Flow cytometric analysis of macrophage oxidative metabolism using DCFH.
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Animals↗
Biomedical subjects
L Kobzik
Publications and source records attributed to L Kobzik.
Suppression of herpes simplex virus type 1 (HSV-1)-induced pneumonia in mice by inhibition of inducible nitric oxide synthase (iNOS, NOS2).
Intranasal Herpes simplex virus type 1 (HSV-1) infection of mice caused pneumonia. Manifestations of the disease included: histological pneumonitis, pulmonary influx of lymphocytes, decreased pulmonary compliance, and decreased survival. Immunohistochemical staining demonstrated iNOS induction and the nitrotyrosine antigen in the lungs of infected, but not uninfected mice, suggesting that nitric oxide contributes to the development of pneumonia. To elucidate the role of nitric oxide in the pathogenesis of HSV-1 pneumonia, infected mice were treated either with the inhibitor of nitric oxide synthase activity, N(G)-monomethyl-L-arginine (L-NMMA), or, as a control, with PBS or D-NMMA. L-NMMA treatment decreased the histological evidence of pneumonia and reduced the bronchoalveolar lavage lymphocyte number to one-quarter of the total measured in control-treated mice. L-NMMA treatment significantly improved survival and pulmonary compliance of HSV-1-infected mice. Strikingly, the L-NMMA-mediated suppression of pneumonia occurred despite the presence of a 17-fold higher pulmonary viral titer. Taken together, these data demonstrated a previously unrecognized role of nitric oxide in HSV-1-induced pneumonia. Of note, suppression of pneumonia occurred despite higher pulmonary virus content; therefore, our data suggest that HSV-1 pneumonia is due to aspects of the inflammatory response rather than to direct viral cytopathic effects.
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Comparison of efficacy of antisense oligomers directed toward TNF-alpha in helper T and macrophage cell lines.
The authors investigated the use of antisense oligomers specific for TNF-alpha (AS-2) and nonsense control oligomers (NS) in T cells (HT2) and macrophages (RAW264.7), comparing three distinct chemical formulations. Phosphorothioate antisense (S-AS) caused sequence-specific inhibition of TNF-alpha production by activated HT2s (0.5 microM S-AS 2 vs S-NS: 31.4 +/- 1.2%, 4.2 +/- 3.2% inhibition, respectively). In contrast, S-AS were ineffective in RAW264.7, despite greater uptake as measured with fluorescent S-oligonucleotides. Furthermore, differences in efficacy of S-AS (HT2 > RAW) were not attributable to differences in the pinocytic (HT2 = RAW) or adsorptive endocytic (RAW > HT2) pathways implicated in oligonucleotide uptake, suggesting an important role for intracellular events after antisense uptake. Morpholino oligomers (M-AS), in contrast, were more effective in RAW264.7 than in HT2 (32.6 +/- 2.6% vs 12.3 +/- 0.5% inhibition), consistent with uptake experiments using fluorescent M-oligomers. Phosphodiester oligonucleotides were ineffective in both cell types. It was concluded that antisense efficacy in leukocytes varies according to type of oligomer, cell target and intracellular processing event(s).
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Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin.
We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-gamma (IFN-gamma, 25 U/ml). Using both microplate-based fluorometry and flow cytometric analysis, AMs treated with l (but not d)-arginine showed a dose- and time-dependent increase in DCFH oxidation above buffer control (e.g., DCF production (nM): control, d-arginine 100 microM, l-arginine 100 microM respectively; 91+/-9, 76+/-11, 396+/-45, mean +/- SE, n = 6, 120 min). Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of the l-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (microM): 2.2, 3.6, 7.1; for 5, 10, 50 microM l-arginine, respectively). In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated with l-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 microM). We conclude that DCFH is a useful probe for quantitation of NOS-2 activity in activated rat lung macrophages.
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Persistence of pulmonary pathology and abnormal lung function in IL-3/GM-CSF/IL-5 beta c receptor-deficient mice despite correction of alveolar proteinosis after BMT.
Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor (beta cR KO) develop lung disease similar to that seen in human pulmonary alveolar proteinosis (PAP) which includes lymphocytic infiltration around airways and vessels and the progressive accumulation of surfactant and macrophages within the alveolar space. We investigated bone marrow transplantation (BMT) as a curative treatment of PAP in beta cR KO mice by semiquantitative histologic analysis and evaluation of pulmonary function. BMT from wild-type (WT) donors into lethally irradiated beta cR KO recipients (WT --> KO) led to the complete resolution of alveolar protein accumulation and to normalization of BAL fluid cellularity and macrophage morphology. However, detailed microscopic analysis of lung tissue revealed the persistence of significant cellular infiltrates in WT --> KO recipients which were equivalent to those seen in KO --> KO animals. Evaluation of pulmonary function demonstrated that only dynamic compliance (Cdyn) and not airway conductance (G[L]) was significantly improved in the WT --> KO group compared to KO --> KO animals and that both of these measurements remained significantly abnormal when compared to WT --> WT controls. We conclude, that although BMT for PAP reverses alveolar macrophage and protein accumulation, it does not decrease the interstitial inflammatory component of this disease. The importance of this residual pathology is demonstrated by the incomplete correction of alveolar function (Cdyn) and lack of improvement in increased airway resistance (G[L]). These findings may have important implications with regard to the extent that BMT can be considered a potential curative procedure for this clinical disorder.
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Development of a model of low-inoculum Streptococcus pneumoniae intrapulmonary infection in infant rats.
We have developed a model of low-inoculum Streptococcus pneumoniae infection in infant rats. We challenged 4-day-old Sprague-Dawley pups via intraperitoneal or intrapulmonary injection of S. pneumoniae serotypes 1, 3, 4, 5, 6b, 7f, 9v, 14, 19f, and 23f. To achieve bacteremia with low inocula, it was necessary to passage the isolates in rats. Inocula of the 10 S. pneumoniae serotypes producing bacteremia in 50% or more animals ranged from 1 to 400 CFU. Virulence was similar by intraperitoneal and intrapulmonary routes. Lung specimens from animals challenged by the intrapulmonary route grew S. pneumoniae and demonstrated histologic evidence of focal infection. Meningitis was detected in 20 to 50% of bacteremic animals, and mortality invariably followed bacteremia within 24 to 48 h. This model of intrapulmonary infection uses low inocula of S. pneumoniae and results in bacteremia, meningitis, and death in infant rats.
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alpha 1-Acid glycoprotein reduces local and remote injuries after intestinal ischemia in the rat.
The aim of this study was to look at the role of alpha 1-acid glycoprotein as a natural anti-inflammatory agent with particular respect to its antineutrophil and anticomplement activity. A recombinantly engineered form of sialyl Lewisx (sLe(x))-bearing alpha 1-acid glycoprotein (sAGP) was administered intravenously to pentobarbital-anesthetized rats after 50 min of intestinal ischemia just before 4 h of reperfusion. A non-sLe(x)-bearing form of AGP (nsAGP) was used as control. sAGP-treated animals had a 62% reduction (P < 0.05) in remote lung injury, assessed by 125I-albumin permeability, compared with those treated with nsAGP (permeability index of 3.61 +/- 0.15 x 10(-3) and 5.18 +/- 0.67 x 10(-3), respectively). There was a reduction in pulmonary myeloperoxidase levels in sAGP-treated rats compared with nsAGP-treated rats. Complement-dependent intestinal injury, assessed by 125I-albumin permeability was reduced by 28% (P < 0.05) in animals treated with sAGP (7.58 +/- 0.63) compared with those treated with nsAGP (10.4 +/- 0.54). We conclude that sAGP ameliorates both complement- and neutrophil-mediated injuries.
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S-nitrosothiols inhibit neuronal norepinephrine transport.
