Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca2+ handling

Peter J. Darby, C. Y. Kwan, and Edwin E. Daniel

Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca2+-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca2+. We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca2+ channels and Ca2+ binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca2+ pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca2+ pump, or the type 1 inositol 1,4,5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca2+ pump to the cell periphery, whereas the SR Ca2+ pump was located deeper in the cell. The presence of L-type Ca2+ channels, the PM Ca2+ pump, and the Ca2+ bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca2+ handling.

caveolin; L-type calcium channels; plasma membrane calcium pump; calsequestrin; calreticulin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RELATIVE ROLES AND INTERACTIONS between the plasma membrane (PM) and the sarcoplasmic reticulum (SR) in controlling intracellular Ca2+ have been areas of intense study in smooth muscle (as well as other cell types) for many years. Studies in canine tracheal smooth muscle have demonstrated a direct refilling of the SR with Ca2+ that enters from the extracellular space via a "preferred pathway," independent of contraction. This refilling could be enhanced by raising extracellular Ca2+ or opening L-type Ca2+ channels with BAY K 8644 or blocked by the addition of an L-type Ca2+ channel blocker (3, 4). This pathway did not require a functional SR Ca2+ pump because cyclopiazonic acid did not block the refilling (3, 4). Contractility studies (34, 35) with canine bronchial smooth muscle demonstrated a sustained agonist-induced contraction in a Ca2+-free medium containing 50 µM EGTA that could be reproduced several times, with only the magnitude of the contraction reduced on subsequent contractions. The sustained phase of the contraction was prevented or abolished by the addition of nifedipine or by raising the EGTA concentration to millimolar levels (35), suggesting that the source of Ca2+ was extracellular but protected from the extracellular medium. Although others have suggested (23, 38, 39) that the source of this Ca2+ may be the cartilage, this is unlikely because pretreatment with cyclopiazonic acid resulted in only transient contractions, suggesting that a functional SR is required for sustained contraction (35). Additionally, studies of 45Ca2+ uptake into and efflux from cartilage revealed very rapid exchange, too fast to supply Ca2+ for long periods (34). The identity of this protected extracellular source of Ca2+ remains unknown, but we postulate that caveolae may be this source.

A growing body of evidence implicates caveolae in various cellular pathways in many cell types, including a role in cell signaling (reviewed in Ref. 2). Immunofluorescence microscopy and immunoelectron microscopy studies in smooth muscle have demonstrated the PM Ca2+ pump (14) and an inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor-like protein (16, 17) in mouse ileum. Biochemical studies that isolated caveolae with the use of detergent treatment (6, 12) or detergent-free isolation methods [OptiPrep (12) or Na2CO3 (24) treatment methods] have demonstrated the presence of glycosyl phosphatidylinositol (GPI)-linked proteins (6), G protein subunits [both alpha - and beta - subunits (6, 12)], and B2 bradykinin (12) and angiotensin II type 1 (24) receptors.

In addition to these various cell signaling proteins, caveolae are also enriched in caveolin, which is the only true marker protein for caveolae. Caveolin, a 22-kDa protein, forms the protein coat on the cytoplasmic surfaces of caveolae. The protein is inserted into the membrane so that both the NH2 and COOH terminals are located on the cytoplasmic surface, with none of the protein in contact with the luminal surface of the caveolae (18, 19, 26). In addition to forming the protein coat on the cytoplasmic surface of the caveolae, caveolin also has a unique sequence that allows it to bind to aromatic-rich sequences on adjacent caveolin proteins as well as on other proteins (29). This caveolin binding domain gives caveolin a role in the clustering of various proteins to caveolae. The list of proteins associated with caveolin includes G proteins, various receptors, and nitric oxide synthase (for extensive list, see Ref. 8). Some investigators have taken advantage of the interaction between caveolin and other proteins and have conducted immunoprecipitation experiments with antibodies to caveolin to determine which proteins are also localized to caveolae (7, 12, 13).

There have been no previous studies in airway smooth muscle that have used either biochemical or histochemical methods to investigate the proteins present in caveolae. We developed a method of isolating caveolin-enriched membranes from canine tracheal smooth muscle with detergent treatment based on the methods of Sargiacoma et al. (43) and Chang et al. (6). We asked, "are proteins necessary for a role in the Ca2+ handling associated with caveolae?" These include the PM Ca2+ pump, L-type Ca2+ channels, and Ca2+ binding proteins. To confirm the results of detergent treatment, immunoprecipitation experiments were conducted with PM-enriched microsomal membranes, and immunocytochemistry experiments were conducted with fixed tracheal smooth muscle cryosections.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of microsomes from tracheal smooth muscle. Mongrel dogs (20-40 kg) of either sex were euthanized with a pentobarbital sodium overdose (100 mg/kg body wt). All procedures were approved by the McMaster University (Hamilton, ON) Animal Care Committee. The tracheae were removed and immediately placed in ice-cold Ringer solution except for the tissues used for the histochemistry experiments, which were placed in 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). For preparation of microsomes, the tracheal smooth muscle was dissected from the cartilage and epithelium and then cleaned of any connective tissue and blood vessels with fine scissors. Once cleaned, the muscle was placed in ice-cold sucrose-magnesium-MOPS buffer (SMB; 250 mM sucrose, 10 mM MgCl2, and 25 mM MOPS, pH 7.4) for 5-10 min, blotted dry, weighed, and frozen at -20°C until used for membrane preparation. Typically, tissue from six to eight dogs was pooled for one membrane preparation.

