Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115; and Cardiovascular and Pulmonary Research Institute, Allegheny University of the Health Sciences, Pittsburgh, Pennsylvania 15212
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ABSTRACT |
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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 -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
-adrenergic receptor stimulation in the
heart.
caveolae; G protein; isoproterenol; ventricular myocardium
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INTRODUCTION |
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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-1, -1
, -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
-adrenergic
receptor/Gs
/adenylyl cyclase
signaling (15). Ultrastructural immunolocalization studies demonstrated
the internalization of
-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
- and
-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 -adrenergic receptor/Gs
/adenylyl
cyclase-signaling pathway (for review see Ref. 7). Chronic
isoproterenol infusion to mice induced significant downregulation of
the
-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.
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MATERIALS AND METHODS |
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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.
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(-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.
-Adrenergic binding study.
-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.
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RESULTS |
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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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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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Infusion of (+)-isoproterenol.
To address the -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
-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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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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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
-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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DISCUSSION |
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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 -adrenergic stimulation. We found that
these changes were accompanied by decreases in the
-adrenergic receptor density (49% decrease) and
isoproterenol-stimulated adenylyl cyclase activities (45% decrease),
findings that classically characterize desensitization and
downregulation of
-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
-adrenergic receptors may be internalized via caveolae (4, 22), our
data suggest that caveolae containing
-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
-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 -adrenergic receptor
sequestration remains to be addressed.
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ACKNOWLEDGEMENTS |
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We thank J. Olivo for editorial work.
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FOOTNOTES |
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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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