Although it has been recently shown that nitric oxide (NO) and its congeners (NO(x)), including nitrosothiols, may modify catecholamine turnover in the brain, it is not known whether NO(x) affect norepinephrine (NE) uptake by sympathetic neurons. The nitrosothiol NO donor S-nitroso-acetylpenicillamine (SNAP, 100 microM for 1 h) elicited a concentration-dependent reduction in desipramine-sensitive [3H]NE uptake into PC-12 cells (66 +/- 3%; P < 0.01) or cultured rat superior cervical ganglia (74 +/- 5%; P < 0.001), whereas desipramine-insensitive [3H]NE uptake was unaffected, indicating a selective effect on uptake-1-mediated transport. Short-term coculture of PC-12 cells with microvascular endothelial cells expressing the cytokine-inducible NO synthase (NOS2) also exhibited a reduction in [3H]NE uptake (33 +/- 3%, P < 0.001) that could be prevented by the addition of the NOS inhibitor N-monomethyl-L-arginine (L-NMMA, 1 mM). Endogenous production of NO(x) by nerve growth factor-pretreated PC-12 cells also exhibited an L-NMMA-inhibitable reduction in [3H]NE uptake. Whereas SNAP resulted in a 10-fold elevation of PC-12 guanosine 3',5'-cyclic monophosphate (cGMP) content (P < 0.01), its effect on [3H]NE uptake was not mimicked by exposure to 8-bromo-cGMP. However, the inhibitory effect of SNAP on uptake-1-mediated [3H]NE transport could be attenuated by 1 mM cysteine, a sulfhydryl compound that could act as a sink for NO(x)-mediated nitrosation reactions, although cysteine did not affect the increase in intracellular cGMP with SNAP. These data suggest that an endogenous NO(x) source(s) modifies the activity of the uptake-1 catecholamine transporter in postganglionic sympathetic neurons, which, as we demonstrate, express both NOS1 and NOS3 isoforms, possibly by S-nitrosothiol-mediated nitrosation of regulatory sites on the transporter.
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Contribution of type I NOS to expired gas NO and bronchial responsiveness in mice.
Nitric oxide (NO) can be measured in the expired gas of humans and animals, but the source of expired NO (F(E)NO) and the functional contribution of the various known isoforms of NO synthase (NOS) to the NO measured in the expired air is not known. F(E)NO was measured in the expired air of mice during mechanical ventilation via a tracheal cannula. F(E)NO was significantly higher in wild-type B6SV129J +/+ mice than in mice with a targeted deletion of type I (neural) NOS (nNOS, -/-) (6.3 +/- 0.9 vs. 3.9 +/- 0.4 parts/billion, P = 0.0345, for +/+ and -/- mice, respectively), indicating that approximately 40% of the NO in expired air in B6SV129 mice is derived from nNOS. Airway responsiveness to methacholine (MCh), assessed by the log of the effective dose of MCh for a doubling of pulmonary resistance from baseline (ED(200)R(L)), was significantly lower in the -/- nNOS mice than in the wild-type mice (logED(200)R(L), 2.24 +/- 0.07 vs. 2.51 +/- 0.06 microg/kg, respectively; P = 0.003). These findings indicate that nNOS significantly contributes to baseline F(E)NO and promotes airway hyperresponsiveness in the mouse.
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Experimental murine acid aspiration injury is mediated by neutrophils and the alternative complement pathway.
Acid aspiration may result in the development of the acute respiratory distress syndrome, an event associated with significant morbidity and mortality. Although once attributed to direct distal airway injury, the pulmonary failure after acid aspiration is more complex and involves an inflammatory injury mediated by complement (C) and polymorphonuclear leukocytes. This study examines the injurious inflammatory cascades that are activated after acid aspiration. The role of neutrophils was defined by immunodepletion before aspiration, which reduced injury by 59%. The injury was not modified in either P- or E-selectin-knockout mice, indicating that these adhesion molecules were not operative. C activation after aspiration was documented with immunochemistry by C3 deposition on injured alveolar pneumocytes. Animals in which C activation was inhibited with soluble C receptor type 1 (sCR1) had a 54% reduction in injury, similar to the level of protection seen in C3-knockout mice (58%). However C4-knockout mice were not protected from injury, indicating that C activation is mediated by the alternative pathway. Finally, an additive effect of neutrophils and C was demonstrated whereby neutropenic animals that were treated with sCR1 showed an 85% reduction in injury. Thus acid aspiration injury is mediated by neutrophils and the alternative C pathway.