Microsomal membranes (MIC I) were prepared according to the method of Grover et al. (21) and were those membranes that pelleted at 100,000 g in 45 min. All steps were carried out at 4°C. The MIC I microsomal pellet was further separated by resuspension in SMB and centrifugation at 10,000 g for 10 min. This resulting pellet was MIT II and consisted mainly of synaptosomes and mitochondria. The PM-enriched supernatant was called MIC II.

Preparation of detergent-treated microsomes. The MIC II fraction was divided, with one half diluted with SMB and left at 4°C and the other half diluted with SMB containing Triton X-100 (1% final concentration) and incubated at 4°C with occasional mixing. After 1 h, the control and detergent-treated microsomes were centrifuged at 100,000 g for 45 min at 4°C. The resulting pellets were resuspended in SMB and were called M3C and M3T for control and detergent-resistant membranes, respectively.

Electrophoresis and immunoblotting. Isolated membrane fractions were boiled for 5 min in Laemmli (28) sample buffer containing 62.5 mM Tris · HCl (pH 6.8), 3% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue and separated by SDS-PAGE according to the method of Laemmli with 7.5 or 12% separating gels. For immunodetection of caveolin, 5-10 µg of protein were loaded in each lane. For immunodetection of all other proteins, 60-80 µg were loaded in each lane. Detection limits for most proteins, including calreticulin and calsequestrin, were >= 20 µg of protein. For caveolin, the detection limit was much less. After electrophoresis, the proteins were transferred overnight onto 0.45-µm nitrocellulose according to the method of Towbin et al. (47).

For immunoblotting, all incubations and washes were done at room temperature with constant shaking. The nitrocellulose was first incubated with 5% milk powder in 20 mM Tris · HCl, pH 7.5, 500 mM NaCl [Tris-buffered saline (TBS)] and 0.05% Tween 20 (TBS-T) for 1 h to block nonspecific protein binding before incubation with the primary antibodies for 1 h, diluted in the same solution (TBS-T and 5% milk powder). The blots were washed for 30 min with three changes of TBS-T before they were incubated for 1 h with the horseradish peroxidase (HRP)-linked secondary antibodies (diluted in TBS-T and 5% milk powder). Antibody dilutions were 1:2,500 for the anti-mouse and 1:4,000 for the anti-rabbit secondary antibodies. The blots were washed for 30 min with three changes of TBS-T and then washed for 10 min with two changes of TBS (20 mM Tris · HCl and 0.5 M NaCl, pH 7.5,) before immunodetection with enhanced chemiluminescence (Amersham) according to the manufacturer's directions. Proteins were detected by exposure of the immunoblots to enhanced chemiluminescence Hyperfilm (Amersham) for various time periods (10 s to 30 min).

The immunoblots were cut to show only the band of interest, which allowed the figures to be presented in condensed format. In all cases, a corresponding positive control was run in one lane along with the samples to confirm the identity of the band of interest. Additionally, a negative control was also run in one lane (usually from the soluble fraction produced from the centrifugation step to pellet MIC I). No band corresponding to a protein of interest found in the soluble fraction was found. Some extra bands of varied intensities were present in both the experimental fractions and the soluble fraction, especially when polyclonal antibodies were used. With one exception, these bands were much more diffuse than the band of interest. The exception was the antibody to the alpha 1-chain of the L-type Ca2+ channel, which had numerous bands present, some of which were quite intense. For this reason, this antibody was not used for immunocytochemistry. In other cases, the extra diffuse bands could usually be attributed to the secondary antibody used; i.e., when the primary antibody was omitted and only the polyclonal secondary antibody was used, the diffuse bands were present on both the experimental and soluble lanes in nearly all cases.

Immunoprecipitation. Immunoprecipitation experiments were conducted with MIC II membranes under nondenaturing conditions according to the method of Feron et al. (13). All steps were carried out at 4°C. Briefly, ~200-400 µg of protein were incubated in a fourfold dilution of [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) buffer (50 mM Tris · HCl, pH 7.4, 20 mM CHAPS, 125 mM NaCl, 2 mM dithiothreitol, 0.1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride) for 30 min with constant agitation. Anti-caveolin antibody (4 µg) was added and incubated for 60 min with constant agitation. The solution was centrifuged at 10,000 g for 30 min on a desktop centrifuge to pellet any insoluble membranes. The supernatant was removed to a 1.5-ml Eppendorf tube containing 50 µl of a 50% slurry of protein A Sepharose beads (in CHAPS buffer) and incubated with constant agitation for 60 min. The beads were then pelleted (10,000 g for 30 s), the supernatant was aspirated, and the pellet was washed six times with a small volume (150 µl) of CHAPS buffer. After the final wash, the beads were pelleted (10,000 g for 4 min), and all of the supernatant was removed. The samples were prepared for immunoblotting by adding 50 µl of Laemmli buffer to the pelleted beads, boiling for 5 min and briefly centrifuging, and then loading the supernatant onto a gel.

