Oxidized Low Density Lipoprotein Displaces Endothelial Nitric-oxide Synthase (eNOS) from Plasmalemmal Caveolae and Impairs eNOS Activation
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.32512
ISSN1083-351X
AutoresAlison Blair, Philip W. Shaul, Ivan S. Yuhanna, Patricia A. Conrad, Eric J. Smart,
Tópico(s)Ion Transport and Channel Regulation
ResumoHypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 μg/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis. Hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by a decrease in the bioavailability of endothelium-derived nitric oxide. Endothelial nitric-oxide synthase (eNOS) associates with caveolae and is directly regulated by the caveola protein, caveolin. In the present study, we examined the effects of oxidized low density lipoprotein (oxLDL) on the subcellular location of eNOS, on eNOS activation, and on caveola cholesterol in endothelial cells. We found that treatment with 10 μg/ml oxLDL for 60 min caused greater than 90% of eNOS and caveolin to leave caveolae. Treatment with oxLDL also inhibited acetylcholine-induced activation of eNOS but not prostacyclin production. oxLDL did not affect total cellular eNOS abundance. Oxidized LDL also did not affect the palmitoylation, myristoylation or phosphorylation of eNOS. Oxidized LDL, but not native LDL, or HDL depleted caveolae of cholesterol by serving as an acceptor for cholesterol. Cyclodextrin also depleted caveolae of cholesterol and caused eNOS and caveolin to translocate from caveolae. Furthermore, removal of oxLDL allowed eNOS and caveolin to return to caveolae. We conclude that oxLDL-induced depletion of caveola cholesterol causes eNOS to leave caveolae and inhibits acetylcholine-induced activation of the enzyme. This process may be an important mechanism in the early pathogenesis of atherosclerosis. nitric oxide nitric-oxide synthase(s) low density lipoprotein oxidized LDL endothelial NOS high density lipoprotein polyacrylamide gel electrophoresis thiobarbituric acid reactive substances phosphate buffer phosphate-buffered saline native LDL porcine pulmonary artery endothelial lipoprotein-deficient serum protein kinase C Studies in animal models and in humans have shown that hypercholesterolemia-induced vascular disease and atherosclerosis are characterized by an early, selective impairment of endothelium-derived relaxation. This impairment is due to a decrease in bioavailable endothelium-derived nitric oxide (NO)1 (1Vane J.R. Anggard E.E. Botting R.M. N. Engl. J. Med. 1990; 323: 27-36Crossref PubMed Scopus (1771) Google Scholar). In the initial phase of the disease process, there is impaired responsiveness to receptor-dependent stimuli, such as acetylcholine, whereas responsiveness to receptor-independent stimuli such as the calcium ionophore A23187 is not altered. As such, the early pathogenesis involves attenuated endothelial NO production in response to extracellular stimuli, but the capacity for maximal enzyme activation and the breakdown of NO are not affected. As the disease progresses, there is nonspecific inhibition of NO bioavailability. Although this later inhibition is most likely multifactorial in origin, it is at least partly due to enhanced inactivation of NO by superoxide anions (2Cohen R.A. Prog. Cardiovasc. Dis. 1995; 38: 105-128Crossref PubMed Scopus (261) Google Scholar, 3Harrison D.G. Basic Res. Cardiol. 1994; 89 Suppl. 1: 87-102PubMed Google Scholar, 4Flavahan N.A. Circulation. 1992; 85: 1927-1938Crossref PubMed Scopus (443) Google Scholar). These processes result in increased neutrophil adherence to the endothelium, thereby promoting a key step in the pathogenesis of atherosclerosis (5Lefer A.M. Ma X.L. Arterioscler. Thromb. 1993; 13: 771-776Crossref PubMed Scopus (150) Google Scholar). The chronic inhibition of NO synthesis in rabbit models of hypercholesterolemia accelerates the development of vascular dysfunction and intimal lesions, providing additional evidence that the impairment of NO synthesis promotes atherogenesis (6Cayatte A.J. Palacino J.J. Horten K. Cohen R.A. Arterioscler. Thromb. 1994; 14: 753-759Crossref PubMed Scopus (464) Google Scholar). In vitro investigations have further shown that oxidized LDL (oxLDL) inhibits NO-mediated responses. Antioxidants reduce both the formation of free radicals and the oxidative modification of LDL that lead to impaired NO-related responses (7Plane F. Jacob M. McManus D. Bruckdorfer K.R. Atherosclerosis. 