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Am J Physiol Heart Circ Physiol 288: H2457-H2464, 2005. First published December 30, 2004; doi:10.1152/ajpheart.01090.2004
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Decline in caveolin-1 expression and scaffolding of G protein receptor kinase-2 with age in Fischer 344 aortic vascular smooth muscle

William E. Schutzer,1,2 John F. Reed,1 and Scott L. Mader1,2

1Portland Veterans Affairs Medical Center, Research Service, and 2Oregon Health and Science University, School of Medicine, Portland, Oregon

Submitted 2 October 2004 ; accepted in final form 16 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
{beta}-Adrenergic receptor ({beta}-AR)-mediated vasorelaxation declines with age in humans and animal models. This is not caused by changes in expression of {beta}-AR, G{alpha}s, adenylyl cyclase, or protein kinase A but is associated with decreased cAMP production. Expression and activity of G protein receptor kinase-2 (GRK-2), which phosphorylates and desensitizes the {beta}-AR, increases with age in rat aortic tissue. Caveolin scaffolds the {beta}-AR, GRK, and other proteins within "signaling pockets" and inhibits GRK activity when bound. We questioned the effect of age on caveolin-1 expression and interaction between caveolin-1 and GRK-2 in vascular smooth muscle (VSM) isolated from 2-, 6-, 12-, and 24-mo-old male Fischer 344 rat aorta. Western blot analysis found expression of caveolin-1 declined with age (6-, 12- and 24-mo-old rat aortas express 92, 50, and 42% of 2-mo-old rat aortas, respectively). Results from density-buoyancy analysis showed a lower percentage of GRK in caveolin-1-specific fractions with age (6-, 12- and 24-mo-old rat aortas express 95, 56, and 12% of 2-mo-old rat aortas, respectively). Coimmunoprecipitation confirmed this finding; density of GRK in caveolin-1 immunoprecipitates was 97, 30, and 21% of 2-mo-old aortas compared with 6-, 12- and 24-mo-old animals, respectively. Immunohistocytochemistry and confocal microscopy confirmed that GRK-2 and caveolin-1 colocalize in VSM. These results suggest that in nonoverexpressed, intact tissue, the decline in {beta}-AR-mediated vasorelaxation may be caused by both a reduction in caveolin-1 expression and a reduction in binding of GRK-2 by caveolin-1. This could lead to an increase in the fraction of free GRK-2, which could phosphorylate and desensitize the {beta}-AR.

aging; aorta; {beta}-adrenergic; blood vessel; caveolae; hypertension

CAVEOLAE ARE DISTINCT cholesterol- and glycosphingolipid-enriched plasma membrane vesicular structures (34). Classical understanding describes their function as mediators of transcytosis, potocytosis, and pinocytosis (18). Recently, caveolae have also been shown to anchor multiple signaling molecules. Therefore, caveolae can serve as a scaffolding platform and thus a mediator for intracellular signal transduction (1). For instance, {beta}-adrenergic receptors ({beta}-AR) (19, 31), G proteins (14, 15), adenylyl cyclase (23, 32, 36), and G protein-coupled receptor kinases (GRK) (3) along with other signaling factors (20) have all been localized within caveolae. The major protein constituent of caveolae is caveolin, a 22- to 24-kDa protein that has been identified in nearly all cell types. Four distinct molecular subtypes of caveolin have been identified to date: caveolin-1{alpha}, -1{beta}, -2, and -3 (1). Caveolin can also function as a negative regulator of many proteins; GRK-2-mediated phosphorylation, for example, is inhibited when bound to caveolin-1 or -3 (3).

Changes in caveolin could be related to signal transduction changes seen during aging. {beta}-AR-mediated vasorelaxation and related cAMP accumulation decline with advancing age (28), whereas adenylyl cyclase-mediated vasorelaxation remain intact in the mammalian vasculature (5). Low intracellular cAMP accumulation is associated with enhanced vascular smooth muscle proliferation, which may promote atherosclerosis (33). Also, declining {beta}-AR-mediated vasorelaxation with preserved {alpha}-AR-mediated vasoconstriction yields a chronic vasoconstricted state that can be associated with both hypertension and orthostatic hypotension (35). Therefore, these biochemical changes may manifest clinically as an age-related increase in the prevalence of hypertension, atherosclerosis, and orthostatic hypotension (38). Studies to understand the biochemical/molecular basis of these findings show little, if any, age-related change in abundance of the {beta}-AR (37), G proteins include G{alpha}s (12, 17, 30), the effector enzyme adenylyl cyclase (16), or protein kinase A (7). However, Gurdal et al. (10) found that with advancing age, the number of {beta}-AR in the high-affinity state declines. This finding suggests an age-related increase in {beta}-AR phosphorylation, a mechanism of its desensitization (24).

