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Caveolae facilitate muscarinic receptor-mediated intracellular Ca²⁺ mobilization and contraction in airway smooth muscle

Departments of ¹Physiology, ²Internal Medicine, ⁶Pediatrics and Child Health, and ⁸Human Anatomy and Cell Science, University of Manitoba, ³Biology of Breathing Group, Manitoba Institute of Child Health, ⁴Canadian Institutes of Health Research National Training Program in Allergy and Asthma, University of Manitoba, and ⁹Section of Thoracic Surgery, University of Manitoba, Winnipeg, Manitoba, Canada; ⁵Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands; and ⁷Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada

Submitted 4 August 2007 ; accepted in final form 20 September 2007

	ABSTRACT

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Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca²⁺ homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca²⁺ ([Ca²⁺]_i) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M₃ receptors (M₃R) and G_q/11 cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with β-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-β-cyclodextrin (mβCD) reduced sensitivity but not maximum [Ca²⁺]_i induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mβCD disrupted the colocalization of caveolae-1 and M₃R, but [N-methyl-³H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca²⁺]_i flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mβCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca²⁺]_i mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca²⁺]_i mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

caveolin; G protein-coupled receptor; asthma; histamine; G_q

Several studies support a possible role for caveolae and caveolins in GPCR-mediated smooth muscle contraction. In vascular and gastrointestinal smooth muscle, caveolae harbor a number of GPCR-regulated signaling proteins, including -subunits of trimeric G proteins, PLC, the monomeric G protein RhoA and its downstream effector Rho kinase, and Ca²⁺-sensitive PKC isoforms (4, 6, 38, 48, 50, 52, 55). Somara et al. (50) recently showed that ectopic overexpression of caveolin-1 in aged rabbit colonic smooth muscle restored muscarinic receptor-mediated contraction, suggesting a facilitator role. Consistent with this observation, disruption of caveolae using cholesterol-depleting agents leads to reduced contraction of vascular smooth muscle in response to endothelin-1 or serotonin (2, 12). Using a cell-permeable CSD peptide that blocks caveolin-1 interactions with signaling proteins but leaves caveolae intact, Je et al. (28) reported a significant suppression in contractile responses of ferret aortic smooth muscle to -adrenergic agonist. Notably, caveolin-1 has tissue- and receptor-specific effects on smooth muscle contraction; using tissue from caveolin-1 knockout mice, Shakirova et al. (48) observed that ileum longitudinal muscle had reduced contraction to endothelin-1 with no change in response to 5-HT or carbachol, whereas femoral arterial muscle contraction was increased in response to ₁-adrenergic receptor stimulation. Collectively, these observations point to agonist- and tissue-specific role for caveolae and caveolin-1 in modulating receptor-mediated smooth muscle contraction.

In contractile smooth muscle cells, caveolae distribution is highly ordered, being concentrated in longitudinal plasma membrane arrays that lie in close proximity to intracellular sarcoplasmic reticulum and mitochondria, thereby forming nanospaces for Ca²⁺ homeostasis (17, 18, 40). Ultrastructural studies confirm this spatial orientation in airway smooth muscle (ASM; Ref. 31), and biochemical fractionation of caveolae from canine trachealis has revealed that caveolae are enriched in number of Ca²⁺-handling proteins, including L-type Ca²⁺ channels, the Ca²⁺-binding proteins calsequestrin and calreticulin, and the plasma membrane Ca²⁺ pump (9). In a recent electrophysiological study using isolated rat cerebral resistance arterial myocytes, Kamishima et al. (29) showed that caveolin-1 inhibition with CSD peptide or selective antibodies markedly slowed Ca²⁺ removal rate after stimulation to physiologically relevant cytoplasmic Ca²⁺ concentrations. Collectively, these studies suggest that effects of caveolae disruption or caveolin-1 depletion on GPCR-mediated smooth muscle contraction may be underpinned, in part, by changes in intracellular Ca²⁺ ([Ca²⁺]_i) flux.

Despite the association of Ca²⁺-handling proteins with caveolae in ASM and compelling evidence from other smooth muscle tissues that GPCR-mediated contraction is modulated by caveolae and caveolin-1, the role of caveolae in functional excitation-contraction coupling in ASM has not yet been reported. Therefore, in the current study, we tested the hypothesis that caveolin and caveolin-1 modulate [Ca²⁺]_i mobilization and associated contractile responses induced by physiologically relevant agonists in human and canine ASM. We used biochemical fractionation of cells and tissues to assess subcellular distribution of muscarinic M₃ receptors (M₃R) and G_q/11 subunits that transduce ligand-induced cell responses. We also used cholesterol depletion, small interfering RNA (siRNA) knockdown of caveolin-1, and treatment with cell-permeable caveolin-1 CSD peptide to determine the functional role of caveolae and caveolin-1 in muscarinic receptor-mediated Ca²⁺ flux in primary cultured ASM and contraction of intact tracheal smooth muscle (TSM). Our results are significant, as they describe for the first time a novel role for caveolae and caveolin-1 in ASM contractile responses, and thus they are of relevance to understanding mechanisms that control airway resistance in health and disease.

