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Effects of ethanol on the lipid composition of bovine vascular smooth muscle cells in culture
Int. J. Biochrm. Vol. 17, No. 8, pp. 863-866, 1985 Punted in Great Britain. All rights reserved
0020-711X/85 $3.00 + 0.00
CopyrIght ‘:LB1985 Pergamon Press Ltd
EFFECTS OF ETHANOL ON THE LIPID OF BOVINE VASCULAR SMOOTH CELLS IN CULTURE DAVID
Department
A.
COOK, PETER
of Biochemistry.
University (Received
A. WILCE and of Queensland, 2 I Not)enlher
BRIAN
St Lucia,
C.
COMPOSITION MUSCLE
SHANLEY*
Queensland
4067, Australia
1984)
Abstract-l. Ethanol (50 mM) had no effect on the growth rate or viability of arterial smooth muscle cells over 3.5 days. 2. The cholesterol:phospholipid ratio of the cells was unchanged after 7 days exposure. 3. The major phospholipid components phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were unchanged by ethanol exposure. Sphingomyelin content fell significantly within 12 hr. 4. There were major changes in the fatty acid composition of the phospholipids with a reduction in saturated fatty acids and an increase in unsaturated fatty acids.
INTRODUCTION
75 cm’ culture flasks (Corning Industries) using Dulbecco’s modified Eagles medium. Medium was changed every three days or sooner. Cells were subcultured during logarithmic growth as the culture approached confluence. For viability studies cells were plated onto 2.5 cm diameter culture dishes at a density of 5000 viable cells/cm’. After allowing time for the cells to adhere (6 hr) the cultures were grown in 2 ml of medium containing ethanol (0, 25, 50, 100 or 200 mM) and harvested at specified times (0, 12. 36, 60 or 84 hr). Cell counting was performed with a Coulter Counter Model ZF. The enzymatic assay of Bonnichsen (1965) was used to monitor ethanol concentration in the medium.
The involvement of ethanol in the pathogenesis of ischaemic heart disease is complex. Epidemiological evidence, supported by some animal studies, suggests moderate consumption of ethanol may have a protective effect by impeding the development of atherosclerotic plaques (La Porte, 1980). In contrast high ethanol consumption is recognised as a causal agent in hypertension and atherosclerosis (Mathews, 1979). It is not known whether these changes are due to indirect
effects,
e.g. through
alcohol-induced
changes
in lipoprotein metabolism or to direct effects on cells of the arterial wall. Chronic exposure of various tissues to ethanol has been observed to result in alterations in membrane lipid composition, both in whole animals (Chin et al., 1978; Waring et al., 1981; Wing et al., 1982) and in cell cultures (Ingram rt ul., 1978; Keegan et al., 1983). Since changes in membrane lipids can alter the activity of membrane-bound enzymes (Kimelberg, 1975; Farias et al., 1975) and the binding characteristics of receptors (Heron et ul., 1980, Bakardjieva et NI., 1979; Hanski et al., 1979) it is possible that changes in smooth muscle function, particularly receptor-mediated uptake of lipoproteins, may be influenced by ethanol-induced changes in the lipid bilayer. In this paper we report the results of an investigation into the effects of ethanol (50 mM) on the lipid composition of smooth muscle cells maintained in culture.
