The focal adhesion protein paxillin regulates contraction in canine tracheal smooth muscle

Abstract

The adapter protein paxillin localizes to the focal adhesions of adherent cells and has been implicated in the regulation of cytoskeletal organization and cell motility. Paxillin undergoes tyrosine phosphorylation in response to the contractile stimulation of tracheal smooth muscle. We therefore hypothesized that paxillin may be involved in regulating smooth muscle contraction. Tracheal smooth muscle strips were treated with paxillin antisense oligonucleotides to inhibit the expression of paxillin protein selectively. Paxillin antisense or sense was introduced into muscle strips by reversible permeabilization and strips were incubated with antisense or sense for 3 days. Paxillin antisense selectively depressed paxillin expression, but it did not affect the expression of vinculin, focal adhesion kinase, myosin light chain kinase, myosin heavy chain or myosin light chain. Tension development in response to stimulation with ACh or KCl was markedly depressed in paxillin-depleted muscle strips. Active force and paxillin protein expression were restored by incubation of antisense-treated strips in the absence of oligonucleotides. The depletion of paxillin did not inhibit the increase in intracellular free Ca²⁺, myosin light chain phosphorylation or myosin ATPase activity in response to contractile stimulation. The concentration of G-actin was significantly lower in unstimulated paxillin-depleted smooth muscle tissues than in normal tissues. While stimulation with acetylcholine caused a decrease in G-actin in normal muscle strips, it caused little change in the G-actin concentration in paxillin-depleted muscle strips, suggesting that paxillin is necessary for normal actin dynamics in smooth muscle. We conclude that paxillin is required for active tension development in smooth muscle, but that it does not regulate increases in intracellular Ca²⁺, myosin light chain phosphorylation or myosin ATPase activity during contractile stimulation. Paxillin may be important in regulating actin filament dynamics and organization during smooth muscle contraction.

Paxillin is a multidomain adapter protein that binds to structural and signalling proteins at the focal adhesion sites of substrate-adherent cells (Turner et al. 1990; Burridge & Chrzanowska-Wodnicka, 1996; Turner, 2000). Paxillin has been implicated in the regulation of cytoskeletal organization, cell migration and cell motility (Burridge et al. 1992; Turner et al. 1999; Ito et al. 2000; Nakamura et al. 2000; Petit et al. 2000; West et al. 2001). The actin cytoskeleton of smooth muscle cells connects to extracellular matrix proteins at membrane sites referred to as dense plaques, which are structurally similar to the focal adhesion sites of cultured cells (Burridge & Chrzanowska-Wodnicka, 1996). At these membrane plaque sites, the extracellular domains of transmembrane integrins engage with extra- cellular matrix proteins, and the cytoplasmic domains of β-integrin subunits connect with actin filaments via a series of linker proteins that include talin, vinculin and α-actinin (Small, 1985; Drenckhahn et al. 1988; Burridge & Chrzanowska-Wodnicka, 1996). These cytoskeletal structures have been proposed to mediate the transmission of force between the contractile apparatus and the extracellular matrix (Wang et al. 1993).

Paxillin has been implicated in the regulation of cytoskeletal dynamics in a number of cell types (Turner et al. 1999; Ito et al. 2000; Nakamura et al. 2000; Petit et al. 2000; West et al. 2001). It is a substrate for focal adhesion kinase (FAK) and Src kinase, which are tyrosine kinases that localize to focal adhesion sites of cultured substrate-adherent cells (Bellis et al. 1995; Richardson et al. 1997; Thomas et al. 1999). In tracheal smooth muscle, the tyrosine phosphorylation of both FAK and paxillin can be stimulated by muscarinic activation (Wang et al. 1996; Tang et al. 1999; Tang & Gunst, 2001a). Both FAK and paxillin phosphorylation can be mediated by a Ca²⁺-independent signalling pathway in this tissue (Mehta et al. 1998; Tang et al. 1999). The depletion of FAK by antisense oligonucleotides depresses the contractile activation of tracheal smooth muscle by inhibiting agonist-induced Ca²⁺ activation and myosin light chain phosphorylation, and FAK depletion also depresses the tyrosine phosphorylation of paxillin (Tang & Gunst, 2001a). Since paxillin is a substrate for FAK, we evaluated the possibility that paxillin might also be involved in regulating contraction.

The contractile activation of tracheal smooth muscle stimulates the polymerization of actin, which is necessary for active tension development in this tissue (Jones et al. 1999; Mehta & Gunst, 1999). Actin polymerization also occurs in other smooth muscle tissues and has been shown to regulate tension development (Cipolla & Osol, 1998; Barany et al. 2001). We and others have previously proposed that the organization of the actin cytoskeleton is plastic and that remodelling of the actin cytoskeleton enables smooth muscle cells to adapt their contractile response to changes in cell shape induced by external mechanical stimuli (Gunst et al. 1995; Cipolla & Osol, 1998; Mehta & Gunst, 1999; Gerthoffer & Gunst, 2001; Gunst & Wu, 2001; Cipolla et al. 2002). We therefore speculated that paxillin might play a role in regulating actin dynamics and cytoskeletal organization in smooth muscle.

In the present study, we evaluated the function of paxillin in the regulation of smooth muscle contraction by introducing antisense oligonucleotides for paxillin into tracheal smooth muscle tissues to suppress the expression of paxillin protein selectively. We then measured tension generation, intra- cellular Ca²⁺, myosin light chain phosphorylation, myosin ATPase activity and actin dynamics in paxillin-deficient smooth muscle tissues during contractile stimulation. The depletion of paxillin protein inhibited active tension development, but it had little or no effect on intracellular Ca²⁺, myosin light chain phosphorylation or myosin ATPase activity, indicating that paxillin does not regulate contractile protein activation or cross-bridge cycling. However, the depletion of paxillin disrupted normal actin dynamics, indicating that paxillin may be required for the regulation of cytoskeletal organization and actin filament remodelling in differentiated smooth muscle during contractile stimulation and tension development.

METHODS

Preparation of smooth muscle tissues

Mongrel dogs (20-25 kg) were anaesthetized with pentobarbitone sodium (30mgkg⁻¹, I.V.) and quickly exsanguinated. All experiments were carried out according to the guidelines of Institutional Animal Care and Use Committee, Indiana University School of Medicine. A 12-15 cm segment of extra-thoracic trachea was immediately removed and immersed in physiological saline solution (PSS) at 22 °C (composition (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 tracheal muscle 0.6-0.7 mm in diameter and 8-10 mm in length were dissected from the trachea after removal of the epithelium and connective tissue layer. The use of an appropriately sized muscle strip was critical for maintaining muscle contractility during the incubation period and for the successful introduction of oligonucleotides throughout the muscle strips. Each muscle strip was placed in PSS at 37 °C in a 25 ml organ bath and attached to a Grass force transducer (Grass Medical Instruments, Quincy, MA, USA). At the beginning of each experiment, the optimal length for muscle contraction was determined by increasing muscle length progressively until the force of active contraction in response to a contractile stimulus reached a maximum.