[3H]PN200-110 binding. [3H]PN200-110 binding was carried out on the various membrane fractions in 1.5-ml Eppendorf tubes at 25°C in 50 mM Tris · HCl, pH 7.4, with 1.0 nM [3H]PN200-110 for 60 min. This concentration of PN200-100 exceeds the dissociation constant (Kd) value by >= 10-fold. The reaction was started by the addition of 5-50 µg of protein and terminated by placing the tubes on ice. The tubes were then immediately centrifuged at 10,000 g for 60 min at 4°C. The supernatant (usually the soluble fraction produced from the centrifugation step to pellet MIC I) was removed, and the bottom of the Eppendorf tube containing the pelleted proteins was cut off, placed in a scintillation vial, and counted for 3H in a Beckman model LS 6800 scintillation counter. Binding was done in reduced lighting by covering the water bath with a cardboard box. Nonspecific binding was defined as [3H]PN200-110 binding in the presence of 1 µM nitrendipine. Specific binding was defined as total [3H]PN200-110 binding minus nonspecific binding (<= 10%).

Protein and enzyme assays. Protein was determined by the use of colorimetric assays based on the methods of Lowry et al. (30) or Bradford (5) with BSA as standard. The specific activities of 5'-nucleotidase and Mg2+-ATPase (markers for smooth muscle PM) were determined spectrophotometrically based on the amount of inorganic phosphate liberated from 5'-AMP over 60 min (for 5'-nucleotidase) or 5'-ATP over 15 min (for Mg2+-ATPase) on all membrane fractions according to the method of Matlib et al. (31).

Immunocytochemistry. Preparation of cryosections and immunocytochemistry were performed according to the methods of Salapatek et al. (42). The tracheae from euthanized dogs were removed and placed in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde for 3 h at room temperature. The fixed tissues were washed with 0.1 M phosphate buffer and then placed in 30% sucrose in 0.1 M phosphate buffer for cryoprotection overnight at 4°C. Small pieces of tissue were sectioned at 10-µm thickness in a cryostat (Leitz 1720 digital) and collected on glass slides coated with gelatin.

The slides with the attached cryostat sections were washed (3 × 15 min) with 0.1 M phosphate buffer containing 0.4% Triton X-100, incubated for 1 h in 0.1 M phosphate buffer containing 0.4% Triton X-100 and 3% BSA (to block nonspecific binding; both steps at room temperature), and incubated overnight in the same buffer containing the various antibodies at 4°C in a humid chamber. The slides were then washed (3 × 15 min) with 0.1 M phosphate buffer containing 0.4% Triton X-100 and incubated with the various secondary antibodies [FITC-labeled goat anti-rabbit IgG and indocarbocyanide (Cy3)-labeled anti-guinea pig or anti-mouse IgG; all from Jackson ImmunoResearch] for 1-2 h in the dark in a humid chamber at room temperature. The slides were washed under reduced lighting, first with 0.1 M phosphate buffer (3 × 15 min) and then with 4 mM Na2CO3 (10 min). The tissues were mounted in 80% glycerol in 0.1 M phosphate buffer (pH 10) and viewed with a Leitz microscope equipped with a fluorescence epi-illuminator and an I2 filter for Cy3 and a N2 filter for FITC. Kodak ISO 400/27° 35-mm film was used for color photography.

Antibodies. The antibodies used for immunoblotting, immunoprecipitation, and immunocytochemistry experiments are listed, along with the dilution used, the source and specificity, and the immunogen used for production. Anti-caveolin-1 (polyclonal, dilution 1:1,000 for immunoblots, 1:400 for immunohistochemistry; Transduction Laboratories, Lexington, KY) recognizes caveolin-1 and was generated from amino acids 1-97 of human caveolin-1. In other studies (Daniel EE and Wang YF, unpublished observations), we showed that caveolin-3 is present in much smaller amounts than caveolin-1 in canine smooth muscle. Anti-Ca2+ channel alpha 1-subunit (polyclonal, dilution 1:100 for immunoblots, a gift from Dr. Arnold Schwartz, University of Cincinnati, Cinncinnati, OH) recognizes both the cardiac and smooth muscle Ca2+ channel alpha 1-subunit. Anti-PM Ca2+-ATPase (monoclonal, dilution 1:1,000 for immunoblots, 1:200 for histochemistry; clone 5F10, Affinity Bioreagents, Golden, CO) recognizes all four isoforms of the PM Ca2+ pump and specifically recognizes an epitope between amino acids 724 and 783 of the human erythrocyte Ca2+ pump. Anti-sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2; monoclonal, dilution 1:2,500 for immunoblots, 1:500 for histochemistry; clone IID8, Affinity Bioreagents) recognizes both isoforms of the SERCA2 Ca2+ pump and was generated with purified canine cardiac SR. Anti-calreticulin (polyclonal, dilution 1:1,000 for immunoblots, 1:100 for histochemistry; Affinity Bioreagents) recognizes the protein from various animal and tissue sources and was generated from recombinant human calreticulin. Anti-canine cardiac calsequestrin (polyclonal, dilution 1:2,000 for immunoblots, 1:100 for histochemistry; Upstate Biotechnology, Lake Placid, NY) recognizes the cardiac but not the skeletal muscle isoform of calsequestrin and was generated from amino acids 39-48 of canine cardiac calsequestrin. Anti-connexin 43 (monoclonal, dilution 1:250 for immunoblots, Transduction Laboratories) recognizes connexin 43 from various species and was generated from amino acids 252-270 of rat connexin 43. Anti-Ins(1,4,5)P3 receptor (polyclonal, dilution 1:500 for immunoblots, Calbiochem, San Diego, CA) recognizes the Ins(1,4,5)P3 receptor from various cell types and animal species and was generated from a short synthetic peptide corresponding to the COOH-terminal cytoplasmic domain.