Lipid atruction
MATERIALS AND METHODS Ccl1 culture Cells were obtained from a primary explant of foetal bovine thoracic aorta (Ross. 1971). Cultures were grown in *Author
to whom
reprint
requests
should
and anui~.~is
Lipid analyses were performed on cells grown until the 8th passage. At this time, the medium was changed to one containing ethanol and maintained for up to 7 days. Cell cultures were harvested. cooled to 4 C and pelleted. Lipids were extracted from the cell pellets according to the method of Bligh and Dyer (1959) utilizing the salt solution of Folch el al. (1956) for the upper aqueous phase. The lipid extract was dissolved in chloroform and stored under nitrogen at -20’C in glass vials with Teflon-silicone septa until analysis. Butylated hydroxytoluene (250 g/ml) was used as an anti-oxidant throughout. Extraction efficiency of lipids was calculated by monitoring the recovery of [‘HIcholesterol and [“Clphosphatidylcholine. Mean recoveries were 63 and SSP,, respectively. Cholesterol was assayed by isothennic gas liquid chromatography as described by Horwitz L’I ul. (1978). Total lipid phosphorus was measured by the method of Rouser et al. (1965). The quantities of cholesterol and phospholipid contained in the samples were calculated with respect to cell protein (Lowry et cd., 1951) and adjusted according to extraction efficiency. Phospholipid classes were separated by unidimensional thin layer chromatography. A 0.3 mm thick layer of silica gel H containing lo”, (w/w) magnesium trisilicate was air dried and activated for 2 hr at 110 C prior to use. Plates were developed with a solvent system comprising chloroform: methanol: petroleum spirit (b.p. 4&60 C): acetic acid (40:20: 30: IO) containing 0.9”;, borate (w/v). Lipid spots
be addressed. 863
were visualized with iodinc vapour and tdentificd hq comparison with R, values ol‘ standards. Areas of silica gel corresponding to each class were quantrtatively collected and inorganic phosphorus was determined according to Rouser PI &. ( 1966). For acyl group analysis. phosphoiipids wcrc isolated under N2 as a hulk group by thin layer ~hr~~rn~tog~~phy on Glica gel G devclopcd in ;t petrolcum spirit (b.p. 40.-60 C):diethyl ether:acetic acid (90: IO: I) according to Marinette (1967). The phospholipids. which remained at the origin were scraped oft‘ and extracted twice with chloroform:mcthanol (I : I ). The dried eluates were methylsted by the method of Morrison and Smith (1964) and analyscd on a 1.5 m glass column packed with lo”,, (w:w) SF’330 on 100/l 70 Chromosorh WAW (Supelco Inc.. Pennsylvania, U.S.A.) in a Shimadzu hC-6AM chromatograph using N2 as carrier. Individual fatty acid methyl esters were dctectcd by Bane ionization.
All ~aiues are expressed as mean + SEM detcrmin~t~ons. Signi~~~ncc was determined r-test.
of nt least 4 by Student’s
RESL!LTS
Doubling times for cells grown in ethanol up to 200 mM were determined. Semi-logarithmic plots of cell density vs time were linear at all concentrations, implying that viability and logarithmic growth were maintained. There was. however, a significantly increased doubling time at an ethanol concentration of 200 mM (26.9 hr compared to 21.4 hr in controls. P < 0.05). 50 mM ethanol was used for subsequent experiments. This gave a doubling time of 22.8 hr. Loss of ethanol from the cultures was estimated at 6’1,, per day.
The cholesterol and phospholipid concentration of arterial smooth muscle cells (4.6 I: 0.24 ~mol~rng protein and 9.8 +_0.5 jLmol/mg protein respectively), remained unchanged after incubation of cells with ethanol for up to 48 hr. Similarly, the cholesterol: phospholipid ratio (0.48 & 0. I) was unchanged after 48 hr exposure. To investigate alterations in the relative proportions of the major phospholipid classes, smooth muscle cells were grown in medium containing
c
s s
II
1 I 1234
I
I
1
t
t
5
6
7
Days Fig. 1. Retativc proportions of the phospholipid classc~ from smooth muscle cells grown in medium containing ethanol (SOmM). Points represent mean of 4 dcterminations. Error bars have been omitted for ctarity (0) Phosphatidylcholint. (m) phusphatidylethanolamine. (r) phosph~tid~lserin~ and phospllatidrlinosltol. (0 t sphingomyciin. Values fur sphingor~ly~lln at 1. 7 days were signtiicantly diffrrent from control I P c 0.051.
