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• W2246457688 abstract "Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37°C with a unit cell periodicity of 34 Å coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binar y lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol–phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.—Tulenko, T. N., M. Chen, P. E. Mason, and R. P. Mason. Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. J. Lipid Res. 1998. 39: 947–956. Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37°C with a unit cell periodicity of 34 Å coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binar y lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol–phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.—Tulenko, T. N., M. Chen, P. E. Mason, and R. P. Mason. Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis. J. Lipid Res. 1998. 39: 947–956. Sterols are obligatory lipids in plasma membranes of nearly all eukaryotic cell lines. In mammals, this sterol requirement is met exclusively by unesterified (free) cholesterol (1Bloch K. Sterol structure and membrane function.Crit. Rev. Biochem. 1983; 14: 47-92Google Scholar). The content of cholesterol in membranes, expressed as the cholesterol to phospholipid (C/PL) mole ratio, is high for plasma membranes compared to intracellular membranes and is relatively fixed for any given cell line, but varies from ≈0.5 to 1.0 between cell lines. The importance of maintaining a constant cholesterol content in the membrane is illustrated by studies which show marked alterations in the activity of transmembrane proteins and cell function following alterations in the C/PL mole ratio (2Broderick R. Bialecki R.A. Tulenko T.N. Cholesterol-induced changes in arterial sensitivity to adrenergic stimulation.Am. J. Physiol. 1989; 257: H170-H178Google Scholar, 3Bialecki R.A. Tulenko T.N. Excess membrane cholesterol alters calcium channels in arterial smooth muscle.Am. J. Physiol. 1989; 257: C306-C314Google Scholar). Cholesterol-induced changes in membrane protein activity have been suggested to mediate cholesterol-induced changes in cell function in vivo (4Chen M. Mason R.P. Tulenko T.N. Atherosclerosis alters composition, structure and function of arterial smooth muscle plasma membranes.Biochim. Biophys. Acta. 1995; 1272: 101-112Google Scholar). The mechanism for cholesterol's effects on membrane and cell function presumably results, in part, from its ability to modulate the biophysical properties of the membrane bilayer (5Yeagle P.L. Cholesterol and cell membranes.Biochim. Biophys. Acta. 1985; 822: 267-287Google Scholar). In studies utilizing artificial lipid bilayers, increasing the membrane cholesterol content reduces the order parameter at temperatures below the lipid bilayer phase temperature (Tc) and increases it above Tc (6Leonard A. Dufourc E.J. Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach.Biochimie. 1991; 73: 1295-1302Google Scholar). Moreover, incorporation of cholesterol into lipid bilayers increases the modulus of compressibility (K) of the liquid-crystalline state (7Evans E. Needham D. Physical properties of surfactant bilayer membranes: thermal transition, elasticity, rigidity, cohesion and colloidal interactions.J. Phys. Chem. 1987; 91: 4219-4228Google Scholar, 8Needham D. Nunn R.S. Elastic deformation and failure of lipid bilayer membranes containing cholesterol.Biophys. J. 1990; 58: 997-1009Google Scholar). These studies demonstrate that changes in membrane cholesterol content alter the physical properties of the lipid bilayer which may, in turn, modulate membrane protein function, and therefore, cell function. Small angle X-ray diffraction studies have provided direct evidence of physical interactions of cholesterol with membranes, suggesting that cholesterol perturbs the structure of the membrane lipid bilayer (9McIntosh T.J. The effect of cholesterol on the structure of phosphatidylcholine bilayers.Biochim. Biophys. Acta. 1978; 513: 43-58Google Scholar, 10Mason R.P. Moisey D.M. Shajenko L. Cholesterol alters the binding of Ca2+ channel blockers to the membrane lipid bilayer.Mol. Pharmacol. 1992; 41: 315-321Google Scholar), including hydrocarbon core width, in a concentration-dependent manner (4Chen M. Mason R.P. Tulenko T.N. Atherosclerosis alters composition, structure and function of arterial smooth muscle plasma membranes.Biochim. Biophys. Acta. 1995; 1272: 101-112Google Scholar). In model membrane bilayers, an increase in the cholesterol content to a level of 50 mole% of total phospholipid was shown to produce an immiscible cholesterol monohydrate phase with a periodicity of 34 Å coexisting with the liquid crystalline lipid bilayer (11Ruocco M.J. Shipley G.G. Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes.Biophys. J. 1984; 46: 695-707Google Scholar). The periodicity of 34 Å corresponds to a tail-to-tail cholesterol bilayer as the long axis of an individual cholesterol molecule is 17 Å in the crystalline state (12Craven B.M. Crystal structure of cholesterol monohydrate.Nature. 1976; 260: 727-729Google Scholar). Separate domains of cholesterol in monolayer lipid systems have also been observed using magnetic resonance and fluorescence microscopy as a function of temperature and lateral pressure (13Mantripragada B.S. Thompson T.E. Cholesterol-induced fluid-phase immiscibility in membranes.Proc. Natl. Acad. Sci. USA. 1991; 88: 8686-8690Google Scholar, 14Rice P. A McConnell H.M. Critical shape transitions of monolayer lipid domains.Proc. Natl. Acad. Sci. USA. 1989; 86: 6445-6448Google Scholar). However, evidence for the formation of cholesterol monohydrate domains in either biological membranes or membranes reconstituted from native phospholipid molecules has not been reported. Regarding vascular cells, epidemiological studies have provided convincing evidence that an elevation in serum cholesterol represents a primary and independent risk factor for the development of atherosclerotic vascular disease (15Kannel W.B. Castelli W.P. Gordon T. Cholesterol in the prediction of atherosclerotic disease. New perspectives based on the Framingham study.Ann. Intern. Med. 1979; 90: 85-91Google Scholar, 16Kannel W.B. Neaton J.D. Wentworth D. Overall coronary heart disease mortality rate in relation to major risk factors in 325,348 men screened for the MRFIT.Am. Heart. J. 1986; 112: 825-836Google Scholar). Smooth muscle cells (SMC) in dietary atherosclerosis are characterized by an increase in cytosolic calcium levels which may be related to an alteration in the plasma membrane cholesterol content (4Chen M. Mason R.P. Tulenko T.N. Atherosclerosis alters composition, structure and function of arterial smooth muscle plasma membranes.Biochim. Biophys. Acta. 1995; 1272: 101-112Google Scholar, 17Stepp D.S. Tulenko T.N. Alterations in basal and serotonin-stimulated Ca2+ movements and vasoconstriction in atherosclerotic aorta.Arterioscler. Thromb. 1994; 14: 1854-1859Google Scholar). In the present study, we examined changes in the membrane physical properties of SMC during the genesis of early fatty streak atherosclerotic lesions in the rabbit. A highly enriched SMC plasma membrane fraction was isolated from SMC enzymatically dispersed from aortas of New Zealand rabbits fed a normal or cholesterol-enriched (2%) diet for up to 13 weeks. Changes in the structure and lipid organization of SMC plasma membranes were observed in SMC from cholesterol-fed animals that correlated with an elevated C/PL mole ratio. These observations were reproduced in model C:PL binary lipid bilayers. The results of this study indicate that fundamental changes in SMC plasma membrane structure and lipid organization occur as a direct result of physical interactions of cholesterol with neighboring membrane phospholipid molecules in vivo during the development of dietary atherosclerosis, which may account, in part, for some of the changes in the cell biology of SMC in this disease. All chemicals used were reagent grade or better and prepared in ultra-pure deionized water. Bovine cardiac phosphatidylcholine (BCPC), dimyristoyl phosphatidylcholine (DMPC), and cholesterol powders were purchased from Avanti Polar Lipids (Alabaster, AL) and stored at -80°C. Thin-layer chromatography was used to ascertain the presence of any degradative products in the lipid and cholesterol suspensions. The primary fatty acid components of the BCPC lipid molecules were determined by the method of Christie (18Christie W.W. A simple procedure for rapid transmethylation of glycerolipids and cholesteryl esters.J. Lipid. Res. 1982; 23: 1072-1075Google Scholar) using fatty acid methyl esters. Gas–liquid chromatographic analysis showed the following constituents: 18:2 linoleic acid (29.9%), 16:0 palmitic acid (22.5%), 18:1 oleic acid (13.3%), 20:4 arachidonic acid (11.4%), and 20:3 homogamma linoleic acid (4.6%), with small amounts (<1% each) of palmitoleic acid (16:1), linolenic acid (18:3), 11,14-eicosadienic acid (20:2), and myristic acid (14:0). Freshly dispersed SMC were obtained from the thoracic aorta as previously described (19Gleason M.M. Medow M.S. Tulenko T.N. Excess membrane cholestorel alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells.Circ. Res. 1991; 69: 216-227Google Scholar). Briefly, following surgical excision, the thoracic aorta was cut open longitudinally and pinned intimal site up to a wax substrate submerged in physiological salt solution. The intimal surface was scraped with a scalpel to remove endothelial cells and lesions, and the medial smooth muscle layer was peeled free by stripping. The medial layer was minced and incubated for 1–2 h in minimum essential medium (MEM) containing 275 units/ml collagenase, 0.425 units/ml elastase, and 0.12% soybean trypsin inhibitor. Dispersed cells were washed in MEM and pelleted at 350 × g for 10 min and used immediately for isolation of membranes. To confirm cell lineage, an aliquot of cells (≈104 cells) was placed in culture medium and incubated overnight to permit cell attachment, followed by exposure to cell-specific monoclonal antibodies and immunostaining performed with an avidin–biotin system conjugated with horse-radish peroxidase. Conformation of smooth muscle cell identity was accomplished by immunostaining with the muscle actin-specific HHF-35 monoclonal antibody (Enzo Biochemicals, NY) (20Tsukada T. Tippens D. Gordon D. Ross R. Gown A.M. HHF-35, a muscle-actin-specific monoclonal antibody.Am. J. Pathol. 1987; 126: 51-60Google Scholar) and macrophage-specific monoclonal antibody RAM-11 (gift from A. Gown) (21Tsukada T. Rosenfield M.E. Ross R. Gown A.M. Immunocytochemical analysis of cellular components in atherosclerotic lesions. Use of monoclonal antibodies with the Watanabe and fat-fed rabbits.Arteriosclerosis. 1986; 6: 601-613Google Scholar). In cells from both normal and atherosclerotic animals, uniform positive staining with the HHF-35 antibody, and absence of staining with the RAM-11 antibody was observed, confirming SMC lineage. SMC microsomal membranes were isolated using a modification of a procedure previously described (19Gleason M.M. Medow M.S. Tulenko T.N. Excess membrane cholestorel alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells.Circ. Res. 1991; 69: 216-227Google Scholar). Cell dispersions freshly isolated from the aortic medial layer were suspended in cold (4°C) hypoosmolar sucrose (0.25 m) containing 10 mm Tris (pH 7.4) for 6 min, followed by disruption with a tissue homogenizer (Tekmar #60) using 3 homogenization cycles. An equal volume of 0.4 m sucrose solution containing 10 mm Tris, pH 7.4, was then added to the disrupted cells. After removal of an aliquot for chemical and enzymatic analysis, the resultant crude homogenate was centrifuged at 268 g for 20 min. The resulting pellet (nuclear/unbroken cells) was resuspended in 0.25 m sucrose, 10 mm Tris, pH 7.4 (as were all subsequent pellets) and the supernatant was centrifuged at 10,950 g for 10 min. The resulting pellet (mitochrondrial/lysosomal) was resuspended and the supernatant was centrifuged at 144,000 g for 90 min. The final pellet (microsomal) was resuspended after removal of the post microsomal supernatant (soluble fraction). All fractions were frozen for chemical and enzymatic analysis at a later time with the exception of cytochrome-oxidase activity which was measured on the day of cell fractionation. The following markers were assayed in all subcellular fractions by the indicated procedures: Na+/K+ ATPase (22O'Neill R.G. Dubinsky W.P. Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence.Am. J. Physiol. 1984; 247: C314-C320Google Scholar), alkaline phosphodiesterase (23Beaufay G. Amar-Costesec A. Feytmans E. Thines-Semponx D. Wibo M. Berthet J. Analytical studies of microsomes and isolated subcellular membranes from rat liver.J. Cell Biol. 1974; 61: 188-200Google Scholar) and free cholesterol (plasma membrane); cytochrome-oxidase (mitochondria) (24Cooperstein S. Lazarow A. A microspectrophotometric method for the determination of cytochrome oxidase.J. Biol. Chem. 1951; 189: 665-670Google Scholar) and N-acetyl-β-glucosaminidase (lysosomes) (25Harrison E.H. Bowers W.E. Characterization of rat lymphocyte cell membranes by analytical isopynic centrifugation.J. Biol. Chem. 1983; 258: 7134-7140Google Scholar); DNA (26Labarka C. Paigen K. A simple, rapid and sensitive DNA assay procedure.Anal. Biochem. 