Relationship between paxillin and myosin phosphorylation during muscarinic stimulation of smooth muscle

Dolly Mehta, Zhonglin Wang, Ming-Fang Wu, and Susan J. Gunst

Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The tyrosine phosphorylation of paxillin increases in association with force development during tracheal smooth muscle contraction, suggesting that paxillin plays a role in the contractile activation of smooth muscle [Z. L. Wang, F. M. Pavalko, and S. J. Gunst. Am. J. Physiol. 271 (Cell Physiol. 40): C1594-C1602, 1996]. We compared the Ca2+ sensitivity of the tyrosine phosphorylation of paxillin and myosin light chain (MLC) phosphorylation in tracheal muscle and evaluated whether MLC phosphorylation is necessary to induce paxillin phosphorylation. Ca2+-depleted muscle strips were stimulated with 10-7-10-4 M acetylcholine (ACh) in 0, 0.05, 0.1, or 0.5 mM extracellular Ca2+. In the absence of extracellular Ca2+, 10-4 M ACh induced a maximal increase in paxillin phosphorylation without increasing MLC phosphorylation or force. Increases in extracellular Ca2+ concentration did not further increase paxillin phosphorylation. However, during stimulation with 10-6 M ACh, paxillin phosphorylation increased with increases in extracellular Ca2+ concentration. We conclude that the tyrosine phosphorylation of paxillin can be stimulated by signaling pathways that do not depend on Ca2+ mobilization and that the activation of contractile proteins is not required to elicit paxillin phosphorylation.

myosin light chain phosphorylation; cytoskeleton; focal adhesion proteins; smooth muscle contraction

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE CYTOSKELETAL PROTEINS paxillin and talin have been localized to the focal adhesion sites of cultured cells (5, 27) as well as to the membrane-associated dense plaque (MADP) sites of smooth muscle cells (9, 28). These proteins are thought to play a role in linking actin filaments to transmembrane integrins to enable force transmission across the membrane (4, 31). In cultured cells, talin and paxillin undergo phosphorylation during integrin-mediated cell adhesion and during stimulation by a variety of mitogens and growth factors (6, 16, 26, 29, 33). The phosphorylation of these proteins has been correlated with the assembly of actin stress fibers and with focal adhesion formation (6,7, 23, 29). It has been proposed that the phosphorylation of actin-membrane linker proteins may regulate the interaction of actin filaments with the cytoplasmic domains of transmembrane integrins in focal adhesions and smooth muscle dense plaques (5, 7, 18, 29).

In previous studies we reported that the contraction of tracheal smooth muscle with muscarinic agonists results in a three- to fourfold increase in the phosphorylation of paxillin and talin (17, 32). The increase in paxillin phosphorylation occurs on tyrosine residues, whereas talin is phosphorylated on serine-threonine residues. The changes in the phosphorylation of these MADP proteins occur with a time course similar to force development, suggesting that they may play a role in the contractile activation of smooth muscle. We have postulated that the stimulation of smooth muscle cells with contractile agonists may initiate active processes that regulate the organization of the actin cytoskeleton and the attachment of actin filaments to the membrane at MADP sites and that these events may occur in parallel to the activation of contractile proteins (17). The remodeling of the organization of actin filaments could serve to optimize force development to the physical conformation of the smooth muscle cell at the time of contractile activation.

If the tyrosine phosphorylation of paxillin plays a role in smooth muscle contraction, regulation of the phosphorylation of paxillin and of myosin light chains (MLCs) may be interdependent and may occur through the stimulation of a common signaling pathway. This is suggested by studies of cultured fibroblasts, in which the activation of contractile proteins was found to be prerequisite to the tyrosine phosphorylation of paxillin, the assembly of actin stress fibers, and focal adhesion formation (4, 8). Alternatively, the activation of contractile and cytoskeletal proteins may be parallel events elicited through distinct signaling pathways.

Agonists that activate smooth muscle tissues via receptor-coupled pathways can also stimulate pathways that increase the Ca2+ sensitivity of the serine-threonine phosphorylation of the regulatory MLC (10, 13, 20). It has been proposed that the mechanisms that regulate the Ca2+ sensitivity of contractile activation in smooth muscle may involve protein tyrosine phosphorylation (25).