Oligodeoxynucleotides (ODNs) dissolved in Tris-EDTAbuffer were introduced into muscle strips according to experimental procedures described below. Muscle strips were then incubated for 3 days with ODNs in Dulbecco's modified Eagle's medium (DMEM). The strips were then returned to PSS at 37 °C in 25 ml organ baths and attached to Grass force transducers for the measurement of isometric force. For biochemical analysis, muscle strips were frozen using liquid N₂-cooled tongs and then pulverized under liquid N₂ using a mortar and pestle.

Introduction of paxillin antisense or paxillin sense ODNs into tracheal smooth muscle tissues

Antisense ODNs with the following sequence were designed to suppress paxillin expression selectively in canine tracheal smooth muscle: 5′-GCCATTTAGGGCCTCACT-3′. In some protocols, the following sequence of sense oligonucleotides was used as a control: 5′-AGTGAGGCCCTAAATGGC-3′. According to sequence matching results obtained from The National Center for Biotechnology Information, these sequences are not homologous to sequences of any other contractile proteins or cytoskeletal proteins. The ODNs were fully phosphorothiolated to enhance their intracellular stability in smooth muscle cells. The antisense molecule targeted to a region of mRNA that is unique to paxillin (Turner & Miller, 1994). The ODNs were synthesized and purified by Life Technologies (Rockville, MD, USA) or Integrated DNA Technologies (Coralville, IA, USA). A homologous region was confirmed for canine paxillin mRNA by using reverse transcription-polymerase chain reaction (RT-PCR) to amplify a 147 bp fragment from canine tracheal mRNA. The 5′-primer used for RT-PCR was 5′-TCCACCACCTCGCATATCTCT-3′. The 3′-primer used for RT-PCR was 5′-GCCATTTAGGGCCTCACT- GGA-3′.

The ODNs were introduced into the smooth muscle strips by reversible permeabilization using methods we have previously used for the introduction of focal adhesion kinase into tracheal muscle strips (Tang & Gunst, 2001a). After determination of the optimal length, muscle strips were attached to a metal mount at the appropriate length. The strips were placed in 0.5 ml tubes and incubated successively in each of the following solutions: Solution 1 (at 4°C for 120 min), containing 10 mm EGTA, 5 mm Na₂ATP, 120 mm KCl, 2 mm MgCl₂ and 20 mm N-tris(hydroxy- methyl)methyl-2-aminoethanesulphonic acid (Tes); Solution 2 (at 4°C overnight), containing 0.1 mm EGTA, 5 mm Na₂ATP, 120 mm KCl,2 mm MgCl₂,20 mm Tes and 10 μm antisense or sense ODNs;Solution3 (at4^°Cfor30min),containing0.1 mmEGTA, 5 mm Na₂ATP, 120 mm KCl, 10 mm MgCl₂ and 20 mm Tes; and Solution 4 (at 22 °C for 60 min), containing 110 mm NaCl, 3.4 mm KCl, 0.8 mm MgSO₄, 25.8 mm NaHCO₃, 1.2 mm KH₂PO₄ and 5.6 mm dextrose. Solutions 1-3 were maintained at pH 7.1 using Tes buffer and aerated with 100 % O₂. Solution 4 was maintained at pH 7.4 using a bicarbonate buffer and was aerated with 95 % O₂-5 % CO₂. After 30 min in Solution 4, CaCl₂ was added gradually to reach a final concentration of 2.4 mm. The strips were then incubated for 3 days in DMEM containing 5 mm Na₂ATP, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and 10 μm antisense or 10 μm sense oligonucleotides, which were kept at 37 °C, 5 % CO₂. The media were changed every other day.

Analysis of protein expression

Pulverized muscle strips were mixed with 50 ml of extraction buffer containing: 20 mm Tris-HCl at pH 7.4, 2 % Triton X-100, 0.2 % sodium dodecyl sulphate (SDS), 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 phenylmethylsulphonyl fluoride (PMSF)). Each sample was boiled and centrifuged for the collection of supernatant. Muscle extracts were boiled in sample buffer (1.5 % dithiothreitol, 2 % SDS, 80 mm Tris-HCl (pH 6.8), 10 % glycerol and 0.01 % Bromophenol Blue) for 5 min and then separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose, after which the membrane was cut into several parts for immunoblotting of different proteins. The nitrocellulose membrane was blocked with 5 % milk for 1 h and probed with monoclonal antibody to paxillin (clone 349, BD Biosciences (Transduction Labs), San Diego, CA, USA) or myosin light chain kinase (courtesy of Dr P. Gallagher, Indiana University) followed by horseradish peroxidase (HRP)- conjugated anti-mouse immunoglobulin (Ig) (Amersham Life Sciences, Arlington Heights, IL, USA). Nitrocellulose membranes were then stripped of bound antibodies. Focal adhesion kinase was probed using a monoclonal antibody to focal adhesion kinase (clone 77, Transduction Labs) and then with HRP-conjugated anti-mouse Ig (Amersham Life Sciences). Polyclonal antibodies to vinculin, myosin heavy chain and myosin light chain (BABCO, Richmond, CA, USA) were used to probe each of these proteins, followed by HRP-conjugated anti-rabbit IgG (ICN, Irvine, CA, USA). Proteins were visualized by enhanced chemiluminescence (ECL) and quantitated by scanning densitometry. Densitometric values of paxillin, FAK, vinculin, myosin light chain kinase (MLCK), myosin heavy chain and myosin light chain were determined for sense-treated and antisense-treated muscle strips and normalized to those of strips not treated with ODNs. The ratios of these proteins were calculated to verify that changes in protein expression were selective for paxillin.