ethanol for periods of up to 7 days. Figure I shows the relative proportions of the major phospholipid classes during the course of the experiment. The major phosphol~pid component, phosphatidyl” choline, varied from 46.24 & 1.Wo to 5 1.8Yf 3.68”,, of the lipid phosphorus. Phosphatidylethanolamine varied between 26.68 t_ 0.56”U and 31.35 + 0.71”,, of the total phospholipid. Phosphatidylserine and phosphatidylinositol together comprised between 13.97 _t 0.3!‘!,, and 16.22 iii.(X4”,, The changes in the cellular content of these lipids during the course of the experiment were not statistically significant. By contrast significant differences were obvious in the proportion of sphingomyelin. After 12 hr exposure trl ethanol the proportion of sphingomyclin had fallen from LO.23 + 1.29”,, to 3.79 + 0.97”,, (P < 0.01) and remained low at approximately S”,, of the total phospholipids for the duration of the experiment. Table 1 shows the fatty acid composition of phos-
Ethanol and smooth muscle cells pholipids from smooth muscle cells grown for up to 7 days in medium containing ethanol (50 mM). The percentage of 14:O and 16:O fell significantly within 24 hr and remained depressed for 7 days. The concentration of 18: I declined over the initial 12 hr and maintained this lower level for the duration of the experiment. These changes were compensated by an increase in the proportion of 16: I, 22: 1 and 22: 6 within I2 hr exposure to ethanol. There was no change in the proportion of the other major fatty acids. The nature of the changes can be readily appreciated when alterations in degree of saturation are examined. There was a fall in the proportion of fatty acids that are fully saturated from 42.83 f 0.02:‘; to 38.47 f 0.05)‘” (P < 0.001) after 7 days exposure to ethanol. The double bond index @[percentage of each unsaturated fatty acid x number of its double bonds]) is an index of the degree of fatty acid unsaturation (Farias PI u/., 1975). It increased by 20”); in the first 12 hr and maintained this higher level ’ throughout the incubation. The increased degree of unsaturation shown by the higher double bond index after ethanol exposure is consistent with the fall observed in the percentage of saturated fatty acids. DISCUSSION
Smooth muscle cells proliferated in the presence of ethanol at higher concentration than can be achieved in cir?o. The doubling time of smooth muscle cells grown in medium containing ethanol (200 mM) was significantly longer than that of controls (26.9 hr compared to 21.4 hr). However, these cells still maintained logarithmic growth. Keegan et cd. (1983) found that ethanol (86 mM) had no effect on the growth rate of HeLa cells. Li et al. (1980) exposed Chinese hamster ovary cells (HA-l) to 660mM ethanol for 45 min and demonstrated no loss of cell viability. In the present study subsequent experiments were carried out with medium containing 50 mM ethanol. This concentration did not impede cell proliferation and can be readily achieved in rice (Victor and Adams, 1983). We found no change in the cholesterol:phospholipid ratio of smooth muscle cells exposed to ethanol (50mM) for 48 hr. This agrees with the results of Keegan rt u/. (1983) who found no alteration in the cholesterol: phospholipid ratio after HeLa cells were exposed in culture to ethanol (86mM) for up to 8 days. The relative proportions of each phopsholipid class found by us in bovine vascular smooth muscle cells in culture were very similar to those of cultured embryonic chick muscle cells (Kent et d., 1974) and cultured monkey vascular smooth muscle cells (Bates rt UI., 1978). The decrease in the proportion of sphingomyelin observed when bovine vascular smooth muscle was grown in the presence of ethanol (50 mM, Fig. 1) was similar to changes found by Ingram e/ crl. (1978) in Chinese hamster ovary cells grown in the presence of ethanol (139 mM). However, Ingram et ul. (1978) also found a decrease in the percentage of lysophosphatidylcholine and an increase in phosphatidylcholine. Keegan et rtl. (1983) studying HeLa
865
cells grown in ethanol (86 mM), found rapid increases in the proportion of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine accompanied by a decrease in phosphaditic acid content. It is obvious, therefore, that the phospholipid changes which occur in response to ethanol exposure are peculiar to the cell type under study. Both the present study and previous work (Keegan et ul.. 1983), however, suggest that the changes in phospholipid class distribution are an acute effect, occurring within the first day of exposure to ethanol. These changes may represent attempts by the cell to counter ethanolinduced membrane fluidization; alternatively, they may reflect direct effects of ethanol on membrane phospholipid turnover. A high concentration of phospholipid has been reported in the medial tissue of atherosclerotic vessels in rabbits and humans (Eisenberg ct al., 1969a: Bottcher and Van Gent, 1961). Over 70”,, of the increase in phospholipid was due to a raised sphingomyelin concentration, with only slight increases in phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylserine. Two possible mechanisms have been suggested to account for this phenomenon. Eisenberg et ul. (I 969b) found a significantly lower activity of sphingomyelinase in the media of atheromatous vessels in humans, suggesting that a decrease in sphingomyelin breakdown may account for the higher sphingomyelin level. Seth and Newman (1975) found no increase in sphingomyelin synthesis, but an increase in sphingomyelin uptake from serum lipoproteins, large enough to account for the higher medial sphingomyelin concentration. It is quite possible that the protective effect of moderate alcohol intake against atherosclerosis may be partly related to the lowering of vascular smooth muscle sphingomyelin concentration as observed in the present study. The fatty acid composition of smooth muscle cell phospholipids was altered by the presence of ethanol in the culture medium. We found a significant increase in the degree of unsaturation of the phospholipid acyl groups. Mechanisms to account for such alterations have been suggested on the basis of studies of the regulation of prokaryote membrane composition. Ingram (1982) postulated that the biosynthesis of saturated or unsaturated fatty acids in E. co/i may be related to the strength of hydrophobic interactions between membrane lipids and membrane-bound lipid-synthesizing enzymes, The presence of ethanol weakens hydrophobic interactions and thus causes increased synthesis of polyunsaturated fatty acids. This thesis is supported by the observation of Buttke and Ingram (1978) that the presence of ethanol decreased the amount of saturated fatty acid synthesized by E. coli, and so decreased the saturated fatty acid available for incorporation into phosphohpids. Melchior and Stein (1976) suggested a mechanism to account for these changes. They point out that the enzymes responsible for catalysing transfer of acyl groups from acyl CoA to phospholipid are embedded in the lipid bilayer and. although the enzymes have no intrinsic ability to differentiate between the various acyl derivatives of CoA, regulation of their catalytic function occurs according to the physical state of the supporting
DAVII) A COOK rt cd
866
membrane. The changes in phospholipid fatty acid composition observed in the present study could thus be due to a change in catalytic function of the acyl CoA-phospholipid acyl transferase group of enzymes. through altered substrate specificity. .4~,~noll,/~djiemmtThis study was supported National Heart Foundation of Australia.
