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1 The John B. Pierce Laboratory, 2 Department of Cellular and Molecular Physiology, 3 Department of Pharmacology, and 4 Molecular Cardiobiology Division, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06519
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In isolated cell systems, nitric oxide synthase (NOS) activity is regulated by caveolin (CAV), a resident caveolae coat protein. Because little is known of this interaction in vivo, we tested whether NOS and caveolin are distributed together in the intact organism. Using immunohistochemistry, we investigated the localization of constitutive neuronal (nNOS) and endothelial (eNOS) enzyme isoforms along with caveolin-1 (CAV-1) and caveolin-3 (CAV-3) throughout the systemic vasculature and peripheral tissues of the hamster. The carotid artery, abdominal aorta, vena cava, femoral artery and vein, feed artery and collecting vein of the cheek pouch retractor muscle, capillaries and muscle fibers of retractor and cremaster muscles, and arterioles and venules of the cheek pouch were studied. In endothelial cells, eNOS and CAV-1 were present throughout the vasculature, whereas nNOS and CAV-3 were absent except in capillaries, which reacted for nNOS. In smooth muscle cells, nNOS and CAV-1 were also expressed systemically, whereas eNOS was absent; CAV-3 was present in the arterial but not the venous vasculature. Both nNOS and CAV-3 were located at the sarcolemma of skeletal muscle fibers, which were devoid of eNOS and CAV-1. These immunolabeling patterns suggest functional interactions between eNOS and CAV-1 throughout the endothelium, regional differences in the modulation of nNOS by caveolin isoforms in vascular smooth muscle, and modulation of nNOS by CAV-3 in skeletal muscle.
nitric oxide; endothelium; vascular smooth muscle; signal transduction
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE (NO) is implicated in the regulation of numerous cellular functions throughout the systemic circulation. The pervasive nature of its influence includes the regulation of smooth muscle tone (14), microvascular resistance (18) and permeability (22), platelet aggregation (33) and leukocyte adhesion (21), and remodeling of the vascular wall (35). Physical exercise promotes blood flow to the periphery and the delivery of oxygen to active skeletal muscle, where NO can govern mitochondrial respiration (20, 40) as well as force production (1, 19). Both of these processes influence the magnitude and distribution of blood flow through the systemic circulation (34).
The constitutive production of NO from L-arginine is mediated by two primary isoforms of nitric oxide synthase (NOS) (36). Endothelial NOS (type III, eNOS) has been found in endothelial cells throughout the cardiovascular system (2, 32). The expression of neuronal NOS (type I; nNOS) was first observed in the brain (6) and has since been found in skeletal muscle fibers (19, 27) and vascular smooth muscle cells (5). The enzymatic activity of constitutive NOS is stimulated by a rise in intracellular Ca2+ concentration, which promotes the binding of calmodulin to the enzyme and disrupts the association between NOS and caveolin (17, 26). Conversely, the allosteric binding of caveolin to NOS suppresses NO production in endothelial cells (15) as well as in muscle fibers (42). Caveolin-1 (CAV-1) has been found to be expressed in endothelial cells (16) and caveolin-3 (CAV-3) in skeletal muscle (42). The interaction between NOS and caveolin proteins has been studied extensively in isolated cell systems (15-17, 26), which have provided insight into the molecular mechanisms underlying the regulation of NO production. Nevertheless, little is known of how these respective enzymatic and regulatory proteins are distributed in the intact organism.
The purpose of this study was to examine the systemic distribution of CAV-1, CAV-3, eNOS, and nNOS expression throughout a full spectrum of blood vessels and in peripheral tissues of the hamster using immunohistochemistry. We reasoned that if caveolin modulates the constitutive activity of NOS under physiological conditions, then these proteins should be found together systematically. To investigate this relationship, representative samples were taken of conduit arteries and veins, resistance arteries and collecting veins of the cheek pouch retractor muscle, and microvessels within several tissues that have been established as microcirculatory preparations (3, 10, 28). On the basis of collective findings from several laboratories (16, 17, 26, 42), we hypothesized that CAV-1 would be found codistributed with eNOS in endothelium and CAV-3 with nNOS in skeletal muscle.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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All procedures were approved by the Animal Care and Use Committee of The John B. Pierce Laboratory and were performed in accord with the Guide for the Care and Use of Laboratory Animals of the National Research Council.