1980; 102: 344-352Google Scholar) and protein (soluble fraction) (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). Total phospholipid mass (total membrane) was determined in each fraction using a phospholipid phosphorus assay (28Sokoloff L. Rothblat G.H. Sterol to phospholipid molar ratio of L-cell with qualitative and quantitative variations of cellular sterol.Proc. Soc. Exp. Biol. Med. 1974; 146: 1166-1172Google Scholar). For cholesterol feeding studies, rabbits were maintained on a cholesterol-rich diet for up to 13 weeks. The diet was a commercially prepared, calibrated rabbit chow (Buckshire Feeds) supplemented with cholesterol (2%). The chow consisted of 30.1% protein, 5.5% fat, 21.1% fiber, and 43.3% carbohydrate (with or without added cholesterol). Control and diet rabbits were housed separately, but in the same room, throughout the feeding period. Control rabbits were fed batch-matched standard chow without added cholesterol. SMC plasma membrane samples were oriented for small-angle X-ray scattering by stacking using a modification of our previously described technique (29Chester D.W. Herbette L.G. Mason R.P. Joslyn A.F. Triggle D.J. Koppel D.E. Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multilayers.Biophys. J. 1987; 52: 1021-1030Google Scholar). Briefly, SMC plasma membrane vesicles were spun in an SW-28 rotor (Beckman Instruments, Inc., Fullerton, CA) at 35,000 g for 1 h at 5°C in Lucite sedimentation cells, each containing an aluminum foil substrate. On completion of the spin, supernatants were removed and each sample was mounted on a curved glass support. The samples, composed of 250 mg phospholipid, were equilibrated overnight in glass vials by suspending the samples over, but not in contact with, a saturated salt solution that served to define a specific relative humidity of 95% at 5°C (ZnSO4). Oriented membrane samples were then placed in sealed brass canisters containing aluminum foil in which temperature and relative humidity were controlled. The effects of cholesterol content on membrane bilayer structure were directly examined in model membranes using vesicular lipid bilayer preparations. Lipid vesicles for these studies were prepared from dimyristoyl phosphatidylcholine (DMPC) or bovine cardiac phosphatidylcholine (BCPC) and cholesterol reconstituted in chloroform at various cholesterol-to-phospholipid mole ratios (0:1 to 0.6:1). The lipids were dissolved in redistilled chloroform at a concentration of 10.0 mg/ml. Samples containing DMPC or BCPC in the absence and presence of cholesterol were dried down with a stream of nitrogen gas to a thin film on the sides and bottom of 13 × 100 mm glass test tubes while vortexing. Residual solvent was removed by vacuum. A volume of buffer (0.5 mm HEPES, 2.0 mm NaCl, pH 7.3) was added to the other dried lipid preparation, yielding a final phospholipid concentration of 5.0 mg/ml. Multilamellar vesicles were formed by vortexing the buffer and lipids above the thermal phase transition temperature for 3 min. Oriented membrane samples were prepared by centrifugation, as described above for the SMC samples. Small-angle X-ray scattering was carried out by aligning the membrane samples at near-grazing incidence with respect to the X-ray beam. The radiation source was a collimated, monochromatic X-ray beam (CuKα, λ = 1.54Å) from a Rigaku RU200 (Danvers, MA) rotating anode microfocus generator. The fixed geometry beamline utilized a single Franks mirror providing nickel-filtered radiation (Kα1 and Kα2 unresolved) at the detection plane. The beam height at the sample was ≈1 mm. Small-angle X-ray diffraction data from the oriented membrane multilayer samples were recorded on a one-dimensional position-sensitive electronic detector (Innovative Technologies, Inc., Newburyport, MA). In addition to direct calibration of the detector system, cholesterol monohydrate was used to verify the calibration. The unit cell periodicity, or d space, of the membrane lipid bilayer is the measured distance from the center between one bilayer to the next, including surface hydration. The d-space for the membrane multilayer samples was calculated by Bragg's Law: nλ=2dsinΘ in which n is the diffraction order number, λ is the wavelength of the X-ray radiation (1.54Å), d is the membrane lipid bilayer unit cell periodicity, and θ is the Bragg angle equal to one-half of the angle between the