in part by the
REFERENCES
Bakardjieva A., Gabba J. H. and Helmriech E. J. M. (1979) Modulation of the receptor adenylate cyclase interactions in the cultured Chang liver cells by phospholipid enrichment. Biodwmis/~~~ 18, 30 I c-3023. Bates S. R. (197X) Phospholipid metabolism of monkey smooth muscle cells grown in hyperlipidemic serum. Bioc+ir~. hiophj~.s. Actu 530, 175-l 87. Bligh E. G. and Dyer W. J. (1959) A rapld method of total lipid extraction and purilication. Ctm. J. Biochcm. Physid. 37, 91 l-917. Bonnichsen R. (1965) Ethanol determination with alcohol dehydrogenase and DPN. In Methods ofEnqwwric And~si.\ (Edited by Bergmeyer H.), 2nd edn. p. 2X5. Academic Press. New York. Bottchcr C. J. F. and Van Gent C. M. (1961) Changes in the composition of phospholipids and of phospholipid fatty acids associated with the atherosclerosis in the human aortic wall. J. Atheroder. Rex. I, 36-43. Buttke T. M. and Ingram L. 0. (1978) Mechanism of ethanol induced changes in lipid composition of E. coli: Inhibition of saturated fatty acid synthesis in r,iro. Bioc~hc’nii.s/~>~ 17, 639 644. Chin J. H.. Parsons L. M. and Goldstein D. B. (1978) Increased cholesterol content of erythrocyte and brain membranes in ethanol tolerant mice. Biochim. hiophv.s. Ac,rc/ 513, 35X 363. Eisenberg S.. Stein Y. and Stein 0. (1969a) Phospholipases m arterial tissue (III). Biochim. hiophys. Actor 176, 557 569. Eisenbcrg S.. Stein Y. and Stein 0. (1969b) Phospholipases in arterial tissue (IV). J. din. Inrest. 48, 2320-2329. Farias R. N.. Bloj B.. Morero R. D.. Sineriz F. and Trucco R. E. (1975) Regulation of allosteric membrane-bound enzymes through changes in membrane lipid composition. Biodwn. hiophw. Acrrr 415, 23 l-235. Folch J.. Lees M. and Sloane Stanleu G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. hiol. Cllem. 226, 497-509. Hanski E.. Rimon G. and Lcvitzki A. (1979) Adenylate cyclase activation by adrenergic receptors as a diffusion controlled mocess. Biwhem. 18(5), 846X53. Ileron D. S.. ‘Shinitzky M., Hershkowitz M. and Samuel D. (19X0)Lipid Iluidity markedly modulates the binding of aerotonin to mouse brain membranes. Proc,. n&/r. AL.&. Sci. I ‘..~.A. 77, 746%7467. Horwitl A. F.. Wight A.. Ludwig P. and Cornell R. (1978) Interrelated lipid alterations and their influence on the proliferation and fusion of cultured myogenic cells. J. (‘
Ingram L. 0. (19X2) Regulation of fatty acid compocitlon of E. co/i: a proposed common mechanism for change\ involved by ethanol. chaototrophic agents and a rcduction in growth temperature. J. Bud. 149, 160 172. Ingram L. 0.. Ley K. D. and Holt‘man E. M. c19iS1 Drug-induced changes in lipid composition of E. ~j/i and of mammalian cells in culture: ethanol. pentobarbltol l~nd chlorpromazine. Life Sci. 22, 489 -494. Keegan R.. Wilce P. A.. Ruczkal-Pictrrak E. and Shanlc! B. C. (1983) Effect of ethanol on cholesterol and phc~\pholipid composition of HeLa cells. Biodwn~. h/rydr~.\ Res. Commun. 114, 985-990. Kent C.. Schimmel S. D. and Vagclos P. R. (1974) l.lp~~l composition of plasma membranes from dcvcloplng chick muscle cells in culture. Bimhim. Bioplr~,\. .i cfci 360. 312. 321.
