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CARDIOVASCULAR

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Department of Physiology, Medical College of Georgia, Augusta, Georgia

Received January 12, 2005; accepted March 17, 2005.

	Abstract

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Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMP- and cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), and by 8-bromo-cGMP was impaired in the presence of methyl--cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl--cyclodextrin. Methyl--cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

eNOS has been shown to be localized to small invaginations of the plasma membrane called caveolae (Feron et al., 1996; Garcia-Cardena et al., 1996; Shaul et al., 1996). Biochemically, caveolae represent a plasma membrane subdomain enriched with cholesterol, glycosphingolipids, and the structural proteins caveolins that interact, in vitro, with a variety of signal-transducing molecules. Caveolin-1, a member of the protein caveolin family, is the major coat protein of caveolae (Anderson, 1998).

eNOS catalyzes the production of NO in response to a rise in endothelial intracellular calcium concentration (Sanders et al., 2000). The increase in the intracellular calcium promotes the dissociation of eNOS from the protein caveolin-1, necessary to its activation (Feron et al., 1996; Gratton et al., 2000). This suggests that caveolae may function as a site of integration of events linking extracellular stimuli and intracellular effectors. Caveolae exist in most cell types and are particularly abundant in endothelial cells and smooth muscle cells (Galbiati et al., 1998; Voldstedlund et al., 2001). The absence of this organelle impairs NO and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone (Blair et al., 1999; Darblade et al., 2001; Je et al., 2004).

Cholesterol depletion leads to a marked decline in acetylcholine (ACh)-induced eNOS activation and smooth muscle relaxation (Blair et al., 1999; Darblade et al., 2001). ACh-induced relaxation in rat aorta is mainly attributed to NO (Shimokawa et al., 1996). Colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation. It has been shown that NO is more soluble in a hydrophobic environment than in water (Goss et al., 1999), making us hypothesize that downstream molecules in the NO cascade must be in close relationship to the endothelial caveolae in the membrane.

The exciting possibility that caveolae provide a platform for interactions between eNOS activation and smooth muscle relaxation is emerging. This study tested the hypothesis that endothelial caveolae are the platform for sGC and PKG activation contributing to rat aortic smooth muscle relaxation. Because cAMP-dependent protein kinase (PKA) also can be activated by cGMP (Li et al., 2003), thereby contributing to smooth muscle relaxation, localization of PKA to endothelial caveolae was also evaluated.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (200–224 g) were maintained on a 12-h light/dark cycle with rat chow and water ad libitum and were anesthetized with 50 mg/kg sodium pentobarbital, and the thoracic aorta was excised. All experiments were conducted in accordance with the Medical College of Georgia's Animal Use for Research and Education Committee.

In Vitro Measurement of Isometric Force Generation in Aortic Rings. After removal of fat and connective tissue, aortic rings (4 mm in length) were mounted in an organ chamber for isometric tension recordings and bathed in physiological salt solution (PSS) of the following composition: 130.0 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.6 mM CaCl₂, 14.9 mM NaHCO₃, 0.03 mM EDTA, and 5.5 mM glucose, which was maintained at 37°C and bubbled with 95% O₂, 5% CO₂. Some of the experiments were performed in denuded aortas to avoid the interference of endothelial factors and endothelial caveolae. The endothelium was mechanically removed by gently rubbing the intimal surface with stainless steel wires. The aortic rings were set at 3.5- to 4.0-g passive tension. Under this tension, optimal contractile responses were previously observed in rat aorta. After a 1-h equilibration period, vessels were contracted with 0.1 µM phenylephrine (Sigma-Aldrich, St. Louis, MO) and subsequently treated with 10 µM ACh (Sigma-Aldrich) to test tissue viability and the presence or absence of endothelium. The failure of ACh to relax denuded aortic rings was considered proof of endothelium disruption. Endothelium-intact aortic rings were considered as those that developed more than 70% of relaxation to ACh. The vessels were rinsed, and phenylephrine was added again until reproducible contractions were obtained. Methyl--cyclodextrin (1, 6, or 10 mM; Sigma-Aldrich) was added 60 min before phenylephrine. Control responses were obtained in experiments where methyl--cyclodextrin was replaced by vehicle (PSS). The contractile response induced by phenylephrine in 10 mM methyl--cyclodextrin-treated aortic segments did not differ significantly from those not treated with methyl--cyclodextrin [maximal force development: (endothelium-intact: 1453 ± 110 mg, n = 26, control versus 1626 ± 99 mg, n = 15, after methyl--cyclodextrin; P > 0.05) (endothelium-denuded: 1537 ± 56 mg, n = 27, control versus 1566 ± 72 mg, n = 15, after methyl--cyclodextrin; P > 0.05)].