Vessel and Tissue Specimens
Male golden hamsters (80-110 g; Charles River Laboratories, Kingston, NY) were anesthetized with pentobarbital sodium (65 mg/kg ip). A tracheotomy was performed (PE-190 tubing; Clay Adams) to ensure airway patency, and the left femoral vein was catheterized (PE-50) for infusing additional anesthetic (10-15 mg/kg bolus; as needed). The specimens excised for study were carotid artery, abdominal aorta and vena cava, femoral artery and vein, feed artery and collecting vein of the retractor muscle, cremaster muscle, retractor muscle, and cheek pouch. Tissue specimens were rinsed in phosphate-buffered saline (pH 7.4), pinned to dental wax to approximate in vivo dimensions, and then covered with cryomatrix compound (Shandon and Lipshaw, Pittsburgh, PA) for rapid freezing by immersion in a solution of isopentane and dry ice. Specimens were stored at
70°C until sectioned
(typically within 48 h). Hamsters were exsanguinated or given an
intravenous overdose of pentobarbital.
Immunohistochemistry
Specimens were sectioned (thickness, 10 µm) at20°C using
a microtome (model 500, Bright's Instruments, Fairfield, NJ). Serial sections were collected on
poly-L-lysine (Sigma, St. Louis,
MO)-coated glass slides, dried at room temperature, stored at
20°C, and then processed in parallel. On the day of
staining, sections were fixed in cold (4°C) 2%
paraformaldehyde-phosphate-buffered saline (15 min) and permeabilized
(20 min) in 0.1% Triton-X in 0.05 M Tris-saline solution (TSS).
Sections were then exposed (20 min, 25°C) to 3.0% hydrogen peroxide
in 60% methanol-40% TSS to quench endogenous peroxidase activity. To
block nonspecific binding of antibodies, sections were incubated for 4 h (25°C) in 0.1 M TSS containing 2% bovine serum albumin (BSA; no.
10856, Amersham, Arlington Heights, IL)-10% normal serum (goat or
donkey), which was then aspirated from each section. Primary antibodies
for eNOS (monoclonal; Transduction Laboratories, Lexington, KY) and for nNOS (polyclonal; Zymed Laboratories, South San Francisco, CA), CAV-1,
and CAV-3 (polyclonal; Transduction Laboratories) were then added to
serial sections and incubated overnight at 4°C. Final
concentrations (µg/ml) of primary antibodies during incubations of
vessel and tissue sections, respectively, were as follows: 10, 20 for
eNOS; 0.5, 1.0 for nNOS; 0.1, 0.05 for CAV-1; and 0.125, 0.125 for
CAV-3.
To label bound primary antibody, sections were incubated for 30 min (25°C) with biotinylated secondary antibody (polyclonal rabbit IgG; Vector Laboratories, Ingold, CA, for polyclonal primary antibodies; monoclonal mouse IgG; Jackson ImmunoResearch, West Grove, PA for monoclonal primary antibodies). Final working concentrations of secondary antibodies (diluted in 0.1 M TSS with 2% BSA-10% serum) were 1.5 and 1.7 µg/ml, respectively. Sections were then incubated for 60 min with avidin-biotinylated enzyme complex (ABC) solution (Vectastain kit, Vector). Between each step in the labeling protocol, sections were rinsed in TSS (3 × 5 min). Sections were then exposed to nickel-free diaminobenzidine tetrahydrochloride (DAB; Vector) solution for 1 min, rinsed in deionized water, permanently mounted with Biomedia gel/mount (Electron Microscopy Sciences, Fort Washington, PA), and protected with a no. 1 coverslip.