Changes in GRK, caveolin, or both could explain this age-related increase in {beta}-AR desensitization. Our studies, as well as others, have shown that expression of GRK-2 and -3 increase with advancing age (9, 29); however, there are only a few reports examining age-related changes in caveolin. Ostrom and Insel (22) suggest that a change in caveolin expression could alter cell function with aging, and Kawabe et al. (13) show variable changes in caveolin-1, -2, and -3 expression with aging and maturation in heart, lung, and skeletal muscle. To our knowledge, no studies have examined age-related changes in vascular smooth muscle, although, Bakircioglu et al. (2) found a decline in expression of caveolin-1 with advancing age in penile smooth muscle.

In the present study, we ask whether the age-related decline in {beta}-AR-mediated vasorelaxation could be partially explained by an age-related change in caveolin-1 expression and scaffolding. A decline in caveolin expression could yield intracellular changes that would allow a loss of {beta}-AR signaling due to a lack of scaffolding and thus a loss of appropriate protein-protein interactions. A decrease in caveolin expression could also allow for a decline in caveolin-mediated GRK inhibition, which would increase GRK activity and increase {beta}-AR desensitization over that already occurring by the age-related increase in GRK expression. The data presented focus on caveolin-1 and GRK-2 because: 1) our previous findings that GRK-2 expression increases with advancing age (29), 2) data by others show that caveolin-1 is the predominant isoform expressed in vascular smooth muscle (11), and 3) caveolin-1 forms a regulatory complex with GRK-2 (3). Results show that in intact, native aortic vascular smooth muscle of male Fischer 344 rats, caveolin-1 and GRK-2 colocalize and expression of both caveolin-1 and caveolin-1-mediated scaffolding of GRK-2 declines with advancing age.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and solutions. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Before some assays, antibodies were conjugated to various moieties as shown in Table 1. Total protein determinations were conducted with bicinchonic acid (BCA) (Pierce Biotechnology, Rockford, IL) analysis. Homogenization buffer (HB) contained 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 20 µg/ml leupeptin, 20 µg/ml benzamidine, and 40 µg/ml phenylmethylsulfonyl fluoride. TE buffer contained 25 mM Tris and 5 mM EDTA. Laemmli's loading buffer (LB) contained 62 mM Tris, 10% glycerol, 2% SDS, 5% {beta}-mercaptoethanol, 0.01% bromophenyl blue. In Western blot analysis, Tris-buffered saline plus Tween 20 (WB-TBS-T) contained 10 mM Tris, 150 mM NaCl, and 0.05% Tween 20. Immunoprecipitation (IP) buffer (IPB) contained HB with 0.5% Triton and 60 mM octyl glucoside detergents. IP wash buffer (IPWB) contained (in mM) 25 Tris, 300 NaCl, 1 EDTA, and 60 octyl glucoside detergents plus 0.5% Triton. Morpholinoethanesulfonic acid-buffered saline (MBS) contained (in mM) 250 sodium carbonate, 25 2-morpholinoethanesulfonic acid (pH 6.5), and 150 NaCl. Immunohistochemistry TBS (IHC-TBS) contained 20 mM Tris and 137 mM NaCl.


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  		Table 1. Antibodies utilized, manufacturer catalog numbers, indicated conjugations, and operating concentrations per assay

 
Animals and tissue preparation. Male Fischer 344 rats (2, 6, 12, and 24 mo old) obtained from the National Institute on Aging colony at Harlan Sprague Dawley (Indianapolis, IN) were killed by pentobarbital sedation and exsanguination in accordance with the procedures approved by the Institutional Animal Care and Use Committee at the Portland Veterans Affairs Medical Center. Thoracic aortas were collected and placed into ice-cold PBS (in mM: 2.7 KCl, 137 NaCl, 4.3 Na2HPO4, 1.4 KH2PO4). After aortas were split lengthwise and the endothelial layer was removed with gentle rubbing with a sterile cotton swab, the medial layer composed predominantly of vascular smooth muscle was collected, immediately frozen under liquid nitrogen, and stored at –80°C until use. For some assays, it was necessary to pool aortas from multiple animals for a single experiment to ensure adequate protein content.