To examine the effects of methyl--cyclodextrin on the endothelium-dependent relaxation induced by ACh, concentration-effect curves to ACh (10 nM–0.5 mM) were constructed in endothelium-intact rat aortic rings precontracted with 0.3 µM phenylephrine in the presence and absence of methyl--cyclodextrin (1, 6, and 10 mM, 60 min). The effects of methyl--cyclodextrin on the relaxation induced by YC-1 (Sigma-Aldrich), 8-bromo-cGMP (8-Br-cGMP) (Sigma-Aldrich), and nifedipine (Sigma-Aldrich) were also investigated. Concentration-effect curves to nifedipine (1 nM–50 µM) were constructed in endothelium-intact aortic rings precontracted with 0.3 µM phenylephrine in the presence and absence of 10 mM methyl--cyclodextrin (60 min). Concentration-effect curves to YC-1 (1 nM to 30 µM) and 8-Br-cGMP (0.1 µM–0.3 mM) were constructed in endothelium-intact and -denuded rat aortic rings precontracted with phenylephrine (0.3 and 0.1 µM, respectively) in the presence and absence of 10 mM methyl--cyclodextrin (60 min). Phenylephrine was used in these concentrations to achieve the same level of force development in endothelium-intact and -denuded aortic rings.

Immunohistochemistry. Endothelium-intact and -denuded rat aortic segments isolated in PSS were embedded in OCT compound (Sakura Finetek USA, Inc., Torrance, CA) in the presence and absence of 10 mM methyl--cyclodextrin (60 min). Fresh-frozen sections (8 µm in thickness) were thaw-mounted onto precleaned glass slides (Fisher Scientific Co., Pittsburgh, PA) and kept overnight in a desiccator at 4°C. After washing in phosphate-buffered saline (PBS) and fixation in acetone, slides were blocked with 10% normal goat serum (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) in PBS for 30 min at room temperature. The slices were then incubated with mouse monoclonal anti-caveolin-1 (Research Diagnostics, Flanders, NJ; final dilution 1:500) and rabbit polyclonal anti-sGC (1 subunit) (Cayman Chemical, Ann Arbor, MI; final dilution 1:500), or 4 µg/ml anti-PKA (Abcam, Inc., Cambridge, MA), or 4 µg/ml anti-PKG (Abcam, Inc.) antibodies. After washing in PBS, the fluorescent secondary antibodies, goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (Molecular Probes, Eugene, OR; final dilution 1:1000), were applied and incubated for 1 h at room temperature. After washing in PBS, the slides were coverslipped with anti-fading mounting medium (Gel/Mount medium; Biomeda, Foster City, CA) and allowed to desiccate overnight at 4°C. Sections were viewed by confocal microscopy (Carl Zeiss, Thornwood, NY).