Control Experiments
For each antibody, the specificity of immunolabeling was evaluated by omitting the primary antiserum (i.e., incubation with secondary antibody and ABC alone) and omitting both primary and secondary antisera (incubation with ABC alone). Additional positive controls (data not shown) included incubation with antibodies to von Willebrand's factor (polyclonal; 2.0 µg/ml; Dako, Carpinteria, CA) as an endothelial cell marker and to smooth muscle
-actin (monoclonal; 6.9 µg/ml; Sigma) as a smooth muscle cell marker. To
further test the specificity of the nNOS antibody, it was incubated (undiluted) with excess nNOS protein (0.5 µg/ml; purified from HEK-293 cells expressing rat nNOS cDNA). This preabsorbed antibody was
then diluted to working concentrations and applied to serial sections
as described above.
Data analysis. Sections processed for
DAB staining were visualized through a Nikon E800 microscope with Plan
Fluor infinity objectives (magnification, numerical aperture:
×20, 0.50; ×40, 0.75; ×60, 0.85) using differential
interference contrast with Köhler illumination. Relative staining
intensity (, if absent; +, if apparent; ++, if definite; +++, if
bold) and localization (endothelial, myocyte) were evaluated by a
trained observer who developed the working conditions for these
experiments. Evaluations of specific vessels (Table
1) and tissues (Table
2) were based on at least eight samples
from as many hamsters. The apparent codistribution of protein isoforms
was based on the similarity of staining in serial sections.
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Photomicrography
Photomicrographs were acquired digitally from the E800 microscope using a Spot Camera (Diagnostics Instruments, Sterling Heights, MI) coupled to a Matrox (Dorval, PQ, Canada) Millenium II frame grabber (resolution, 1,200 × 1,600 pixels) housed in a Pentium-based personal computer. Images were arranged for display using Adobe Photoshop (v. 4.01).| |
RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Summary data are presented for vessels in Table 1 and for tissues in Table 2.
Systemic Distribution of NOS and Caveolin Isoforms
eNOS was present in endothelial cells of all conduit vessels, microvessels, and capillaries. CAV-1 was present in endothelial and smooth muscle cells throughout the vasculature, including capillaries. nNOS was present in smooth muscle throughout the vasculature, in the periphery of skeletal muscle fibers, and in capillaries of skeletal muscle. CAV-3 was present in smooth muscle cells of the arterial vasculature and the periphery of skeletal muscle fibers, yet was strikingly absent from smooth muscle cells throughout the venous vasculature.Codistribution of NOS and Caveolin Isoforms According to Cell Type
In endothelial cells, eNOS and CAV-1 were present throughout arterial and venous vasculature; however, neither nNOS nor CAV-3 was apparent. In smooth muscle, nNOS and CAV-1 were present systemically, with no staining for eNOS. Whereas smooth muscle of arteries and arterioles showed CAV-3 to be present, it was distinctly absent from smooth muscle of veins and venules. In skeletal muscle fibers, nNOS and CAV-3 were codistributed near the sarcolemma, with no evidence of eNOS or CAV-1. In capillaries supplying muscle fibers, both eNOS and nNOS were found intermittently with CAV-1.Expression of NOS and Caveolin Isoforms in Specific Vessels and Tissues
Conduit vessels. In the carotid artery (not shown), abdominal aorta (Fig. 1), and femoral artery (Fig. 2), endothelial cells labeled for eNOS and CAV-1, whereas the surrounding smooth muscle cells labeled for CAV-1, nNOS, and CAV-3 (Figs. 1 and 2). In the femoral vein (Fig. 2) and vena cava (Fig. 3), endothelial cells labeled for eNOS and CAV-1. Venous smooth muscle cells were positive for nNOS and CAV-1 but did not label for eNOS or CAV-3 (Table 1).
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Microvessels. In the feed artery of
the retractor muscle (Fig. 4) and
arterioles of the cheek pouch (Fig. 5),
endothelial cells labeled for eNOS and CAV-1. The surrounding smooth
muscle cells labeled for CAV-1, nNOS, and CAV-3. In the collecting vein and venules (Figs. 4 and 5), smooth muscle cells labeled for CAV-1 and
nNOS but not for CAV-3. These patterns of distribution reflect those
observed for conduit arteries and veins, respectively.
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Skeletal muscle. Striated muscle
fibers examined in cross section labeled at or near the sarcolemma for
both nNOS and CAV-3 in the retractor muscle (Fig.