Western blot analysis. Frozen tissues were pulverized and prepared with glass-glass motor-driven disruptors in HB with 1% Triton and 60 mM octyl glucoside detergents. Homogenates were centrifuged at 500 g for 15 min at 4°C, and a fraction of this was used for total protein content determination. Aliquots of each preparation had their protein content equalized (per age group) by dilution in TE buffer, and LB was added. Separation of proteins occurred with SDS-PAGE (12%), and electrotransfer to polyvinylidene difluoride membranes (Bio-Rad; Hercules, CA) followed. Membranes were blocked overnight at 4°C in WB-TBS-T with 5% nonfat dry milk, washed extensively with WB-TBS-T, incubated with primary antibodies A, D, or F (see Table 1) for 2 h at room temperature, washed again extensively in WB-TBS-T, and then exposed to secondary antibody G in TBS-T with 3% nonfat dry milk for 1 h. Localization of secondary antibody G occurred with 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate diammonium salt (Molecular Probes, Eugene, OR) and detection with a Molecular Dynamics Typhoon scanner (excitation at 633 nM, and emission at 670 nM). Membranes exposed to primary antibody B (see Table 1) were incubated for 2 h at room temperature and then washed extensively in WB-TBS-T; detection occurred with excitation at 488 nM and emission at 520 nM. All gels were loaded with 4 µg of a pooled aortic vascular smooth muscle extract that served as both an intra- and an interassay control. This extract was prepared as described above. Some membranes were stripped (Re-Blot; Chemicon, Temecula, CA) and reprobed with antibody F (Table 1) to verify equal loading of protein in each lane. Appropriate molecular mass for bands were verified with 5 µl of a 132- to 23-kDa blot-detectable ladder (Cruz Marker; Santa Cruz Biotechnology). Image analysis was performed with the ImageJ (v1.32; http://rsb.info.nih.gov/ij/) gel analysis algorithm. Further details are described in Analysis and statistics.

Coimmunoprecipitation. Frozen tissues were pulverized and prepared with glass-glass motor-driven disruptors in IPB. Protein from this homogenate (500 µg) was incubated with primary antibody C (25 µg; Table 1) while being gently rotated overnight at 4°C. This immunoprecipitate was collected with centrifugation at 3,000 g for 1 min at room temperature and washed three times with IPWB. After a second centrifugation, the immunoprecipitate was collected via processing with a Seize IP Kit (Pierce) per the manufacturer's instructions. The purified immunoprecipitate was concentrated to ~20 µl with Microcon YM10 spin columns (Millipore, Bedford, MA), combined with LB, and separated with SDS-PAGE. Western blot analysis and visualization occurred as described in Western blot analysis with primary antibodies B and D and secondary antibody G (Table 1).

Density-buoyancy analysis. Separation of preparations into caveolin-specific and nonspecific fractions was accomplished as described by Song et al. (34). Briefly, aortic medial layers were homogenized in 500 mM sodium carbonate (pH 11) with glass-glass motor-driven disruptors, sonicated three-times for 20 s each on ice, incubated on ice for 1 h, and centrifuged at 500 g for 15 min at 4°C. A fraction of the supernatant from this preparation was used for total protein concentration determination. An equal (age-to-age) concentration of protein extracts was combined in an ultracentrifuge tube with an equal volume of 90% sucrose in 2x MBS, yielding a 45% sucrose MBS solution. A discontinuous step gradient was then formed by layering 35 and 5% sucrose in MBS. After overnight centrifugation at 188,000 g at 4°C, eight 250-µl fractions were collected from the bottom of the tube. The pellet was also resuspended in 250 µl 5% sucrose MBS. Equal volumes of each fraction (and pellet) were diluted in LB and screened with SDS-PAGE and Western blot analysis for caveolin-1 (antibody B) and GRK-2 (antibodies D and G; Table 1) as described under Western blot analysis.