Electron Microscopy. Endothelium-intact aortic segments treated with 10 mM methyl--cyclodextrin or vehicle (PSS) for 1 h were fixed overnight at 4°C in a solution consisting of 2% glutaraldehyde, 2% paraformaldehyde dissolved in 0.1 mol/l sodium cacodylate buffer containing sucrose. Samples were washed with sodium cacodylate buffer (0.1 mol/l) and then postfixed in 4% osmium tetroxide for 1 h. Samples were dehydrated through a graded series of alcohol and embedded in Epon 812 with araldite. Ultrathin sections were double stained with alcoholic uranyl acetate and lead citrate and examined with a JEOL-1010 transmission electron microscope.

Data Analysis. Sensitivity was expressed as pD₂ = -log EC₅₀ calculated by nonlinear regression. Data were analyzed by Student's t test for paired comparisons. Additional statistical analyses were performed using one-way analysis of variance followed by the Student-Newman-Keuls post hoc test for multiple comparisons. A value of P < 0.05 was considered statistically significant. Isometric force generation data were measured as a contraction in milligrams or as a percentage of phenylephrine-induced tone and expressed as mean ± S.E.M.

	Results

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Effect of Methyl--Cyclodextrin on the Relaxing Response to Acetylcholine. ACh induced concentration-dependent relaxation of phenylephrine-induced contraction (pD₂ = 6.06 ± 0.4; n = 10) (Fig. 1). At a concentration of 1 mM, methyl--cyclodextrin (60 min) had no effect on ACh-induced relaxation (pD₂ = 6.6 ± 0.16; n = 6; P > 0.05) (Fig. 1), whereas prior exposure of tissues to 6 mM methyl--cyclodextrin (60 min) inhibited the relaxation induced by 1 µM ACh (56.7 ± 6.4%, n = 10, control versus 32.4 ± 7.3%, n = 4, after methyl--cyclodextrin; P < 0.05). Furthermore, 10 mM methyl--cyclodextrin (60 min) inhibited ACh-induced relaxation to a greater extent and significantly decreased the maximal response to ACh (E_max = 95.6 ± 5.4%; n = 10, control versus 61.0 ± 9%, n = 6, after methyl--cyclodextrin; P < 0.01) (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Methyl--cyclodextrin inhibits ACh-induced relaxation in a concentration-dependent way. Endothelium-intact aortic rings treated for 1 h with 1, 6, and 10 mM methyl--cyclodextrin (CD) or vehicle (control) were contracted with 0.3 mM phenylephrine to the plateau phase and then treated with increasing concentrations of ACh (10 nM–0.5 mM). Data are expressed as mean ± S.E.M. of the percentage of relaxation of phenylephrine-induced force generation. *, P < 0.05; **, P < 0.01 versus control; n 4.

Effect of Methyl--Cyclodextrin on the Relaxing Response to YC-1. The relaxation induced by YC-1 was inhibited in the presence of 10 mM methyl--cyclodextrin (60 min) in endothelium-intact and -denuded rat aortic rings (Fig. 2). YC-1-induced relaxation in endothelium-intact (pD₂ = 6.6 ± 0.14; n = 4) was significantly more inhibited in the presence of 10 mM methyl--cyclodextrin (pD₂ = 5.31 ± 0.08; n = 4; P < 0.001) (Fig. 2, top) than in endothelium-denuded aortic rings (pD₂ = 5.8 ± 0.01, n = 4, control versus pD₂ = 5.3 ± 0.1, n = 4, after methyl--cyclodextrin; P < 0.01) (Fig. 2, bottom). Methyl--cyclodextrin had no effect on nifedipine-induced relaxation (E_max = 82.4 ± 4.7%, pD₂ = 6.9 ± 0.5, n = 6, control versus E_max = 81.0 ± 2.2%, pD₂ = 7.0 ± 0.3, n = 6, after methyl--cyclodextrin; P > 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Methyl--cyclodextrin inhibits YC-1-induced relaxation. Endothelium-intact (top) and -denuded (bottom) aortic rings treated for 1 h with 10 mM methyl--cyclodextrin (CD) or vehicle (control) were contracted with phenylephrine to the plateau phase and then treated with increasing concentrations of YC-1 (1 nM–30 µM). Data are expressed as mean ± S.E.M. of the percentage of relaxation of phenylephrine-induced force generation. **, P < 0.01; ***, P < 0.001 versus control; n = 4.