	MATERIALS AND METHODS

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Cell culture. Canine and human ASM cells were used for cell culture studies. Primary cultured canine airway myocytes were established from dissociated canine trachealis as we (25, 37) have described previously. Human bronchial smooth muscle cell lines immortalized by stable expression of human telomerase reverse transcriptase (hTERT) were prepared as described previously (21). The primary cultured human bronchial smooth muscle cells used to generate hTERT ASM cells were prepared from macroscopically healthy segments of 2nd-to-4th generation main bronchus obtained after lung resection surgery from patients with a diagnosis of adenocarcinoma. All procedures were approved by the Human Research Ethics Board of the University of Manitoba.

Cells were grown to confluence using DMEM supplemented with 10% fetal bovine serum, 50 U/ml streptomycin, and 50 µg/ml penicillin. Cultures were maintained in a humidified incubator at 37°C-5% CO₂, and media was changed every 2 days. For the induction of contractile phenotype myocytes, confluent cultures of canine (passage 0 or 1) or human ASM cell (passages 9-15) were serum-deprived for 4–10 days using Ham's F-12 medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium (ITS) as described previously (21, 25).

Isolation of caveolae-enriched membrane fractions. Cells grown on uncoated 150-mm dishes were washed with ice-cold PBS and lysed in 500 mM sodium carbonate (pH 11.0) supplemented with 1 mM Na₃VO₄, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, and 1 mM PMSF. After homogenization by sonification, 2 ml of homogenate was mixed with an equal volume of a solution containing 150 mM NaCl, 25 mM MES (pH 6.5), and 80% (wt/vol) sucrose and placed in the bottom of a centrifuge tube. A stepwise sucrose density gradient (45%, 30%, 20%, and 5%) was then carefully layered on top of the homogenate. Thereafter, the samples were centrifuged at 210,000 g for 18 h, and then 1-ml fractions were collected from the top of the gradient. Samples were stored at –80°C until further use.

Preparation of total protein lysates from ASM tissue. Intact ASM tissue was isolated from human bronchial specimens or canine trachealis by microdissection at 4°C. Thereafter, tissues were homogenized by sonification in ice-cold in RIPA buffer and subjected to centrifugation (760 g, 5 min), and the supernatant was stored at –80°C for subsequent protein assay and immunoblot analyses.

Western blot analysis. Protein content in supernatant samples was determined using the Bio-Rad protein assay with BSA as a reference (Bio-Rad, Hercules, CA). Equal amounts of protein from sucrose density-isolated fractions or total protein lysates were subjected to electrophoresis, transferred to nitrocellulose membranes, and analyzed for the proteins of interest using specific primary and horseradish peroxidase (HRP)-conjugated secondary antibodies as we (25) have previously described. Bands were subsequently visualized on film using ECL reagents.

Transmission electron microscopy. The ultrastructure of intact canine trachealis was assessed. Specimens consisting of two cartilage rings with intact trachealis were prepared from the cervical segments using a sharp scalpel. Specimens were incubated at 37°C in oxygenated Krebs-Henseleit solution (KH; 117.5 mM NaCl, 5.6 mM KCl, 1.18 mM MgSO₄, 2.5 mM CaCl₂, 1.28 mM NaH₂PO₄, 25 mM NaHCO₃, and 5.55 mM D-glucose, gassed with 5% CO₂ and 95% O₂, 37°C, pH 7.4) for 1 h in the presence or absence of β-cyclodextrin (β-CD; 10 mM), which depletes cholesterol and thereby disrupts lipid rafts including caveolae. Specimens were washed once with fresh KH buffer and fixed in PBS (pH 7.4) containing 4% paraformaldehyde-1.25% glutaraldehyde at room temperature for 48 h. Thereafter, the smooth muscle layer was removed from each ring and subjected to postfixation with 1% osmium tetroxide and embedded in LX-112 acrylic medium. Ultrathin cross-sections of the muscle tissue were then prepared, mounted onto coated grids, and stained with 1% uranyl acetate and lead citrate. Cell ultrastructure was assessed with an electron microscope at an acceleration voltage of 60–80 kV.

Contraction studies. Canine TSM was dissected free from the cartilage, the adventitia, and the submucosal layers. The muscle was then cut into strips 2 mm wide and mounted for isometric recording in water-jacketed organ baths containing KH buffer and allowed to equilibrate at a passive resting force of 1.2 g for 1 h. Buffer was then replaced with isosmotic KH containing 47 mM KCl. The active force generated by KCl exposure was measured three times with 15-min washouts between each stimulation. Muscle strips were then stimulated using a single concentration of acetylcholine (ACh) for each preparation. After ACh exposure, chambers were washed out, and the strips were incubated for 1 h in KH or in KH containing 10 mM β-CD to disrupt caveolae. Thereafter, muscle chambers were washed out repeatedly, and muscle strips were reexposed to the same concentration of ACh used for the initial stimulation. To ensure muscle strip viability, after the final ACh exposure, chambers were washed, and muscle strips were again stimulated with isosmotic KH containing 47 mM KCl. Any muscle strips that exhibited a change of more than 15% in active tension compared with that obtained with last KCl exposure before the ACh protocol were not included for data analysis.