Measurement of [Ca²⁺]i

Tracheal smooth muscle strips were pinned in a dish at a slightly stretched length (1.2 times slack length) and incubated in PSS containing 20 μm fura-2 AM, which was dissolved in 0.5 % dimethyl sulphoxide premixed with 0.01 %pluronic 127. Sonication was used to generate suspended micelles of fura-2 AM that facilitate the entry of fura-2 into the extracellular space of smooth muscle tissues. The tissues were incubated in the fura-2 AM solution for 3.5 h at room temperature. They were then washed in PSS for 30 min to remove extracellular fura-2 AM and to allow time for the hydrolytic conversion of intracellular fura-2 AM to fura-2. Tissues were mounted in a cuvette and attached to a force transducer for the simultaneous measurement of isometric force and fura-2 fluorescence using a Ratio Fluorescence Spectrophotomer (PTI International, Lawrenceville, NJ, USA). The muscle was illuminated alternately at excitation wavelengths of 340 and 380 nm at a frequency of 2 Hz. Emitted light was collected through a single longpass filter (510 nm) and detected with a photomultiplier tube. The fluorescence ratio of 340:380 was continuously computed by a dedicated computer.

Analysis of myosin light chain phosphorylation

Muscle strips were rapidly frozen at desired time points after contractile stimulation and then immersed in acetone containing 10 % (w/v) trichloroacetic acid and 10 mm dithiothreitol (acetone-TCA-DTT) which was precooled with dry ice. Strips were thawed in acetone-TCA-DTT at room temperature and then washed 4 times with acetone-DTT. Proteins were extracted for 60 min in 8 M urea, 20 mm Tris base, 22 mm glycine and 10 mm DTT. Myosin light chains (MLCs) were separated by glycerol-urea polyacrylamide gel electrophoresis and transferred to nitrocellulose. The membranes were blocked with 5 % bovine serum albumin and incubated with polyclonal affinity-purified rabbit myosin light chain 20 antibody. The primary antibody was reacted with ¹²⁵I-labelled recombinant protein A (New England Nuclear, Beverly, MA, USA). Unphosphorylated and phosphorylated bands of MLCs were detected by autoradiography. Bands were cut out and counted in a g-counter. Background counts were subtracted and MLC phosphorylation was calculated as the ratio of phosphorylated MLCs to total MLCs.

Permeabilization of muscle strips with β-escin

After 3 days of incubation, muscle strips were trimmed to a smaller size (0.5 mm wide, 5 mm long). These strips were pinned in Petri dishes and incubated at 22 °C in a relaxing solution composed of (mm): 8.5 disodium adenosine triphosphate (Na₂ATP), 4 potassium ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (K⁺-EGTA), 1 dithiothreitol (DTT), 10 Na creatinine phosphate, 20 imidazole, 8.9 magnesium acetate (MgAc₂), 100.5 potassium acetate (KAc) and 1 mg ml⁻¹ creatine phosphokinase (pH 7.1). After 10 min the strips were then incubated in the same solution with the addition of β-escin (100 mg ml⁻¹, Sigma), 1 μm leupeptin (a protease inhibitor) and 1 μm carbonyl cyanide m- chlorophenylhydrazone (FCCP; a mitochondrial blocker) for another 20-25 min. An algorithm of Fabiato (Fabiato & Fabiato, 1979) was used to calculate the composition of relaxing or contracting solutions containing free Ca²⁺ from pCa 9 to 5. For the measurement of isometric force, permeabilized muscle strips were mounted in tissue baths and attached to Gould GM-2 force transducers (Gould Instruments Inc., Valley View, OH, USA). In each experiment, permeabilization of the strips was verified by contracting the muscles with 10 μm Ca²⁺.

Determination of G-actin concentration and total actin

The concentration of G-actin in smooth muscle extracts was determined by measuring the inhibition of DNase I activity by G-actin (Blikstad et al. 1978; Mehta & Gunst, 1999). Pulverized smooth muscle strips were mixed with extraction buffer (1:3, w/v) and then incubated for 20 min at 4 °C. The extraction buffer contained: 60 mm Pipes, 25 mm Hepes, 10 mm EGTA, 2 mm MgSO4, 0.5 % Triton X-100, 0.1 mm DTT, 0.5 mm PMSF, and protease inhibitors (5 mg ml⁻¹ each of chymostatin, leupeptin, aprotinin and pepstatin). The samples were centrifuged at 16000g for 10 min at 4°C and the supernatant was removed for the measurement of G-actin concentration.

The DNase I inhibition assay was performed at 25 °C. The same time course for protein extraction and G-actin determination was maintained for each sample. One millilitre of DNA solution (100 mg of calf thymus DNA dissolved in 0.1 M Tris-HCl (pH 7.4), 4 mm MgSO₄ and 1.8 mm CaCl₂) was added to 10 ml of DNase I solution (1 mg of enzyme in 0.05 M Tris-HCl (pH 7.4), 0.01 mm PMSF and 0.1 mm CaCl₂). The production of DNA oligo- nucleotides owing to the hydrolysis of DNA was then monitored by recording the hyperchromicity at 260 nm as a function of time using a Beckman UV spectrophotometer. Samples were well mixed with DNase I solution for 10 s before the addition of DNA solution and the reaction rate was followed for up to 1 min. Muscle extract volumes (5-30 ml) were chosen to allow 30-70 % inhibition of DNase activity. DNase activity was also measured in the presence of the same volume of sample buffer without the addition of muscle extract. The level of G-actin in the extract that caused 50 % inhibition of DNase activity was estimated from a standard curve that was determined using known amounts of purified actin (Sigma). The accuracy of the assay in smooth muscle extracts was confirmed by adding known amounts of purified G- actin to samples of muscle extracts and verifying that this resulted in the predicted increase in the inhibition of DNase I activity.

The total actin content in the smooth muscle strips was evaluated from immunoblots. The pulverized frozen muscle strips were quickly mixed with extraction buffer which contained 20 mm Tris-HCl at pH 7.4, 2 % Triton X-100, 150 mm NaCl, 2 % SDS, 2 mm EDTA, phosphatase inhibitors (2 mm sodium ortho- vanadate, 2 mm molybdate and 2 mm sodium pyrophosphate) and protease inhibitors (2 mm benzamidine, 0.5 mm aprotinin and 1 mm phenylmethylsulphonyl fluoride). Each sample was boiled for 5 min to inactivate phosphatases and proteases, after which it was maintained at 4°C for 1 h. The supernatant was collected after centrifugation at 16000g for 25 min at 4°C. The extract was then diluted with H₂O by 1:4. The concentration of protein in each sample was determined using a standard bicinchoninic (BCA) protein assay kit (Pierce, Rockford, IL, USA). Muscle extracts were boiled in sample buffer (1.5 % dithiothreitol, 2 % SDS, 80 mm Tris-HCl (pH 6.8), 10 % glycerol and 0.01 % Bromophenol Blue) for 5 min and then separated by SDS-PAGE. Proteins were transferred to nitrocellulose, probed with monoclonal actin antibody (Sigma) followed by HRP-conjugated anti-mouse Ig (Amersham Life Sciences) for visualization by enhanced chemiluminescence. Total actin was quantified by scanning densitometry.