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Neutral endopeptidase activity in newborn and adult rabbit tracheas.
Neutral endopeptidase (NEP, E.C. 3.4.24.11), a widely distributed ectoenzyme, cleaves and inactivates a variety of biologically active peptides, including the tachykinin, substance P (SP). This study was undertaken to determine whether the modulation of SP airway smooth muscle contraction by NEP is age-dependent. We studied the contractile response of isolated tracheal rings from newborn and 120 day old New Zealand white rabbits. We measured NEP activity and determined immunoreactive NEP content in tracheal membrane preparations. NEP activity was then localized histochemically in sections of rabbit tracheas. In the presence of the NEP inhibitor, SCH 32615, the contractile response of isolated tracheal rings to SP was increased both in the newborn and 120 day old rabbits. However, the increase was greatest in the newborn animals. NEP activity in tracheal membrane preparations increased fivefold between the newborn and 120 day old rabbits. Western blot analysis also revealed a significant increase in the immunoreactive NEP content of these tracheal membrane preparations between the newborn and 120 day old rabbits. NEP activity, localized histochemically, was most intense in the epithelial region of the newborn animals, with a shift of activity to the subepithelial region with age. The prominent epithelial localization of neutral endopeptidase in the tracheas of these 1 day old rabbits, which we have shown to have relatively low neutral endopeptidase activity, suggests that the location of neutral endopeptidase in the airway, including proximity to relevant substance P receptors, may be critical to its function.
Aging↗
Alveolar macrophage interaction with air pollution particulates.
We applied flow cytometric analysis to characterize the in vitro response of alveolar macrophages (AM) to air pollution particulates. Normal hamster AM were incubated with varying concentrations of residual oil fly ash (ROFA) or concentrated ambient air particulates (CAP). We found a dose-dependent increase in AM-associated right angle light scatter (RAS) after uptake of ROFA (e.g., mean channel number 149.4 +/- 6.5, 102.5 +/- 4.1, 75.8 +/- 3.5, and 61.0 +/- 4.6 at 200, 100, 50, and 25 mg/ml, respectively) or CAP. A role for scavenger-type receptors (SR) in AM uptake of components of ROFA and CAP was identified by marked inhibition of RAS increases in AM pretreated with the specific SR inhibitor polyinosinic acid. We combined measurement of particle uptake (RAS) with flow cytometric analysis of intracellular oxidation of dichlorofluorescin. Both ROFA and CAP caused a dose-related intracellular oxidant stress within AM, comparable to that seen with phorbol myristate acetate (PMA) (e.g., fold increase over control, 6.6 +/- 0.4, 3.6 +/- 0.4, 4.6 +/- 0.5, 200 mg/ml ROFA, 100 mg/ml ROFA, and 10(-7) M PMA, respectively). We conclude that flow cytometry of RAS increases provides a useful relative measurement of AM uptake of complex particulates within ROFA and CAP. Both ROFA and CAP cause substantial intracellular oxidant stress within AM, which may contribute to subsequent cell activation and production of proinflammatory mediators.
Air Pollutants↗
Nitric oxide regulation of atrioventricular node excitability.
The role of nitric oxide in the autonomical regulation of atrioventricular (AV) spontaneous action potentials and L-type calcium current (ICa-L) in isolated single AV nodal cells from rabbit heart was examined by using the whole cell patch clamp technique, immunohistochemical staining and single cell reverse transcription polymerase chain reaction analysis. The nitric oxide donor 3-morpholino-sydnonimine (SIN-1) (0.1 mmol/L) suppressed the beta-agonist isoproterenol- (1 mumol/L) stimulated increase in ICa-L and decreased the frequency and amplitude of spontaneous action potentials. In cells in which ICa-L had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 mumol/L), SIN-1 had no additive effect. Intracellular dialysis with the nitric oxide synthase inhibitor N-monomethyl-L-arginine (L-NMMA, 0.5 mmol/L) blocked CCh- but not SIN-1-induced ICa-L attenuation. However, intracellular dialysis with methylene blue (20 mumol/L), which inhibits nitric oxide-mediated activation of guanylyl cyclase and cGMP production blocked the effects of both CCh and SIN-1 on ICa-L. In these cells, neither L-NMMA nor methylene blue affected the CCh-activated potassium current (IK(ACh)). Internal dialysis with cGMP (10 mumol/L) significantly inhibited isoproterenol-stimulated ICa-L without affecting IK(ACh). In AV nodal cells internally perfused with either a nonhydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit ICa-L but still activated IK(ACh). CCh-induced ICa-L attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (20 mumol/L) but not by milrinone (5 mumol/L), which only inhibits the cGMP-inhibited phosphodiesterase isozyme (PDE3). Immunohistochemical staining identified the presence of the endothelial constitutive nitric oxide synthase (NOS3) in both single AV node cells in vitro and in cryostat sections of AV node tissue in situ. These results demonstrate that endogenous nitric oxide is involved in the muscarinic cholinergic attenuation of ICa-L in AV nodal cells; the mechanism likely involves the cGMP-stimulated phosphodiesterase.