Immunocytochemistry. ASM cells were plated onto precleared glass coverslips in six-well culture dishes. Cells were fixed for 15 min at 4°C in cytoskeletal buffer (10 mM MES, 150 mM NaCl, 5 mM EGTA, 5 mM MgCl₂, and 5 mM glucose, pH 6.1) containing 3% paraformaldehyde. Cells were then permeabilized by incubation for 5 min at 4°C in cytoskeletal buffer containing 3% paraformaldehyde and 0.3% Triton X-100. For immunofluorescence microscopy, fixed cells were first blocked for 2 h at room temperature in Cyto-TBS buffer (20 mM Tris base, 154 mM NaCl, 20 mM EGTA, and 20 mM MgCl₂, pH 7.2) containing 1% BSA and 2% normal donkey serum. Incubation with primary antibodies occurred overnight at 4°C in Cyto-TBS containing 0.1% Tween 20 (Cyto-TBST). Incubation with FITC- or Cy5-conjugated secondary antibodies was for 2 h at room temperature in Cyto-TBST. Nuclei were stained with Hoechst 33342 (10 µg/ml). For studies examining the effects of cholesterol depletion on immunocytochemical protein distribution, before fixation, serum-deprived airway myocytes were washed briefly in HBSS/HEPES buffer (1.26 mM CaCl₂, 5.33 mM KCl, 0.44 mM KH₂PO₄, 0.50 mM MgCl₂^.6H₂O, 0.41 mM MgSO₄^.7H₂O, 138 mM NaCl, 4 mM NaHCO₃, 0.30 mM Na₂HPO₄, 5.60 mM glucose, 20 mM HEPES, pH 7.4) and then incubated for 1 h at room temperature in HBSS/HEPES buffer containing 10 mM β-CD. Controls were incubated for the same time period in buffer only.

Measurement of [Ca²⁺]_i concentration. Real-time quantification of cytosolic Ca²⁺ in cultured ASM cells was performed using the Ca²⁺-sensitive ratiometric fluorescent dye fura-2 AM as we (37) have described previously. All measurements were carried out using myocytes grown on glass coverslips or chamber slides. Myocytes were washed briefly with HBSS/HEPES buffer containing 0.1% BSA and then incubated with 5 µg/ml fura-2 AM (37°C, 1 h) in buffer supplemented with 0.01% pluronic acid. Cells were then washed three times and incubated in buffer for a further hour at room temperature to allow for fura-2 AM deesterification. Real-time changes in [Ca²⁺]_i were recorded using an Olympus LX-70 inverted epifluorescent microscope (x20 objective) coupled to a Perkin-Elmer Ultra Pix FSI CCD camera controlled by UltraView imaging software. The system was further coupled to a Sutter Instruments Lambda 10-2 filter wheel and controller with repeated 200–400 ms excitation at 340 and 380 nm; emission at 510 nm was recorded continually for up to 3 min after the addition of contractile agonists. Maximum change in [Ca²⁺]_i was calculated as the average baseline value subtracted from the peak [Ca²⁺]_i response to agonist. The ratio of emission at 510 nm excited by 340- and 380-nm light was converted to [Ca²⁺]_i values from a calibration curve generated using calcium standards and calculated by the method of Grynkiewicz (23).

For studies examining the effects of cholesterol depletion on [Ca]_i mobilization induced by contractile agonists, fura-2-loaded cells were incubated at room temperature for 1–1.5 h in buffer containing 10 mM β-CD or 5 mM methyl-β-cyclodextrin (mβCD). Controls cultures were incubated for the same time period in buffer only. For other experiments, before fura-2 loading, myocytes were incubated (1 h, 37°C) in HBSS/HEPES containing a cell-permeable rhodamine conjugated synthetic peptide (1 µM) that included the human CSD amino acid sequence (residues 82–101) linked at the NH₂ terminus with a 17-residue antennapedia (AT) protein transduction domain (RQIKIWFQNRRRMKWKK-DGIWKASFTTFTVTKYWFYR). For control experiments, cells were incubated with a rhodamine-conjugated synthetic peptide containing AT sequence linked to the NH₂-terminal residue of a scrambled CSD sequence (RQIKIWFQNRRRMKWKK-WGIDKAFFTTSTVTYKWFRY).

Muscarinic receptor-binding assay. Cells were grown on uncoated 150-mm dishes and subjected to 4 days of serum deprivation. Cultures were incubated in HEPES-HBSS for 1 h at 37°C with or without 5 mM mβCD and then washed with ice-cold PBS and lysed on ice in 25 mM Tris, 2.5 mM CaCl₂ (pH 7.4), supplemented with 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, and 1 mM PMSF. After 10 strokes in a Dounce homogenizer, the homogenate was centrifuged for 5 min (1,000 g). The supernatant was transferred to a new tube and centrifuged for 30 min (150,000 g). The pellet-containing membrane was resuspended by sonification and stored at –80°C until further use. Binding of [N-methyl-³H]scopolamine ([³H]NMS) was subsequently determined by incubating equal amounts of membrane (90 µg per sample) with [³H]NMS (0.0001–1,000 nM) for 60 min at room temperature. Nonspecific binding was determined by competing with 100 nM atropine. Bound and unbound [³H]NMS was quantified by liquid scintillation counting; maximal binding capacity (B_max) and dissociation constant (K_d) were subsequently calculated using Scatchard analysis.

siRNA transfection. Transfection to suppress caveolin-1 protein expression was carried out as we (21) have previously described for studies in which total caveolin-1 protein abundance was reduced by 50–60% 72 h after siRNA transfection. Cells were grown to 50–70% confluence in chamber slides and transiently transfected in serum-free DMEM containing antibiotics with a 21-bp, double-stranded siRNA targeted against a sequence between residues 529–589 of the caveolin-1 transcript (Qiagen, Mississauga, Ontario, Canada). Cells were transfected using 2.5 µg/ml siRNA in combination with 6 µl/µg siRNA of RNAiFect transfection reagent (Qiagen). In all studies, control transfections were performed using a nonsilencing control 21-bp siRNA (Qiagen). Cells were washed with fresh DMEM 72 h after transfection and used immediately for experiments to assess changes in cytosolic Ca²⁺ concentration in response to ACh.