IHC. Paraformaldehyde-fixed aorta were sectioned (10 µm), placed on glass slides, deparaffinized in xylene, rehydrated through graded ethanols to water, and treated by steaming in 10% antigen retrieval solution (CITRA; BioGenex, San Ramon, CA). After protein was blocked with 0.3% BSA diluted in IHC-TBS for 1 h, sections were incubated overnight at 4°C with the indicated (Table 1) concentration of primary antibody (fluorescent-labeled antibodies kept in dark conditions). The slides were then washed extensively in IHC-TBS with 0.05% Tween 20 and localization occurred with diamminobenzine staining per manufacturer's instructions (Vectastain ABC-Elite system; Vector Labs, Burlingame, CA). Slides were counterstained with Harris modified hematoxylin and dehydrated through ascending ethanol solutions and xylene, and coverslips were applied with Cytoseal 60 (Richard-Allan Scientific; Kalamazoo, MI). Visualization occurred with a Zeiss Axioskop microscope connected to a Micropublisher 3.3 digital camera (QImaging; Burnaby, BC, Canada) interfaced with a Macintosh G5 computer (Apple, Cupertino, CA) utilizing QCapture software (QImaging). For two-color confocal microscopy, slides were washed extensively in TBS with 0.05% Tween 20, sealed with Prolong Antifade (Molecular Probes) and allowed to dry overnight in the dark. A Leica SP laser-scanning microscope was used for visualization with appropriate excitation and emission parameters. Images were generated by using the "blending layers" function in Photoshop 7.0 (Adobe; San Jose, CA). Control experiments (no primary antibody, normal rabbit serum, and primary antibody titer) were conducted to establish specificity and optimize signal and background.

Analysis and statistics. Western blot analysis was performed by using the gel analysis algorithm in ImageJ to determine the density of each band after background subtraction. Lane-to-lane (loading) normalization occurred in combination by loading equalized total protein content on the basis of BCA analysis as well as by reporting the ratio (for density) between caveolin-1 and actin. Blot-to-blot normalization occurred by determining the density for each blot's internal standard band and using this value to generate a multiplier for each blot; subsequently, the raw density for each band on each blot was adjusted accordingly. For coimmunoprecipitation (co-IP) experiments, the ratio between normalized density of caveolin-1 and GRK-2 bands was determined. For density-buoyancy experiments, normalized densities were pooled into four fractions: pellet, high density, caveolin-enriched, and low density; values are presented as percent per fraction. Statistical differences between age groups for caveolin-1 expression and caveolin-1/GRK co-IP were determined by one-way ANOVA. Statistical differences between age groups for caveolin-1 density-buoyancy (GRK localization) was determined by two-way ANOVA (age-by-fraction). In both cases, Bonferroni's post hoc analysis was used, and a value of P < 0.05 was considered significant. All analyses were performed with Prism 4.0 (GraphPad; San Diego, CA). Data are reported as means ± SE.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Caveolin-1 is expressed in a variety of tissues. Expression of caveolin-1 varies across tissue beds and cultured cells. Figure 1 shows a representative Western blot in which different tissues collected from 6-mo-old male Fischer 344 rats were probed for caveolin-1 expression. In addition, four cultured cell lines were also examined. Of note is the relatively high expression found in the cultured human endothelial cells, which agrees with a defined functions of caveolin-1 in endothelial cell-mediated vesicular transcytosis and signal transduction (18).



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  		Fig. 1. Expression of caveolin-1 (CAV-1) from multiple tissues and cultured cells. Protein extracts (1 µg per lane) were subjected to SDS-PAGE and immunoblotting with an anti-CAV-1 antibody (antibodies A and G; see Table 1). A representative immunoblot is shown from n = 3 independent trials. Analysis was performed by densitometry with a Typhoon fluroimager as described in MATERIALS AND METHODS.

 
Caveolin-1 expression declines with age in vascular smooth muscle. Caveolin is highly expressed in vascular smooth muscle and is known to provide signal transduction scaffolding. Therefore, changes in its expression could alter signal transduction pathways. Expression of caveolin-1 in vascular smooth muscle homogenates from 2-, 6-, 12-, and 24-mo-old animals was assessed by Western blot analysis (Fig. 2). Expression declined with advancing age. In 2-mo-old animals, optical density (normalized to expression of internal standard and actin; see MATERIALS AND METHODS) was 1.2 ± 0.13 arbitrary units (AU); this compared with 1.1 ± 0.1, 0.6 ± 0.08, and 0.5 ± 0.06 AU (means ± SE) for 6-, 12-, and 24-mo-old animals, respectively. When density of 2- and 6-mo-old animals was compared with density values of 12- and 24-mo-old animals, total caveolin expression declined ~1.9-fold.