View larger version (57K): [in this window] [in a new window]   Fig. 3. Caveolin-1 and sGC localization in rat aorta. Endothelium-intact and -denuded rat aortic sections were immunolabeled with antibodies against caveolin-1 (Cav-1) and sGC, as indicated in the figure, and visualized by confocal microscopy. Areas of colocalization (merge) of caveolin-1 and sGC results in yellow. Colocalization of caveolin-1 and sGC observed in the endothelium is absent when the endothelium was removed. Tissue autofluorescence (background) is represented by the signal obtained with the fluorescence secondary antibodies alone. The results are representative of three to four separate experiments.

Effect of Methyl--Cyclodextrin on 8-Br-cGMP-Induced Relaxation. The sensitivity to 8-Br-cGMP was not changed in the presence of 10 mM methyl--cyclodextrin (60 min) in endothelium-intact (pD₂ = 4.3 ± 0.4, n = 8, control versus 4.4 ± 0.1, n = 4, after methyl--cyclodextrin; P > 0.05) and in endothelium-denuded (pD₂ = 4.4 ± 0.1, n = 10, control versus 4.4 ± 0.3, n = 6, after methyl--cyclodextrin; P > 0.05) aortic rings. However, methyl--cyclodextrin significantly impaired the relaxation induced by 10 µM 8-Br-cGMP in endothelium-intact aortic rings (Fig. 4, top), whereas this inhibitory effect was not observed in endothelium-denuded aortic rings (Fig. 4, bottom).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Methyl--cyclodextrin inhibits 8-Br-cGMP-induced relaxation in endothelium-intact rat aortic rings. Endothelium-intact (top) and -denuded (bottom) aortic rings treated for 1 h with 10 mM methyl--cyclodextrin (CD) or vehicle (control) were contracted with phenylephrine to the plateau phase and then treated with increasing concentrations of 8-Br-cGMP (0.1 µM–0.3 mM). Data are expressed as mean ± S.E.M. of the percentage of relaxation of phenylephrine-induced force generation. **, P < 0.01 versus control; n 4.

View larger version (41K): [in this window] [in a new window]   Fig. 5. PKG and PKA colocalization with caveolin-1 in rat aorta. Endothelium-intact and/or endothelium-denuded rat aortic sections were immunolabeled with antibodies against caveolin-1 (Cav-1) and PKG or PKA, as indicated in the figure, and visualized by confocal microscopy. Yellow indicates areas of colocalization of caveolin-1 with PKG or PKA. The results are representative of three to four separate experiments.

Effects of Methyl--Cyclodextrin on Endothelial Caveolae.

View larger version (106K): [in this window] [in a new window]   Fig. 6. Effect of methyl--cyclodextrin on endothelial caveolar structure in rat aortic endothelium. Endothelium-intact rat aortic segments treated with vehicle (A) or 10 mM methyl--cyclodextrin (B) for 60 min were evaluated by transmission electron microscopy. Electron micrograph of the endothelium from aorta showing caveolar structure in A that is disrupted in B. Original magnification, 46,000x.

	Discussion

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In the present study, we observed impaired relaxation stimulated with YC-1 and 8-Br-cGMP in endothelium-intact rat aortic rings preciously treated with the cholesterol sequester methyl--cyclodextrin. We also observed colocalization of sGC, PKG, and PKA with caveolin-1. These results provide evidence of compartmentalization of signaling molecules of the NO cascade involved in smooth muscle relaxation in endothelial caveolae.

Cholesterol-rich membrane domains may possibly be considered as physical platforms for the coding of intracellular signals. Caveolae are small membrane invaginations, rich in cholesterol, involved in signal transduction by ensuring the compartmentalization of several signaling proteins (Doyle et al., 1997; Oh and Schnitzer, 2001; Liu et al., 2002).