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Inhibition of T cell costimulation abrogates airway hyperresponsiveness in a murine model.
Activation of naive T cells requires at least two signals. In addition to the well characterized interaction of the T cell antigen receptor with the antigen/MHC expressed on an antigen-presenting cell, T cell activation also requires costimulation by a second set of signals. The best characterized costimulatory receptor is CD28, which binds to a family of B7 ligands expressed on antigen-presenting cells. In asthma, although activated T cells play a role in the initiation and maintenance of airway inflammation, the importance of T cell costimulation in bronchial hyperresponsiveness had not been characterized. Therefore, we tested the hypothesis that inhibition of the CD28:B7 costimulatory pathway would abrogate airway hyperresponsiveness. Our results show that blockade of costimulation with CTLA4-Ig, a fusion protein known to prevent costimulation by blocking CD28:B7 interactions, inhibits airway hyperresponsiveness, inflammatory infiltration, expansion of thoracic lymphocytes, and allergen-specific responsiveness of thoracic T cells in this murine model of allergic asthma.
Airway Resistance↗
Mac-1 (CD11b/CD18) and the urokinase receptor (CD87) form a functional unit on monocytic cells.
The leukocyte integrin Mac-1 (CD11b/CD18) and the urokinase receptor (uPAR, CD87) mediate complementary functions in myelomonocytic cells. Both receptors promote degradation of fibrin(ogen) and also confer adhesive properties on cells because Mac-1 and uPAR bind fibrin and vitronectin, respectively. Staining of lung biopsy specimens from patients with acute lung injury indicated that fibrin and vitronectin colocalize at exudative sites in which macrophages bearing these receptors accumulate. Because of the parallel roles and physical proximity of Mac-1 and uPAR, the capacity of these receptors to functionally interact was explored. Induction of Mac-1 and uPAR expression on monocytic cell lines by transforming growth factor- beta 1 and 1.25-(OH)2 vitamin D3 conferred urokinase and uPAR-dependent adhesion to vitronectin, which was further promoted by engagement of Mac-1. Vitronectin attachment promoted subsequent Mac-1-mediated fibrinogen degradation threefold to fourfold. In contrast, enhancement of uPAR occupancy by exogenous urokinase or receptor binding fragments thereof inhibited Mac-1 function. Addition of urokinase progressively inhibited Mac-1-mediated fibrinogen binding and degradation (maximal inhibition, 91% +/- 14% and 72% +/- 15%, respectively). Saturation of uPAR with urokinase also inhibited binding of the procoagulant Mac-1 ligand, Factor X. These inhibitory effects of uPAR were reproduced in fresh monocytes, cultured monocytic cells, and in Chinese hamster ovary (CHO) cells transfected with both human Mac-1 and human uPAR. These data show that the procoagulant and fibrinolytic potential of monocytic cells is co-ordinately regulated by ligand binding to both Mac-1 and uPAR and identify uPAR as a regulator of integrin function. Vitronectin-enhanced fibrin(ogen) turnover by Mac-1 may operate as a salvage pathway in the setting of urokinase and plasmin inhibitors to promote clearance of the provisional matrix and subsequent healing.