Analysis of myosin ATPase activity

The hydrolysis of ATP was measured by the NADH (reduced β-nicotinamide adenine dinucleotide) fluorescence method, in which the regeneration of ATP from ADP and phosphoenol- pyruvate (PEP) is catalysed by pyruvate kinase (PK; Kerrick & Hoar, 1994; Zhang & Moreland, 1994; Jones et al. 1999). This reaction is coupled to the oxidation of NADH and reduction of pyruvate to lactate by lactase dehydrogenase (LDH). In this reaction, 1 mol of PEP and 1 mol of NADH (which fluoresces at 470 nM) are used to produce 1 mol of ATP and 1 mol of NAD (a non-fluorescent compound). The rate of decrease in NADH fluorescence is thus proportional to the rate of ATP hydrolysis by the tissue.

Smooth muscle strips (0.6-0.7 mm wide, 8-10 mm long) were permeabilized for 25 min with 10 % Triton X-100 in relaxing solution at pCa 9 (see above). These strips were then attached to a force transducer, placed in a cuvette containing NADH solution at pCa 9 and allowed to equilibrate for 30 min to ensure penetration of all enzymes into cells throughout the tissue. The composition of the NADH solution was: 8.5 mm Na₂ATP, 4 mm K⁺-EGTA, 1 nM Ca²⁺, 1 mm DTT, 20 mm imidazole, 8.9 mm MgAc₂, 100.5 mm KAc, 5 mm PEP, 0.1 mm NADH, 0.2 mm P¹P⁵-di(adenosine-5′)pentaphosphate (AP₅A), 100 units ml⁻ PK¹ and 140 units ml⁻¹ LDH. The solution was excited at a wavelength of 340 nm and the NADH concentration was monitored by measuring light emission at 470 nm. NADH fluorescence and force were simultaneously measured using a Ratio Fluorescence Spectrophotomer (PTI International). After 30 min, fresh NADH solution at pCa 9 was rapidly pulsed into the cuvette and the rate of decline of NADH was monitored to evaluate the basal ATPase activity. Fresh NADH solution containing 10 μm Ca²⁺ was then pulsed into the cuvette to evaluate ATPase activity during the initial phase of contractile activation. The measurements were also repeated using solutions containing 10 μm ouabain (an inhibitor of Na⁺,K⁺-ATPase) and 10 μm cyclopiazonic acid (CPA, a sarcoplasmic reticular Ca²⁺ ATPase inhibitor) to verify that no significant ATPase activity resulted from plasma membrane or sarcoplasmic reticulum ATPases. ATPase activity was quantified in μmol min⁻¹ (gtissue)⁻¹.

Statistical analysis

All statistical analysis was performed using SigmaStat software. Comparison among multiple groups was performed by one-way analysis of variance or Kruskal-Wallis one-way analysis of variance. Differences between pairs of groups were analysed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.

RESULTS

Inhibition of expression of paxillin protein by antisense ODNs

We evaluated the effect of treatment with paxillin antisense ODNs on the expression of paxillin protein in smooth muscle tissues. Protein extracted from smooth muscle strips that had been treated with antisense or sense ODNs or with no ODNs for 3 days was analysed by Western blot to compare the expression of paxillin with that of vinculin, FAK, myosin light chain kinase (MLCK), myosin heavy chain (MHC) and myosin light chain (MLC).

Paxillin expression was lower in muscle strips treated with antisense ODNs than in strips treated with sense ODNs or no ODNs (Fig. 1). Protein expression in sense-treated and antisense-treated strips was normalized to that in strips not treated with ODNs. The expression of paxillin protein relative to that of vinculin, MLCK, FAK, MHC and MLC was significantly lower in antisense-treated strips than in strips not treated with ODNs or in sense-treated strips (Fig. 1B, n = 4-5, P < 0.05). There were no significant differences in the expression of vinculin, MLCK, FAK, MHC and MLC among antisense-treated strips, sense- treated strips and strips treated with no ODNs (n = 4–5, P > 0.05). This indicates that the decrease in paxillin expression was a selective effect of the antisense treatment and that it did not result from general deterioration of the tissue during the incubation period, or from nonselective effects of the antisense on protein synthesis.

Figure 1. Inhibition of paxillin expression by antisense treatment.

A, immunoblots of protein extracts from muscle strips that had each been incubated for 3 days with paxillin antisense ODNs, paxillin sense ODNs or no ODNs. Immunoblots were obtained using antibodies against paxillin, vinculin, focal adhesion kinase (FAK), myosin light chain kinase (MLCK), myosin heavy chain (MHC) and myosin light chain (MLC). Less paxillin was detected in antisense-treated muscle strips than in the strips that were not treated with ODNs or sense-treated muscle strips. Similar amounts of vinculin, FAK, MLCK, MHC and MLC were detected in all three strips. Molecular mass markers (in kDa) are indicated on the left. B, ratios of protein expression obtained from muscle strips treated with paxillin sense () or paxillin antisense (▪). Ratios in sense-treated and antisense-treated strips are normalized to ratios obtained in muscle strips that were not treated with ODNs (no ODNs). Values represent means + S.E.M. (n = 4-5). Asterisk indicates significantly lower ratios in antisense-treated strips relative to strips that were not treated with ODNs and sense-treated muscle strips (P < 0.05).

Effect of paxillin depletion on contractile force and myosin light chain phosphorylation

We evaluated isometric force development in response to acetylcholine (ACh) in muscle strips treated with paxillin sense, paxillin antisense and in strips not treated with ODNs (Figs 2 and 3). Force in response to 10⁻⁵ M ACh was compared before and after the 3 day incubation period. Contractile force in response to stimulation with ACh for 5 min was dramatically reduced in antisense-treated strips (Figs 2A and 3A). In contrast, in strips treated with paxillin sense or without ODN treatment, isometric force in response to stimulation with ACh for 5 min was not significantly different from the preincubation force. There were no differences in tension among the three groups of strips before contractile stimulation.

Figure 2. Active force and protein expression in antisense-treated smooth muscle strips.

