Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis

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 linoleic acid with of acid acid acid and acid dispersed SMC were obtained from the as Tulenko T.N. Excess membrane alters calcium cytosolic calcium and membrane in arterial smooth muscle Res. 1991; Scholar). following the was and up to a in The was with a to cells and and the smooth muscle was by The was and for in containing and cells were in and at for and used for of cell an of cells was in and to cell by to and with an with of smooth muscle cell was by with the muscle T. D. Gordon D. R. a J. 1987; Scholar) and from A. T. R. analysis of components in atherosclerotic of with the and 1986; Scholar). In cells from normal and atherosclerotic with the and of with the was SMC SMC microsomal membranes were isolated using a of a procedure Tulenko T.N. Excess membrane alters calcium cytosolic calcium and membrane in arterial smooth muscle Res. 1991; Scholar). isolated from the aortic were in containing for by with a using of containing was to the of an for and the was at for The resulting was in were all and the was at for The resulting was and the was at for The was of the microsomal All were for and analysis at a with the of activity which was on the of cell The following were in all by the W.P. for activity in J. Physiol. 1984; Scholar), A. E. D. M. J. studies of and isolated membranes from Scholar) and cholesterol A. A method for the of Chem. Scholar) and of cell membranes by Chem. 1983; K. A rapid and Biochem. Scholar) and protein with the Chem. Scholar). phospholipid was determined in fraction using a phospholipid L. Sterol to phospholipid ratio of with and of Med. Scholar). cholesterol feeding rabbits were on a diet for up to 13 weeks. The diet was a with cholesterol The of and or and diet rabbits were but in the the feeding rabbits were fed SMC plasma membrane were for X-ray by using a of Mason R.P. of calcium channel in cardiac lipid J. 1987; Scholar). SMC plasma membrane were in an at for at in cells, containing an On of the were and was on a The of were in by the but not in a that to a of at membrane were in containing in which temperature and were The effects of cholesterol content on membrane bilayer structure were examined in model membranes using lipid bilayer Lipid for these studies were prepared from dimyristoyl phosphatidylcholine or cardiac phosphatidylcholine and cholesterol reconstituted in at mole to The lipids were in at a of containing or BCPC in the and presence of cholesterol were with a of to a on the and of 13 was by A of was to the lipid a phospholipid of were by the and lipids above the thermal phase temperature for membrane were prepared by as above for the SMC X-ray was by the membrane at with to the X-ray The was a X-ray from a The fixed a and at the The at the was X-ray diffraction from the membrane were on a In to direct of the cholesterol monohydrate was used to the The unit cell or of the membrane lipid bilayer is the from the between bilayer to the including The for the membrane was by in which is the diffraction order is the of the X-ray is the membrane lipid bilayer unit cell and is the angle to of the angle between the and membranes isolated from dispersed SMC cells showed a in the plasma membrane was of and in the on the of the plasma membrane the of plasma membrane in this fraction was to for the cells for the and for the The content of these membranes was to that for highly enriched cardiac plasma protein W.B. K. A. L. and properties of highly enriched cardiac from isolated Chem. Scholar). the structure of SMC plasma membranes from control and atherosclerotic X-ray diffraction was In X-ray diffraction order patterns from SMC plasma membranes isolated from control and diet are The was from the diffraction order patterns and a width of Å for the control and Å for the diet In the of the diffraction also in the diet the atherosclerotic SMC membrane was Å than In was evidence for a separate lipid and 13 weeks on the the C:PL mole ratio of the enriched SMC plasma membrane fraction X-ray diffraction from SMC plasma membranes from these animals patterns with separate lipid a membrane liquid crystalline phase and an immiscible cholesterol monohydrate phase and at 37°C The of the membrane liquid crystalline phase was to that for control membranes and less than that for from animals on the cholesterol-enriched diet for less than 9 weeks. These that the membrane lipid bilayer has an for cholesterol to fully above this cholesterol a immiscible cholesterol The presence of the immiscible cholesterol domain was to the temperature of the the were from 37°C to or a lipid bilayer phase was observed and was evidence for a cholesterol On cooling to the immiscible cholesterol domain reappeared, indicating its temperature cooling to the cholesterol domain and the lipid phase demonstrated a increase in width to with