incident beam and scattered beam. Microsomal membranes isolated from freshly dispersed SMC cells showed a 13-fold enrichment in the specific plasma membrane marker, alkaline phosphodiesterase (APD). There was only minor contamination of DNA (2.8%), NABGase (5.7%) and cytochrome oxidase (5.5%) in the same fraction. Based on the recovery of the plasma membrane marker APD, the calculated yield of plasma membrane in this fraction was 44.7 ± 7.6% (compared to 41.7 ± 10.0% for the nuclear/unbroken cells pellet, 8.6 ± 1.9% for the mitochondrial/lysosomal pellet and 5.0 ± 1.3% for the soluble fraction). The lipid-to-protein content of these membranes (2.3 mmol phospholipid/mg protein) was similar to that reported for highly enriched cardiac plasma membrane, 1.98 mmol phospholipid/mg protein (30Weglicki W.B. Owens K. Kennet F.F. Kessner A. Harris L. Wise R.M. Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes.J. Biol. Chem. 1980; 255: 3605-3609Google Scholar). To characterize the structure of SMC plasma membranes from control and atherosclerotic SMC, small-angle X-ray diffraction was used. In Fig. 1, representative X-ray diffraction order patterns from oriented SMC plasma membranes isolated from control and 8-week diet samples are shown. The d-space was computed from the diffraction order patterns and revealed a width of 56.3 Å for the control sample (Fig. 1A) and 60.3 Å for the 8-week diet sample (Fig. 1B). In addition, the relative intensity of the diffraction orders also decreased in the diet samples. Thus, the atherosclerotic SMC membrane was 4 Å larger than control. In both samples, there was no evidence for a separate lipid phase. Between 10 and 13 weeks on the experimental diet, the C:PL mole ratio of the enriched SMC plasma membrane fraction averaged 0.96:1 ± 0.27 (means ± SD, n = 9). Small-angle X-ray diffraction from SMC plasma membranes derived from these animals produced meridonal patterns consistent with two separate lipid phases: a membrane liquid crystalline phase (56 Å) and an immiscible cholesterol monohydrate phase (34 Å-orders 1′ and 2′ at 37°C (Fig. 2). The d-space of the membrane liquid crystalline phase (56–57 Å) was identical to that reported for control membranes and less than that reported for samples from animals on the cholesterol-enriched diet for less than 9 weeks. These data suggest that the membrane lipid bilayer has an upper limit for cholesterol to remain fully miscible; above this limitation, cholesterol forms a separate, immiscible cholesterol phase. The presence of the immiscible cholesterol domain was sensitive to the temperature of the sample. When the samples were warmed from 37°C to 45°C or 50°C, a single lamellar lipid bilayer phase was observed and there was no evidence for a cholesterol domain. On cooling back to 37°C, the immiscible cholesterol domain reappeared, indicating its temperature sensitivity. With further cooling to 20°C, the cholesterol domain remained and the lamellar lipid phase demonstrated a further increase in width (57.4 to 60.6 Å), consistent with an ordering effect of the membrane bilayer at low temperatures. Table 1 reviews these changes in membrane structural dimensions.TABLE 1.X-ray diffraction d-space measurements for atherosclerotic SMC plasma membranes as a function of temperatureSample temperatureMembrane Lipid Bilayer d-spaceCholesterol Phase d-SpaceÅÅ37°C57.43445°C55.4no50°C55.1no37°C57.43420°C60.634Sample C:PL mole ratio was 1.21:1 (13 weeks on cholesterol diet); no, cholesterol phase not observed. Open table in a new tab Sample C:PL mole ratio was 1.21:1 (13 weeks on cholesterol diet); no, cholesterol phase not observed. Results from the small-angle X-ray diffraction experiments demonstrated that the oriented DMPC/cholesterol and BCPC/cholesterol membrane vesicles produced four strong, reproducible diffraction orders at 37°C and 93%. Membrane width, including surface hydration (i.e., d-space or unit cell periodicity), was measured for the samples as a function of cholesterol content. Figure 3A illustrates a typical electron density profile demonstrating the effects of adding cholesterol to phospholipid membranes. In the absence of cholesterol, the DMPC d-space was 50.9 Å. At the highest cholesterol level measured (0.6:1 cholesterol to phospholipid mole ratio), the membrane width increased by 18.3% to 60.2 Å (P < 0.01). In addition, there was a significant, direct relationship between the cholesterol: DMPC mole ratio and