Materials. Cell culture media (DMEM and Ham's F-12), supplements (fetal bovine serum and ITS), and antibiotics (penicillin and streptomycin) were obtained from Invitrogen. Caveolin-1 primary antibody was from BD Biosciences (San Jose, CA). Muscarinic M₃R antibody was kindly provided by Dr. Jurgen Wess (National Institutes of Health, Bethesda, MD). G_q/11 primary antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). FITC- and Cy5-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). β-Actin primary antibody, smooth muscle myosin heavy chain (smMHC) primary antibody, anti-rabbit, anti-mouse, and anti-goat HRP-conjugated secondary antibodies, β-CD, and mβCD were from Sigma-Aldrich (St. Louis, MO). Fura-2 AM dye and Hoechst 33342 nuclear stain were from Molecular Probes (Leiden, The Netherlands). ECL reagent was from Amersham (Oakville, Ontario, Canada). Rhodamine-conjugated synthetic CSD- and scrambled CSD-AT peptides were obtained from AnaSpec (San Jose, CA).

	RESULTS

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Caveolin expression and caveolae association of muscarinic M₃R and G_q/11. We first used immunoblotting to characterize the distribution of caveolins expressed in isolated ASM tissue from trachea or 2nd-to-3rd generation mainstem bronchi. Figure 1A shows that caveolin-1 (24 kDa) is the predominant isoform expressed in canine trachealis, human trachealis, and human bronchial smooth muscle. Although we were unable to detect a distinct band for caveolin-2 in canine trachealis lysates (even when the total protein loaded was increased 5-fold), caveolin-2 was abundant in human ASM lysates. In fact, three distinct bands for caveolin-2, which likely correspond to -, β-, and -isoforms of decreasing molecular weight, respectively, were visible in human ASM. Caveolin-3 was undetectable in human ASM lysates; however, a pale band (21 kDa) for caveolin-3 was evident for canine trachealis in blots where the total protein load was increased by fivefold compared with that used to detect caveolin-1 in the same lysates.

View larger version (28K): [in this window] [in a new window]   Fig. 1. A–C: caveolin (Cav) expression and association of muscarinic M3 receptors (M3R) and Gq/11 with caveolae isolated from airway smooth muscle (ASM) tissue. A: immunoblots showing Cav-1, Cav-2, and Cav-3 expression in ASM tissues. Protein lysates were prepared in RIPA buffer and size-fractionated on 12% SDS acrylamide gels. Fresh canine tracheal smooth muscle (TSM), human TSM (obtained from healthy lung donors), or 2nd-to-3rd order human mainstem bronchial smooth muscle (BSM; obtained from lung resection subjects) were cleaned of nonmuscle tissues by fine dissection under a binocular microscope. For canine TSM blots, 5 µg of total protein was loaded for Cav-1 detection, and 30 µg of total protein was loaded for Cav-2 and Cav-3 lanes. For human TSM and BSM, 15 µg of total protein was added in each lane. B: immunoblots showing distribution of Cav-1, M3R, and Gq in cell fractions obtained from isolated canine TSM homogenized in carbonate buffer and subjected to sucrose-density gradient centrifugation as described in MATERIALS AND METHODS. After centrifugation, 1-ml fractions (designated by numbers above each lane in the gels shown) were collected starting from the top of each centrifuge tube. Equal volumes from each fraction were then subjected to Western blotting; 12% SDS-acrylamide gels were used for Cav-1 and Gq, whereas 10% gels were used for M3R. Representative blots from 3 experiments are shown. C: immunoblots showing distribution of Cav-1, M3R, and Gq in cell fractions obtained from isolated human BSM. Samples were obtained and assessed by immunoblots as described for canine BSM.

Role of caveolae in ACh-induced ASM contraction. We next tested whether the association of muscarinic M₃R and G_q/11 with caveolae was of functional significance. Therefore, we measured active isometric force generated by individual canine TSM strips in response to ACh exposure both before and after incubation with the cholesterol-depleting agent β-CD. When applied at concentrations of 5–10 mM, the hydrophobic core of β-CD and its analog mβCD sequester cholesterol and thereby dramatically reduce membrane cholesterol content (30). We first used transmission electron microscopy to confirm the β-CD protocol we used led to the disruption of caveolae (Fig. 2). Although organized arrays of flask-shaped membrane invaginations characteristic of caveolae were readily apparent in control samples, we were unable to see any evidence of these structures after muscle strips had been incubated in β-CD.