In the present study we have compared the Ca2+ sensitivity of the tyrosine phosphorylation of paxillin with the Ca2+ sensitivity of MLC phosphorylation during the muscarinic stimulation of tracheal smooth muscle strips. We evaluated whether the activation of contractile proteins is required to induce the tyrosine phosphorylation of paxillin in this tissue.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue preparation. Mongrel dogs weighing 20-25 kg were anesthetized with pentobarbital sodium and quickly exsanguinated. A 10- to 15-cm segment of extrathoracic trachea was immediately removed and immersed in physiological saline solution (PSS) composed of (in mM) 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose. The solution was aerated with 95% O2-5% CO2 to maintain a pH of 7.4. Rectangular strips of trachealis muscle 12-15 mm long and 2-3 mm wide were dissected from the trachea after removal of the epithelium and connective tissue layer. Muscle strips were mounted in PSS at 37°C in a 25-ml glass tissue bath and attached to a Grass force transducer at a resting tension of 2-4 g and then equilibrated for ~90 min. Each muscle was then stimulated repeatedly with 10-5 M acetylcholine (ACh; Sigma Chemical). The optimal length for maximal active force (Lo) was determined by increasing the muscle length progressively after each stimulation until the force of active contraction reached a maximum (Fmax).

Ca2+ depletion of muscle strips. After the determination of Lo, muscle strips were depleted of Ca2+ as described previously (10). Briefly, strips were incubated in Ca2+-free PSS containing 0.1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for 10 min for the removal of extracellular Ca2+. No change in resting tension occurred when the bath was changed from PSS to Ca2+-free PSS containing 0.1 mM EGTA. Muscle strips were then stimulated for 10 min by addition of 10-5 M ACh to the Ca2+-free PSS. This step was repeated three to four times with 10-5 M ACh. Between each stimulation the strips were incubated in Ca2+-free PSS containing 0.1 mM EGTA for 10 min. Stimulation with 10-5 M ACh initially produced a force of ~70% Fmax, but subsequent stimulations resulted in progressively smaller contractions. At the end of the depletion protocol, force in response to 10-5 M ACh was <10% of Fmax. The strips did not contract in response to 60 mM KCl after the Ca2+-depletion protocol.

Experimental design. Up to 14 muscle strips from a single trachea were studied concurrently. Duplicate muscle strips were used for each measurement. Ca2+-depleted strips were stimulated with ACh in the absence of extracellular Ca2+ for 5 min and then rapidly freeze clamped with liquid N2-cooled tongs for measurement of the tyrosine phosphorylation of paxillin or MLC phosphorylation. The effects of Ca2+ on paxillin and MLC phosphorylation were also assessed by increasing the extracellular Ca2+ concentration ([Ca2+]o) during stimulation with ACh. Muscle strips not subjected to the Ca2+-depletion protocol were also frozen at rest or after stimulation with 10-4 or 10-6 M ACh. In most cases, separate experiments were performed for the analysis of MLC phosphorylation and paxillin phosphorylation for any given protocol.

Measurement of the tyrosine phosphorylation of paxillin. The tyrosine phosphorylation of paxillin was determined by Western blot as described previously (32). Frozen muscle strips were pulverized under liquid N2, and the powder was transferred to dry-ice-cooled centrifuge tubes. While on dry ice, 300 µl of extraction buffer were added to each of the tubes, and then they were quickly vortexed. The extraction buffer contained 20 mM tris(hydroxymethyl)aminomethane (pH 7.4), 1% Triton X-100, 0.2% sodium dodecyl sulfate, 2 mM EDTA, phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and 2 mM sodium pyrophosphate), and protease inhibitors (2 mM benzamidine, 0.5 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Each sample was then boiled for 5 min to inactivate phosphatases and proteases, and then it was kept at 4°C for 1 h. The concentration of protein in each sample was determined using a standard bicinchoninic acid protein assay kit (Pierce).