6) as well as the cremaster muscle (not
shown). In contrast, there was no evidence of immunoreactivity to eNOS
or to CAV-1 in these myocytes. In both muscles, intermittent capillaries labeled for nNOS as well as eNOS and CAV-1. The
codistribution of nNOS with eNOS was apparent in some but not all of
the labeled capillaries.
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Controls. Endothelial cells in all
locations routinely labeled for von Willebrand's factor as did smooth
muscle cells for -actin (data not shown). No staining was found
following incubation with secondary antibody + ABC (Fig. 7) or with ABC
alone. Preabsorption of the nNOS antibody eliminated staining for nNOS
in vessels and tissues (Fig. 7). Further
evidence for the selectivity of immunolabeling included
1) the expression of nNOS in capillaries (Fig. 6) but not
arterial or venous endothelium (Figs. 1-5); 2) the
absence of CAV-3 throughout the endothelium and from venous but not
arterial smooth muscle (Figs. 1-5); and
3) the absence of CAV-1 in striated (Fig. 6) but not smooth muscle (Figs. 1-5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Immunohistochemistry was used to investigate the location of constitutive isoforms of NOS and caveolin throughout the systemic vasculature and in tissues routinely used to study the microcirculation of the hamster. Without exception, at least one isoform of NOS was codistributed with at least one isoform of caveolin. CAV-1 and eNOS were found together in endothelial cells from arteries to veins and throughout the microcirculation, whereas CAV-3 codistributed with nNOS in arterial and arteriolar smooth muscle cells, as well as in skeletal muscle fibers. These findings support the hypothesis that NOS and caveolin proteins are constitutively coexpressed throughout the peripheral vasculature. However, CAV-3 was distinctly absent from venous smooth muscle, whereas CAV-1 was absent from skeletal muscle. In capillaries supplying muscle fibers, the presence of eNOS was complimented intermittently by that of nNOS, yet only the CAV-1 isoform of caveolin was present. These findings argue against the hypothesis that CAV-1 is exclusively colocalized with eNOS or that CAV-3 is always found with nNOS (16, 17, 26, 42). Nevertheless, based on in vitro studies that show nNOS and eNOS to interact similarly with both isoforms of caveolin (15, 17, 42), we suggest that the constitutive regulation of NO production by caveolin (in conjunction with other modulators) is of physiological importance throughout the intact organism.
Localization and Interaction of NOS and Caveolin Isoforms
The goals of this study were to determine in which endothelial cells and myocytes NOS and caveolin isoforms are expressed physiologically, and whether there was consistent evidence for codistribution of respective proteins. In earlier studies (32), a monoclonal antibody to eNOS was developed and used to show that eNOS was present throughout the endothelium of arteries, veins, and microvessels of bovine and human tissues. Electron microscopy revealed the protein to be associated primarily with the plasma membrane and cytoplasmic vesicles, although a cytosolic component was also evident. In arterioles, venules, and capillaries of guinea pig submucosa, immunoreactivity for eNOS was found in endothelial cells but not in smooth muscle cells (29). Electron microscopy located the staining to membranes of the Golgi apparatus, to intracellular vesicles, and to microdomains of the plasma membrane and rough endoplasmic reticulum (29). Further research has confirmed these observations while revealing how the association of NOS with caveolin can regulate the production of NO (2, 16).In microvascular endothelial cells cultured from bovine lung, eNOS was found to be colocalized with caveolin on discrete regions of plasma membrane that were determined to be caveolae; association of these proteins was present to a lesser extent in the perinuclear region (16). A similar pattern has recently been documented in vascular and endocardial endothelial cells of the rat heart (2), where much of the staining was associated with the Golgi apparatus, as first described for eNOS in bovine aortic endothelial cells (38). This pattern of labeling suggests that eNOS may "traffic" from the Golgi apparatus to specialized regions of the plasma membrane (16). In turn, caveolin has been shown to cycle between plasma and Golgi membranes along microtubules, acting as a molecular "chaperone" for eNOS (8).
