. Average rise time was approximately fivefold longer
for contraction to GST-mPAK3 than for maximal Ca2+
(200 ± 26 min), although more rapid responses occasionally
occurred (Fig. 2C). Variation in fiber thickness might
account for the varied time course of GST-mPAK3 contractions by
altering the diffusion time for large proteins into the fibers.
Catalytically dead GST-mPAK3K297R did not increase force in
fibers, which also displayed normal responses to Ca2+ (Fig.
2, B and D).
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GST-mPAK3 potentiates the response of ASM to
Ca2+.
To determine whether PAK is capable of increasing Ca2+
sensitization of contraction in the skinned fibers, we studied the
effect of mPAK3 on contraction at submaximal as well as maximal
Ca2+ concentrations (Fig. 4).
The canine skinned fibers were initially contracted in buffer
containing a submaximal concentration of Ca2+ (pCa 6.2) and
allowed to relax to baseline. The relaxed fibers were again incubated
in the pCa 6.2 buffer with and without GST-mPAK3. mPAK3 increases force
generation by ~80% at submaximal Ca2+ (n = 6 fibers from 5 animals, P < 0.01 by paired
t-test), demonstrating that PAK enhances Ca2+
sensitivity of contraction (Fig. 4A). However, sensitivity
to Ca2+ was not altered by GST-mPAK3 at maximal
Ca2+ (pCa 4.4, n = 6 fibers from 5 animals;
Fig. 4B).
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Phosphorylation of caldesmon in skinned trachealis.
mPAK3 phosphorylates h-caldesmon at two unique sites,
Ser657 and Ser687, at the calmodulin-binding
sites A and B, respectively (based on chicken gizzard sequence)
(6). Neither site can be phosphorylated by other kinases
that are known to phosphorylate caldesmon, including MAPK, cdc2, casein
kinase II, calmodulin kinase II, and protein kinase C. To determine the
level of caldesmon phosphorylation by PAK3 in the skinned fibers, we
raised specific antibodies against a synthetic peptide corresponding to
residues Glu651 and Gly662 (chicken gizzard
h-caldesmon) containing the pSer657 residue. This antibody
recognized GST-mPAK3-phosphorylated caldesmon but not its
unphosphorylated counterpart (Fig.
5A). Western blots of skinned
and resting intact canine trachealis showed low levels of
phosphorylation of caldesmon at Ser657 (Fig. 5,
B and C), indicating that caldesmon is
phosphorylated at this PAK-specific site in intact ASM. The level of
caldesmon phosphorylation at Ser657 increased significantly
60 and 90 min after mPAK3 treatment at pCa > 9 (Fig.
5D).
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DISCUSSION |
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Interest in the effects of small GTP-binding proteins and their associated kinases on smooth muscle has increased after evidence of the Ca2+-sensitizing properties of ROK (3, 8, 13, 15, 22). ROK and Rho have been the focus of several studies, whereas comparatively little work has examined other members of the Rho subfamily, Cdc42 and Rac, and their associated kinase PAK. In this study, we present evidence that 1) PAK1 is present in ASM across several mammalian species, whereas PAK3 protein is not detected by Western blot analyses; 2) mPAK3 produces Ca2+-independent contraction as well as Ca2+ sensitization of contraction in skinned ASM; 3) the level of mPAK3 phosphorylation of caldesmon increases after PAK-induced contraction in skinned fibers; and 4) mPAK3 phosphorylation of caldesmon is present in resting intact ASM. Given the homology of different PAK isoforms (1) and our use of the constitutively active catalytic domain of mPAK3, the presence of PAK-induced contraction in skinned ASM fiber is likely representative of the potential action of PAK1 within ASM. We speculate that PAK could have a significant role in ASM physiology and might affect nonspecific bronchial hyperresponsiveness.
Western blot analysis demonstrated the expression of PAK1 in the ASM of four species from three different mammalian orders (Rodentia, Ungulata, and Carnivora), suggesting that at least one PAK isoenzyme is ubiquitously expressed in ASM. Dechert et al. (5), who used an anti-PAK1 antibody that cross-reacts with PAK1, PAK2, and PAK3 in Western blot analysis, detected bands corresponding to the expected molecular weight of the three PAK isoenzymes in canine ASM, although PAK3 appears to be a minor component (see Fig. 1B in Ref. 5). However, we did not detect PAK3 in ASM using anti-PAK3-specific antibodies, although PAK3 has been shown to be expressed in guinea pig taenia coli and rat aortic smooth muscle (28). There is little information about the functional diversity of PAK1, PAK2, and PAK3 isoenzymes in various mammalian cell types, although it is likely that they share similar substrate targets considering the highly conserved sequence of the kinase domains. However, we do not rule out subtle differences in PAK isoenzyme functions in vivo as a result of more conspicuous sequence differences at their NH2-terminal regulatory domains, which may lead to a difference in subcellular targeting and localization of the kinases and, therefore, substrate accessibility.