Double-label immunohistochemistry was carried out by exposing sections overnight to complex solutions containing antisera against the two proteins of interest diluted as described above. After the primary antisera were washed away, the appropriate secondary antibodies (diluted 1:50) with the conjugated fluorophores FITC or Cy3 were added. The sections were examined with a Leitz fluorescence microscope equipped with the filters I2 for Cy3 and N2 for FITC.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of detergent-resistant membranes. Caveola-derived membranes were prepared from canine tracheal smooth muscle with detergent treatment based on the methods of Sargiacoma et al. (43) and Chang et al. (6). Their results and the results of others (reviewed in Ref. 2) suggest that detergent treatment is a method that allows the preparation of an enriched fraction of caveola-derived membranes and their associated proteins. To assess whether we had produced a fraction enriched in caveola-derived membranes, Western blotting with antibodies to caveolin was performed. The relative abundance of caveolin in the detergent-resistant membrane fraction (M3T) was compared with that in the control membrane fraction (M3C) and starting material (MIC II fraction), as well as with that in the MIT II fraction. As can be seen in Fig. 1, the detergent-resistant membrane fraction, M3T, is greatly enriched in caveolin compared with the other fractions, suggesting an enrichment of caveolar membranes.


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Fig. 1.   Immunoblots demonstrating the distribution of caveolin and various Ca2+ handling proteins in the canine tracheal smooth muscle membrane fractions. MIT II, 2nd 10,000 g spin; MIC II, plasma-membrane enriched supernatant of MIT II; M3C, pelleted MIC II suspended in sucrose-magnesium-MOPS (control); M3T, detergent-resistant membrane pelleted as for M3C. (For detailed definitions of membrane fractions see METHODS.) There is an enrichment of caveolin, the alpha 1-chain of the L-type Ca2+ channel, calsequestrin, and calreticulin in the detergent-resistant fraction M3T. M3T also appears to contain the plasma membrane (PM) Ca2+ pump, although it is not enriched. Immunoblots represent 4-9 separate experiments.

Presence of Ca2+ handling proteins. Further Western blots with an antibody that recognizes both the cardiac and smooth muscle isoforms of the alpha 1-chain (Fig. 1) revealed that the caveolin-enriched M3T fraction was also enriched in the alpha 1-chain of the L-type Ca2+ channel. The caveolin-enriched fraction also contained the PM Ca2+ pump, although this protein was not enriched in the M3T compared with M3C or MIC II fractions (Fig. 1). Furthermore, the M3T fraction was also enriched in two SR Ca2+ binding proteins, calsequestrin and calreticulin (Fig. 1). Thus the caveolin-enriched membrane fraction contained the components necessary for a role in Ca2+ handling: a Ca2+ pump, Ca2+ channel, and Ca2+ binding proteins. Further experiments were done in an attempt to confirm the localization of these proteins to caveolin-enriched membranes.

[3H]PN200-110 binding. Radioligand binding with the dihydropyridine PN200-110 was conducted on the various membrane fractions to confirm the presence and enrichment of L-type Ca2+ channels in the caveolin-enriched membrane fraction. Preliminary experiments that measured the distribution of binding on the various membrane fractions indicated that [3H]PN200-110-specific binding was highest in the MIT II and MIC II fractions, similar to results in other smooth muscle types with another dihydropyridine, nitrendipine (1, 22, 48). Binding experiments were conducted on the caveolin-enriched membrane fraction M3T and compared with binding in M3C, MIC II, and MIT II fractions. The concentration of ligand corresponds to one 10-fold higher than the Kd measured in other canine smooth muscle tissues (1, 22, 48) and should allow for accurate measurement of total binding in each fraction. As can be seen in Fig. 2, specific binding in the caveolin-enriched fraction was almost double that in the control fraction (M3C; P = 0.09) and three times that in the MIC II fraction (P < 0.05), indicating an enrichment of L-type Ca2+ channels in the detergent-resistant membrane fraction. Nonspecifc binding was <10% of total binding in all fractions tested. Calculations of total recovery of [3H]PN200-110-specific binding in the M3T and M3C fractions, expressed as a percentage of binding in MIC II, indicated that despite a recovery of only one-half of the protein compared with that in the M3C fraction (P < 0.05), there was a near equal recovery of [3H]PN200-110-specific binding (Table 1).