Cyclodextrin, a water-soluble cyclic oligosaccharide formed of seven glucopyranose units able to accept one molecule of cholesterol in its hydrophobic core, is a membrane-impermeable molecule that depletes cellular cholesterol content through solubilization of the plasmalemmal cholesterol (Kilsdonk et al., 1995). This cholesterol-binding agent has been efficiently used as a pharmacological tool to study the role of caveolae in vascular reactivity (Dreja et al., 2002; Kaiser et al., 2002; Je et al., 2004). Indeed, in our experiments we observed by electron microscopy that endothelial caveolae were disassembled after exposure of aortic rings to methyl--cyclodextrin.

Some agonists causing contraction of vascular smooth muscle act on receptors that are believed to be located in caveolae or to aggregate in caveolae upon ligand binding (Chun et al., 1994; de Weerd and Leeb-Lundberg, 1997; Ishizaka et al., 1998; Je et al., 2004). We observed in this study that methyl--cyclodextrin did not change smooth muscle contractile responses to phenylephrine, indicating that this -adrenergic agonist does not require signaling molecules in cholesterol-rich domains to cause contraction. A similar observation was also reported in tail artery (Dreja et al., 2002).

Atherosclerosis, as well as other pathophysiological states, is linked to functional abnormalities of the endothelium. One major aspect of endothelial dysfunction is a reduction of the endothelium-dependent vasodilation induced by ACh. Any decrease in NO bioavailability has major consequences, due to loss of the protective properties of NO against thrombosis and vasospasm. It has been shown that in hypercholesterolemic rabbits, endothelial dysfunction is associated with a decrease in the amount of caveolae in the endothelium (Darblade et al., 2001).

We observed that the relaxation induced by ACh is impaired after treatment with methyl--cyclodextrin, suggesting localization of eNOS in rat aorta endothelial caveolae. Similar results were reported in rabbit aorta (Darblade et al., 2001), whereas in guinea pig aorta, treatment with methyl--cyclodextrin abolished the relaxation induced by ACh (Kaiser et al., 2002). It has been reported that caveolar cholesterol depletion is associated with a marked decline in ACh-induced eNOS activation (Blair et al., 1999). As far as protein composition, caveolae are characterized by the presence of a family of three proteins called caveolins. Caveolin-1 is surely the most important protein involved in the structure and function of caveolae (Anderson, 1998). eNOS is regulated by caveolin-1. By its interaction with caveolin-1, eNOS is maintained in an inactive state (Garcia-Cardena et al., 1996; Ju et al., 1997). Upon stimulation followed by an increase in intracellular calcium concentration, eNOS dissociates from caveolin-1, a necessary step for its activation and NO production (Feron et al., 1996; Gratton et al., 2000). Caveolae are therefore regulatory platforms for the cascade of events resulting in NO production.

Although the natural receptor for NO, sGC has been detected in association with the plasma membrane (Zabel et al., 2002; Venema et al., 2003), and sGC is translocated to caveolar domains to be sensitized by NO (Zabel et al., 2002); the interaction with caveolin-1 and the role of this association with the plasma membrane in blood vessel relaxation are unknown. We observed that the relaxation induced by the sGC activator YC-1 is impaired in the presence of methyl--cyclodextrin in rat aorta. However, methyl--cyclodextrin had no effect on the relaxation induced by nifedipine, indicating that a cGMP-independent relaxing pathway is not altered by methyl--cyclodextrin. We also observed colocalization of caveolin-1 with sGC in aorta endothelium that was abolished in the presence of methyl--cyclodextrin. Caveolar association of sGC has been reported to be sensitive to detergent (Zabel et al., 2002), indicating that this membrane association is labile. It is interesting to note that we did not make use of detergents in our procedures, supporting the concept that the site of colocalization is the membrane. These findings established the compartmentalization of sGC to caveolae due to its association with caveolin-1 in the endothelium introducing a potential therapeutic strategy for cardiovascular diseases related to endothelial dysfunction.