Kimelberg I-1. K. ( 1975) Alteration\ 1n phosphollplcl~ dependent (Na’ ~ K +)-ATPasc activity due to Ilp~(l fluidity. Biochim hioph>~s. .Acfrr 413, 143 156. LaPorte R. E.. Cresanta J. L. and Kullet I. Il. (l9S01 Relationship of alcohol consumption to athero\clcrotlc heart disease. Prer. Merl. 9(l). 22 40. Li G. C.. Shiu E. C. and Hahn CJ. M. (IWO) Smul;trltlc\ 111 cellular inactivation by hyperthermia or ethanol. Kcr~l,~,/ RP.Y. 82, 257-268. Lowry 0. H., Rosebrough N. J.. Farr L. and Randall K. J (I 951 ) Protein measurement with the Folin phenol r-i‘~ agent. J. hid. Chem. 193, 265 -27.5. Marinetti G. V. (Ed.) (1967)I_ipic/ (‘/trollltrr~,,~~[r/~/~/(.Imii y.si.s, Vol. 1. Marcel Dekkcr, New Yorh. Mathews J. D. (1979) Alcohol and hypcrten\ion ,Irc\i. \ / J. Med. 9, I24- 128. Melchior D. L. and Stem 1. M. (1976) 1 hcrmotropl< transitions in biomembranca. -1. Kw. Bicrp/r~~\.R~~w~~ 5. 205%23X. Morrison W. R. and Smith L. M. (1964) Preparation 111 fatty acid methyl esters and dimethylacctal\ 1’rom lipId\ with boron fluoride-methanol. .I. Lipid Rc\. 5. hO0 60X Ross R. (1971) The smooth muscle cell 1n culture II Growth of smooth muscle in culture and I;)rmalic~r 01 elastic tibres. J. Cc// Bid. SO, 172-l X6 Rouser G.. Siakotos A. N. and Flelschcr S. (I9hh) QLLIIItitative analysis of phosphohpid by thin laker cht-o matography and phosphorus analysts of \pot\. (,~/~c~/rI. X5-86. Seth S. K. and Newman H. .A. I. (1975) Sph~ngomqcl~n ,III~I other phospholipid metabolism In rabbit atheromatou\ and normai aoria. C‘irr,. RPY. 36, 294 299. Victor M. and Adams R. D. (19x7) Alcohol. In I~u~,-,\~I/I t
Waring A. J.. Rottenburg H.. OhmshI ‘I’. and RubIn I (1981) Membranes and phospholipids of li\cr mitt>-chondria from chronic alcoholic rats are rcsl\tunt to membrane disordering by alcohol. Pro< m,,t,. 4~~rri.SfJ U.S.A. 78, 2581~35x6. Wmg D. R.. Harvq D. J.. llughes J., Dunbar I’ (1 McPherson K. A. and Paton W. I). M. CIOX?)I‘:ll’ccr~ of chronic ethanol administration on the compo%;ltion (11’ membrane lipids m the mouse. Biodwm. I’/urrmc~~~~d31. 3431-3439.
0020-711X/85 $3.00 + 0.00
CopyrIght ‘:LB1985 Pergamon Press Ltd
EFFECTS OF ETHANOL ON THE LIPID OF BOVINE VASCULAR SMOOTH CELLS IN CULTURE DAVID
Department
A.
COOK, PETER
of Biochemistry.
University (Received
A. WILCE and of Queensland, 2 I Not)enlher
BRIAN
St Lucia,
C.
COMPOSITION MUSCLE
SHANLEY*
Queensland
4067, Australia
1984)
Abstract-l. Ethanol (50 mM) had no effect on the growth rate or viability of arterial smooth muscle cells over 3.5 days. 2. The cholesterol:phospholipid ratio of the cells was unchanged after 7 days exposure. 3. The major phospholipid components phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were unchanged by ethanol exposure. Sphingomyelin content fell significantly within 12 hr. 4. There were major changes in the fatty acid composition of the phospholipids with a reduction in saturated fatty acids and an increase in unsaturated fatty acids.
