Downregulation of caveolin by chronic beta -adrenergic receptor stimulation in mice

Naoki Oka, Kuniya Asai, Raymond K. Kudej, John G. Edwards, Yoshiyuki Toya, Carsten Schwencke, Dorothy E. Vatner, Stephen F. Vatner, and Yoshihiro Ishikawa

Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115; and Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Caveolae, flask-shaped invaginations of cell membranes, are believed to play pivotal roles in transmembrane transportation of molecules and cellular signaling. Caveolin, a structural component of caveolae, interacts directly with G proteins and regulates their function. We investigated the effect of chronic beta -adrenergic receptor stimulation on the expression of caveolin subtypes in mouse hearts by immunoblotting and Northern blotting. Caveolin-1 and -3 were abundantly expressed in the heart and skeletal muscles, but not in the brain. Continuous (-)-isoproterenol, but not (+)-isoproterenol, infusion via osmotic minipump (30 µg · g-1 · day-1) for 13 days significantly downregulated both caveolin subtypes in the heart. The expression of caveolin-1 was reduced by 48 ± 6.1% and that of caveolin-3 by 28 ± 4.0% (P < 0.01, n = 8 for each). The subcellular distribution of caveolin subtypes in ventricular myocardium was not altered as determined by sucrose gradient fractionation. In contrast, the expression of both caveolin subtypes in skeletal muscles was not significantly changed. Our data suggest that the expression of caveolin subtypes is regulated by beta -adrenergic receptor stimulation in the heart.

caveolae; G protein; isoproterenol; ventricular myocardium

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

CAVEOLAE ARE FLASK-SHAPED, non-clathrin-coated invaginations located at the cell surface of most cell types (1, 32). Caveolae are thought to play pivotal roles in transcytosis, potocytosis, and transmembrane signaling, as evidenced by numerous studies (for review see Refs. 2 and 16). Caveolin, a 20- to 25-kDa integral membrane protein, is a structural component of caveolae (15). Recent molecular cloning studies have elucidated the presence of multiple caveolin subtypes (caveolin-1alpha , -1beta , -2, and -3) (6, 24, 25, 29): caveolin-1 and -2 are mainly localized in endothelial cells, adipocytes, smooth muscle cells, and fibroblasts; caveolin-3 is found specifically in muscle tissues. A caveolin subtype within cells, however, may be switched to another with cell differentiation (29). The content of caveolin may be decreased in oncogenically transformed cells (11) or increased with aging and after high-fat feeding (19), suggesting that the expression of caveolin subtypes is subject to changes in cellular environments. Recent data indicate that caveolae are involved in beta -adrenergic receptor/Gsalpha /adenylyl cyclase signaling (15). Ultrastructural immunolocalization studies demonstrated the internalization of beta -adrenergic receptors via non-clathrin-coated invaginations and vesicles (22), which were later identified as caveolae (4). Other G protein-coupled receptors such as muscarinic receptors, various G protein alpha - and beta gamma -subunits, and G protein-modifying bacterial toxins (cholera and tetanus toxin) are enriched in caveolae (3, 13, 18, 23, 27, 28). A histocytochemical study demonstrated elevated adenylyl cyclase activity in endothelial caveolae (31). Thus it has been proposed that caveolae represent a membrane site for concentrating these signaling molecules and regulating their function.

Chronic isoproterenol infusion to experimental animals is a well-established method to induce changes in the beta -adrenergic receptor/Gsalpha /adenylyl cyclase-signaling pathway (for review see Ref. 7). Chronic isoproterenol infusion to mice induced significant downregulation of the beta -adrenergic-adenylyl cyclase system, an increase in left ventricular-to-body weight ratio, and cardiac remodeling (12). In the present study we examined whether the content of each caveolin subtype changes in the heart after chronic isoproterenol infusion.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Isoproterenol infusion. Before the experimental protocol began, 25 female and 25 male CD-1 mice of either sex (Charles River Laboratories, Wilmington, MA) were acclimated for 1 wk. The mice were anesthetized at 50 days of age with an intraperitoneal injection of ketamine (0.0065 mg/g), xylazine (0.013 mg/g), and acepromazine (0.002 mg/g), and a miniosmotic pump (model 2002, Alza, Palo Alto, CA) was implanted subcutaneously via a small intrascapular incision. The miniosmotic pump for isoproterenol-infused mice contained (-)- or (+)-isoproterenol bitartrate (Sigma Chemical, St. Louis, MO) in 0.9% NaCl at a concentration calculated to deliver an average of 30 µg · g-1 · day-1 during the infusion period. Control mice were implanted with pumps filled with 0.9% NaCl.