Caveolae are specialized regions of the plasma membrane that are not only rich in caveolin but also contain a host of molecules involved in signal transduction initiated at the cell surface (30). Such "targeting" of eNOS to the surface of endothelial cells provides an effective mechanism for producing NO in response to hemodynamic forces and luminal receptor activation. Both myristoylation and palmitoylation are necessary to direct eNOS to Golgi membranes (23, 38), whereas palmitoylation is required for targeting to caveolae (16, 39). A rise in intracellular Ca2+ concentration enables calmodulin to disrupt the association of eNOS from the caveolin complex (26); as intracellular Ca2+ concentration returns to basal levels, the interaction between eNOS and caveolin is restored (12). In turn, the turnover of palmitate suggests that eNOS cycles between Golgi membranes within the cell and caveolae at the cell surface (16). Indeed, prevention of eNOS targeting into caveolae or Golgi membranes reduces NO release upon stimulation of the enzyme (24, 38).
The presence of biologically active eNOS in different subcellular compartments suggests that each may be regulated differentially in response to different modes of stimulation. Nevertheless, as shown in vitro, neither acylation nor targeting to caveolae is required for eNOS to interact with caveolin (15, 17, 24). A similar interaction for nNOS and CAV-3 occurs, with either isoform of NOS able to be regulated by either isoform of caveolin (15, 42). Thus present findings in the hamster suggest that the signaling pathways for NOS regulation that have been defined in isolated cell systems (above) may well apply to those of intact tissues.
Coincident Expression of Protein Isoforms
More than a single isoform of each protein is often expressed in individual cells. Both CAV-1 and CAV-3 were present in smooth muscle cells throughout the arterial vasculature (Figs. 1-3 and 5), as previously found in rat cardiac myocytes (11). As expected from previous studies (11, 13, 41), CAV-3 was not observed in endothelial cells, which routinely labeled for CAV-1 (Table 1). The absence of CAV-3 from postcapillary smooth muscle (i.e., venules and veins; Figs. 2-5) was particularly striking in light of its clear presence in precapillary smooth muscle (i.e., arteries and arterioles; Figs. 1, 2, 4, and 5), as well as skeletal muscle (Fig. 6 and Ref. 42) and cardiac myocytes (11, 41).In Northern and Western blot analyses of human skeletal muscle homogenates, nNOS message and protein were highly expressed, with lesser expression of eNOS (27); unfortunately, the cell types actually containing the respective isoforms were not identified. With the use of immunohistochemistry, both nNOS and eNOS have been localized to discrete neural populations of the brain (6, 9), whereas nNOS has been identified in a subpopulation (~5%) of endothelial cells in the rabbit aorta (25) and in capillary cultures (31) that also contained eNOS. These observations are consistent with our findings in skeletal muscle, where at least some capillaries labeled for both NOS isoforms (Fig. 6). It is unlikely that "pericapillary nerves" are responsible for this nNOS labeling. However, we cannot exclude the possibility that pericytes contain nNOS (e.g., as found in smooth muscle cells) and thereby may have contributed to our capillary staining. Nevertheless, the similarity of staining we observed for von Willebrand's factor (data not shown) supports the interpretation that capillary endothelial cells do contain nNOS. As shown by others (13, 19), nNOS in skeletal muscle otherwise appeared restricted to the periphery of myocytes (Fig. 6). Whereas only eNOS was present in the endothelium of the other vessels we studied (Figs. 1-5), the expression of this isoform has been found within striated myocytes (11, 20).
Before the identification of nNOS in the sarcolemma of rats (19) and
human (13) skeletal muscle fibers (and see Fig. 6), eNOS was taken to
be the only major isoform associated with cell membranes (37). In more
recent studies of murine skeletal muscle, nNOS was found localized to
the sarcolemma with dystrophin, a muscle-specific plasma membrane
marker, with a similar relationship apparent for cardiac and aortic
smooth muscle cells of the rat (41). Immunoprecipitation of nNOS with
dystrophin suggested a physical association such that dystrophin
complexes enriched in caveolae were coated with CAV-3 (41). The
mechanism of targeting nNOS to the myocyte membrane is distinct from
the acylation of eNOS (12, 16). Instead, nNOS interacts with
1-syntrophin in the membrane
cytoskeleton of the dystrophin complex, mediated by an
NH2-terminal extension of the
protein that is not found in other NOS isoforms (7). Analogous to CAV-1
in endothelial cells, CAV-3 is proposed to act as a scaffolding protein
within myocyte caveolae, recruiting proteins that interact with
caveolin to the membrane to enable rapid and effective coupling of
membrane events to cellular responses (30, 41).