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Fig. 2.   Distribution of [3H]PN200-110 binding in canine tracheal smooth muscle membrane fractions. Values are means ± SE expressed as fmol [3H]PN200-110/mg protein from 4 separate experiments. Specific binding in M3C is almost double that in MIC II, whereas for the detergent-resistant fraction M3T, the specific binding is 3 times that in the MIC II fraction. Calculation of total recovery of specific binding in M3C and M3T indicates an approximately equal recovery between the 2 fractions.


                              
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Table 1.   Total recovery in fractions isolated from canine tracheal smooth muscle

Plasma membrane enzyme activities. GPI linkage of proteins was formerly considered a marker for caveolae, but this relationship appears to be an artifact (44); however, see Ref. 2. The specific activities of two PM marker enzymes, 5'-nucleotidase, which is a GPI-linked protein, and Mg2+-ATPase, which is not a GPI-linked protein, were compared in the various fractions isolated. The specific activities of 5'-nucleotidase and Mg2+-ATPase in the M3C fraction were increased compared with the MIC II fraction (Table 2). In the M3T fraction, the specific activity of 5'-nucleotidase was approximately threefold higher than in the MIC II fraction (P < 0.001) and significantly higher than in the M3C fraction (P < 0.001), whereas the specific activity of Mg2+-ATPase was almost eliminated (P < 0.01 compared with MIC II and M3C; Table 2). Calculations of total recovery of 5'-nucleotidase activity in the M3C and M3T fractions (expressed as a percentage of activity in MIC II; Table 1) indicate no difference between the two fractions, similar to that seen in the recovery of dihydropyridine binding sites. Further experiments demonstrated that the loss of Mg2+-ATPase activity in the detergent-resistant membrane fraction was due, in part, to inactivation of the enzyme. There was still a small but significant component that was not a result of detergent effects on the enzyme assay, suggesting that the decrease in Mg2+-ATPase activity in the M3T fraction is not solely an artifact of detergent affecting the enzyme but that a certain component of the change is due to the elimination of membranes that contain Mg2+-ATPase and the enrichment of membranes that contain 5'-nucleotidase.

                              
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Table 2.   Summary of enzyme activities and [3H]PN200-110-specific binding in fractions isolated from canine tracheal smooth muscle

Immunodetection of connexin 43 and SR proteins. To confirm that the detergent-resistant membranes were a subset of the PM and did not represent a purification of the total PM, immunoblots were performed with an antibody specific for a protein that should not be localized in caveolae. Connexin 43, a protein localized to and involved in the formation of gap junctions, should not be found in caveolae. With the MIT II, MIC II, M3C, and M3T fractions, connexin 43 was detected in MIT II, MIC II, and M3C but not in the M3T fraction (Fig. 3), even at longer exposures of the nitrocellulose to film (data not shown). This supports the hypothesis that detergent treatment results in the purification of a subset of PM that is enriched in caveolar membranes and not the purification of the PM from noncaveolar membrane sources that contain gap junctions (9).


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Fig. 3.   Distribution of caveolin and noncaveolar proteins in canine tracheal smooth muscle membrane fractions. There is no detectable signal for the inositol 1,4,5-trisphosphate (IP3) receptor in M3T, although it is present in MIT II and MIC II. Similarly, the detergent-resistant fraction M3T does not show detectable levels of the sarcoplasmic reticulum (SR) Ca2+ pump or connexin 43, even at longer exposure times (data not shown). Immunoblots are representative of 4-9 separate experiments.

The presence of calsequestrin and calreticulin in M3T may result because SR membranes are also detergent resistant or because the detergent treatment results in the movement of proteins from SR membranes into the detergent-resistant membranes. To address these issues, immunoblots were performed with antibodies specific for the SR Ca2+ pump (SERCA2b) and Ins(1,4,5)P3 receptor (type 1). As indicated in Fig. 3, although detected in both the originating MIC II fraction and the M3C fraction, neither the SR Ca2+ pump nor the Ins(1,4,5)P3 receptor was detected in the M3T fraction. These results suggest that the SR membranes and their proteins are not detergent resistant or translocated from the SR to detergent-resistant membranes, but they do not exclude the possibility that SR proteins localized in the lumen (such as calsequestrin and calreticulin) may be translocated.

Immunoprecipitation of caveolin and associated proteins. In addition to forming the protein coat on caveolae, caveolin-1 has been shown to bind to numerous proteins localized to caveolae via an interaction between the caveolin-scaffolding domain (amino acids 82-101) and an aromatic-rich domain on the various proteins (8). We took advantage of this interaction, conducting immunoprecipitation experiments under nondenaturing conditions to determine which proteins are associated with caveolin.