Tissue preparation. After 13 days of miniosmotic pump infusion, the mice were anesthetized with pentobarbital sodium (70 µg/g ip). Hearts were rapidly excised and rinsed in iced saline. The atria were removed, and the ventricles were frozen rapidly in liquid nitrogen and stored at -70°C until use. Femoral skeletal muscles and the whole brain were also excised and frozen in the same manner.

Frozen samples were homogenized in a buffer containing (in mM) 50 tris(hydroxymethyl)aminomethane · HCl (pH 8.0), 1 EDTA, 1 dithiothreitol, 200 sucrose, and 1 phenylmethylsulfonyl fluoride with a Polytron (Brinkmann Instruments, Westbury, KY) for 30 s. The homogenate was centrifuged at 500 g for 10 min. The supernatant was further centrifuged at 100,000 g for 40 min, and the pellet was resuspended in the homogenization buffer and used as the membrane fraction. The resultant supernatant was used as the cytosolic fraction. A ground glass homogenizer was used to evenly disperse membranes. Protein concentration was measured with a protein assay system (Bio-Rad, Hercules, CA). Both fractions were stored at -70°C until use.

Immunoblotting. Immunoblotting was performed using monoclonal antibodies to caveolin-1 and -3 (Transduction Laboratories, Lexington, KY). Membrane and cytosolic proteins (30 µg) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (12%) and transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore, Bedford, MA). Nonspecific binding sites on the membrane were blocked by incubation with 5% nonfat dry milk and 5% bovine serum albumin in 1× phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 h at room temperature. After three 5-min washes with 1× PBS containing Tween 20, immunoblotting was performed with a 1:2,000 dilution of antibody to caveolin-1 or -3. Bound caveolin antibodies were detected by a 1:2,000 dilution of anti-mouse immunoglobulin G conjugated to horseradish peroxidase in the enhanced chemiluminescence (ECL) detection system (Amersham, Arlington Heights, IL). Caveolin protein expression was quantitated with use of a computer densitometer (Molecular Dynamics, Sunnyvale, CA).

Northern blotting. Total RNA was extracted from tissues using RNAzol (Biotecx Laboratories, Houston, TX). Ten micrograms of total RNA were employed for Northern blotting. The blot was prehybridized in a solution containing 50% formamide, 5× saline-sodium citrate, 5× Denhardt's solution, 25 mM NaPO4 (pH 6.5), 0.25 mg/ml calf thymus DNA, and 0.1% SDS at 42°C for 2 h before the addition of probe. Hybridization was performed at 42°C for 18 h, and then the blot was washed under increasingly stringent conditions. A 0.5-kb fragment from canine caveolin-1 or rat caveolin-3 cDNA clone (kindly provided by Dr. Michael P. Lisanti, Albert Einstein Medical College), which contained the entire coding sequence, was used as probe. The probes were radiolabeled by the multiprimer random labeling method using [32P]dCTP. Each mRNA expression of caveolin was quantitated using a densitometer (Molecular Dynamics, Sunnyvale, CA). Glyceraldehyde 3-phosphate dehydrogenase was used to standardize each loading.

Cell fractionation. Caveolin-rich fractions were purified from mouse ventricular myocardium by a previously described method with minor modifications (27, 28). Ventricular myocardium was homogenized with a Polytron for 50 s in 500 mmol/l sodium carbonate, pH 11.0, and sonicated for 120 s. The sucrose concentration in tissue extracts was adjusted to 45% by the addition of 1.7 ml of 90% sucrose prepared in MBS [25 mmol/l 2-(N-morpholino)ethanesulfonic acid, pH 6.5, 150 mmol/l NaCl], and the extracts were placed at the bottom of an ultracentrifuge tube. A 5-35% discontinuous sucrose gradient was formed above the extract (4 ml of 35% sucrose-4 ml of 5% sucrose, both prepared in MBS containing 250 mmol/l sodium carbonate) and centrifuged at 39,000 revolutions/min for 16 h at 4°C in a Sorvall TH 641 rotor. A light-scattering band was confined to the 5-35% sucrose interface. From the top of each gradient, a total of 12 fractions (1 ml each) were collected and analyzed by a 12% SDS-polyacrylamide gel and subjected to immunoblotting.