1993; 103: 73-79Abstract Full Text PDF PubMed Scopus (40) Google Scholar). Thus, NO is critically involved in the pathogenesis of atherosclerosis, and the initiating events are characterized by attenuated endothelial NO production in response to extracellular stimuli. A family of nitric-oxide synthases (NOS) are responsible for NO synthesis (8Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). Inducible NOS and neuronal NOS are primarily cytosolic enzymes, whereas the constitutive or endothelial NOS (eNOS) is primarily membrane-bound (8Griffith O.W. Stuehr D.J. Annu. Rev. Physiol. 1995; 57: 707-736Crossref PubMed Google Scholar). Endothelial NOS is both myristoylated and palmitoylated (9Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 10Robinson L.J. Busconi L. Michel T. J. Biol. 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Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar) reported that the caveola protein, caveolin, directly binds eNOS, thereby regulating the generation of NO. Interestingly, Sessa and colleagues (12Liu J. Hughes T.E. Sessa W.C. J. Cell Biol. 1997; 137: 1525-1535Crossref PubMed Scopus (157) Google Scholar, 13Sessa W.C. Garcia-Cardena G. Liu J. Keh A. Pollock J.S. Bradley J. Thiru S. Braverman I.M. Desai K.M. J. Biol. Chem. 1995; 270: 17641-17644Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) demonstrated that eNOS also associates with Golgi membranes in an acylation-dependent manner. The biological function of Golgi-localized eNOS and the relationship between Golgi and caveola pools of the enzyme has not been explored. The caveola microdomains to which eNOS is targeted are found in most cells. Caveolae are highly enriched in cholesterol, sphingomyelin, glycolipids, and known signal transducing molecules (14Smart E.J. Ying Y.-Y. Conrad P.A. Anderson R.G.W. J. Cell Biol. 1994; 127: 1185-1197Crossref PubMed Scopus (377) Google Scholar, 15Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar, 16Liu P. Anderson R.G.W. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Caveolae contain complete signaling complexes from extracellular receptors to intracellular effector systems. For instance, caveolae are enriched with the following (for a review, see Ref. 17Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1719) Google Scholar): heterotrimeric GTP binding proteins; protein kinase C; receptors for endothelin 1 and acetylcholine; an ATP-dependent Ca2+ pump and a form of the inositol 1,4,5-triphosphate-sensitive Ca2+channel; and the small GTP-binding protein Ras. Recent studies further indicate that caveolae play a role not only in the compartmentalization of signaling molecules, but also in the modulation and integration of various responses to extracellular stimuli (11Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 18Feron O. Michel J.B. Sase K. Michel T. Biochemistry. 1998; 37: 193-200Crossref PubMed Scopus (116) Google Scholar, 19Feron O. Smith T.W. Michel T. Kelly R.A. J. Biol. Chem. 1997; 272: 17744-17748Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). In the present study, we hypothesized that oxLDL inhibits eNOS activation by disrupting the association of eNOS with caveolae. We examined the effects of oxLDL on the subcellular location of eNOS, eNOS activation, and caveola cholesterol. The present study demonstrates that oxLDL depletes caveolae of cholesterol and causes eNOS to translocate from the plasma membrane. In addition, oxLDL inhibits the ability of acetylcholine to efficiently activate the enzyme. M199, BME vitamin mix, fetal bovine serum, glutamine, trypsin-EDTA, OptiPrep, endothelial SFM growth medium, and penicillin/streptomycin were from Life Technologies, Inc. The BME amino acid mix, Percoll, and fluorescently labeled 2° antibodies were from Sigma. The analytical silica gel thin layer chromatography plates, heptane, petroleum ether, ethyl ether, acetic acid, and 2-propanol were from Fisher. The lipid standards (cholesterol, sphingomyelin, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine) were from Supelco (Bellefonte, PA). [3H]Acetate (specific activity, 5.21 Ci/mmol), [3H]palmitate (specific activity, 50 Ci/mmol), [3H]myristate (specific activity, 41 Ci/mmol), [3H]arginine (specific activity, 53 Ci/mmol), and [32P]orthophosphate (specific activity, 9000 Ci/mmol) were from DuPont. The anti-caveolin IgG (caveolin-1), anti-PKCα IgG, and anti-eNOS IgG were from Transduction Laboratories (Lexington, KY). The cholesterol determination kit was from Wako (Richmond, VA). Fluoromount G was from Southern Biotechnology Associates, Inc. (Birmingham, AL). Human lipoprotein-deficient serum was prepared as described (20Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Crossref PubMed Scopus (1278) Google Scholar). Sample buffer (5×) consisted of 0.31 mTris, pH 6.8, 2.5% (w/v) SDS, 50% (v/v) glycerol, and 0.125% (w/v) bromphenol blue. TBS consisted of 20 mm Tris, pH 7.6, and 137 mm NaCl. Blotting buffer consisted of TBS plus 0.5% Tween 20 and 5% dry milk. Wash buffer consisted of TBS plus 0.5% Tween 20 and 0.2% dry milk. Tris-saline consisted of 50 mmTris (pH 7.4), 150 mm NaCl. Low passage (<5) porcine pulmonary artery endothelial cells were kindly provided by Dr. Bernhard Hennig (University of Kentucky). The cells were cultured in a monolayer and plated in a standard format. On day 0, approximately 5 × 105 cells were seeded into 100-mm dishes with 10 ml of M199 medium supplemented with 100 units/ml penicillin/streptomycin, 0.5% (v/v) l-glutamine, BME vitamin mix (1 ml/100 ml of M199), BME amino acid mix (1 ml/100 ml of M199), and 10% (v/v) fetal bovine serum. The cells were used on day 6. To radiolabel endogenous lipids, the medium on day 5 was changed to M199 plus 20 mm HEPES (pH 7.4), adding [3H]acetate (30 μCi/dish), and incubating for 18 h at 37 °C. LDL (d = 1.019–1.05 g/ml) and HDL (d = 1.063–1.21 g/ml) were isolated from fresh human plasma by density gradient ultracentrifugation as described previously (21Fisher W.R. Schumaker V.N. Methods Enzymol. 1986; 128: 247-262Crossref PubMed Scopus (30) Google Scholar, 22Kelley J.L. Kruski A.W. Methods Enzymol. 1986; 128: 170-181Crossref PubMed Scopus (87) Google Scholar). The HDL3 subfraction (d = 1.13–1.18 g/ml) was isolated from other HDL subfractions using a density gradient fractionator (ISCO). The HDL3 subfraction was used for all of the experimental treatments described herein. The purity of each lipoprotein fraction was assayed by SDS-PAGE and Coomassie staining. [3H]cholesteryl linoleate was incorporated into LDL as described previously (23Gwynne J.T. Mahaffee D.D. J. Biol. Chem. 1989; 264: 8141-8150Abstract Full Text PDF PubMed Google Scholar). The specific activity of [3H]cholesteryl linoleate-LDL ranged from 32 to 58 dpm/ng of LDL protein. LDL apolipoproteins were iodinated by the iodine monochloride method (24Bilheimer D.W. Eisenberg S. Levy R.I. Biochim. Biophys. Acta. 1972; 260: 212-221Crossref PubMed Scopus (1185) Google Scholar) to a specific activity of 400–600 cpm/ng LDL protein. Oxidized LDL was prepared by incubating fresh LDL with 10 μm of CuSO4 at 37 °C for 16 h. The material was dialyzed against a sterile solution (150 mmNaCl, 1 mm EDTA, 100 μg/ml polymyxin B, pH 7.4) and then sterilized by filtration. The cholesterol, triglyceride (25Krieger M. McPhaul M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1979; 254: 3845-3853Abstract Full Text PDF PubMed Google Scholar), and protein (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) content of the lipoprotein was determined by standard methods. The homogeneity of each preparation was determined by SDS-PAGE. The extent of LDL oxidation was estimated by agarose gel electrophoresis and by measuring the amount of thiobarbituric acid reactive substances (TBARS) generated with a colorimetric assay for malondialdehyde (27Buege J.A. Aust S.D. Methods Enzymol. 1978; 52: 302-310Crossref PubMed Scopus (10683) Google Scholar). Caveolae membranes were isolated as described previously (28Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar, 29Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). This procedure generates a highly purified plasma membrane microdomain that is free from intracellular markers and bulk plasma membrane markers. This method has been used extensively to characterize caveola membranes (9Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G.W. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 16Liu P. Anderson R.G.W. J. Biol. Chem. 1995; 270: 27179-27185Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 29Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 30Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 31Smart E.J. Mineo C. Anderson R.G.W. J. Cell Biol. 1996; 134: 1169-1177Crossref PubMed Scopus (79) Google Scholar, 32Smart E.J. Ying Y. Donzell W.C. Anderson R.G.W. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Galactosyl transferase, NADPH cytochromec reductase, and alkaline phosphatase were assayed using methods adapted from Graham and Higgins (33Graham J. Higgins J. Methods in Molecular Biology (Walker, J. M., ed). 1993; 19Google Scholar). The amount of protein in each subcellular fraction was determined by a Lowry assay (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE and immunoblots were performed as described previously (28Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar,29Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Endothelial cells grown on glass coverslips were washed once with PBS and then incubated at 37 °C in lipoprotein-deficient serum containing 20 mmHEPES (pH 7.4) and 10 μg/ml oxLDL for 60 min. Control and oxLDL-treated cells were then washed in 0.1 m phosphate buffer (PB) and fixed for 10 min with 4% (w/v) paraformaldehyde in PB. After fixation, the cells were rinsed three times with PB, permeabilized with 0.1% Triton X-100 in PB for 2 min, and rinsed three times in PB. Cells were then incubated for 30 min in a 1° antibody mixture containing polyclonal antibody to caveolin and monoclonal antibody to eNOS made up in PB plus 1% bovine serum albumin. Cells were washed three time with PB and then incubated for 30 min in a 2° antibody mixture consisting of rhodamine isothiocyanate-goat anti-rabbit IgG and fluorescein isothiocyanate-goat anti-mouse IgG made up in PB plus 1% bovine serum albumin. The concentration of individual antibodies in the mixtures was 20 μg/ml. Cells were washed three times with PB and once in distilled water and mounted in Fluoromount G containing 2.5% 1,4-diazabicyclo-(2.2.2)octane as an anti-bleach agent. Cells were examined on a Nikon Eclipse E800 microscope, and fluorescence images were recorded using a Hamamatsu Orca CCD camera. NOS activation was determined in whole endothelial cells by measuring l-[3H]arginine conversion to l-[3H]citrulline using methods previously described (34Davda R.K. Chandler L.J. Crews F.T. Guzman N.J. Hypertension. 1993; 21: 939-943Crossref PubMed Scopus (82) Google Scholar). Subconfluent cells were preincubated in PBS for 60 min at 37 °C. After the preincubation period, the incubation for NOS activity was initiated by aspirating the PBS from the wells and replacing it with 400 μl of PBS containing 0.75 μCi/mll-[3H]arginine. The plates were incubated at 37 °C for 15 min in the absence of exogenous stimulation (basal) or in the presence of varying concentrations of acetylcholine (10−10 to 10−4m). The NOS reaction was terminated by adding 500 μl of ice-cold 1 ntrichloroacetic acid to each well. The preincubation and incubation were performed in the presence of either native LDL (nLDL) or oxLDL at a final concentration of 10 μg/ml. The amount ofl-[3H]citrulline generated was measured as described (34Davda R.K. Chandler L.J. Crews F.T. Guzman N.J. Hypertension. 1993; 21: 939-943Crossref PubMed Scopus (82) Google Scholar). In individual experiments performed in 24-well plates, four wells were used for each treatment group. Findings were confirmed in three independent experiments. NOS activation in the intact cells was completely inhibited by 2 mmnitro-l-arginine methyl ester.l-[3H]Arginine uptake was measured following nLDL or oxLDL pretreatment as done in the experiments evaluating eNOS localization and eNOS activation. Uptake was similar in nLDL- and oxLDL-treated cells. NOS enzymatic activity in cell lysates and subcellular fractions was determined by measuring the conversion of [3H]arginine to [3H]citrulline in the presence of excess amounts of all required substrates and cofactors (34Davda R.K. Chandler L.J. Crews F.T. Guzman N.J. Hypertension. 1993; 21: 939-943Crossref PubMed Scopus (82) Google Scholar). Cells were incubated for determinations of prostacyclin production using methods we have previously described (35Jun S.S. Chen Z. Pace M.C. Shaul P.W. J. Clin. Invest. 