Protein (40 µg) from each muscle extract was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose, blocked with 2% gelatin, and probed with mouse monoclonal antiphosphotyrosine antibody (ICN Pharmaceuticals) and then with horseradish peroxidase anti-mouse immunoglobulin (Amersham) for visualization by chemiluminescence. Nitrocellulose membranes were then stripped of bound antibodies and reprobed with mouse monoclonal antibody against paxillin (Transduction Laboratories) to confirm the location of paxillin and to normalize for minor differences in protein loading. Phosphotyrosine-containing proteins and paxillin were visualized by chemiluminescence and quantitated by scanning densitometry. In previous studies, similar increases in paxillin phosphorylation were obtained from phosphotyrosine blots of paxillin immunoprecipitated from muscle extracts and from blots of whole muscle extracts (17, 32).

Measurement of MLC phosphorylation. MLC phosphorylation was measured in separate experiments. Frozen muscle strips were immersed in dry ice-cooled acetone containing 10% (wt/vol) trichloroacetic acid (TCA) and 10 mM dithiothreitol (DTT). Strips were thawed in acetone-TCA-DTT at room temperature and then washed with acetone-DTT. MLCs were extracted for 60 min in 8 M urea, 20 mM tris(hydroxymethyl)- aminomethane, 22 mM glycine, and 10 mM DTT. Proteins were separated by glycerol-urea polyacrylamide gel electrophoresis and blotted to nitrocellulose. MLCs were specifically labeled with polyclonal rabbit anti-MLC 20 antibody. The primary antibody was detected with 125I-labeled recombinant protein A (New England Nuclear). Unphosphorylated and phosphorylated bands of MLCs were localized on nitrocellulose membranes by autoradiography. Bands were cut out and counted in a gamma counter. Background counts were subtracted, and fractional phosphorylation was calculated as the ratio of phosphorylated MLCs to total MLCs.

Statistical analysis. Comparisons among different groups were performed by one-way analysis of variance or Kruskal-Wallis one-way analysis of variance. Differences between pairs of groups were analyzed by Student's t-test or Dunn's method. All statistical analyses were performed using SigmaStat software. Values of n represent the number of experiments used to obtain each value. P < 0.05 was considered to be significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of ACh concentration on MLC phosphorylation and the tyrosine phosphorylation of paxillin in Ca2+-depleted muscle strips. Duplicate muscle strips were depleted of Ca2+ and then stimulated with 10-7-10-4 M ACh in the absence of extracellular Ca2+ for 5 min. They were then quickly frozen for the measurement of MLC phosphorylation and the tyrosine phosphorylation of paxillin. MLC phosphorylation and tyrosine phosphorylation of paxillin were also determined in muscle strips that had not been depleted of Ca2+. (Changes in the tyrosine phosphorylation of paxillin are illustrated in the immunoblot shown in Fig. 1.)


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Fig. 1.   Immunoblots of muscle extracts probed with antiphosphotyrosine antibody (A) and stripped and reprobed with antipaxillin antibody (B) to determine effect of ACh concentration on tyrosine phosphorylation of paxillin (Pax) in Ca2+-depleted tracheal muscle strips. Duplicate Ca2+-depleted strips were quickly frozen in resting state or after stimulation for 5 min with 10-7-10-4 ACh in absence of extracellular Ca2+. Muscle strips that were not depleted of Ca2+ were also frozen at rest or after stimulation for 5 min with 10-4 M ACh in 2.4 mM extracellular Ca2+. Arrows, paxillin. Numbers on left, molecular masses (in kDa) of standard weight markers.

The effects of ACh on active force, MLC phosphorylation, and tyrosine phosphorylation of paxillin in Ca2+-depleted strips from five experiments are shown in Fig. 2. After Ca2+ depletion the contractile response to 10-4 M ACh was 10 ± 2% of the response of undepleted muscle strips to the same stimulus. There was no significant increase in force in Ca2+-depleted muscle strips at lower concentrations of ACh. Paxillin phosphorylation increased significantly over its resting level at 10-5 or 10-4 M ACh. The increase in paxillin phosphorylation caused by 10-4 M ACh in Ca2+-depleted muscle strips was not significantly different from that in non-Ca2+-depleted muscle strips (Fig. 2). Ca2+ depletion also had no significant effect on paxillin phosphorylation in resting muscle strips. In contrast, MLC phosphorylation was not significantly increased over resting levels when Ca2+-depleted strips were stimulated with any concentration of ACh.