With the method of Feron et al. (13), immunoprecipitation experiments were conducted with the MIC II membrane fraction under nondenaturing conditions. Antibodies to caveolin were able to immunoprecipitate caveolin from MIC II membranes isolated from canine tracheal smooth muscle (Fig. 4A, IP), confirming that the immunoprecipitation was successful, although solubilization was incomplete because there was still a large portion of caveolin in the insoluble pellet (Fig. 4A, Pellet). Because the antibody used for detection was the same polyclonal antibody used for immunoprecipitation, a second band was visible on the immunoblots at ~54 kDa, which corresponds to the heavy chain of the caveolin antibody used for immunoprecipitation (Fig. 4, B and C).


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Fig. 4.   Coimmunoprecipitation of caveolin, calsequestrin, and calreticulin. With an antibody to caveolin, proteins from tracheal smooth muscle microsomes (MIC II) were immunoprecipitated under nondenaturing conditions according to the method of Feron et al. (13). A: after incubation with anti-caveolin antibodies and before incubation with protein A Sepharose beads, a 10,000 g centrifugation step was added to pellet any undissolved membranes or proteins (Pellet). IP, proteins immunoprecipitated by anti-caveolin antibodies. Western blots with antibodies directed against caveolin and calsequestrin indicate that caveolin and calsequestrin were both immunoprecipitated. Because the same polyclonal antibody used for immunoprecipitation was used for immunodetection, an additional band is present at 54 kDa, which corresponds to the caveolin antibody heavy chain. This band partially obscures the 2 bands for calsequestrin at 57 and 60 kDa (refer to Fig. 1). However, as seen in B and C, Western blots with caveolin antibodies alone clearly show only a single band at 54 kDa, whereas double staining of the same Western blots with caveolin + calsequestrin (B) or calreticulin (C) clearly shows an additional band at 60 kDa, indicating immunoprecipitation of these proteins with caveolin. Each blot is representative of 4 separate experiments.

Double staining of the immunoblots revealed that in addition to caveolin, calsequestrin (Fig. 4, A and B) and calreticulin (Fig. 4C) were also immunoprecipitated but that connexin 43, the alpha 1-chain of the L-type Ca2+ channel, and the PM Ca2+ pump (data not shown) were not. The presence of bands for calsequestrin and calreticulin, which appear as two bands at ~57 and ~60 kDa experiements (see Fig. 1), was difficult to detect because of the presence of the band at ~54 kDa, which corresponds to the caveolin antibody heavy chain (Fig. 4A, compare IP with Pellet). Comparison of the lanes labeled caveolin (Fig. 4, B and C) with those labeled caveolin + calsequestrin (Fig. 4B) or caveolin + calreticulin (Fig. 4C) clearly show that when stained with caveolin only, the band at ~54 kDa is a single band, whereas double staining shows two overlapped bands at ~54 and ~60 kDa, indicating the presence of calsequestrin and calreticulin in the material immunoprecipitated with caveolin antibodies. The band at ~60 kDa was never present when caveolin antibodies alone were used for immunodetection of immunoprecipitated proteins, even at longer exposures of the nitrocellulose to film (data not shown). These results suggest an interaction between caveolin and calsequestrin or calreticulin and also show that their presence in the detergent-resistant membranes was not an artifact of protein sorting during detergent treatment.

Immunocytochemistry. Immunocytochemistry was performed with cryosections of fixed tracheal smooth muscle (10 µm thick, cut transverse to the smooth muscle bundles). Antibodies to caveolin stained only the periphery of each smooth muscle cell, consistent with its localization to caveolae on the PM (Fig. 5, A and C). No staining was seen deeper in the cells. The staining was particulate, although all regions of the cell surface were stained. As mentioned in the introduction, caveolae of smooth muscle are arranged in rows that run parallel to the long axis of the cells. However, if the rows are not straight or if the transverse sections are not perfectly perpendicular to the long axis of the cells, then caveolae will appear to be located throughout the cell periphery of a 10-µm section as opposed to discrete sites.


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Fig. 5.   Double immunohistochemical staining of canine tracheal smooth muscle cryosections with antibodies to caveolin (A) and the PM Ca2+ pump (B), caveolin (C) and the SR Ca2+ pump (D), calsequestrin (E) and the PM Ca2+ pump (F), and calreticulin (G) and the PM Ca2+ pump (H). Each pair of images corresponds to a single section stained with 2 primary antibodies (and subsequent secondary antibodies). Sections (10 µm thick) were cut transverse to the smooth muscle bundles. Insets, further magnification (approximately ×2 of boxed area). A and B, insets: the punctate nature of caveolin staining frequently seen and the similar localization of the PM Ca2+ pump. C and D, insets: dissimilar localizations of caveolin and the SR Ca pump. E and F, insets, and G and H, insets: partial localization of calsequestrin and calreticulin, respectively, with the PM Ca2+ pump, probably related to close locations of peripheral SR and the PM. Bars, 25 µm.