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  		Fig. 2. CAV-1 expression in aortic vascular smooth muscle preparations from 2-, 6-, 12-, and 24-mo-old rats (n = 10 per age). A: protein extracts (10 µg per lane) were subjected to SDS-PAGE and immunoblotting with an anti-CAV-1 antibody (antibody B; see Table 1). Immunoblots were analyzed by densitometry with a Typhoon fluroimager as described in MATERIALS AND METHODS. B: CAV-1 expression is presented as the ratio of intensity of CAV-1 bands compared with intensity of actin bands (as determined with antibodies F and G; see Table 1) presented as means ± SE. *P < 0.05 vs. 2-mo-old. {dagger}P < 0.05 vs. 6-mo-old.

 
Compartmentalization of GRK within caveolae decline with age. Because GRK-2 is a primary kinase responsible for {beta}-AR desensitization and caveolin-1 negatively regulates GRK-2 activity, evaluating how GRK-2 and caveolin-1 interact is necessary to determine the role of caveolin-1 in age-related {beta}-AR desensitization. Aortic medial-layer strips were fractionated by using an established protocol that isolates caveolae-specific fractions predominantly made up of the protein caveolin with a detergent-free, sodium-carbonate extraction (4, 34). With the use of this scheme, a light-refractive band at the interface of the 35 and 5% sucrose gradients was identified. This area corresponded to fractions 4–5, in which over 95% of caveolin was recovered (see MATERIALS AND METHODS for details, and Fig. 3A). Although this method is not ideal for determining age-related changes in caveolin-1 itself, when all fractions (1–8 and pellet) are considered, expression of caveolin-1 declines with advancing age similar to that shown in Fig. 2. This method is ideal for allowing the determination of other proteins that are isolated within caveolae. Figure 3, B and C shows that the percentage of all detectable GRK-2 localized in caveolin-1-rich fractions significantly declines with advancing age. In caveolin-1-rich fractions, 35.2 ± 4.1% of all detectable GRK-2 was localized in vascular smooth muscle extracts from 2-mo-old animals, whereas 33.6 ± 3.5, 19.8 ± 1.5, and 4.1 ± 2% of all detectable GRK-2 were localized in caveolin-1-rich fractions from vascular smooth muscle extracts from 6-, 12-, and 24-mo-old animals, respectively. GRK-2 was predominantly localized in high-density fractions instead of in caveolin-1-rich fractions with advancing age (55.2 ± 6.5, 58.1 ± 5.6, 72.4 ± 6.6, and 86.7 ± 5.5% for extracts from 2-, 6-, 12-, and 24-mo-old animals, respectively).



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  		Fig. 3. GRK-2 expression in CAV-1-specific fractions from vascular smooth muscle preparations from 2-, 6-, 12-, and 24-mo-old rats (n = 10 per age). Eight fractions (plus a pellet) were prepared, and equal volumes were analyzed with Western blot analysis as described in MATERIALS AND METHODS. A: representative CAV-1 blots utilizing antibody B (Table 1). B: same membranes as in A when exposed to G protein receptor kinase (GRK)-2 analysis (antibodies D and G). Bands from the blots were pooled into pellet (P), high-density (High Den), and low-density (Low Den) regions on the basis of their relationship to the CAV-1-specific region as shown. C: percentage of GRK-2 expression in each pooled fraction as a function of age is shown. Values are presented as means ± SE. *P < 0.05 vs. 2-mo-old rats. {dagger}P < 0.05 vs. 6-mo-old rats, {ddagger}P < 0.05 vs. 12-mo-old rats. Normalization occurred with the internal standard (IS) as defined in MATERIALS AND METHODS.