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An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: I. The roles of minor H antigens and endotoxin.
Idiopathic pneumonia syndrome (IPS) refers to diffuse, non-infectious pneumonia that occurs after allogeneic bone marrow transplantation (BMT). We have developed a model of IPS using a well-characterized murine BMT system (B10.BR-->CBA) in which lung injury after BMT can be induced by minor histocompatibility (H) antigenic differences between donor and host. Lung pathology and broncho-alveolar lavage (BAL) fluid were analyzed in transplant recipients before and after both syngeneic and allogeneic BMT. At 2 weeks after BMT, no specific pathologic abnormalities were noted; at 6 weeks, both pneumonitis and mononuclear cell infiltration around vessels and bronchioles were observed only in mice receiving allogeneic BMT. This injury was associated with elevated BAL fluid levels of endotoxin (lipopolysaccharide [LPS]), neutrophils, and tumor necrosis factor alpha. No pathologic organisms were isolated from the respiratory tract of any animal. We also tested the role of endotoxin in the development of this injury. Injection of LPS 6 weeks after transplantation caused profound lung injury only in mice with moderate graft-versus-host disease; dramatic increases in BAL neutrophils and tumor necrosis factor alpha were observed, with alveolar hemorrhage occurring in 4 of 12 of these mice but in no other group. We conclude that (1) this murine BMT system is a potentially useful model of clinical IPS; (2) minor H differences between donor and recipient can be important stimuli in the pathogenesis of IPS; and (3) endotoxin in BAL fluid is associated with lung injury, and excess endotoxin can cause the development of alveolar hemorrhage in this model.
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Characterization of murine lung interstitial macrophages in comparison with alveolar macrophages in vitro.
The present study was performed to characterize the immunologic potential of interstitial macrophages (INT) in comparison with alveolar macrophages (AL). The data showed that AL, compared with INT, have a more efficient phagocytic potential. In addition, they have a strong microbicidal activity and secrete large amounts of reactive oxygen radicals, nitric oxides, TNF, and IFN on appropriate stimulation. They also exert strong tumoricidal and parasiticidal activities. In contrast, INT are more efficient in releasing immunoregulatory cytokines such as IL-1 and IL-6. As determined by Ab staining, INT express more MHC class II molecules and are more effective in functioning as accessory cells for mitogen-stimulated lymphocyte proliferation compared with AL. Thus, AL appear to be particularly effective as nonspecific first line defense cells against infectious agents, whereas INT are equipped to cooperate with interstitial lymphocytes in inducing a specific immune reaction.
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Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells.
The endothelial isoform of nitric oxide synthase (eNOS) modulates cardiac myocyte function and is expressed in the particulate subcellular fraction. We have previously shown that eNOS is targeted to plasmalemmal caveolae in endothelial cells. Caveolae, specialized domains of the plasma membrane, may serve to sequester signaling proteins; a family of transmembrane proteins, the caveolins, form a key structural component of these microdomains. Caveolae in cardiac tissues contain the muscle-specific isoform caveolin-3, and caveolae in endothelial cells contain the widely expressed isoform caveolin-1, which shares limited sequence identity with caveolin-3. Our immunohistochemical analyses of rat cardiac muscle used isoform-specific caveolin antibodies to reveal prominent caveolin-3 staining in myocyte sarcolemmal membranes and at intercalated discs, whereas caveolin-1 staining was prominent in the vascular endothelium. Caveolin or eNOS antibodies were utilized to immunoprecipitate cardiac myocyte or cultured aortic endothelial cell lysates, which then were analyzed in immunoblots. In endothelial cells, we found that eNOS is quantitatively immunoprecipitated by antibodies to caveolin-1. In cardiac myocyte lysates, nearly all the eNOS is immunoprecipitated instead by antibodies to caveolin-3 and, conversely, eNOS antiserum immunoprecipitated primarily caveolin-3. These studies establish expression of eNOS in cardiac myocyte caveolae and document tissue-specific and quantitative associations of eNOS with caveolin. These findings may have important implications for the regulation of eNOS by caveolin isoforms and by other signaling proteins targeted to caveolae.
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