Na-K-ATPase assay. Na-K-ATPase assay of the cardiac membrane was performed according to the method of Jones and Besch (9). Briefly, crude membranes (20 µg) were pretreated with SDS and added to a solution (900 µl) containing 50 mM histidine, 3 mM MgCl2, 111 mM NaCl, 11 mM KCl, 3.3 mM phosphoenolpyruvate, 75 µg pyruvate kinase, 11 mM NaN3, and 1.1 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid with or without 1.11 mM ouabain. The reaction was started by addition of 100 µl of 30 mM ATP, and then membranes were incubated at 37°C for 30 min. Reactions were terminated by the addition of 2.5 ml of stop solution containing 30 g/l sodium bisulfate, 10 g/l p-methylaminophenol sulfate, 25 g/l molybdic acid, and 0.14× H2SO4. After 15 min at room temperature, absorbance at 660 nm was determined.

beta -Adrenergic binding study. beta -Adrenergic antagonist binding studies were performed using eight concentrations of 25 µl of 125I-cyanopindolol ranging from 0.02 to 1.0 nmol/l, 25 µl of isoproterenol (0.1 mmol/l), or buffer and 100 µl of the membranes (10 µg/assay). Assays were incubated at 37°C for 30 min, terminated by rapid filtration on GF/C filters (Whatman Laboratory Products, Clifton, NJ), and counted in a gamma counter (TM Analytic, Elkgrove Village, IL) for 1 min.

Data analysis and statistics. Values are means ± SE. Data from control and isoproterenol-infused animals were compared with Student's t-test. P < 0.05 was considered indicative of a significant difference.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of caveolin subtypes in mouse tissues. As previously reported (28, 29), caveolin-1 and -3 were detected in the membrane fraction of skeletal muscle and ventricular myocardium (Fig. 1). Caveolin-1 and -3 migrated as a single band of 22-25 and 20-22 kDa, respectively. Neither subtype was detected in the cytosol fractions of skeletal muscles and ventricular myocardium (data not shown). No caveolin was detectable in the brain.


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Fig. 1.   Caveolin subtypes in mouse tissues. Membrane proteins (30 µg) prepared from each tissue (skeletal muscle, ventricular myocardium, and brain) were separated by a 12% SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with antibodies to caveolin-1 (Cav-1, A) or caveolin-3 (Cav-3, B). Molecular markers (in kDa) are shown on left.

Expression of caveolin protein in (-)-isoproterenol-infused mice. In the next experiment we investigated changes in the caveolin expression in the hearts of (-)-isoproterenol-infused mice (Fig. 2). Although the expression of both subtypes in the membrane fraction was significantly decreased in these mice, the degree of decrease was greater (P < 0.05, paired Student's t-test) for caveolin-1 (decreased by 48 ± 6.1%, P < 0.01, n = 8) than for caveolin-3 (decreased by 28 ± 4.0%, P < 0.01, n = 8). No caveolin was detectable in the cytosol fraction of ventricular myocardium of isoproterenol-infused mice (data not shown).


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Fig. 2.   Caveolin protein expression in hearts of (-)-isoproterenol-infused and control mice. A: membrane proteins (30 µg) prepared from (-)-isoproterenol-infused or control hearts were separated by a 12% SDS-PAGE and analyzed by immunoblot with antibody to caveolin-1 (a) or caveolin-3 (b). Molecular markers are shown on left. Results from 8 control (1-8) and 8 (-)-isoproterenol-infused mice (9-16) are shown. A representative immunoblot from four experiments is shown. B: caveolin subtype expression in A quantified by a densitometer. Open bars, control mice; solid bars, (-)-isoproterenol-infused mice. Values are means ± SE (n = 8 for each). * P < 0.01 compared with control.

We also measured Na-K-ATPase activity of the membrane to ensure that the amount of the membrane protein was similar between the two groups. In contrast to the changes in caveolin expression, Na-K-ATPase activity was not significantly different in ventricular myocardium of (-)-isoproterenol-infused mice: 2.57 ± 0.24 and 2.43 ± 0.27 µmol Pi · h-1 · mg protein-1 in control and (-)-isoproterenol-treated mice, respectively (n = 8).