View larger version (100K): [in this window] [in a new window]   Fig. 2. Treatment of TSM strips with β-cyclodextrin (β-CD) depletes membrane caveolae. A and B: transmission electron microscopy images of control canine TSM tissue [A; incubated in Krebs-Henseleit solution (KH) at 37°C for 1 h], and canine TSM after incubation with KH containing β-CD (B; 10 mM, 1 h, 37°C). Organized arrays of caveolae are abundant in the plasma membrane of TSM (arrows in A) but are depleted or markedly modified (arrowheads in B) by β-CD treatment. Bar in each image = 1 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Loss of caveolae reduces sensitivity to ACh-induced contraction of canine ASM tissue. A: ACh concentration-response curves obtained for canine TSM strips before (ACh1 = control) and after (ACh2) treatment with β-CD (10 mM, 1 h, 37°C). Canine trachealis strips were mounted for isometric recording, and responses were normalized on the basis of maximum force elicited by isotonic KH containing 47 mM KCl. β-CD reduced sensitivity to ACh (control EC50 = 0.34 µM; β-CD EC50 = 0.8 µM; P < 0.0001), whereas maximum active force was unchanged. Curves are constructed from experiments using individual strips from at least 4 different animals at each concentration. Data show means ± SE. *P < 0.01 for control vs. β-CD (Wilcoxon test for matched pairs). B: contractile responses in groups of individual canine TSM strips exposed to 10–8 or 10–7 M ACh before (ACh1) and after (ACh2) 1 h incubation in KH buffer (control) or β-CD (10 mM, 1 h, 37°C). β-CD treatment reduced contractions to ACh (bottom), whereas there was no difference between ACh1 and ACh2 responses in individual control strips (top). Horizontal bars indicate the mean isometric force for each group of responses. Data for experiments using 8 individual strips are shown. *P < 0.05; **P < 0.01 compared with 1st ACh response (Wilcoxon test for matched pairs).

View larger version (103K): [in this window] [in a new window]   Fig. 4. A–D: distribution of muscarinic M3R and Cav-1 in cultured, contractile phenotype canine tracheal myocytes. Primary canine TSM cells were grown to confluence on glass coverslips and subjected to 7–10 days of culture in serum-deficient conditions. Thereafter, myocytes were fixed, double-labeled for smooth muscle myosin heavy chain (smMHC) and Cav-1 or M3R, and imaged by fluorescent microscopy before (A and B) or after (C and D) incubation in media containing β-CD (10 mM, 1 h, 37°C). The left and middle panels in A–D are matched fields of similar magnification. The insets highlight areas of Cav-1 or M3R labeling that are shown as an enlarged image on the right of each set of images. Nuclei were counterstained with Hoechst 33342 (10 µg/ml).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Loss of caveolae reduces ACh-induced Ca2+ responses of canine ASM cells. A: representative tracings from a typical experiment using canine ASM cells grown to confluence on coverslips and serum-deprived in insulin-supplemented media for 7–10 days. The tracings are the mean from 4 to 6 elongate cells identified in a single field. ACh (1 µM) was applied to fura-2-loaded cells to induce an increase in intracellular Ca2+ ([Ca2+]i) (ACh1); after a subsequent washout, cells were either treated with β-CD (10 mM, 1 h) to remove membrane cholesterol or with HEPES-HBSS (vehicle) control buffer. Cells were then washed again with HEPES-HBSS and exposed for a 2nd time to 1 µM ACh (ACh2). B: the mean ratio of 2nd (ACh2) and 1st (ACh1) responses for individual cells from control and β-CD-treated cultured are shown. Data represent the means ± SE from 35 cells measured in at least 3 different experiments. ***P < 0.001 (Wilcoxon test for matched pairs).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Loss of caveolae reduces ACh-induced [Ca2+]i release in human ASM cells. Human ASM cells were grown on chamber slides, maintained in serum-free media for 3 days, and then loaded with the Ca2+-sensitive dye fura-2. Before measuring [Ca2+]i responses elicited by different contractile agonists, cultures were incubated (1 h, room temperature) in HBSS/HEPES (control) or HBSS/HEPES-HBSS containing 5 mM methyl-β-cyclodextrin (mβCD). A: concentration-response curve for the muscarinic receptor agonist, ACh. B: concentration-response curve for the partial muscarinic receptor agonist, pilocarpine. C: concentration-response curve for histamine. D: concentration-response curve for bradykinin. For all curves, data points represent means ± SE from at least 20 cells studied in 3 ASM cell lines. *P < 0.05; ***P < 0.001 compared with untreated cells (ANOVA with Bonferroni post hoc comparison).