Smooth muscle strips that were not treated with ODNs, sense-treated or antisense-treated smooth muscle strips were incubated for 3 days and the contractile responses were then evaluated. These strips were then subjected to an additional 3 days incubation in the absence of ODNs. The contractile responses and protein expression were then evaluated. A, representative tracings of 3 muscle strips from 1 experiment show that paxillin antisense inhibited ACh-induced contraction in smooth muscle strips after 3 days incubation in the presence of oligonucleotides. ACh-induced contraction of antisense-treated strip was restored with an additional 3 days incubation in the absence of ODNs. The contractile responses of strips that were not treated with ODNs or sense-treated muscle strips were similar to preincubation responses after 3 days incubation and also after an additional 3 days incubation in the absence of ODNs. B, the expression of paxillin in antisense-treated strips is restored with 3 days additional incubation. The expression of myosin light chain kinase (MLCK), myosin heavy chain (MHC) and myosin light chain (MLC) is similar in strips not treated with ODNs, sense-treated or antisense-treated smooth muscle tissues after an additional 3 days incubation in the absence of oligonucleotides. The immunoblots are representative of 4 similar experiments.

Figure 3. Effect of paxillin depletion by antisense on contractile force and myosin light chain phosphorylation stimulated by ACh.

A, smooth muscle strips were contracted with 10⁻⁵ M ACh before and after 3 days incubation with paxillin antisense (•), paxillin sense (▪) or without ODNs (○). Mean active force in response to 10⁻⁵ M ACh was quantified as a percentage of ACh-induced force in each strip before incubation. Values are means ± S.E.M. Asterisk indicates significantly lower response compared to muscles withoutODNs (n = 11-12,P < 0.05). B, myosin light chain phosphorylation in muscle strips incubated with paxillin antisense (•), sense (▪) orwithoutODNs (○). Myosin light chain phosphorylation was measured in smooth muscle strips 1 or 5 min after stimulation with 10⁻⁵ M ACh. Differences between antisense- treated strips, sense-treated strips and strips that were not treated with ODNs were not statistically significant (P > 0.05). Values shown are means ± S.E.M. (n = 6).

In an additional set of muscle strips, the incubation period was extended for an additional 3 days to test the reversibility of the effects of antisense on force (Fig. 2A). With further incubation of antisense-treated strips in the absence of oligonucleotides, the contractile response of these strips was restored to 95.4 ± 4.3 % of preincubation force (n = 4). The contractile responses of strips not treated with ODNs or sense-treated strips with the extended incubation in the absence of oligonucleotides were 94.6 ± 5.6 (n = 4) and 93.7 ± 4.6 % (n = 4) of preincubation force, respectively. Analysis by Western blot indicated that the expression of paxillin was also restored in paxillin antisense-treated tissues after further incubation in the absence of oligonucleotides (Fig. 2B).

A subset of the strips treated with paxillin antisense or sense ODNs, or with no ODNs was frozen for the analysis of myosin light chain phosphorylation. Although force production was dramatically depressed, ACh stimulated a significant increase in myosin light chain phosphorylation in strips treated with paxillin antisense. The mean increases in myosin light chain phosphorylation during contractile stimulation in the strips not treated with ODNs, sense-treated and antisense-treated tissues were not statistically different 5 min after stimulation (Fig. 3B).

The effect of treatment with paxillin antisense on contractile force and myosin light chain phosphorylation in response to stimulation with 60 mm KCl was also evaluated. Force and myosin light chain phosphorylation were determined 5 min after contractile activation. By this time, both force and myosin light chain phosphorylation in response to KCl stimulation have reached a steady state (Mehta et al. 1996). Active force in response to KCl was depressed to approximately 30 % of preincubation force in strips incubated with paxillin antisense, whereas the contractile response to KCl of strips not treated with ODNs was not depressed at all (Fig. 4). However, the depletion of paxillin did not significantly affect KCl-stimulated myosin light chain phosphorylation (0.27 ± 0.03 versus 0.30 ± 0.02 molPi (molMLC)⁻¹ in paxillin-depleted tissues and in undepleted tissues respectively, n = 6).

Figure 4. Effect of paxillin depletion by antisense on contractile force and MLC phosphorylation stimulated by KCl.

A, smooth muscle strips were contracted with 60 mm KCl before and after 3 days incubation with paxillin antisense or without ODNs. Mean active force in response to 60 mm KCl was quantified as a percentage of KCl-induced force in each strip before incubation. Values are means + S.E.M. Asterisk indicates significantly lower response compared to muscles without ODNs (n = 6; P < 0.05). B, MLC phosphorylation in muscle strips incubated with paxillin antisense or without ODNs. MLC phosphorylation was measured in smooth muscle strips 5 min after stimulation with 60 mm KCl. Differences between antisense- treated strips and strips that were not treated with ODNs were not statistically significant. Values shown are means + S.E.M. (n = 6).

Effect of paxillin depletion on myosin ATPase activity

Smooth muscle strips that had been treated with antisense ODNs, sense ODNs or no ODNs were permeabilized with Triton X-100 and ATPase activity in the strips was monitored by the NADH fluorescence method. The average basal rate of ATP hydrolysis at pCa 9 was not different in strips without oligonucleotide treatment, sense-treated tissues and antisense-treated tissues (n = 5, P > 0.05). The magnitude of increase in ATPase activity in response to an increase in Ca²⁺ to pCa 5 was also not significantly different in strips not treated with ODNs, sense-treated or antisense-treated strips (n = 5, P > 0.05). However, the contractile response of the antisense-treated strips was significantly lower than that of sense-treated strips or strips not treated with ODNs (Fig. 5).

Figure 5. Myosin ATPase activity in paxillin-depleted smooth muscle tissues stimulated with calcium.

Smooth muscle strips that had been treated for 3 days with antisense, sense or no ODNs were permeabilized with Triton X-100 and ATPase hydrolysis was determined by the NADH fluorescence method during the initial phase of contractile activation. A, ATPase activity was similar in strips that were not treated with ODNs, sense-treated or antisense-treated smooth muscle strips at calcium concentrations of pCa 9 or pCa 5. Values shown are means + S.E.M. (n = 5, P > 0.05). B, pCa 5-stimulated force was significantly depressed in paxillin antisense-treated strips (n = 5). Active force in sense- or antisense-treated groups is normalized to the force obtained in strips that were not treated with ODNs at pCa 5. Asterisk indicates values that are significantly different from strips not treated with ODNs and sense-treated muscle strips (P < 0.05).

To confirm that neither the Na⁺,K⁺-ATPase nor the sarcoplasmic reticular Ca²⁺-ATPase contributed signif- icantly to the total ATPase activity, we used the Na⁺,K⁺-ATPase inhibitor ouabain and the sarcoplasmic reticular Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA). In the presence of 10 μm ouabain and 10 μm CPA, ATP hydrolysis in response to an increase in Ca²⁺ to pCa 5 was 1.86 ± 0.12 μmol min g⁻¹ (n = 4), which was not significantly different from the ATPase activity (1.90 ± 0.15 μmol min⁻¹ g⁻¹, n = 4) in the absence of the inhibitors (P > 0.05).