INTRODUCTION
75 cm’ culture flasks (Corning Industries) using Dulbecco’s modified Eagles medium. Medium was changed every three days or sooner. Cells were subcultured during logarithmic growth as the culture approached confluence. For viability studies cells were plated onto 2.5 cm diameter culture dishes at a density of 5000 viable cells/cm’. After allowing time for the cells to adhere (6 hr) the cultures were grown in 2 ml of medium containing ethanol (0, 25, 50, 100 or 200 mM) and harvested at specified times (0, 12. 36, 60 or 84 hr). Cell counting was performed with a Coulter Counter Model ZF. The enzymatic assay of Bonnichsen (1965) was used to monitor ethanol concentration in the medium.
The involvement of ethanol in the pathogenesis of ischaemic heart disease is complex. Epidemiological evidence, supported by some animal studies, suggests moderate consumption of ethanol may have a protective effect by impeding the development of atherosclerotic plaques (La Porte, 1980). In contrast high ethanol consumption is recognised as a causal agent in hypertension and atherosclerosis (Mathews, 1979). It is not known whether these changes are due to indirect
effects,
e.g. through
alcohol-induced
changes
in lipoprotein metabolism or to direct effects on cells of the arterial wall. Chronic exposure of various tissues to ethanol has been observed to result in alterations in membrane lipid composition, both in whole animals (Chin et al., 1978; Waring et al., 1981; Wing et al., 1982) and in cell cultures (Ingram rt ul., 1978; Keegan et al., 1983). Since changes in membrane lipids can alter the activity of membrane-bound enzymes (Kimelberg, 1975; Farias et al., 1975) and the binding characteristics of receptors (Heron et ul., 1980, Bakardjieva et NI., 1979; Hanski et al., 1979) it is possible that changes in smooth muscle function, particularly receptor-mediated uptake of lipoproteins, may be influenced by ethanol-induced changes in the lipid bilayer. In this paper we report the results of an investigation into the effects of ethanol (50 mM) on the lipid composition of smooth muscle cells maintained in culture.
Lipid atruction
MATERIALS AND METHODS Ccl1 culture Cells were obtained from a primary explant of foetal bovine thoracic aorta (Ross. 1971). Cultures were grown in *Author
to whom
reprint
requests
should
and anui~.~is
Lipid analyses were performed on cells grown until the 8th passage. At this time, the medium was changed to one containing ethanol and maintained for up to 7 days. Cell cultures were harvested. cooled to 4 C and pelleted. Lipids were extracted from the cell pellets according to the method of Bligh and Dyer (1959) utilizing the salt solution of Folch el al. (1956) for the upper aqueous phase. The lipid extract was dissolved in chloroform and stored under nitrogen at -20’C in glass vials with Teflon-silicone septa until analysis. Butylated hydroxytoluene (250 g/ml) was used as an anti-oxidant throughout. Extraction efficiency of lipids was calculated by monitoring the recovery of [‘HIcholesterol and [“Clphosphatidylcholine. Mean recoveries were 63 and SSP,, respectively. Cholesterol was assayed by isothennic gas liquid chromatography as described by Horwitz L’I ul. (1978). Total lipid phosphorus was measured by the method of Rouser et al. (1965). The quantities of cholesterol and phospholipid contained in the samples were calculated with respect to cell protein (Lowry et cd., 1951) and adjusted according to extraction efficiency. Phospholipid classes were separated by unidimensional thin layer chromatography. A 0.3 mm thick layer of silica gel H containing lo”, (w/w) magnesium trisilicate was air dried and activated for 2 hr at 110 C prior to use. Plates were developed with a solvent system comprising chloroform: methanol: petroleum spirit (b.p. 4&60 C): acetic acid (40:20: 30: IO) containing 0.9”;, borate (w/v). Lipid spots
be addressed. 863
were visualized with iodinc vapour and tdentificd hq comparison with R, values ol‘ standards. Areas of silica gel corresponding to each class were quantrtatively collected and inorganic phosphorus was determined according to Rouser PI &. ( 1966). For acyl group analysis. phosphoiipids wcrc isolated under N2 as a hulk group by thin layer ~hr~~rn~tog~~phy on Glica gel G devclopcd in ;t petrolcum spirit (b.p. 40.-60 C):diethyl ether:acetic acid (90: IO: I) according to Marinette (1967). The phospholipids. which remained at the origin were scraped oft‘ and extracted twice with chloroform:mcthanol (I : I ). The dried eluates were methylsted by the method of Morrison and Smith (1964) and analyscd on a 1.5 m glass column packed with lo”,, (w:w) SF’330 on 100/l 70 Chromosorh WAW (Supelco Inc.. Pennsylvania, U.S.A.) in a Shimadzu hC-6AM chromatograph using N2 as carrier. Individual fatty acid methyl esters were dctectcd by Bane ionization.