Functional Implications
In the rat heart, the subcellular localization of eNOS favored the plasma membrane of postcapillary endothelial cells and the Golgi apparatus in precapillary endothelial cells (2). Such heterogeneity of protein targeting may convey subtle differences to the regulation of NO production in endothelial cells according to the particular requirements of the local milieu; e.g., flow control in arterioles (18) and transvascular macromolecule transport in postcapillary venules (22). In turn, the presence of both constitutive isoforms of NOS in capillary endothelial cells (Fig. 6) suggests a unique role for (and regulation of) NO production in capillaries of skeletal muscle (40). In biopsies of human quadriceps muscle, immunohistochemical staining for eNOS was restricted to microvascular endothelial cells (13). Whereas staining for nNOS was most apparent at the periphery of muscle fibers, punctate nNOS staining was also present throughout fiber cross sections, with an intracellular pattern similar to that for mitochondrial enzymes (13). Such intracellular localization of nNOS is consistent with a role for NO in governing respiration (20, 40) as well as force production (1, 19).When the present findings are taken in light of previous studies, it is now apparent that NO can be generated physiologically from vascular smooth muscle cells (5), endothelial cells (32), and skeletal muscle (4, 19). Therefore, studies that have inhibited all constitutive NOS activity (e.g., by treating an entire animal or tissue with an L-arginine analog), or that have measured NO production and release from an intact tissue (4) [or its homogenate (27)], overlook the subtleties of NO generation from the respective cellular "elements" of a tissue. This raises the question of where NO production that has previously been attributed to arterial (14) and arteriolar (18) endothelial cells, skeletal muscle capillaries (40), or venular endothelium (21, 22) may actually occur in vivo. Refining experimental approaches to these questions should provide valuable new insight into the regulation and action(s) of NO in the intact system.
Summary and Conclusion
Immunohistochemical studies of the constitutive expression of NOS have focused on where eNOS (2, 29, 32), nNOS (5, 6, 19, 27), or both [(13, 31) and present study] are localized in particular tissues and cells. The corresponding expression of caveolin has received relatively less attention (2, 11, 41). In isolated cell systems, studies have focused on elucidating the molecular interaction and biochemical regulation of NOS isoforms with and by caveolin isoforms (11, 15, 38). These efforts have provided fundamental insight into a signaling pathway that is central to many physiological processes, not only for the cells involved, but also for homeostasis of the organism as a whole. Nevertheless, there has been a paucity of information concerned with whether (and if so, where) NOS and caveolin may systematically interact in vivo. In the present study NOS and caveolin were consistently codistributed in endothelial cells and myocytes throughout the hamster, albeit with differential expression of respective isoforms. In turn, findings that eNOS and nNOS are inhibited similarly by CAV-1 or CAV-3 (15, 42) imply that the regulation of NOS by caveolin occurs throughout the intact system, regardless of which of the respective isoforms are present. Regional heterogeneity in the distribution of one and/or the other type of NOS or caveolin protein may reflect differences not only in the influence of the local milieu on gene expression but also in the subtleties of physiological control effected through a common signaling pathway.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Jill E. Hungerford for critical review of this paper.
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FOOTNOTES |
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This project was supported by National Heart, Lung, and Blood Institute Grants HL-57665, HL-56786, HL-41026, and HL-61371 and by the Yale Diabetes and Endocrinology Research Center Grant P30 DK45735. S. S. Segal and W. C. Sessa are Established Investigators of the American Heart Association.
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. §1734 solely to indicate this fact.
Address for reprint requests: S. S. Segal, The John B. Pierce Laboratory, Yale Univ. School of Medicine, 290 Congress Ave., New Haven, CT 06519 (E-mail: sssegal{at}jbpierce.org).
Received 10 February 1999; accepted in final form 12 April 1999.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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