Effect of paxillin depletion by antisense on intracellular Ca²⁺

Tracheal smooth muscle strips that had been incubated for 3 days with paxillin antisense or sense, or without ODNs were loaded with fura-2 AM and then stimulated with 10⁻⁴ M ACh. Results were compared to Ca²⁺ signals obtained in fresh tracheal muscle strips. Ca²⁺ signals in all of the treated strips were similar to the Ca²⁺ signals obtained in fresh strips. The ACh-induced increase in [Ca²⁺]_i in strips treated with antisense was similar to the levels in muscle strips that had been treated with sense ODNs or no ODNs (Fig. 6). The magnitude of the Ca²⁺signals after 1 or 5 min stimulation with ACh was compared statistically among different treatment groups. There were no significant differences of the ACh-induced Ca²⁺ signals in fresh strips and muscle strips that had been treated with no ODNs, sense or antisense (n = 4–5, P > 0.05). However, as previously observed, the contractile force in response to ACh stimulation was significantly lower in antisense-treated strips than in strips not treated with ODNs or in sense-treated tissues (n = 4–5, P < 0.05).

Figure 6. ACh-induced increase in intracellular calcium in paxillin-depleted muscle strips.

Mean traces of fura-2 fluorescence and force in response to ACh stimulation are shown for fresh undepleted muscle strips and muscle strips that have been treated with no ODNs or with paxillin sense or antisense (n = 4-5). Shaded areas represent standard errors for each trace. The intracellular calcium signals in response to ACh were similar in fresh muscle strips, strips that were not treated with ODNs, sense-treated and antisense-treated muscle strips. Force in the antisense-treated strips was depressed relative to strips not treated with ODNs and sense-treated strips. Fura-2 fluorescence is quantified as an increase in the ratio of fluorescence at excitation wavelengths of 340 and 380 over baseline.

Effect of paxillin depletion on the contractile response of β-escin-permeabilized muscle strips to activation with Ca²⁺

Tracheal muscle strips were incubated for 3 days with paxillin antisense or without ODNs, after which the strips were permeabilized with β-escin and stimulated with increasing concentrations of intracellular Ca²⁺ (Mehta et al. 2000; Tang & Gunst, 2001a, b). At all Ca²⁺ concentrations, active force induced by increasing intracellular Ca²⁺was dramatically depressed in muscle strips that had been depleted of paxillin (Fig. 7A). Myosin light chain phos- phorylation was not significantly different in paxillin- depleted and in undepleted muscle strips stimulated with 10 μm Ca²⁺ for 15 min (Fig. 7B and C).

Figure 7. Effect of paxillin depletion on Ca²⁺-induced contraction and MLC phosphorylation in β-escin-permeabilized muscle strips.

Permeabilized smooth muscle strips that had been incubated with paxillin antisense or with no ODNs were stimulated with increasing concentrations of Ca²⁺. A, Ca²⁺-stimulated force was significantly depressed in paxillin antisense-treated strips (n = 6). Active force in both groups is normalized to the force obtained in strips that were not treated with ODNs at pCa 5. Asterisk indicates values that are significantly different in antisense-treated muscle strips and in strips that were not treated with ODNs (P < 0.05). B, representative immunoblot of 20 kDa MLC in muscle strips treated with antisense or with no ODNS. Paxillin depletion did not inhibit MLC phosphorylation in the permeabilized strips stimulated with pCa 5 for 15 min. C, mean values of MLC phosphorylation in the permeabilized muscle strips stimulated with intracellular Ca²⁺ (pCa 5). Values shown are means + S.E.M. MLC phosphorylation increased significantly in response to pCa 5 in strips treated with antisense and in strips not treated with ODNs, but there were no significant differences between the antisense- treated strips and muscle strips not treated with ODNs. Asterisk indicates values that are significantly different from values at pCa 7 (n = 6,P < 0.05).

Effect of paxillin depletion on actin in smooth muscle strips

The DNase I inhibition assay was used to measure the G-actin concentration in the soluble fraction of extracts from paxillin-depleted smooth muscle strips. The G-actin concentration was measured both in unstimulated strips and in strips stimulated with 10⁻⁵ M ACh for 5 min. In the strips depleted of paxillin, the G-actin level was markedly reduced in the absence of contractile stimulation; G-actin concentration in paxillin-depleted strips was only 52.7 % of that in undepleted muscle strips (Fig. 8). In muscle strips not treated with ODNs, the G-actin concentration decreased significantly in response to ACh stimulation (Fig. 8), as previously demonstrated (Mehta & Gunst, 1999). Stimulation with ACh did not cause a further reduction in the G-actin concentration in the antisense-treated strips. The G-actin concentration in ACh-stimulated muscles was not significantly different in undepleted and paxillin- depleted muscle strips.

Figure 8. Effect of paxillin depletion on G-actin content.

Tracheal smooth muscle strips subjected to treatment with paxillin antisense ODNs or no ODNs for 3 days were stimulated with 10⁻⁵ MAChfor5 min. G-actin content was assessed by the DNase I inhibition assay. Values represent G-actin concentration in the soluble fraction of muscle extracts as a percentage of the concentration in unstimulated strips incubated for 3 days with no ODNs. Asterisk indicates that the concentration of G-actin in ACh-stimulated strips is significantly lower than the value for corresponding unstimulated strips (n = 4, P < 0.05).

We evaluated the amount of total actin in paxillin- depleted and undepleted smooth muscle strips (Fig. 9). In undepleted strips, mean total actin in response to ACh was 98.5 ±5.6 % of that in unstimulated tissues (n = 4, P > 0.05). When paxillin was depleted, mean total actin in unstimulated and ACh-stimulated strips was 102.5 ± 6.12 and 101.2 ± 4.0 %, respectively, of the level in undepleted, unstimulated strips (n = 4, P > 0.05). The depletion of paxillin did not affect the total amount of actin in either unstimulated or ACh-stimulated muscle strips. Thus, the lower levels of G-actin in the paxillin-depleted muscle strips did not result from a reduction in total actin.

Figure 9. Total actin content in paxillin-depleted smooth muscle tissues.

Representative immunoblots of muscle extracts were prepared from 4 muscle strips subjected to treatment with paxillin antisense or with no ODNs for 3 days. The depletion of paxillin does not affect total actin.