We also observed that the relaxation induced by 8-Br-cGMP is impaired in endothelium-intact aortic rings previously treated with methyl--cyclodextrin. The differences between the inhibition by methyl--cyclodextrin to YC-1- and 8-Br-cGMP-induced relaxation may be related to the additional effects attributed to YC-1. YC-1 has been shown to not only activate sGC but also to potentiate the stimulatory action of NO (Friebe and Koesling, 1998; Schmidt et al., 2001). YC-1 also affects cGMP metabolism, inhibiting cGMP breakdown and the activity of phosphodiesterase (Galle et al., 1999), and it stimulates NO synthesis and release in endothelial cells independently of elevation in cGMP levels (Wohlfart et al., 1999).

cGMP generated upon sGC activation in the microdomain underlying endothelial caveolae may activate PKG. Indeed, we observed colocalization of caveolin-1 and PKG in rat aorta endothelium. Cross-talk between cGMP and cAMP has been reported (Dhanakoti et al., 2000; White et al., 2000). cGMP can also activate PKA (Li et al., 2003).

It was recently reported that membrane cholesterol content regulates PKA activity (Burgos et al., 2004) and that the catalytic subunit of PKA was associated with cell surface caveolae (Heijnen et al., 2004). In our study, we observed colocalization of caveolin-1 and PKA in rat aortic endothelium prevented after cholesterol depletion with methyl--cyclodextrin.

Recently, it was reported that 8-Br-cGMP mediates relaxation of tracheal smooth muscle through PKA activation (Algara-Suarez and Espinosa-Tanguma, 2004). Unpublished data from our laboratory indicate similar results in rat aorta. Together, these results indicate that cGMP activates PKA in rat aortic endothelial caveolae contributing to smooth muscle relaxation. It has been shown that eNOS activity can be modulated by cyclic nucleotide-elevating vasodilators (Zou et al., 2002; Kaufmann et al., 2003; Schafer et al., 2003). Of the known phosphorylation sites on eNOS responsible for its activation, serine 1179 has been characterized most extensively (Chen et al., 1999; Fulton et al., 2002). Serine 1179 phosphorylation by PKA leads to activation of eNOS (Butt et al., 2000). We hypothesize that the mechanisms by which NO-cGMP-PKA pathway in the endothelium mediates vascular smooth muscle relaxation involve NO production through eNOS phosphorylation by PKA. The role of the association of PKG with caveolin-1 in rat aortic endothelial caveolae remains unclear and requires further investigation.

In summary, we propose that sGC and PKA are compartmentalized in endothelial caveolae as a necessary spatial organization to facilitate NO actions. Our results provide new insight by which the endothelium mediates vascular smooth muscle relaxation.

	Acknowledgements

 
We thank Darren R. Baker and Penny Roon for valuable technical assistance in the confocal and transmission electron microscopy, respectively.

	Footnotes

 
This work was supported by National Institutes of Health Grants HL71138, HL74167 (to R.C.W.), and DC-005811 (to L.P.M.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.083634.

ABBREVIATIONS: PKG, cGMP-dependent protein kinase; GC, guanylyl cyclase; sGC, soluble guanylyl cyclase; pGC, particulate guanylyl cyclase; NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; ACh, acetylcholine; PKA, cAMP-dependent protein kinase; PSS, physiological salt solution; YC-1, 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole; 8-Br-cGMP, 8-bromo-cGMP; PBS, phosphate-buffered saline; Cav-1, caveolin-1.

Address correspondence to: Dr. A. Elizabeth Linder, Medical College of Georgia, Department of Physiology (CA-3101), 1120 Fifteenth St., Augusta, GA 30912-3000. E-mail: elinder{at}mcg.edu

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