 
Interaction between caveolin-1 and GRK decline with age. Co-IP can be used to directly detect the interaction between proteins of interest. Fischer 344 aortic medial layer homogenates were incubated with a caveolin-specific antibody for IP. Western blot analysis with a GRK-2 antibody was then used to detect whether GRK-2 was found in caveolin IPs (Fig. 4). Caveolin is known to be relatively insoluble to most biochemical detergents, and thus co-IP is problematic. However, Carman et al. (3) found that the addition of 60 mM octylglucoside allows for accurate co-IP between caveolin-1 and GRK-2. With the use of this buffer, plus high stringency washes, the efficiency of the IP was ~60%. This value was determined by evaluating the density of the caveolin-1 band from Western blots of the IP supernatant versus the IP pellet (data not shown). Efficiency of IP increased in preparations from older age groups, which is consistent with the data shown in Fig. 2, in which total expression of caveolin-1 declined with advancing age. The value for the GRK-2, caveolin-1 ratio for extracts prepared from aortic vascular smooth muscle from 2- and 6-mo-old animals was 0.33 ± 0.06 AU and 0.32 ± 0.05 AU, respectively. This compared with a significant decrease for extracts from 12-mo-old animals (0.17 ± 0.03 AU) and a further significant decrease for extracts from 24-mo-old animals (0.07 ± 0.03 AU). These results demonstrate that GRK-2 is bound to caveolin in aortic medial layer homogenates. In addition, results substantiate the finding of the density-buoyancy assay (Fig. 3) and validate that with advancing age, the amount of GRK-2 bound to caveolin significantly declines.



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  		Fig. 4. GRK-2 expression in CAV-1 immunoprecipitates from vascular smooth muscle preparations from 2-, 6-, 12-, and 24-mo-old rats (n = 10 per age). Representative images are shown from n = 4 experiments per age. CAV-1 immunoprecipitates (IP) were prepared with antibody C and collected with a Seize IP Kit (Pierce). Western blot analysis (WB) was performed with antibody B for CAV-1 and antibodies D and G for GRK-2 as described in MATERIALS AND METHODS and shown in A (see also Table 1). Analysis was performed by determining the density ratio between GRK-2 and CAV-1 as shown in B. Values are presented as means ± SE. *P < 0.05 vs. 2-mo-old rats. {dagger}P < 0.05 vs. 6-mo-old rats, {ddagger}P < 0.05 vs. 12-mo-old rats. Normalization occurred with the extract (Ex) as defined in MATERIALS AND METHODS. A normal rabbit IgG primary antibody (NI; cat. no. sc-2027; Santa Cruz Biotechnology) conjugated in the same fashion as antibody C was used as a negative IP control.

 
Caveolin-1 and GRK-2 colocalize in vascular smooth muscle. To further demonstrate that caveolin-1 and GRK-2 localize within aortic vascular smooth muscle, a series of IHC experiments were performed (Fig. 5). Confocal microscopy revealed that both caveolin-1 (green) and GRK-2 (red) are highly expressed in both vascular smooth muscle and endothelial cells of aorta from rats of each age. In addition, there is a high degree of colocalization between these proteins in the smooth muscle layer (yellow); colocalization was reduced in the endothelial cell layer, in which caveolin-1 appears to predominate. IHC with diamminobenzine staining validate the findings from confocal microscopy. Caveolin-1 and GRK-2 are expressed in the aortic vascular smooth muscle and endothelial cell layers of all ages. For both confocal and diamminobenzine analysis, control experiments (no primary antibody or incubation with a normal rabbit IgG primary antibody) yielded little or no signal (data not shown).



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  		Fig. 5. CAV-1 and GRK-2 colocalization determination by immunohistochemistry in aortic sections from 2-, 6-, 12-, and 24-mo-old rats (n = 4 per age). Slides were prepared containing an aortic section from each age. For confocal analysis, slides were incubated with antibodies B and E, concomitantly. Visualization was performed as described in MATERIALS AND METHODS with CAV-1 expressing green and GRK-2 expressing red. The overlay is shown in yellow. For diamminobenzine analysis, slides were incubated in either antibody A or D, independently, and visualization occurred as described in MATERIALS AND METHODS. Both endothelial (E) and vascular smooth muscle cell (VSM) layers express CAV-1 and GRK-2.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study examines age-related changes in caveolin-1 expression and the interaction between caveolin-1 and GRK-2 in intact, native vascular smooth muscle isolated from aorta of 2-, 6-, 12-, and 24-mo-old male Fisher 344 rats. Caveolin-1 is a protein known to provide a scaffolding platform for the {beta}-AR signaling cascade (31), as well as negatively regulate numerous proteins, including GRK-2 (3). These studies were undertaken to provide an explanation for the observation that {beta}-AR-mediated vasorelaxation declines with advancing age, whereas adenylyl cyclase-mediated vasorelaxation is unchanged (5). Results presented show that expression of caveolin-1 is variable across tissue beds in Fischer 344 rats (Fig. 1) and that caveolin-1 expression declines with advancing age (Figs. 2 and 3A). Also, caveolin-1 and GRK-2 colocalize in aortic vascular smooth muscle (Figs. 3– 5), and there appears to be an age-associated decline in the interaction between caveolin-1 and GRK-2 (Figs. 3 and 4).