an effect of the membrane bilayer at these changes in membrane diffraction for atherosclerotic SMC plasma membranes as a function of Lipid C:PL mole ratio was weeks on cholesterol cholesterol phase not observed. in a C:PL mole ratio was weeks on cholesterol cholesterol phase not observed. from the X-ray diffraction demonstrated that the and membrane diffraction at 37°C and width, including or unit cell was for the as a function of cholesterol a the effects of cholesterol to phospholipid In the of the was the cholesterol level cholesterol to phospholipid mole the membrane width by to Å In was a direct between the mole ratio and membrane the was as determined by the A between cholesterol content and membrane width was observed for The BCPC membrane by from Å in the of cholesterol to Å at a mole ratio with was a direct between the mole ratio and membrane width, with a of In to the of membrane the separation core for these was as a function of cholesterol The separation was from from the diffraction in to that observed for the membrane was a between membrane cholesterol content and the phospholipid bilayer The separation as a function of cholesterol content from Å in the of cholesterol to Å at a cholesterol:phospholipid mole ratio, an increase of the BCPC separation with increasing cholesterol content from Å to an increase of the immiscible cholesterol phase be reproduced in a reconstituted lipid bilayer of membrane cholesterol binary lipid were of native from cardiac and and The reconstituted membrane lipid bilayers were at and C:PL mole to normal and cholesterol-enriched the results of these at 37°C and control levels of cholesterol C:PL mole the a phase with a of with the periodicity for and reconstituted cardiac plasma membranes T. R.A. studies of cardiac organization of the membrane as determined by microscopy and X-ray Biophys. Acta. 1985; Scholar, R.P. of an in lipid J. 1989; Scholar). of cholesterol C:PL mole separate were a liquid crystalline lipid bilayer phase and an immiscible cholesterol monolayer phase These were to above for SMC plasma membrane cholesterol-enriched at Moreover, in the presence of the cholesterol monohydrate the membrane lipid bilayer phase a periodicity that was less than that for cardiac lipid bilayers with a C:PL mole ratio of These that the presence of the cholesterol phase results in a in cholesterol in the lipid bilayer the cardiac membrane C:PL mole ratio was elevated to was the of the lipid bilayer of the to that for control the on cholesterol an immiscible cholesterol domain Å angle X-ray diffraction of atherosclerotic SMC plasma membranes and reconstituted cardiac lipid Lipid diet SMC plasma SMC plasma membrane SMC plasma membrane cardiac lipid cardiac lipid cardiac lipid temperatures were 37°C for SMC plasma membrane and for reconstituted lipid cholesterol phase not observed. in a The temperatures were 37°C for SMC plasma membrane and for reconstituted lipid cholesterol phase not observed. on the in this we have a model to the effects of cholesterol on membrane bilayer structure that occurs in vivo in the plasma membrane of SMC during early which is illustrated in increasing cholesterol content of the membrane that occurs during the early of cholesterol feeding, the cell membrane width increases a high cholesterol diet is to the of cholesterol monohydrate bilayers in the of the cell demonstrated by in model bilayers for tail-to-tail cholesterol in Scholar), the cholesterol molecules to in a tail-to-tail In this study, we examined the lipid organization and structure of SMC plasma membranes from rabbits in which atherosclerosis was by cholesterol feeding and compared these findings to cholesterol-induced alterations in model lipid bilayers. X-ray changes in membrane lipid organization were for the the formation of a physical cholesterol domain in In changes were also observed with an increase in membrane bilayer width, and on cholesterol effects on bilayer structure during (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 observed alterations were with a marked increase in the membrane C:PL mole ratio in these and were reproduced in reconstituted lipid bilayer cells, cholesterol is exclusively in membranes it is between the intracellular and plasma The plasma membrane is enriched with cholesterol and is to up to of the total unesterified cholesterol membranes the phospholipid and of the cholesterol and in Chem. 1989; Scholar). In of the plasma membrane with cholesterol has been shown to modulate the activity of transmembrane including calcium channels R.A. Tulenko T.N. Excess membrane cholesterol alters calcium channels in arterial smooth muscle.Am. J. Physiol. 