1998; 102: 176-183Crossref PubMed Scopus (123) Google Scholar). Briefly, cells grown in 24-well plates were preincubated in RPMI 1640 medium containing either nLDL or oxLDL under the conditions used in the studies of eNOS localization and eNOS activation. Incubations for prostacyclin production were then performed over 15 min in the continued presence of nLDL or oxLDL. Basal (nonstimulated) prostacyclin production and that stimulated by 10−5m bradykinin or 10−5m acetylcholine were evaluated. Samples of incubation medium were assayed for the stable metabolite of prostacyclin, 6-ketoprostaglandin F1α, by radioimmunoassay (35Jun S.S. Chen Z. Pace M.C. Shaul P.W. J. Clin. Invest. 1998; 102: 176-183Crossref PubMed Scopus (123) Google Scholar). In all experiments, n = 4 for each determination, and findings were replicated in three independent studies. Biosynthetic labeling with [3H]palmitate, [3H]myristate, and [32P]orthophosphate was carried out exactly as described previously (10Robinson L.J. Busconi L. Michel T. J. Biol. Chem. 1995; 270: 995-998Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 36Michel T. Li G.K. Busconi L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6252-6256Crossref PubMed Scopus (305) Google Scholar). For the acylation experiments, the gels were soaked in 1 m Tris, pH 7.5, or 1 mhydroxylamine, pH 7.5, for 14 h before being processed for fluorography. Palmitoyl groups are attached by a thioester linkage and are susceptible to hydrolysis by hydroxylamine, whereas myristate is insensitive to hydroxylamine. The immunoprecipitation studies were done as described previously (28Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar, 29Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Thin layer chromatography (28Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar, 29Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) and cholesterol mass measurements were performed as described previously (37Graf G.A. Connell P.M. van der Westhuyzen D.R. Smart E.J. J. Biol. Chem. 1999; 274: 12043-12048Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). We first determined if oxLDL alters the subcellular distribution of eNOS. Porcine pulmonary artery endothelial (PPAE) cells were incubated with 100% human lipoprotein-deficient serum (LPDS) containing 20 mm HEPES (pH 7.4) only or with 10 μg/ml HDL, 10 μg/ml nLDL, or 10 μg/ml oxLDL (15–20 nmol/mg TBARS) for 1 h at 37 °C before they were washed and subfractionated to isolate caveolae (28Smart E.J. Ying Y.-S. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar, 29Uittenbogaard A. Ying Y. Smart E.J. J. Biol. Chem. 1998; 273: 6525-6532Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Table I demonstrates that the caveola fraction was not significantly contaminated with galactosyl transferase (Golgi) or NADPH cytochrome creductase (endoplasmic reticulum) but contained a significant amount of the alkaline phosphatase (plasma membrane) activity. Equal amounts of protein from each subcellular fraction were analyzed for the presence of eNOS and caveolin by immunoblotting. Caveolae isolated from control cells (Fig. 1 A, LPDS) and cells treated with nLDL or HDL (Fig.1 A, nLDL and HDL) were highly enriched in eNOS. Caveolin, a caveola marker protein, was also highly enriched in the caveola fraction (Fig. 1 B, LPDS, nLDL, and HDL). Dramatically, treatment with oxLDL induced eNOS and caveolin to move to the internal membrane fraction (Fig. 1, A and B). The internal membrane fraction contains ER, Golgi, mitochondria, and numerous other intracellular organelles, so the exact location(s) of the eNOS and caveolin was not revealed. oxLDL treatment did not cause PKCα or GM1, which are additional molecules shown to be enriched in caveolae (38Parton R.G. J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (452) Google Scholar, 39Smart E.J. Ying Y.-S. Anderson R.G.W. J. Cell Biol. 1995; 131: 929-938Crossref PubMed Scopus (158) Google Scholar, 40Smart E.J. Foster D.C. Ying Y.