DISCUSSION

Effect of paxillin depletion on protein expression and contractility

Our results demonstrate that paxillin plays an essential role in the cellular processes that mediate active tension development in smooth muscle. We used antisense oligo- nucleotides to deplete tracheal smooth muscle tissues selectively of paxillin protein. Analysis of protein expression by Western blot demonstrated that the antisense ODNs for paxillin suppressed the expression of paxillin protein to about 30 % of that in tissues not treated with ODNs. Force development in the paxillin-depleted tissues was markedly inhibited, to less than 15 % of that of tissues not treated with ODNs, while it was not inhibited in muscle strips incubated for 3 days with paxillin sense ODNs or without ODNs.

Our results cannot be attributed to a general decline in protein expression or to a decline in tissue viability caused by the period of organ culture. The expression of vinculin, FAK, MLCK, MHC and MLC were not altered by the procedures used to introduce paxillin antisense into the tissue or by the 3 day period of organ culture. Vinculin and FAK are cytoskeletal proteins that bind to paxillin (Turner et al. 1990; Thomas et al. 1999), while MLCK, MLC and MHC are known components of the contractile apparatus. Thus the depletion of paxillin did not affect the expression of cytoskeletal proteins closely associated with paxillin or proteins involved in mediating smooth muscle contraction. Furthermore, both the contractility and the expression of paxillin protein in the antisense-treated tissues could be restored by incubating the paxillin-depleted tissues for an additional 3 days in the absence of oligonucleotides. The results demonstrate that depression of contraction caused by the treatment of muscle strips with paxillin antisense results specifically from the absence of paxillin protein and that it can be reversed by extended incubation of the tissues without oligonucleotides.

Role of paxillin in the regulation of contractile protein activation and crossbridge cycling

Based on the molecular interactions of paxillin with other proteins, a number of possible mechanisms can be postulated by which paxillin might be involved in the contractile activation of smooth muscle. Paxillin functions as a molecular adaptor or scaffold protein that provides multiple docking sites for an array of signalling and structural proteins. The tyrosine kinase FAK, in association with the tyrosine kinase pp60^c-^Src (Src), phosphorylates paxillin at two main sites, tyrosine 31 and tyrosine 118 (Bellis et al. 1995; Schaller & Parsons, 1995; Richardson et al. 1997; Thomas et al. 1999). In substrate-adherent cells, paxillin tyrosine phosphorylation is associated with the formation of focal adhesions and stress fibres and is important for regulating the dynamics of these structures and the associated actin network (Richardson et al. 1997; Nakamura et al. 2000). In previous studies, we have reported that paxillin and FAK undergo tyrosine phosphorylation coordinately in response to stimulation with contractile agonists in tracheal smooth muscle (Wang et al. 1996; Tang et al. 1999). We have also found that the depletion of FAK from tracheal smooth muscle tissues by antisense oligonucleotides depresses paxillin tyrosine phosphoryl- ation (Tang & Gunst, 2001a). The depletion of FAK protein from tracheal muscle is associated with an inhibition of active tension generation, the suppression of the agonist-induced increase in intracellular Ca²⁺ and the inhibition of MLC phosphorylation (Tang & Gunst, 2001a). Phosphorylation of the 20 kDa light chain of myosin by Ca²⁺-calmodulin-regulated myosin light chain kinase is widely recognized as a major cellular event in the initiation of cross-bridge cycling and smooth muscle contraction (Kamm & Stull, 1989). Thus, in tracheal smooth muscle, FAK appears to be involved in mediating the increase in intracellular Ca²⁺ that regulates the activation of myosin.

Paxillin has been directly implicated in the regulation of intracellular Ca²⁺ in vascular myocytes. In these cells, the introduction of paxillin antibodies depresses the a5b1-integrin-mediated calcium influx through the L-type Ca²⁺ channel (Wu et al. 2001). We therefore considered the possibility that paxillin, like FAK, might play a role in the regulation of agonist-induced Ca²⁺ activation in tracheal smooth muscle.

In the present study, we evaluated a possible role for paxillin in agonist-induced Ca²⁺ mobilization by introducing fura-2 into paxillin-depleted tracheal smooth muscle tissues to measure intracellular Ca²⁺ transients in response to ACh. We found that the intracellular Ca²⁺ transient elicited in response to ACh was not significantly depressed in paxillin- deficient tracheal tissues. We further evaluated the possibility that paxillin was interfering with Ca²⁺ signalling by directly stimulating permeabilized paxillin-depleted tracheal smooth muscle tissues with Ca²⁺; however, increasing intracellular Ca²⁺ in the paxillin-depleted tissues did not restore their contractility. Thus, unlike FAK, paxillin is not involved in regulating agonist-induced Ca²⁺mobilization in tracheal smooth muscle.

We also determined the effect of paxillin depletion on MLC phosphorylation. Paxillin depletion did not significantly affect the increase in MLC phosphorylation in tissues stimulated with either ACh or KCl, although the tension development in response to both stimuli was significantly depressed. The increase in MLC phosphorylation in paxillin-deficient, β-escin-permeabilized muscle strips stimulated by Ca²⁺ was also similar to that in undepleted tissues. This evidence indicates that the inhibition of contraction caused by paxillin depletion results from mechanisms that are independent of the regulation of Ca²⁺-activated MLC phosphorylation.

A number of mechanisms have been proposed whereby cross-bridge cycling might be regulated by actin-associated proteins independently of MLC phosphorylation (Morgan & Gangopadhyay, 2001). We therefore evaluated the possibility that paxillin regulates cross-bridge cycling by a mechanism that is independent of MLC phosphoylation. Myosin ATPase activity was determined in paxillin-depleted and undepleted Triton-permeabilized tracheal muscle strips using the NADH fluorescence method (Kerrick & Hoar, 1994; Zhang & Moreland, 1994; Jones et al. 1999). The stimulation of undepleted tracheal muscle strips with 10 μM Ca²⁺ caused the ATPase rate to increase from a basal level of 0.86 to 1.90 μmol min⁻¹ g⁻¹, about a twofold increase. Although contractile stimulation caused a similar change in the ATPase rate of paxillin-depleted tissues, tension development was dramatically depressed in these tissues. Thus, the presence of paxillin had no apparent effect on the cross-bridge cycling rate, as indicated by this analysis of myosin ATPase activity. We believe that these values reflect primarily myosin ATPase activity, since neither the Na⁺,K⁺-ATPase inhibitor ouabain nor cyclopiazonic acid, an inhibitor of Ca²⁺-ATPase activity, had any measurable effect on the rate of change of NADH fluorescence either before or after stimulation with Ca²⁺. Although it is possible that kinase and phosphatase activities also contributed to the total ATPase activity, based on analysis performed in other smooth muscle tissues (Zhang & Moreland, 1994), we believe that their contribution is likely to be small. Similar magnitudes of change in ATPase activity in response to contractile stimulation have been reported in previous studies of smooth muscle (Krisanda & Paul, 1983; Paul, 1983; Jones et al. 1999).