View larger version (14K): [in this window] [in a new window]   Fig. 7. Small interfering RNA (siRNA) knockdown of Cav-1 reduces ACh-induced Ca2+ responses of human ASM cells. Cultures of human ASM cells were grown on chamber slides; at subconfluence, cultures were transfected with a Cav-1 siRNA or a nonsilencing (control) siRNA. A: Western blot analysis showing that transfection with Cav-1 siRNA markedly suppressed Cav-1 protein within 24 h, and this was maintained for at least 3 days after transfection in cultures grown in serum-free media. Nonsilencing control siRNA had no effect on Cav-1 protein abundance. Blots were also probed for β-actin as a control for equal protein loading. B: 3 days after transfection with control siRNA or Cav-1 siRNA, cells were loaded with fura-2, and after exposure to sub-EC50 concentrations of ACh (0.01 and 0.1 µM), changes in [Ca2+]i were measured. Cav-1 protein knockdown significantly reduced peak [Ca2+]i induced by ACh, whereas control siRNA had no effect. C: in cultures treated with Cav-1 or control siRNA, we also counted the number of cells that were recruited to mount an increase in [Ca2+]i in response to 0.01 or 0.1 µM ACh. Recruitment frequency was significantly suppressed after Cav-1 knockdown. Data for each condition shown are the means ± SE of 25–45 cells from at least 3 different human ASM cell lines. *P < 0.05 and ***P < 0.001 for Cav-1 siRNA vs. nonsilencing control siRNA transfection (unpaired Student's t-test).

View larger version (41K): [in this window] [in a new window]   Fig. 8. Interference of the Cav-1 scaffolding domain (CSD) with a cell-permeable peptide mimetic reduces muscarinic receptor-mediated cytosolic Ca2+ release in human ASM cells. Cultures of human ASM cells were grown to confluence on chamber slides and then subjected to 3 days of serum deprivation. Before measuring cell responses, cultures were incubated with cell-permeable rhodamine-conjugated antennapedia-CSD peptide (AT-CSD) or a rhodamine-conjugated AT-scrambled-CSD (1 µM, 1 h, 37°C). Micrographs of matched fields of difference interference contrast (DIC; left) and merged DIC/rhodamine fluorescence (right) are shown for human ASM cells loaded with rhodamine AT-CSD (A) or rhodamine AT-scrambled-CSD (B). For both peptides, >95% of the cells exhibited rhodamine fluorescence, indicating effective uptake of the peptides. C: after peptide uptake, cells were loaded with fura-2, and concentration-response characteristics for methacholine (MCh)-induced [Ca2+]i were measured. Typical temporal tracings of the mean [Ca2+]i in response to 1 µM MCh in cells loaded with AT-CSD or AT-scrambled-CSD peptide are shown. For each condition, tracings represent an averaged curve derived from 12 to 15 different cells in a single region of interest. D: concentration-response characteristics of human ASM cells to MCh after loading with AT-CSD or AT-scrambled-CSD peptide. Data shown are the means ± SE of 24 fields of cells from at least 3 different cell lines. ***P < 0.001 compared with AT-scrambled-CSD (unpaired Student's t-test).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

The potential for caveolae to serve as foci for Ca²⁺ homeostasis in smooth muscle cells is a long-established concept stemming from early observations that caveolae contain high concentrations of Ca²⁺ (43). Large conductance Ca²⁺-activated K⁺ channels, plasma membrane Ca²⁺ pump, and TRPC class transient receptor potential channels all localize to caveolae (2, 5, 9, 16). An important role in Ca²⁺ handling is also supported by ultrastructural studies, including those in ASM, that show caveolae are in close proximity to sarcoplasmic reticulum and mitochondria (18, 31). Kamishima et al. (29) recently reported that caveolae and caveolin-1 modulate the reuptake of Ca²⁺ by the sarcoplasmic reticulum in isolated arteriolar myocytes after [Ca²⁺]_i mobilization is induced. Interestingly, cerebral resistance arterioles from caveolin-1 knockout mice exhibit a marked reduction in the frequency of spontaneous transient outward currents (STOCs; Ref. 11), possibly due to spatial separation of caveolae from the sarcoplasmic reticulum where ryanodine receptor-mediated Ca²⁺ sparks that trigger STOCs originate (3, 29, 39). Our electron microscopy analyses confirm that plasma membrane invaginations characteristic of caveolae are depleted from ASM tissue after β-CD treatment, supporting the possibility that spatial disruption of Ca²⁺ nanospaces could contribute to suppressed contractile responses to ACh. Similarly, caveolae depletion caused concomitant disorganization of caveolin-1 and M₃R in ASM cells, an effect that correlated with reduced mobilization of cytosolic Ca²⁺ in response to sub-EC₅₀ concentrations of ACh. It did not appear that the sarcoplasmic reticulum was compromised as cholesterol depletion was without effect on resting [Ca²⁺]_i, and Ca²⁺ release induced by bradykinin was refractory to caveolae disruption (Fig. 6D). Interestingly, our experiments using cell-permeable CSD peptide, which competes with binding of caveolin-1 to effector proteins but does not deplete caveolae, revealed a suppression in ACh-induced [Ca²⁺]_i that was quantitatively and qualitatively similar to that caused by cholesterol depletion. These findings indicate that, in the absence of spatial separation of caveolae from sarcoplasmic reticulum and mitochondria, caveolin-1 can play a direct role in regulating Ca²⁺ release triggered by ACh. To clearly discriminate the extent to which the suppression in cytosolic [Ca²⁺]_i we observed after caveolae deletion may have been caused by spatial disruption of Ca²⁺ nanospaces will require future studies using high resolution, real-time assessment of local Ca²⁺ concentration, inositol trisphosphate (IP₃) synthesis, and the distribution of IP₃ receptor, PLCβ, and G_q/11.