All ~aiues are expressed as mean + SEM detcrmin~t~ons. Signi~~~ncc was determined r-test.
of nt least 4 by Student’s
RESL!LTS
Doubling times for cells grown in ethanol up to 200 mM were determined. Semi-logarithmic plots of cell density vs time were linear at all concentrations, implying that viability and logarithmic growth were maintained. There was. however, a significantly increased doubling time at an ethanol concentration of 200 mM (26.9 hr compared to 21.4 hr in controls. P < 0.05). 50 mM ethanol was used for subsequent experiments. This gave a doubling time of 22.8 hr. Loss of ethanol from the cultures was estimated at 6’1,, per day.
The cholesterol and phospholipid concentration of arterial smooth muscle cells (4.6 I: 0.24 ~mol~rng protein and 9.8 +_0.5 jLmol/mg protein respectively), remained unchanged after incubation of cells with ethanol for up to 48 hr. Similarly, the cholesterol: phospholipid ratio (0.48 & 0. I) was unchanged after 48 hr exposure. To investigate alterations in the relative proportions of the major phospholipid classes, smooth muscle cells were grown in medium containing
c
s s
II
1 I 1234
I
I
1
t
t
5
6
7
Days Fig. 1. Retativc proportions of the phospholipid classc~ from smooth muscle cells grown in medium containing ethanol (SOmM). Points represent mean of 4 dcterminations. Error bars have been omitted for ctarity (0) Phosphatidylcholint. (m) phusphatidylethanolamine. (r) phosph~tid~lserin~ and phospllatidrlinosltol. (0 t sphingomyciin. Values fur sphingor~ly~lln at 1. 7 days were signtiicantly diffrrent from control I P c 0.051.
ethanol for periods of up to 7 days. Figure I shows the relative proportions of the major phospholipid classes during the course of the experiment. The major phosphol~pid component, phosphatidyl” choline, varied from 46.24 & 1.Wo to 5 1.8Yf 3.68”,, of the lipid phosphorus. Phosphatidylethanolamine varied between 26.68 t_ 0.56”U and 31.35 + 0.71”,, of the total phospholipid. Phosphatidylserine and phosphatidylinositol together comprised between 13.97 _t 0.3!‘!,, and 16.22 iii.(X4”,, The changes in the cellular content of these lipids during the course of the experiment were not statistically significant. By contrast significant differences were obvious in the proportion of sphingomyelin. After 12 hr exposure trl ethanol the proportion of sphingomyclin had fallen from LO.23 + 1.29”,, to 3.79 + 0.97”,, (P < 0.01) and remained low at approximately S”,, of the total phospholipids for the duration of the experiment. Table 1 shows the fatty acid composition of phos-
Ethanol and smooth muscle cells pholipids from smooth muscle cells grown for up to 7 days in medium containing ethanol (50 mM). The percentage of 14:O and 16:O fell significantly within 24 hr and remained depressed for 7 days. The concentration of 18: I declined over the initial 12 hr and maintained this lower level for the duration of the experiment. These changes were compensated by an increase in the proportion of 16: I, 22: 1 and 22: 6 within I2 hr exposure to ethanol. There was no change in the proportion of the other major fatty acids. The nature of the changes can be readily appreciated when alterations in degree of saturation are examined. There was a fall in the proportion of fatty acids that are fully saturated from 42.83 f 0.02:‘; to 38.47 f 0.05)‘” (P < 0.001) after 7 days exposure to ethanol. The double bond index @[percentage of each unsaturated fatty acid x number of its double bonds]) is an index of the degree of fatty acid unsaturation (Farias PI u/., 1975). It increased by 20”); in the first 12 hr and maintained this higher level ’ throughout the incubation. The increased degree of unsaturation shown by the higher double bond index after ethanol exposure is consistent with the fall observed in the percentage of saturated fatty acids. DISCUSSION
Smooth muscle cells proliferated in the presence of ethanol at higher concentration than can be achieved in cir?o. The doubling time of smooth muscle cells grown in medium containing ethanol (200 mM) was significantly longer than that of controls (26.9 hr compared to 21.4 hr). However, these cells still maintained logarithmic growth. Keegan et cd. (1983) found that ethanol (86 mM) had no effect on the growth rate of HeLa cells. Li et al. (1980) exposed Chinese hamster ovary cells (HA-l) to 660mM ethanol for 45 min and demonstrated no loss of cell viability. In the present study subsequent experiments were carried out with medium containing 50 mM ethanol. This concentration did not impede cell proliferation and can be readily achieved in rice (Victor and Adams, 1983). We found no change in the cholesterol:phospholipid