-S. Kamen B.A. Anderson R.G.W. J. Cell Biol. 1994; 124: 307-313Crossref PubMed Scopus (155) Google Scholar), to translocate out of caveolae (Fig. 1 C). In addition, the noncaveola protein, transferrin receptor, was excluded from the caveola fraction with all treatment conditions (Fig.1 C and data not shown). Importantly, the same data were obtained when the cells were treated with the protein synthesis inhibitor, cycloheximide (50 μg/ml) (data not shown). This indicates that oxLDL causes eNOS and caveolin to translocate to an intracellular membrane and that the presence of intracellular eNOS and caveolin is not due to de novo synthesis. In addition, the removal of oxLDL and continued incubation in the presence of cycloheximide (50 μg/ml for 2 h) permitted eNOS and caveolin to return to the caveolae fraction (Fig. 1, A and B, Recovery).Table ICharacterization of PPAE subcellular fractionsSubcellular fractionProteinGalactosyl transferaseNADPH cytochromec reductaseAlkaline phosphataseμg%%%Postnuclear supernatant4223 ± 223100 ± 5.0100 ± 8.3100 ± 6.1Cytosol2011 ± 1637 ± 0.514 ± 6.86 ± 3.4Intracellular membranes1658 ± 10590 ± 8.784 ± 9.816 ± 8.0Plasma membranes563 ± 561.1 ± 0.21.5 ± 0.576 ± 5.8Caveola membranes31 ± 5NDND27 ± 3.4Nontreated PPAE cells were subfractionated as described under "Experimental Procedures." Galactosyl transferase, NADPH cytochromec reductase, and alkaline phosphatase activities were measured in each fraction and normalized with respect to the values obtained for the postnuclear supernatant fractions. Presented data are the mean ± S.E. from three independent experiments (n = 3).aN.D. means that the level of enzymatic activity was below the sensitivity of the assay. Open table in a new tab Nontreated PPAE cells were subfractionated as described under "Experimental Procedures." Galactosyl transferase, NADPH cytochromec reductase, and alkaline phosphatase activities were measured in each fraction and normalized with respect to the values obtained for the postnuclear supernatant fractions. Presented data are the mean ± S.E. from three independent experiments (n = 3). aN.D. means that the level of enzymatic activity was below the sensitivity of the assay. We used indirect immunofluorescence to confirm the subcellular fractionation data. PPAE cells were treated with LPDS, nLDL, or oxLDL as described above. The cells were co-labeled with anti-caveolin IgG (polyclonal) and anti-eNOS mAb (monoclonal). Fig.2 shows that caveolin and eNOS co-localize at the plasma membrane in cells treated with LPDS and nLDL. However, oxLDL caused both caveolin and eNOS to translocate to an intracellular location. Caveolin and eNOS remain co-localized even after leaving the plasma membrane. These data confirm the redistribution of caveolin and eNOS in subcellular fractions shown in Fig. 1. Because oxLDL induced the redistribution of eNOS, we tested the ability of acetylcholine to activate eNOS in intact cells exposed to oxLDL. NOS activity was determined in whole PPAE cells by using methods previously described (see "Experimental Procedures") (41Lantin-Hermoso R.L. Rosenfeld C.R. Yuhanna I.S. German Z. Chen Z. Shaul P.W. Am. J. Physiol. 1997; 273: L119-L126PubMed Google Scholar). This procedure provides a direct assessment of the acute activation of existing eNOS while keeping signal transduction mechanisms intact (34Davda R.K. Chandler L.J. Crews F.T. Guzman N.J. Hypertension. 1993; 21: 939-943Crossref PubMed Scopus (82) Google Scholar). Treatment of cells with oxLDL (Fig. 3, ●) significantly reduced acetylcholine-stimulated eNOS activation compared with nLDL controls (Fig. 3, ○). Oxidized LDL suppressed the activation of eNOS at all concentrations of acetylcholine tested, resulting in a shift in the dose-response curve to the right by 100-fold. The value obtained when cells were incubated in LPDS (no added lipoproteins) and 10−4m acetylcholine was 49.2 ± 7 fmoll-[3H]citrulline/well. In addition, to rule out the possibility of nonspecific disruption of signaling pathways, we demonstrated that oxLDL treatment did not affect bradykinin- or acetylcholine-stimulated prostacyclin production (TableII).Table IIoxLDL does not inhibit receptor-mediated stimulation of prostacyclin synthesisTreatment6-keto-prostaglandin F1αpg/wellBasal, nLDL271 ± 14Basal, oxLDL280 ± 19Bradykinin, 10−5m, nLDL377 ± 52ap < 0.005 compared with basal.Bradykinin, 10−5m, oxLDL478 ± 71ap < 0.005 compared with basal.Acetylcholine, 10−5m, nLDL388 ± 36ap < 0.005 compared with basal.Acetylcholine, 10−5m, oxLDL435 ± 53ap < 0.005 compared with basal.Cells were incubated for determinations of prostacyclin production using methods we have previously described (35Jun S.S. Chen Z. Pace M.C. Shaul P.W. J. Clin. Invest. 1998; 102: 176-183Crossref PubMed Scopus (123) Google Scholar). In all experiments, n = 4 for each determination, and findings were replicated in three independent studies.a p < 0.005 compared with basal.
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