Role of paxillin in the regulation of the actin cytoskeleton

In a previous study, we demonstrated that contractile activation stimulates actin polymerization in tracheal smooth muscle tissues and that actin polymerization is necessary for active tension development in this tissue (Mehta & Gunst, 1999). The stimulation of tracheal muscle strips with ACh results in a decrease in the concentration of G-actin, consistent with the incorporation of G-actin monomers into F-actin. The treatment of tracheal muscle strips with latrunculin A, which binds to G-actin and prevents its incorporation into F-actin, inhibits tension development without inhibiting MLC phosphorylation (Mehta & Gunst, 1999). There is also evidence that actin polymerization occurs in response to the contractile activation of other smooth muscle tissues (Cipolla & Osol, 1998; Barany et al. 2001).

In the present study, we evaluated the effect of paxillin depletion on the G-actin concentration in resting muscles and in muscles stimulated with acetylcholine. In muscle strips not depleted of paxillin, stimulation with acetyl- choline caused a decrease in the concentration of G-actin, as we have previously demonstrated (Mehta & Gunst, 1999). However, in paxillin-depleted tracheal muscle strips, the concentration of G-actin in the absence of contractile stimulation was significantly lower than that in undepleted strips, suggesting that relatively more of the actin may be maintained in a polymerized state (F-actin) under resting conditions in paxillin-depleted tissues. Furthermore, stimulation with ACh did not cause a significant further reduction in the concentration of G-actin in the paxillin-depleted strips, indicating that additional polymerization of actin did not occur in response to contractile stimulation in the absence of paxillin. These results are consistent with a role for paxillin in regulating cytoskeletal organization or cytoskeletal dynamics in smooth muscle.

Paxillin has been implicated in regulating cytoskeletal organization and the dynamics of the actin cytoskeleton in other cell types (Abedi & Zachary, 1997; Ito et al. 2000; Nakamura et al. 2000). Recent studies from our laboratory and others have suggested that the cytoskeletal organization of smooth muscle cells is dynamic and that it is regulated during contractile stimulation (Gunst et al. 1995; Pratusevich et al. 1995; Cipolla & Osol, 1998; Mehta & Gunst, 1999; Gerthoffer & Gunst, 2001; Gunst & Wu, 2001; Cipolla et al. 2002). This may enable smooth muscle cells to adapt their cytoskeletal organization to changes in cell shape that occur as a result of changes in their external environment and thereby optimize their contractility. We have hypothesized that this reorganization may involve both the remodelling of actin filaments and reorganization of the attachment of the actin cytoskeleton to the membrane.

Paxillin is known to be a component of the focal adhesion sites of cultured cells, which consist of a complex of proteins that link extracellular matrix proteins to the actin cytoskeleton. The dense plaques of differentiated smooth muscle cells contain many of the same proteins that are found in focal adhesions (Burridge & Chrzanowska-Wodnicka, 1996; Gunst & Tang, 2000). Transmembrane integrin proteins mediate the linkage between the extra- cellular matrix and the actin cytoskeleton by binding to extracellular matrix proteins via their extracellular domains, and to cytoskeletal proteins via short cytoplasmic tails on the inside of the cell (Burridge & Chrzanowska-Wodnicka, 1996). The proteins talin and α-actinin have been shown to bind to the cytoplasmic tails of β-integrins and also to actin filaments, suggesting that these proteins might mediate the linkage between integrin proteins and the actin cytoskeleton (Pavalko et al. 1991; Otey et al. 1993; Calderwood et al. 1999). The cytoskeletal protein vinculin also binds to talin and actin and forms a complex with α-actinin and phosphatidylinositol-4,5-bisphosphate (PIP2). PIP₂ has been shown to regulate the binding of vinculin to talin and actin (Gilmore & Burridge, 1996). PIP₂ is a potent stimulator of actin bundle formation by α-actinin, and the amount of PIP₂ bound to α-actinin has been proposed to modulate actin polymerization (Fukami et al. 1992). Vinculin also binds to paxillin and colocalizes with paxillin in focal adhesions (Turner et al. 1990; Wood et al. 1994). The binding of vinculin to paxillin has been proposed as a mechanism for the recruitment of vinculin to focal adhesions (Wood et al. 1994).

Thus, at the dense plaques of smooth muscle, paxillin may play a critical role in regulating the organization of cytoskeletal molecules that function to link actin filaments to integrins. These proteins are also implicated in regulating the polymerization state of actin. Paxillin has also been implicated in p21 GTPase-regulated actin cytoskeletal remodelling through its interaction with p21-activated kinase (PAK; Turner et al. 1999) and also with Crk, an adaptor protein that regulates activation of the small GTPase Rac I (Klemke et al. 1998; Petit et al. 2000). Paxillin may thereby act as an upstream regulator of other signalling pathways involved in the modulation of actin dynamics. The absence of paxillin may disrupt the organization of dense plaque molecular complexes, preventing tension transmission from the actin cytoskeleton to the extra- cellular matrix and interrupting processes critical to the regulation of actin polymerization. This could account for our observations that, while contractile protein activation and cross-bridge cycling appear relatively undisturbed in paxillin-depleted tissues, tension generation is markedly depressed and the balance between G-actin and F-actin is abnormal.

Conclusions

In tracheal smooth muscle, the depletion of paxillin protein by antisense oligonucleotides dramatically depresses active tension generation in response to contractile stimulation without significantly inhibiting intracellular Ca²⁺ signals, MLC phosphorylation or myosin ATPase activity. However, the depletion of paxillin results in an abnormally low level of G-actin in resting muscles and prevents the reduction in G-actin associated with actin polymerization that occurs with the contractile stimulation of smooth muscle. We conclude that paxillin plays a critical role in the generation of active tension in tracheal smooth muscle, but that it does not regulate the activation of contractile proteins or cross-bridge cycling during contractile stimulation. Paxillin may be important in regulating the organization of molecular complexes that mediate the attachment of actin filaments to transmembrane integrins for the transmission of tension from the contractile apparatus to the extra- cellular matrix. Paxillin may also interact with proteins in these complexes that regulate the polymerization and depolymerization of actin in smooth muscle.