Addition of exogenous GST-mPAK3 produced Ca2+-independent contractions of Triton-skinned dog ASM. Although previous studies have demonstrated Ca2+-independent contraction of guinea pig taenia coli (a model phasic smooth muscle) in response to GST-mPAK3 (28), to our knowledge this is the first report of the effect of PAK on ASM (a model tonic smooth muscle). A role for PAK in control of ASM tone is of considerable interest because of the importance of smooth muscle in airway responsiveness and asthmatic disease. This may be especially relevant given a recent report that shows that PAK induces migration of cultured canine tracheal smooth muscle in response to platelet-derived growth factor (5). If PAK is activated in the airway remodeling response associated with asthma, our data also raise the possibility that it may also contribute to enhanced contractile function.
In addition to Ca2+-independent contractions, GST-mPAK3 enhanced the response to moderate Ca2+ concentrations (i.e., caused Ca2+ sensitization). This is a novel finding, because previous studies (using nonmuscle cells) have shown inactivation of MLCK by PAK in baby hamster kidney-21 and endothelial cells (8, 17), an action that would inhibit Ca2+-calmodulin contractions. We found no evidence for reduced contractility. Endothelial cells and ASM cells express different isoforms of MLCK (29), and this may alter the actions of PAK in these cell types. Furthermore, the more organized contractile machinery in smooth muscle than in nonmuscle cells may prevent access of PAK to MLCK. The different effects of PAK on Ca2+-induced contractions of skinned smooth muscle and permeabilized endothelial cells could easily be due to the different cell types studied and are suggestive of different roles for PAK in muscle and nonmuscle.
Although the mechanism of action for PAK-induced smooth muscle contraction is unknown, MLC20 is not believed to be involved, because its phosphorylation state does not change in skinned taenia coli exposed to PAK (28). Van Eyk et al. (28) showed that PAK phosphorylates several proteins in Triton-skinned fibers from guinea pig taenia coli, including desmon and caldesmon. Caldesmon is phosphorylated by PAK at two unique sites (Ser657 and Ser687) that are not phosphorylated by other known kinases (6). Phosphorylation of caldesmon by GST-mPAK3 reduces the inhibitory action of caldesmon on actin-stimulated myosin ATPase activity (6), which could produce contraction by allowing a greater interaction between actin and unphophorylated myosin. PAK may also have a significant role in ASM cell migration and airway remodeling through a p38 MAPK pathway (5). The role of PAK in smooth muscle is unknown; however, the presence of PAK in ASM from several species and the actions of exogenous PAK on skinned fibers suggest a role for PAK in contraction or Ca2+ sensitization. In intact ASM, however, PAK could be compartmentally separated from its target in skinned fibers and unable to influence contraction. Our result showing phosphorylation of caldesmon at a unique PAK phosphorylation site (Ser657) in intact tissue indicates that endogenous PAK does gain access to the thin filaments and contractile machinery of intact ASM. To our knowledge, this is the only evidence implicating caldesmon as a target of PAK in vivo.
In addition to the normal physiological role of PAK in ASM, it could also be associated with the enhanced contractility and hyperresponsiveness associated with asthma. We clearly demonstrated the presence of PAK in ASM from several species and that PAK can induce contraction of ASM. Thus enhanced expression or activation of PAK in ASM could lead to enhanced smooth muscle responsiveness.
In conclusion, monomeric G proteins of the Rho subfamily and their associated kinases are a group of signal transduction proteins, the function of which has not been fully explored in ASM. We have found evidence of at least one PAK isoenzyme (PAK1) in ASM from many species, suggesting that PAK has a regulatory function in ASM. Exogenous mPAK3 induced Ca2+-independent contractions and Ca2+ sensitization in skinned ASM fibers, which is associated with phosphorylation of a PAK-specific site of caldesmon. These findings implicate PAK in the control of contractile function of the airways. Caldesmon phosphorylation at a PAK-specific site in intact ASM suggests that endogenous PAK can and does phosphorylate contractile filament proteins. The mechanism of action of PAK and the physiological role of PAK in contraction remain to be explored. We speculate that PAK and other monomeric G protein-associated kinases have the potential to contribute to airway hyperresponsiveness or other pathological states of enhanced smooth muscle contraction.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Strauss for help with the skinned fiber technique and Dr. Brian Foster and Nina Buscemi for help in producing GST-mPAK3.
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FOOTNOTES |
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This work was supported by the Canadian Institutes for Health Research, the Ontario Thoracic Society, and the Ontario Heart and Stroke Foundation. P. K. McFawn was supported by a Canadian Lung Association/Canadian Institutes of Health Research/Glaxo-Welcome Fellowship.
Address for reprint requests and other correspondence: J. T. Fisher, Dept. of Physiology, Rm. 234 Botterell Hall, Queen's University, Kingston, ON, Canada K7L 3N6 (E-mail: fisherjt{at}post.queensu.ca).
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.
First published January 3, 2003;10.1152/ajplung.00068.2002
Received 25 February 2002; accepted in final form 2 December 2002.
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