Antibodies directed against the PM Ca2+ pump also stained the periphery of the smooth muscle cells (Fig. 5, B, F, and H). Numerous "hot spots" were seen on the cell periphery, apparently randomly distributed. These hot spots were visible only on the cell periphery. Double staining of cryosections with antibodies to caveolin and the PM Ca2+ pump suggested colocalization of staining on the periphery of the same smooth muscle cells (Fig. 5, A and B) within the limits of the microscope resolution. As a control for nonspecific staining, either the caveolin or PM Ca2+ pump antibody was omitted. This resulted in a lack of staining with their respective secondary antibodies. Similar control experiments were done for all double-staining experiments.

In contrast to the results with the caveolin or PM Ca2+ pump antibodies, antibodies directed against the SR Ca2+ pump did not stain the cell surface selectively. Instead, staining was found also in the cell interior, perhaps staining of SR around the nucleus (Fig. 5D). There was also intense staining in regions at or just below the cell surface, which may correspond to peripheral SR. Double staining with antibodies to caveolin and the SR Ca2+ pump indicated little colocalization of staining. Comparison of Fig. 5, C and D, demonstrates that in regions where caveolin staining is in focus, the staining for the SR Ca2+ pump is not, and, conversely, in regions where staining for the SR Ca2+ pump is in focus, the staining for caveolin is not in focus. This would suggest that staining for caveolin is on the cell surface, whereas staining for the SR Ca2+ pump may be located at regions just below the cell surface, on the peripheral SR.

Immunocytochemistry with antibodies directed against calsequestrin or calreticulin, especially the latter, produced more general staining of the smooth muscle cells, with no structures clearly delineated when viewed through the microscope. The photo images appear to show stronger staining at or just below the cell surface (Fig. 5, E and G). Double staining with antibodies to the PM Ca2+ pump and calsequestrin or calreticulin indicated staining for the PM Ca2+ pump on the periphery of the cell surface (Fig. 5, F and H), whereas staining for the Ca2+ binding proteins was more dispersed throughout the smooth muscle cells (Fig. 5, E and G). In some areas (Fig. 5, E and G, insets), there seemed to be overlap with the staining for the PM Ca2+ pump. This staining may include areas at or just below the cell surface.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This paper describes for the first time the isolation of a caveola-enriched, detergent-resistant membrane fraction from canine tracheal smooth muscle. Furthermore, it demonstrates the presence of L-type Ca2+ channels, the PM Ca2+ pump, calsequestrin, and calreticulin in this caveolin-enriched fraction. The presence of these proteins is consistent with our model of caveola involvement in Ca2+ handling in airway smooth muscle. These results were confirmed with the use of other techniques. The association of calsequestrin and calreticulin with caveolae was confirmed by immunoprecipitation experiments in which both calsequestrin and calreticulin were coimmunoprecipitated with caveolin, the marker protein of caveolae. Immunocytochemistry experiments confirmed, as expected, the presence of caveolin and the PM Ca2+ pump at the periphery of the cell surface, whereas the SR Ca2+ pump was located deeper within the cell. However, the limits of microscope resolution make it impossible to determine if the caveolin and PM Ca2+ pump staining are both located at the same sites on the cell surface.

The enrichment of 5'-nucleotidase in the detergent-resistant membrane fraction was consistent with the results of other investigators, demonstrating an enrichment of GPI-linked proteins in detergent-resistant, caveolin-enriched membranes (6; reviewed in Ref. 2). Some (15, 32) have argued that the presence of GPI-linked proteins is an artifact due to either protein sorting during the isolation procedure or the presence of GPI-linked proteins in noncaveolar, detergent-resistant membranes, whereas others (2) have argued that it is not an artifact. The results of the Mg2+-ATPase assays indicated that the loss of Mg2+-ATPase activity was, at least in part, due to the loss of some membranes that contained Mg2+-ATPase activity. Furthermore, the absence of any signal for connexin 43 in the detergent-resistant membrane fraction would suggest that the detergent treatment resulted in the loss of those PM-derived membranes that contain gap junctions. Thus our results are consistent with the hypothesis that detergent treatment resulted in the purification or enrichment of caveola-derived membranes.

The presence of L-type Ca2+ channels in the detergent-resistant membrane fraction was consistent with our model of Ca2+ handling. Indeed, calculation of the total recovery of the dihydropyridine binding indicates equal amounts in the detergent-resistant membrane fraction (M3T) and corresponding control membranes (M3C), suggesting that all of the L-type Ca2+ channels are located in the caveolae. However, it must be noted that a large proportion of the L-type Ca2+ channels was unaccounted for (see Table 1). This may be due to inactivation of the dihydropyridine binding site or loss of L-type Ca2+ channels during the isolation procedure. The large proportion of L-type Ca2+ channels not found in either the M3T or M3C fraction certainly allows for the possibility that caveolae are not the only sites on the PM for L-type Ca2+ channels. Although there may be an enrichment in caveolae consistent with the enrichment seen in Western blots (Fig. 1), it is more likely that L-type Ca2+ channels are located throughout the PM.