1989; 257: C306-C314Google Scholar), channels M. P. between channels in smooth muscle cells isolated from normal and atherosclerotic R. 1991; Scholar) and the (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). In intracellular calcium levels have been shown to be elevated in cholesterol-enriched SMC Tulenko T.N. Excess membrane alters calcium cytosolic calcium and membrane in arterial smooth muscle Res. 1991; Scholar). All these alterations have also been demonstrated to occur in SMC in vivo in cholesterol-fed rabbits (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). Cholesterol of the cell membrane has effects on membrane protein activity including activity in a of cells including cells P. Cholesterol modulation of activity in the Biophys. Acta. 1983; Scholar), cells Cholesterol in 1994; Scholar), and cells P.L. J. D. of cholesterol on activity in Scholar). In a channel in channels reconstituted into lipid bilayers demonstrated an increase in lipid bilayer lateral by cholesterol that the state of the channel H.M. R. Mason R.P. R. of channel and by an using as a 1995; Scholar). This effect of cholesterol on channel to result from the membrane Taken together, these studies indicate that cholesterol levels in the membrane have effects on membrane protein activity in turn, may cell function. The primary of the present study is that at membrane C/PL mole above in SMC obtained from animals fed cholesterol for than 9 weeks, separate to a liquid crystalline lipid bilayer and related to a cholesterol monohydrate phase this separate phase represents a cholesterol monohydrate phase is based on studies the formation of tail-to-tail interactions of cholesterol in model systems for tail-to-tail cholesterol in Scholar, lipid domains and membrane New Scholar). The present findings indicate a in the of the biological membrane to cholesterol in a fully state the of cholesterol a an immiscible cholesterol phase is phase separation of cholesterol into a domain has been shown in model bilayers using a of of lipid domains in biological Biophys. 1992; Scholar, M. of factors on membrane cholesterol of binding of cholesterol in Biophys. Acta. 1991; Scholar, A. M. formation in 1995; Scholar, M. and of membrane domain 1995; Scholar, of domains and domain in and 1995; Scholar). the present is the to demonstrate the formation of physical cholesterol domains in biological membranes in vivo cholesterol The of this membrane cholesterol domain to the of cholesterol in the membrane of of this as during serum (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 formation of immiscible cholesterol domains in the membrane to result from a direct physical interaction between cholesterol's highly structure with membrane phospholipid fatty This is from which the formation of immiscible cholesterol domains in model binary lipids containing cholesterol and This is by the that the to the cholesterol cooling to 37°C this effect the increase in membrane bilayer width that the formation of these domains with cholesterol was also reproduced in a model binary lipid and the that the effect of cholesterol on membrane bilayer width is a direct result of a physical interaction of cholesterol with the membrane phospholipid fatty evidence for cholesterol domains has been from obtained in cells including cells, cells, and aortic smooth muscle cells membrane cholesterol and the of cholesterol Lipid. Res. 1992; Scholar, J. T.J. cholesterol cholesterol domains and Med. 1991; Scholar, E.J. L. in membrane cholesterol domain and intracellular cholesterol Med. Scholar). In these separate cholesterol domains were based on the of unesterified cholesterol from cells in The formation of cholesterol immiscible domains demonstrated in the present study may the of a cholesterol in the cell membrane and of cholesterol and Biophys. Acta. Scholar) immiscible cholesterol be to be to E.J. L. in membrane cholesterol domain and intracellular cholesterol Med. Scholar). The biological of these alterations in SMC membranes is not fully at this in is these cholesterol domains a for the formation of cholesterol monohydrate cholesterol long with atherosclerotic In increases in membrane width were the development of early fatty streak lesions in cholesterol-fed suggesting this membrane may be in the alterations with the of this disease. cholesterol of the SMC membrane has been shown to calcium and in to in SMC in vivo during R.A. Tulenko T.N. Excess membrane cholesterol alters calcium channels in arterial smooth muscle.Am. J. Physiol. 1989; 257: C306-C314Google Scholar, 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, R.A. Tulenko T.N. Cholesterol increases basal and calcium in vascular smooth 1991; 88: Scholar). In cholesterol of the SMC membrane also in these cells T.N. L. Mason R.P. calcium and atherogenesis. a for calcium channel blockers in J. Scholar). atherosclerosis is to result from a it is to that membrane alterations of the in this may be to this The to to and for in this for this was provided in by of and from the and of the M. was by an smooth muscle cells cardiac phosphatidylcholine dimyristoyl phosphatidylcholine acid