In addition to being integral to the spatial organization of Ca²⁺ nanospaces, caveolae and caveolin-1 may also participation in excitation-contraction coupling by sequestering and regulating effector proteins. The results of our experiments provide important new insight in this area. First, we show that both M₃R and its associated G protein effector subunit, G_q/11, are sequestered to caveolae microdomains in human and canine ASM. Second, whereas Darby et al. (9) used biochemical fractionation of canine trachealis to show caveolae are enriched in Ca²⁺-handling proteins, including L-type Ca²⁺ channels, calsequestrin, calreticulin, and plasma membrane Ca²⁺ pump, our experiments are the first to provide a functional link between caveolae and GPCR-induced Ca²⁺ release in human and canine ASM. Moreover, we show this effect is correlated with a similar suppression in contractile responses. Third, our studies with siRNA confirm that caveolin-1 mediates the facilitator role of caveolae in ACh-induced Ca²⁺ release, and with cell-permeable peptides we have also established that the NH₂-terminal CSD is of central importance to this effect. Collectively, these data provide functional confirmation of prior descriptive associations made between caveolae and Ca²⁺ homeostasis in smooth muscle cells.

A key finding of our studies is that caveolae and caveolin-1 appear to be most important in regulating the sensitivity of ASM responses to physiologically relevant concentrations of ACh. A specific role for caveolae under physiologically relevant conditions, in which [Ca²⁺]_i rarely exceeds 400 nM, has also been revealed in studies using caveolin blocking antibodies or CSD peptides to investigate Ca²⁺ reuptake by the sarcoplasmic reticulum in vascular myocytes (29). In our own experiments, ACh at concentrations below 1 µM induced [Ca²⁺]_i that was less than 400 nM, and it was only at these points that we observed a suppression in contraction and/or Ca²⁺ release after cholesterol depletion or treatment with caveolin-1 siRNA or CSD peptide (Fig. 6A). Conversely, at concentrations of ACh greater than 1 µM, which induced maximum increases in [Ca²⁺]_i equal to or greater than 600 nM, we observed no effect of caveolae or caveolin-1 interventions. A likely explanation for the differential effects of caveolae disruption on EC₅₀ and maximum response to ACh is the presence of muscarinic receptor reserve that has been documented in ASM (24, 36), rendering responses to higher ACh concentrations refractory to any reduction in coupling to G proteins and/or other downstream signaling effectors. As our [H]³-NMS-binding assays suggested there was no change in receptor number after caveolae depletion, we tested the functional effects of mβCD on pilocarpine, a partial muscarinic agonist that requires recruitment of all M₃R to induce maximum [Ca²⁺]_i. Unlike our observations with ACh, both the EC₅₀ and maximum response induced by pilocarpine were suppressed by caveolae depletion. This supports the conclusion that even though disrupting caveolae inhibits ACh EC₅₀, receptor reserve protects against effects on maximum ACh-induced responses. These experiments also appear to negate the possibility that divergent mβCD-sensitive and -insensitive pools of M₃Rs exist, as might have otherwise been predicted for receptors localized to caveolae-rich or caveolae-deficient membranes, respectively. If caveolae disruption had selective effects on only a portion of receptors, a partial agonist such as pilocarpine would display a biphasic dose-response relationship; at lower concentrations, the agonist would first use fully functional mβCD-insensitive receptor populations but would then only recruit mβCD-sensitive receptors when much higher concentrations were added. Figure 6B demonstrates that this is not the case. Collectively, these data suggest that caveolae and caveolin-1 are likely to have significant effects on in vivo responses of smooth muscle encircling the airways. Moreover, this provides solid rationale for future investigation of the role of caveolae and caveolin-1 in ASM function in pathological conditions such as during airway inflammation associated with bronchial asthma.

Receptor- and ligand-specific effects of caveolae and caveolins on smooth muscle contractile responses have been reported. In general, for vascular smooth muscle, caveolae and caveolin appear to facilitate contraction induced by physiological agonists (2, 12, 28, 48). There are conflicting reports of the effects of caveolae on muscarinic receptor-mediated responses in gastrointestinal smooth muscle; one study showed no change in carbachol-induced responses of tissue from caveolin-1 knockout mice (48), whereas a recent study showed that ectopic overexpression of caveolin-1 was sufficient to restore muscarinic receptor-mediated contractions in tissue from aged rabbits (50). Our studies indicate that caveolae and caveolin-1, via its CSD domain, facilitate responses of ASM to low concentration of ACh; however, we also saw that bradykinin-induced mobilization of cytosolic Ca²⁺ was resistant to caveolae disruption, whereas responses to all concentrations of histamine were suppressed by cholesterol depletion. As the receptors directly involved with mediating Ca²⁺ mobilization by each of these ligands are coupled to G_q/11, our findings suggest that receptor-specific differences in caveolae regulation may exist. The refractory nature of bradykinin to caveolae depletion markedly contrasts effects on ACh- or histamine-induced Ca²⁺ release. We have reported that in serum-derived canine ASM cultures, responsiveness to ACh is restricted to elongate, contractile phenotype myocytes, whereas bradykinin responses are restricted to myocytes that do not exhibit the contractile phenotype (37). As our current study shows that caveolin-1 is abundant and organized into linear arrays only in elongate, contractile myocytes, it is possible that lack of dependency of bradykinin receptors on caveolae in our cultures may relate to their more prominent functional role in noncontractile myocytes. Divergence in effects of caveolae could also be related to dynamic and variable relationships between GPCRs with caveolae, as has been demonstrated in different cells. For example, on ligand binding, β₂-adrenergic receptors exit caveolae in cardiomyocytes (41), endothelin ET_A receptors remain localized to caveolae in a number of cell lines (35), and in CHO and HEK cell lines, ectopically expressed B1 and B2 bradykinin receptors translocate into caveolae (47). Perhaps even more interesting, the same report revealed that although upon binding ligand both bradykinin receptor subtypes can traffic to caveolae and activate PLCβ via G_q/11, B2 receptors are rapidly internalized and desensitized, whereas B1 receptors remain active and subsequently activate Ras-dependent pathways (47). Thus there is suggestive evidence that caveolar trafficking of GPCRs, which can be associated with heterologous desensitization, may play a role in defining intracellular signaling responses (14, 38, 41, 47). In light of the differences we observed for different GPCRs in response to caveolae depletion, future experiments that address this issue in ASM are clearly warranted.