membrane width; the correlation coefficient was 0.972 (P < 0.05) as determined by the Pearson Product Moment Correlation (Fig. 3B). A similar correlation between cholesterol content and membrane width was observed for BCPC/cholesterol samples (Fig. 4). The BCPC membrane d-space increased by 10.5% (P < 0.05) from 52.5 Å in the absence of cholesterol to 58.0 Å at a 0.6:1 mole ratio (Fig. 4A). As with DMPC, there was a significant, direct relationship between the cholesterol:BCPC mole ratio and membrane width, with a correlation coefficient of 0.953 (P < 0.05) (Fig. 4B). In addition to the measurement of membrane d-space, the intrabilayer phosphorus headgroup separation (hydrocarbon core width) for these samples was calculated as a function of cholesterol content. The phosphorus head-group separation was measured directly from one-dimensional electron density profiles calculated from the diffraction data in identical resolution (Fig. 5). Similar to that observed for the membrane d-space, there was a significant positive correlation between membrane cholesterol content and the measured phospholipid bilayer head-group separation. The DMPC intrabilayer headgroup separation increased as a function of cholesterol content (Fig. 5A) from 35.6 Å in the absence of cholesterol to 43.0 Å at a 0.6:1 cholesterol:phospholipid mole ratio, an increase of 20.8% (P < 0.001). Under identical conditions, the BCPC intrabilayer headgroup separation increased with increasing cholesterol content (Fig. 5B) from 37.2 Å to 42.1 Å, an increase of 13.2% (P < 0.001). To test whether the immiscible cholesterol phase could be reproduced in a reconstituted lipid bilayer system under similar conditions of membrane cholesterol content, binary lipid mixtures were formed composed of native phospholipids derived from cardiac (BCPC) tissue (see Methods and Materials) and cholesterol. The reconstituted membrane lipid bilayers were formed at 0.5:1 and 1:1 C:PL mole ratios to reproduce normal and cholesterol-enriched conditions. Figure 6 illustrates the results of these experiments conducted at 37°C and 93% relative humidity. At control physiologic levels of cholesterol (0.5:1 C:PL mole ratio), the oriented samples produced a single lamellar phase with a d-space of 58Å (Fig. 6A), consistent with the periodicity measured for intact and reconstituted cardiac plasma membranes (31Herbette L.G. MacAlister T. Ashavaid T.F. Colvin R.A. Structure–function studies of canine cardiac sarcolemmal membranes. II. Structural organization of the sarcolemmal membrane as determined by electron microscopy and lamellar X-ray diffraction.Biochim. Biophys. Acta. 1985; 812: 609-623Google Scholar, 32Mason R.P. Chester D.W. Diffusional dynamics of an active rhodamine-labeled 1,4-dihydropyridine in sarcolemmal lipid multilayers.Biophys. J. 1989; 56: 1193-1201Google Scholar). Under conditions of cholesterol enrichment (1:1 C:PL mole ratio), two separate lamellar phases were observed: a liquid crystalline lipid bilayer phase (56 Å) and an immiscible cholesterol monolayer phase (34 Å) (Fig. 6B). These d-space measurements were identical to those reported above for SMC plasma membrane samples under cholesterol-enriched conditions at 37°C. Moreover, in the presence of the cholesterol monohydrate phase, the membrane lipid bilayer phase had a periodicity that was actually less than that measured for cardiac lipid bilayers with a C:PL mole ratio of 0.5:1. These data suggest that the presence of the cholesterol phase actually results in a reduction in cholesterol in the miscible lipid bilayer phase. Only when the cardiac membrane C:PL mole ratio was elevated to 2:1, was the d-space of the lipid bilayer component of the two-phase system identical to that reported for control membrane. Table 2 summarizes the data on cholesterol enrichment inducing an immiscible cholesterol domain (34 Å repeat structure).TABLE 2.Small angle X-ray diffraction d-space measurements of atherosclerotic SMC plasma membranes and reconstituted cardiac lipid bilayersSampleC:PL Mole RatioMembrane Lipid Bilayer d-SpaceCholesterol Phase d-SpaceÅÅControl diet SMC plasma membrane0.4:156noAtheroscleroti" @default.
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• W2246457688 date "1998-05-01" @default.
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• W2246457688 title "Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis" @default.
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