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Fig. 2.   Changes in force, myosin light chain (MLC) phosphorylation, and tyrosine phosphorylation of paxillin (means ± SE) during stimulation of tracheal muscle strips with 10-7-10-4 M ACh for 5 min. Filled bars, strips stimulated with ACh; open bars, unstimulated (resting) strips. Ca2+ depletion had no effect on resting values of MLC phosphorylation or paxillin phosphorylation. Data shown for force (n = 10) include measurements on muscle strips used to assay MLC phosphorylation (n = 5) and paxillin phosphorylation (n = 5). Force is normalized to maximal force obtained in response to 10-4 M ACh in muscles not subjected to Ca2+ depletion and maintained in 2.4 mM extracellular Ca2+. * Significantly different from resting values (P < 0.05).

Ca2+ sensitivity of paxillin phosphorylation and MLC phosphorylation in response to ACh. Ca2+-depleted muscle strips were stimulated with 10-4 or 10-6 M ACh in Ca2+-free buffer. After 1 min in ACh, 0.05, 0.1, or 0.5 mM extracellular Ca2+ was added or strips were maintained in 0 mM extracellular Ca2+. Muscle strips were frozen 5 min after the addition of extracellular Ca2+, and MLC phosphorylation and the tyrosine phosphorylation of paxillin were determined (Fig. 3).


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Fig. 3.   Immunoblots of muscle extracts probed with antiphosphotyrosine antibody (A) and stripped and reprobed with antipaxillin antibody (B) to determine effect of increases in extracellular Ca2+ concentration ([Ca2+]o) on tyrosine phosphorylation of paxillin in response to stimulation of Ca2+-depleted muscle strips with 10-4 M ACh. Ca2+-depleted strips were quickly frozen at rest or after stimulation with 10-4 M ACh in Ca2+-free buffer. After 1 min in ACh, 0.05, 0.1, or 0.5 mM extracellular Ca2+ was added for 5 min or strips were maintained in 0 mM extracellular Ca2+ for 5 min. Arrows, paxillin. Numbers on left, molecular masses (in kDa) of standard weight markers.

Stimulation with 10-4 M ACh increased paxillin phosphorylation to the same extent in Ca2+-depleted and non-Ca2+-depleted muscle strips (Fig. 4). Paxillin phosphorylation in response to 10-4 M ACh was not increased further when extracellular Ca2+ was added to the Ca2+-depleted muscle strips. In contrast, MLC phosphorylation did not increase significantly in response to 10-4 M ACh until [Ca2+]o reached 0.1 mM. MLC phosphorylation remained significantly lower in Ca2+-depleted than in non-Ca2+-depleted muscle strips, even when [Ca2+]o was increased to 0.5 mM (Fig. 4).


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Fig. 4.   Changes in force, MLC phosphorylation, and tyrosine phosphorylation of paxillin (means ± SE) in Ca2+-depleted tracheal muscle strips in response to readdition of extracellular Ca2+ during stimulation with 10-4 M ACh. Filled bars, strips stimulated with ACh; open bars, unstimulated (resting) strips. Data shown for force (n = 10) include measurements on muscle strips used to assay MLC phosphorylation (n = 5) and paxillin phosphorylation (n = 5). Force is normalized to maximal force obtained in response to 10-4 M ACh in muscles not subjected to Ca2+ depletion and maintained in 2.4 mM extracellular Ca2+. * Significantly different from resting values (P < 0.05).

Ca2+-depleted muscle strips stimulated with a lower concentration of ACh (10-6 M) also showed an increase in the tyrosine phosphorylation of paxillin, but the effect was not statistically significant. However, under these conditions the phosphorylation of paxillin increased with increasing [Ca2+]o (Fig. 5). At 0.5 mM extracellular Ca2+, stimulation with 10-6 M ACh increased paxillin phosphorylation to the same extent as in undepleted muscle tissues at 2.4 mM extracellular Ca2+. As with 10-4 M ACh, stimulation with 10-6 M ACh did not increase MLC phosphorylation in the absence of extracellular Ca2+; however, increases in [Ca2+]o caused significant increases in MLC phosphorylation. After stimulation with 10-6 M ACh, MLC phosphorylation remained significantly lower in Ca2+-depleted muscles treated with 0.5 mM extracellular Ca2+ than in non-Ca2+-depleted muscles maintained in 2.4 mM extracellular Ca2+.