Aging is associated with a pronounced decline in {beta}-AR-stimulated cAMP production and subsequent function, whereas adenylyl cyclase-mediated cAMP production is unchanged (5). Generally, this age-related decrease is conserved across tissue beds; {beta}-AR responsiveness to agonist in blood vessels, heart, brain, parotid gland, and lung all decline with advancing age (28). A major finding in vasculature that explains this impairment is that there is an age-related increase in the quantity of {beta}-AR found in the desensitized state (10). Further support that {beta}-AR desensitization may be a primary factor, rather than a change in other signaling molecules, is that neither G{alpha}s protein (17) nor mRNA (12) expression changes with advancing age, and G{alpha}s overexpression only slightly enhances {beta}-AR-mediated vasorelaxation in aorta (30). Also supportive of a primary role for {beta}-AR desensitization is that neither adenylyl cyclase expression nor protein kinase A activity or expression change with age (7). One protein family that has been shown to exhibit age-related increased expression and activity in vascular smooth muscle are GRKs that phosphorylate {beta}-ARs and thus cause their desensitization (24). We have shown that expression of GRK-2 and GRK-3, but not GRK-5, increase with advancing age, and there is an age-related increase in total GRK activity (29).

We propose that the age-related impairment in {beta}-AR signaling results, in part, from changes in caveolin-1 function. Rybin et al. (27) determined the {beta}-AR cascade is localized and regulated in caveolae, and Carman et al. (3) demonstrated caveolin-1 binds GRK-2, which inhibits its kinase activity. Therefore, caveolin-1 can function as a dual-purpose protein; it can act as a scaffolding protein maintaining the {beta}-AR cascade signaling pocket, and it can also act as a negative regulator of certain kinases, including GRK-2. Both of these actions of caveolin-1 would enhance {beta}-AR signaling. Both Figs. 2 and 3A document an age-related decline in caveolin-1 expression. The decline shown in Fig. 2 provides quantitation for the entire vascular preparation. The decline in Fig. 3A is similar to that of Fig. 2 when all fractions are considered. This finding in itself suggests that age-related changes in caveolin-1 could be implicated in affecting {beta}-AR signaling. A reduction in caveolin-1 expression would cause a decrease in caveolae (8) and thus could yield ineffective {beta}-AR signaling pockets (27). Therefore, {beta}-AR-mediated vasorelaxation could be impaired with advancing age, regardless of the expression of {beta}-ARs and other proteins in the cascade.

A second question posed by this study is whether there is a change in the interaction between caveolin-1 and GRK-2. Caveoiln-1 is highly expressed in the vascular smooth muscle layer (Fig. 1), and caveolin-1 and GRK-2 colocalize in the vascular smooth muscle (Fig. 5). However, it has not been determined whether there is an age effect on this interaction. De Luca et al. (6) demonstrated that {beta}-AR signaling components (G{alpha}s and others) shifted into caveolin-rich fractions after stimulation of cardiac tissue with norepinephrine and interpreted this shift as a treatment-mediated change in caveolin-1-mediated scaffolding. In this study, we used similar experimental techniques (Fig. 3) and found that another {beta}-AR signaling component GRK-2 is distributed differentially between caveolin-1-specific and nonspecific fractions with advancing age. A greater percentage of GRK-2 is localized within caveolin-1-rich fractions in preparations isolated from younger animals. Therefore, we too suggest that advancing age could affect the interaction between caveolin and GRK-2. Thus the age-related changes in the interaction between caveolin-1 and GRK-2 may also be important in regulation of signaling.