A number of studies suggest a facilitator role for caveolae in GPCR-mediated contraction in vascular smooth muscle (2, 12) and in smooth muscles from the gastrointestinal tract (13), the urogenital tract (32), and the myometrium (33). Our results showing that caveolae affect the sensitivity of ASM tissue to ACh-induced contraction are consistent with these reports. Although we measured the ability of caveolae and caveolin-1 to facilitate cytosolic Ca²⁺ mobilization, our studies did not directly assess other mechanisms that could also contribute to the effects of caveolins and caveolae on ASM contraction. For example, caveolin-1 reportedly facilitates the interaction of some GPCRs with G subunits (4), and it appears to preferentially bind GDP-G_q/11 or -G_i to its CSD (34, 35). This suggests that activated, GTP-bound G subunits dissociate from caveolin-1. Although we did observe localization of G_q to caveolae microdomains, we did not measure the effects of caveolae depletion or caveolin-1 knockdown on M₃R-induced G_q/11 activation. Also of relevance to GPCR agonists, phosphatidylinositol turnover, which is induced by G_q/11 activation of PLCβ₁ and leads to Ca²⁺ release from the sarcoplasmic reticulum, is highly compartmentalized to caveolae microdomains (6, 42). Future studies examining the effects of caveolin-1 and caveolae on ACh-induced IP₃ production in ASM may reveal more precise understanding of the mechanisms regulating Ca²⁺ release and contraction. Last, the recruitment of activated RhoA and PKC, both of which play an important role in Ca²⁺-independent contractile responses of smooth muscle, can be blocked by CSD peptides, suggesting an additional pathway to be assessed in future investigations of the role of caveolae in contractile responses of ASM to physiological agonists (51).

Our observation that muscarinic M₃R are localized to and functionally dependent on caveolae is of significant importance. Muscarinic M₃R play an important role in airway physiology and in the pathophysiology of asthma and COPD (22). Muscarinic M₃R are the primary muscarinic receptor subtype responsible for contraction of human central and peripheral ASM (45). In addition, in ASM, M₃R is the only muscarinic subtype that mediates IP₃ accumulation and is the primary receptor mediating ACh-induced contraction (15, 46). More recent work also indicates a prominent role for muscarinic M₃R in regulating airway myocyte proliferation and maturation (19, 20). Caveolae and caveolins could modulate muscarinic receptor signaling associated with these responses and thus contribute to more chronic aspects of asthma and COPD (21).

In summary, our results reveal that muscarinic M₃R and G_q/11 are localized to caveolae in ASM. Furthermore, caveolae and caveolin-1 facilitate muscarinic receptor-mediated [Ca²⁺]_i signaling and ASM contraction induced by physiologically relevant concentrations of agonist. These effects are mediated by the CSD of caveolin-1, are not associated with alterations in receptor availability, and thus are likely the result of orchestration of key signaling effectors such as G_q, second messenger producing enzymes, and ion channels in caveolae. Notably, we observed that caveolae contribute differentially to the facilitation of GPCR signaling in ASM cells, suggesting that unique mechanisms related to specific receptor-ligand interactions are a determinant of the functional significance of caveolae. This indicates that caveolae and caveolin-1 may play a complex role in ASM biology and function in health and disease.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by grants from the Sick Children Foundation/Institute of Human Development, Child and Youth Health (XG05-011), Canadian Institutes of Health Research, and the Manitoba Institute of Child Health. This work was undertaken, in part, thanks to funding from the Canada Research Chairs Program and Canada Foundation for Innovation. A. J. Halayko holds a Canada Research Chair in Airway Cell and Molecular Biology. R. Gosens received support from the National Training Program in Allergy and Asthma (NTPAA) and is the recipient of a Marie Curie Outgoing International Fellowship from the European Community (008823).

	ACKNOWLEDGMENTS

 
Dr. Zohair Al-Hariri and Craig T. Hillier contributed during the initial phases of this study.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. J. Halayko, Dept. of Physiology, Univ. of Manitoba, 715 McDermot Ave., Rm. 547, John Buhler Research Centre, Winnipeg, Manitoba, Canada, R3E 3P4 (e-mail: ahalayk{at}cc.umanitoba.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.

^* R. Gosens and G. L. Stelmack contributed equally to this work.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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