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Fig. 5.   Changes in force, MLC phosphorylation, and tyrosine phosphorylation of paxillin (means ± SE) in Ca2+-depleted tracheal muscle strips in response to readdition of extracellular Ca2+ during stimulation with 10-6 M ACh. Filled bars, strips stimulated with ACh; open bars, unstimulated (resting) strips. Data shown for force (n = 10) include measurements on muscle strips used to assay MLC phosphorylation (n = 5) and paxillin phosphorylation (n = 5). Force is normalized to maximal force obtained in response to 10-4 M ACh in muscles not subjected to Ca2+ depletion and maintained in 2.4 mM extracellular Ca2+. * Significantly different from resting values (P < 0.05).

The effects of increases in [Ca2+]o during stimulation with 10-4 and 10-6 M ACh are compared in Fig. 6. At either concentration of ACh, increases in [Ca2+]o resulted in increases in MLC phosphorylation. In contrast, paxillin phosphorylation was unaffected by increases in [Ca2+]o during stimulation with 10-4 M ACh. However, paxillin phosphorylation increased with increases in [Ca2+]o during submaximal stimulation with 10-6 M ACh. Stimulation with ACh under all conditions increased paxillin phosphorylation proportionally more than MLC phosphorylation.


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Fig. 6.   Comparison of increases in MLC phosphorylation (n = 5) and tyrosine phosphorylation of paxillin (n = 5) induced by 10-4 M ACh (A) and 10-6 M ACh (B) in absence of extracellular Ca2+ or in presence of 0.05, 0.1, or 0.5 mM extracellular Ca2+. Increases in phosphorylation of MLC and in phosphorylation of paxillin are quantified as percentage of increase in phosphorylation that occurs in response to ACh in non-Ca2+-depleted muscle strips maintained in 2.4 mM extracellular Ca2+. * Significantly different (P < 0.05).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Our results show that maximal muscarinic stimulation of Ca2+-depleted tracheal smooth muscle strips in the absence of extracellular Ca2+ elicits a maximal increase in the tyrosine phosphorylation of paxillin. Under the same conditions, muscarinic receptor stimulation does not cause contraction of the muscle, nor does it produce a significant increase in MLC phosphorylation. Thus the tyrosine phosphorylation of paxillin can occur in the absence of the activation of contractile proteins, indicating that the tyrosine phosphorylation of paxillin and MLC phosphorylation can be elicited by independent signaling pathways. These observations demonstrate that contractile protein activation is not prerequisite to the tyrosine phosphorylation of paxillin in tracheal smooth muscle.

Our results also suggest a Ca2+-independent pathway for the tyrosine phosphorylation of paxillin in tracheal smooth muscle in addition to a Ca2+-activated pathway. When Ca2+-depleted tracheal smooth muscle strips were maximally stimulated with ACh in the absence of extracellular Ca2+, a maximal increase in paxillin phosphorylation was observed that was unaffected by increases in [Ca2+]o. This indicates that maximal paxillin phosphorylation can be elicited through a pathway that does not require Ca2+ mobilization. Tyrosine phosphorylation of paxillin was also elevated during submaximal stimulation with 10-6 M ACh in the absence of extracellular Ca2+. However, at this ACh concentration, significant increases in paxillin phosphorylation were elicited when [Ca2+]o was increased. Thus, although the muscarinic stimulation of tracheal smooth muscle in the absence of extracellular Ca2+ can stimulate a maximal increase in the tyrosine phosphorylation of paxillin, a Ca2+-activated mechanism for stimulating paxillin tyrosine phosphorylation is also present. When the muscarinic stimulation of tracheal smooth muscle is submaximal, the Ca2+-activated increase in paxillin phosphorylation adds to the paxillin phosphorylation that occurs in the absence of extracellular Ca2+. Therefore, although independent pathways for the phosphorylation of MLC and paxillin appear to be present, a common Ca2+-activated pathway may also be able to stimulate the phosphorylation of both proteins. These results are consistent with previous observations in cultured fibroblasts in which the tyrosine phosphorylation of paxillin can be stimulated in the absence of Ca2+ mobilization (24, 33). In some cultured cells, paxillin phosphorylation has also been shown to be sensitive to Ca2+ activation (14).