Decline of GRK-2 in caveolin-1-specific fractions (Fig. 3, B and C) could be explained by the fact that there is simply less caveolin expressed with advancing age, and therefore less GRK-2 should be found in this fraction. However, co-IP analysis shows that when the concentration of caveolin-1 is held constant across age groups (Fig. 4A, bottom), GRK-2 binding declines. The age-related decline in caveolin-1 expression could disturb the scaffolding of the {beta}-AR cascade, and second, the loss of scaffolding of GRK-2 to caveolin-1 could enhance GRK-2-mediated desensitization of the {beta}-AR with age. Also, GRK-2-mediated {beta}-AR desensitization could be further exasperated due to the age-related increase in expression of GRK-2 (9, 29). Together, these changes could cause a significant reduction in {beta}-AR-mediated vasorelaxation.

Caveolin is a regulator of vascular function. Caveolin-1 null mice have been produced by two independent groups (8, 25). These animals are viable but exhibited multiple abnormalities. For instance, the steady-state maximal tension induced by phenylephrine (an {alpha}-AR agonist) was significantly impaired. Also, these animals display hyperproliferation in certain cell types, suggesting (but not documented) a decline in cAMP production. Neither group investigated any changes in {beta}-AR-, cAMP-, or age-related effects in the null mice. However, the data clearly indicate that caveolin is an important modulator of vascular function. In agreement, Je et al. (11) demonstrated that caveolin-1 is a critical coordinator of signaling for regulation of contractility in vascular smooth muscle. Of further interest is the temporal relationship among age-related changes in {beta}-AR-mediated vasorelaxation, caveolin-1 expression, and GRK expression. Age-related changes in caveolin-1-, GRK-2 (data presented herein and in Ref. 29)-, and {beta}-AR-stimulated vasorelaxation (5) are similar; they are greatest in preparations from 12- and 24-mo-old animals.

Potential shortcomings of the present data are that the buoyancy-density studies (Fig. 3) rely on biochemical isolation of nonspecific cellular fractions and thus may not isolate caveolae alone; such fractions may also contain other microdomains of the cell, such as lipid rafts (21). Western blot analysis did verify significant enrichment of caveolin-1 in specific fractions. Nearly 95% of all expressed caveolin-1 was localized within fractions 4 and 5 (see Fig. 3). Also, co-IP studies are presented to help support these findings (Fig. 4). However, the solubility of caveolin-1 in our IP buffer (that contained multiple detergents) was not complete, and therefore these data (Fig. 4A) represent ~60% of the total cellular content of caveolin-1. In addition, electron microscopy analysis was not conducted that would verify presence of and potentially further quantitate age-related changes in caveolae. However, Wiener et al. (39) found caveolae in rat thoracic aorta with electron microscopy. Future studies could use antibodies combined with electron microscopy to validate the present results. Another shortcoming of the present data is related to its limited scope. We chose to focus only on two proteins, caveolin-1 and GRK-2, primarily related to the reasons outlined above; expression of GRK-2 in the vasculature increases with advancing age, caveolin-1 is the predominantly expressed caveolin isoform in vascular smooth muscle, and caveolin-1 and GRK-2 are known to interact. Determining the age-related changes caveolin-2 and -3, as well as examining the effect of aging on the recently identified dimerization of caveolin isoforms (26), deserves further study.

Our data are the first to examine changes in caveolin-1 across age groups in vascular smooth muscle. In addition, we used intact tissue that was not overexpressed or manipulated in any fashion. Therefore, these data depict changes that we propose occur in the steady state and have come about specifically due to aging. The decline in caveolin-1 expression with advancing age could allow for two potential biochemical affects; both could be additive in terms of affecting the {beta}-AR cascade. First, decreasing caveolin-1 allows for less effective signaling pockets, thus impairing {beta}-AR-mediated signaling. Second, decreasing caveolin-1 alters the stoichiometry between caveolin-1 and GRK-2. In summary, these changes, along with the previous finding that GRK-2 expression and activity increases with advancing age, could provide a mechanism to explain the age-related decline in {beta}-AR-mediated vasorelaxation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Research Service, Department of Veterans Affairs and the Northwest Health Foundation, Portland, OR (to S. L. Mader).


   ACKNOWLEDGMENTS
 
The authors thank the laboratory of Dr. Sharon Anderson (Oregon Health & Science University, Division of Nephrology and Hypertension, Portland OR), including Dr. Radko Komers and Terry T. Oyama and Jessie Lindsley, for technical expertise and editorial comments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. L. Mader, Portland Veterans Affairs Medical Center, Research Service—R&D 26, 3710 SW US Veterans Hospital Rd., Portland, OR 97201 (E-mail: scott.mader{at}med.va.gov)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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