ratio of smooth muscle cells exposed to ethanol (50mM) for 48 hr. This agrees with the results of Keegan rt u/. (1983) who found no alteration in the cholesterol: phospholipid ratio after HeLa cells were exposed in culture to ethanol (86mM) for up to 8 days. The relative proportions of each phopsholipid class found by us in bovine vascular smooth muscle cells in culture were very similar to those of cultured embryonic chick muscle cells (Kent et d., 1974) and cultured monkey vascular smooth muscle cells (Bates rt UI., 1978). The decrease in the proportion of sphingomyelin observed when bovine vascular smooth muscle was grown in the presence of ethanol (50 mM, Fig. 1) was similar to changes found by Ingram e/ crl. (1978) in Chinese hamster ovary cells grown in the presence of ethanol (139 mM). However, Ingram et ul. (1978) also found a decrease in the percentage of lysophosphatidylcholine and an increase in phosphatidylcholine. Keegan et rtl. (1983) studying HeLa
865
cells grown in ethanol (86 mM), found rapid increases in the proportion of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine accompanied by a decrease in phosphaditic acid content. It is obvious, therefore, that the phospholipid changes which occur in response to ethanol exposure are peculiar to the cell type under study. Both the present study and previous work (Keegan et ul.. 1983), however, suggest that the changes in phospholipid class distribution are an acute effect, occurring within the first day of exposure to ethanol. These changes may represent attempts by the cell to counter ethanolinduced membrane fluidization; alternatively, they may reflect direct effects of ethanol on membrane phospholipid turnover. A high concentration of phospholipid has been reported in the medial tissue of atherosclerotic vessels in rabbits and humans (Eisenberg ct al., 1969a: Bottcher and Van Gent, 1961). Over 70”,, of the increase in phospholipid was due to a raised sphingomyelin concentration, with only slight increases in phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and phosphatidylserine. Two possible mechanisms have been suggested to account for this phenomenon. Eisenberg et ul. (I 969b) found a significantly lower activity of sphingomyelinase in the media of atheromatous vessels in humans, suggesting that a decrease in sphingomyelin breakdown may account for the higher sphingomyelin level. Seth and Newman (1975) found no increase in sphingomyelin synthesis, but an increase in sphingomyelin uptake from serum lipoproteins, large enough to account for the higher medial sphingomyelin concentration. It is quite possible that the protective effect of moderate alcohol intake against atherosclerosis may be partly related to the lowering of vascular smooth muscle sphingomyelin concentration as observed in the present study. The fatty acid composition of smooth muscle cell phospholipids was altered by the presence of ethanol in the culture medium. We found a significant increase in the degree of unsaturation of the phospholipid acyl groups. Mechanisms to account for such alterations have been suggested on the basis of studies of the regulation of prokaryote membrane composition. Ingram (1982) postulated that the biosynthesis of saturated or unsaturated fatty acids in E. co/i may be related to the strength of hydrophobic interactions between membrane lipids and membrane-bound lipid-synthesizing enzymes, The presence of ethanol weakens hydrophobic interactions and thus causes increased synthesis of polyunsaturated fatty acids. This thesis is supported by the observation of Buttke and Ingram (1978) that the presence of ethanol decreased the amount of saturated fatty acid synthesized by E. coli, and so decreased the saturated fatty acid available for incorporation into phosphohpids. Melchior and Stein (1976) suggested a mechanism to account for these changes. They point out that the enzymes responsible for catalysing transfer of acyl groups from acyl CoA to phospholipid are embedded in the lipid bilayer and. although the enzymes have no intrinsic ability to differentiate between the various acyl derivatives of CoA, regulation of their catalytic function occurs according to the physical state of the supporting
DAVII) A COOK rt cd
866
membrane. The changes in phospholipid fatty acid composition observed in the present study could thus be due to a change in catalytic function of the acyl CoA-phospholipid acyl transferase group of enzymes. through altered substrate specificity. .4~,~noll,/~djiemmtThis study was supported National Heart Foundation of Australia.
in part by the
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