Infusion of (+)-isoproterenol. To address the beta -adrenergic receptor specificity of the above findings, we also examined changes in caveolin expression in hearts of mice that were infused with (+)-isoproterenol instead of (-)-isoproterenol. In contrast to the trophic changes observed in (-)-isoproterenol-infused mice, body weight [34.9 ± 2.1 and 36.4 ± 1.7 (SE) g in control and (+)-isoproterenol-treated mice, respectively], heart weight [140.9 ± 5.3 and 155.7 ± 9.2 mg in control and (+)-isoproterenol-treated mice, respectively], and heart weight-to-body weight ratio [4.1 ± 0.2 and 4.3 ± 0.1 in control and (+)-isoproterenol-treated mice, respectively] were not significantly increased in (+)-isoproterenol-infused mice (P not significant, n = 8 for each). Similarly, the expression of both caveolin subtypes was not changed [caveolin-1: 1,365 ± 158 and 1,341 ± 102 arbitrary units in control and (+)-isoproterenol-treated mice, respectively; caveolin-3: 1,397 ± 63 and 1,458 ± 56 arbitrary units in control and (+)-isoproterenol-treated mice, respectively; P not significant, n = 6 for each]. Na-K-ATPase activity was not changed: 2.97 ± 0.59 and 2.82 ± 0.47 µmol Pi · h-1 · mg protein-1 in control and (+)-isoproterenol-treated mice, respectively. Therefore, neither changes in the expression of caveolin subtypes nor trophic changes were induced when (+)-isoproterenol was infused, confirming the specificity of the beta -adrenergic receptor stimulation to reduce the caveolin expression in mice.

Expression of caveolin mRNA levels in (-)-isoproterenol-infused mice. To support changes in the protein content of caveolin subtypes, we performed Northern blotting to measure the mRNA expression levels of caveolin-1 and -3 (Fig. 3). The mRNA levels of caveolin-1 as standardized by glyceraldehyde 3-phosphate dehydrogenase showed a 31% decrease in (-)-isoproterenol-infused mice: 30.8 ± 7.6% decrease from control (n = 7 for each, P < 0.05). Caveolin-3 mRNA levels showed a tendency to decrease; however, they did not reach a statistically significant level: 21.7 ± 4.0% decrease from control (n = 7 for each, P not significant).


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Fig. 3.   Caveolin mRNA expression in hearts of (-)-isoproterenol-infused and control mice. Total RNA (10 µg) prepared from (-)-isoproterenol-infused (solid bars) or control (open bars) hearts were analyzed by Northern blot. Caveolin-1 or -3 mRNA levels were determined using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading standard. Values are means ± SE (n = 7 for each). * P < 0.05 compared with control.

Subcellular localization of caveolin in the heart. We also examined whether subcellular localization of caveolin subtypes in ventricular myocardium is altered after isoproterenol infusion using the nondetergent sucrose gradient method (27, 28) (Fig. 4). Both caveolin isoforms were separated from most cellular proteins (fractions 9-12) and confined to fraction 5, as previously described in other tissues and cell lines (14, 28). The distribution of caveolin subtypes did not differ between isoproterenol-infused and control ventricular myocardium, thereby suggesting that the total amount of caveolin decreased without changes in subcellular distribution.


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Fig. 4.   Subcellular distribution of caveolin subtypes in (-)-isoproterenol-infused and control mice. Ventricular myocardial homogenates of control (A) or (-)-isoproterenol-infused mice (B) were fractionated by sucrose gradient method. An equal volume from each fraction was analyzed by SDS-PAGE and immunoblotting for caveolin-1 and -3. Positions of molecular markers are shown on left. Three mice from each group were selected randomly and subjected to analysis. A representative result from each group is shown.

Caveolin subtypes in skeletal muscles. We also examined the expression of caveolin subtypes in skeletal muscle. In contrast to the findings in the heart, there was no significant decrease in either caveolin subtype in skeletal muscle: 13 ± 9% (caveolin-1) and 9 ± 8% (caveolin-3) decrease compared with control (P not significant, n = 6 for each; Fig. 5). In contrast, the skeletal muscle beta -adrenergic receptors as determined by 125I-cyanopindolol-binding assays showed a significant downregulation without changes in dissociation constants [maximum binding: 6.6 ± 0.7 and 2.4 ± 0.3 fmol/mg in control and (-)-isoproterenol-treated mice, respectively, P < 0.01; dissociation constant: 0.047 ± 0.006 and 0.038 ± 0.004 nM in control and (-)-isoproterenol-treated mice, respectively, n = 6 for each].