The lack of enrichment of the PM Ca2+ pump in Western blots was surprising considering that Fujimoto (14) demonstrated an enrichment of the PM Ca2+ pump in mouse ileum caveolae by immunoelectron microscopy. However, there have been no other reports of an enrichment of the PM Ca2+ pump in caveolae isolated from smooth muscle by biochemical methods. The failure of experiments to immunoprecipitate the PM Ca2+ pump with caveolin, despite the presence of a caveolin binding domain, is consistent with a lack of enrichment of the pump in caveolin-enriched membranes. It is likely that the PM Ca2+ pump is distributed more evenly throughout the PM.

The absence of an Ins(1,4,5)P3 receptor or an Ins(1,4,5)P3 receptor-like protein is also inconsistent with the results of Fujimoto et al. (17), although it is consistent with our model of Ca2+ handling in airway smooth muscle. It should be pointed out that our experiments investigated the presence of the type 1 Ins(1,4,5)P3 receptor. A more recent study (10) suggested that the type 3 Ins(1,4,5)P3 receptor is the one localized to the PM and, possibly, caveolae (10). Alternatively, the Ins(1,4,5)P3 receptor localized to the PM may not be a functional Ca2+ channel but may play a structural role as suggested by Fujimoto et al. (16).

The presence of calsequestrin and calreticulin in the detergent-resistant membrane fraction and their coimmunoprecipitation with caveolin was surprising. Although these proteins are generally agreed to be localized to the lumen of the SR, there is some evidence that they may be located in other regions of the cell or beyond. Calreticulin has been detected in circulating plasma at low levels (46). It has been identified on the surface of a cultured mouse melanoma cell line (51) and on the surface of cultured fibroblasts (20), and it binds to the extracellular surface of endothelial cells in vitro and in vivo (27). In addition to its extracellular localization, calreticulin has been localized to other (non-SR) intracellular locations such as the nuclear envelope and cytoplasm (reviewed in Refs. 25, 37).

Biochemical studies (33, 40, 49, 50) in smooth muscle that localized calsequestrin and calreticulin to SR have studied isolated SR membranes and have not investigated whether they are also localized on PM-derived membranes. Immunoelectron microscopy of smooth muscle localized both calsequestrin and calreticulin to the SR (49). However, some micrographs showed gold particles attributed to peripheral SR located on circular structures just below the PM. These structures could be caveolae in sections where the plane of the section missed the necks of the caveolae. Similar structures have been seen in another electron microscopy study of smooth muscle (11) and have been shown to be connected to the extracellular space by the use of extracellular tracers and identified as caveolae. Furthermore, a study by Moore et al. (36) investigating the distribution of the Na+/Ca2+ exchanger Na+-K+- ATPase and calsequestrin in toad stomach smooth muscle cells by immunocytochemical methods colocalized all three proteins to discrete sites at or near the PM. Although the authors concluded that these sites were consistent with localization to caveolae (for the Na+/Ca2+ exchanger and Na+-K+-ATPase) and closely associated peripheral SR (for calsequestrin), an alternate interpretation is that all three proteins were localized to caveolae. Our own immunocytochemistry studies lacked the resolution to resolve the locations of these Ca2+ binding proteins but did allow for the possibility that each was associated, in part, with the PM and caveolae.

The coimmunoprecipitation of calsequestrin and calreticulin with caveolin strongly suggests that a fraction of each of these proteins is located on the cytoplasmic surface of caveolae. Analysis of the available protein sequences for calsequestrin (45) and calreticulin (41) indicates that both have a sequence that corresponds to the caveolin binding domain. The experimental methods used allowed only a qualitative analysis, and so we were unable to compare the relative amounts of calsequestrin or calreticulin associated with detergent-resistant membranes or immunoprecipitated caveolin. Thus it is unknown if all of the calsequestrin and calreticulin associated with caveolae was located on the cytoplasmic surface or if some was also located on the extracellular or luminal surface. Furthermore, no experiments were done to test if calsequestrin or calreticulin was located on the extracellular surface of the cell.

The presence of calsequestrin and calreticulin in the detergent-resistant membrane fraction and with the immunoprecipitated caveolin makes it tempting to speculate on the role of the two proteins on the cytoplasmic surface of caveolae. They may be involved in the interaction or association of caveolae with peripheral SR or may be one of the components of the electron-dense material that can occur between PM and junctional SR. However, it is also possible that the presence of calsequestrin and calreticulin is an artifact of the isolation procedures. The treatment with detergent (either Triton X-100 or CHAPS) may release calsequestrin and calreticulin from SR-derived membranes, allowing the two proteins to interact with caveolin. Further experiments that do not involve detergent treatment of isolated membranes are necessary. One such experiment is immunoelectron microscopy of smooth muscle cryosections, investigating the colocalization of caveolin and calsequestrin or calreticulin in caveolae.


    ACKNOWLEDGEMENTS

This work was supported by the Medical Research Council of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: E. E. Daniel, Dept. of Medicine, HSC 4N51, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: daniele{at}fhs.csu.mcmaster.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.

Received 3 September 1999; accepted in final form 20 June 2000.


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