Our present results are similar to our previous observation that a significant increase in MLC phosphorylation occurs in Ca2+-depleted canine tracheal smooth muscle strips in response to muscarinic stimulation only after [Ca2+]o is increased to 0.05 mM (10). In this previous study we also measured intracellular Ca2+ in Ca2+-depleted tracheal muscle strips. We found that muscarinic stimulation elicited a very small increase in intracellular Ca2+, even in the absence of extracellular Ca2+. Thus in the present study the maximal increase in paxillin phosphorylation elicited by ACh in the absence of extracellular Ca2+ may require a small increase in intracellular Ca2+; however, this increase in intracellular Ca2+ is insufficient to induce MLC phosphorylation.

The results of studies in cultured fibroblasts contrast with ours, in that they suggest that MLC phosphorylation and contractile protein activation are required for the tyrosine phosphorylation of the cytoskeletal proteins paxillin and pp125 focal adhesion kinase (FAK) (4, 8). There is evidence that FAK is the tyrosine kinase that phosphorylates paxillin in cultured smooth muscle cells and fibroblasts (3, 30). The activation of fibroblasts by a number of agents, including vasopressin, endothelin, and lysophosphatidic acid, stimulates the formation of stress fibers and the assembly of focal adhesions. These events are associated with a marked increase in the tyrosine phosphorylation of paxillin and FAK and with MLC phosphorylation (2, 4, 8, 19, 21, 22). The small GTP-binding protein rho has been shown to mediate these events (2, 4, 8, 19, 21, 22). The inhibition of fibroblast contractility and MLC phosphorylation by a number of mechanisms prevents rho-induced paxillin phosphorylation and FAK activation, and it also prevents actin stress fiber formation and focal adhesion assembly (4, 8).

The receptor-mediated activation of tracheal and vascular smooth muscles by agonists such as histamine, phenylephrine, or carbachol has been shown to induce a Ca2+ sensitization of MLC phosphorylation and force (10, 13, 20). This enhanced sensitivity of contractile activation appears to be G protein mediated (13). The small G protein rho has been implicated in the Ca2+ sensitization of contractile protein activation (11) by mediating the inhibition of MLC phosphatase (12, 15) or by stimulating the phosphorylation of MLC on serine-threonine residues by rho kinase (1). Inasmuch as rho is also implicated in cellular processes associated with cytoskeletal organization in cultured fibroblasts (21), receptor activation by contractile agonists in smooth muscle may initiate processes leading to cytoskeletal reorganization as well as to the sensitization of contractile protein activation (25). Although our data indicate that MLC phosphorylation is not prerequisite to paxillin phosphorylation in tracheal smooth muscle, it remains possible that the tyrosine phosphorylation of cytoskeletal proteins plays a role in mediating the Ca2+ sensitization of contractile proteins in this tissue.

In conclusion, the results of this study demonstrate that the muscarinic stimulation of tracheal smooth muscle can activate signaling pathways for the tyrosine phosphorylation of paxillin that appear to be independent of Ca2+ mobilization. In the absence of extracellular Ca2+, muscarinic stimulation elicits high levels of tyrosine phosphorylation on paxillin without eliciting significant increases in MLC phosphorylation or contraction. This observation demonstrates that MLC phosphorylation is not required to stimulate paxillin phosphorylation in tracheal smooth muscle; thus the activation of paxillin can occur independently of contractile protein activation. This is consistent with our previous suggestion that the activation of MADP proteins and contractile proteins may be parallel events in the contractile activation of smooth muscle. Paxillin phosphorylation may play a role in regulating the organization of actin filaments in smooth muscle tissues in response to contractile stimulation. Cytoskeletal proteins may also be involved in regulating the sensitivity of contractile proteins to activation by Ca2+.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-29289 and by a postdoctoral fellowship from the American Heart Association, Indiana Affiliate, to D. Mehta.

    FOOTNOTES

Address for reprint requests: S. J. Gunst, Dept. of Physiology and Biophysics, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5126.

Received 5 May 1997; accepted in final form 13 November 1997.

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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 274(3):C741-C747
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