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Fig. 5.   Caveolin subtype expression in skeletal muscle tissues. Membrane proteins (30 µg) were separated by a 12% SDS-PAGE and analyzed by immunoblot with antibody to caveolin-1 or -3. Caveolin subtype expression was quantified by a densitometer. Open bars, control mice; solid bars, (-)-isoproterenol-infused mice. Values are means ± SE (P not significant, n = 6 for each).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In another study (12) we reported cardiac hypertrophy and attenuation of responses to isoproterenol challenge in left ventricular contractility (fractional shortening and ejection fraction) in our mouse model after chronic beta -adrenergic stimulation. We found that these changes were accompanied by decreases in the beta -adrenergic receptor density (49% decrease) and isoproterenol-stimulated adenylyl cyclase activities (45% decrease), findings that classically characterize desensitization and downregulation of beta -adrenergic receptor signaling (12). An increase in cardiac (42%) and skeletal muscle tissue mass (15%) was also observed.

Caveolin-3, a structural component of muscle caveolae, was downregulated (28%) in our desensitization model. Because beta -adrenergic receptors may be internalized via caveolae (4, 22), our data suggest that caveolae containing beta -adrenergic receptors were also downregulated. Na-K-ATPase activity of the cardiac membrane was not significantly decreased, suggesting that the decrease in caveolin-3 was not a simple reflection of increased total cellular protein in hypertrophied myocytes. In contrast, caveolin expression in skeletal muscle was not significantly decreased, despite the downregulation of the beta -adrenergic receptor, suggesting that the role of caveolin differs among tissues. In support of this idea, a recent study demonstrated that the subcellular distribution of caveolin-3 was different between cardiac and skeletal myocytes (21). It is also well known that caveolae are abundant in myocytes, fibroblasts, endothelial cells, and adipocytes; however, their specificmor phology varies significantly, even when the caveolin subtype expression is similar. Because caveolin-3 plays a role in muscle cell membrane biology, such as interaction with dystrophin (28), a decrease in the content of caveolin-3 may contribute to pathological changes in the membrane function of hypertrophied cardiac myocytes.

Caveolin-1 is a major subtype in nonmuscle cells, including endothelial cells, fibroblasts, and adipocytes (24). It is known that the remodeling of muscle and vascular structure in the heart occurs with cardiac hypertrophy, including dedifferentiation of various cell types (8, 30). Caveolin-1 expression may have decreased as a result of cardiac remodeling that includes changes in cell types, because caveolin-1 expression is known to change on phenotypic changes of cells (11, 29). Furthermore, caveolin-1 may play a role in nitric oxide signaling by targeting nitric oxide synthase to caveolae and optimizing its activation and extracellular release (5, 26). In cardiac hypertrophy, endothelial cell function is damaged, leading to impaired responses to various vasoactive substances and disturbed production of endothelium-derived factors, such as nitric oxide (17, 20), which may be a reflection of altered caveolin-1 expression in endothelial cells. In skeletal muscles no significant changes in caveolin-1 and -3 were observed in the same model, indicating that these changes were specific to the heart.

Although caveolae were identified more than four decades ago (32), their role in cell biology has not been well characterized. Rapidly accumulating evidence in the past few years has suggested that caveolin forms an evolutionally conserved multigene family and that caveolae serve as microdomains of the cell membrane and, what is more important, regulate the function of various signaling molecules, including those in catecholamine signaling (1, 15). The major finding of the current investigation is that chronic catecholamine stimulation can regulate the expression of caveolin subtypes in the heart. Whether caveolin is involved in the molecular mechanism of beta -adrenergic receptor sequestration remains to be addressed.

    ACKNOWLEDGEMENTS

We thank J. Olivo for editorial work.

    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grants HL-54895, HL-59139, HL-33107, HL-33065, and HL-37404 and by American Heart Association Grant 13-533-945. C. Schwencke was supported by the Deutsche Forschungsgemeinschaft.

Address for reprint requests: Y. Ishikawa, Cardiovascular and Pulmonary Research Institute, Allegheny University, Pittsburgh, PA 15212.

Received 26 February 1997; accepted in final form 29 July 1997.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(6):C1957-C1962
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