Relative specificities of inhibition of acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase in cultured rabbit aortic smooth muscle cells by esterastin and cholesteryl oleyl ether

Biochimica et Biophysica Acre, 1004(1989) 139-142

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BBA Report

Relative specificities of inl'fibifion of acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase in cultured rabbit aortic smooth muscle cells by esterasfin and cholesteryl oleyl ether R o b e r t J. M o f i n a n d S h i - K a u n g P e n g Department of Pathology, Harbor.UCLA Medical Center, Torrance, CA (U.S.A.)

(Received 8 DeceL,ber1988) Key words: Acidcholesterylester hydrolase;Neutral cholesterylester hydrolase; Cholesteryloleylether; Esterastin;(Aorticsmooth musclecell) Rabbit aortic smooth muscle cells in culture were incubated with 0.04-500 M estela~an. Acid cholesteryl ester hydrolase (ACEH) and neutral cholesteryl ester hydrolase (NCEH) activities were inhibited to a comparable degree, with 50% inhibition occurring in the range of 0.4 M esterasfin. Cells incubated with cholesteryl oleyl ether showed $0% inhibition of NCEH at 5.0 M, but no inhibition of ACEH over a concentration range of 0.2-20 M. This relative specificity of cholesteryl oleyl ether for NCEH can be employed to study the relative roles of ACEH vs. NCEH [n preventing cellular cholesteryl ester accumulation.

Atherosclerosis is characterized by a marked increase in the content of cholesteryl esters in the arterial wall. It appears probable that cholesterol enters arterial cells in the free and ester forms via receptor-mediated endocytosis of plasma low-density lipoproteins [1]. Hydrolysis of cholesteryl esters occurs within the lysosomes, and the free cholesterol released is subsequently esterified by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT). The relative activities of ACAT vs. cholesteryl ester hydrolases may determine the net accumulation of cholesteryl esters by arterial cells. There appear to be at least two distinct enzymatic mechanisms for hydrolysis of cholesteryl esters in arterial cells. One is an acid cholesteryl ester hydrolase (ACEH), which has been identified in lysosomes of rabbit, rat, guinea pig, monkey and human arteries [2-6]. This enzyme has a pH optimum between 4 and 5, is not cofaetor-dependent, and probably functions primarily to hydrolyze cholesteryl esters of the plasma lipoproteins that are internalized by arterial cells. When normal or acetylated LDL was used as the substrate for rat arterial wall homogenates, a single peak hydrolytic

Abbreviations: ACEH, acid cholesterylester hydrolase; NCEH, neutral cholesteryl ester hydrolase; ACAT, acyl-CoA:cholesterol acyltransferase. Correspondence: R.J. Morin, Department of Pathology, HarborUCLA MedicalCenter, 1000W. Carson Street, Torrance, CA 90509, U.S.A.

activity was noted at pH 4.5; very tittle hydrolysis occurred at neutral or alkaline pH [3]. Another enzyme, with an optimum pH in the reutral range (6.5-7.5), has been identified in rat, rabbit, monkey and human aortas [7-9] and has been termed neutral cholesteryl ester hydrolase (NCEH). This NCEH is located in the cytosol portion of the cell, appears to be hormone-sensitive, and is activated by glucagon and cyclic AMP [9]. The latter activation appears to be d-~: to an ATP- and Mg2+-dependent protein kina~¢. In arterial lipid-laden foam cells, this NCEH is bound to cytoplasmic cholesteryl-ester-rich lipid droplet,~. ACEH activity seems to increase 2-4-fold in rabbit or pigeon atherosclerosis, whereas NCEH is decreased in the latter species [10-13]. The relative roles ,of ACEH and NCEH in possibly preventing accumulation of cholesteryl esters in arteries are not yet understood. If means were available to selectively inhibit one or both of these enzymes, this would be a useful tool in establishing their relative significance in atherogenesis. Inhibition of ACEH in pig aortic smooth muscle cells in culture by esterastin, has previously been reported [14], but its effects on NCEH were unknown. Cholesteryl oleyl ether has been shown to be a potent NCEH inhibitor in rat liver cells [15], but its effects on ACEH are unknown. The present study was designed to determine the relative specificities of inhibition of ACEH vs. NCEH by esterastin and cholesteryl oleyl ether. Aortas of New Zealand white young adult rabbits (2-3 kg) were aseptically removed using the ventral

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140 approach. After dissection of adventitial fat, aortic segments were immersed in basal culture medium and cut into approx. 1 mm square pieces. All fragments were treated with 0.25 collagenase, incubated at 37 °C for 15 min to remove endothclium, washed twice with growth medium, and then transferred into 30 ml Falcon flasks.These explants were nourished in Eagle's medium supplemented with 105 fetal calf serum and antibiotics. Medium in the flasks was changed three times a week. After growth to confluence (3 weeks), the cells were washed with calcium.and magnesium-free phosphatebuffered saline solution. 2 ml of 0.205 trypsin was added to each flask and then decanted off 2 rain later. After incubation for 10 rain, with occasional agitation, all cells were detached from the flasks. The remaining small amount of trypsin was inactivated by the addition of 10 nd of culture medium containing 105 fetal calf serum. "l'he cells were transferred into new 75 sq. cm. Falcon flasks. Confluent monolayers of aortic smooth muscle cells developed within 2 weeks. Cell cultures were incubated in lipoprotein-deficient media (sera delipidized as described by Brown et aL [16] for 24 h prior to ACAT assay. Esterastin (5-(N-acetyl-L-asparaglnyloxy)-2.hexyl.3.hydroxy-7,.10-hexadecadienoic iactone) prepared from Streptomyces lavendulae strain MD~C1 [17] was obtained from the Institute of Microbial Chemistry, Tokyo, Japan. Varying concentrations of esterastin in ethanol or ethanol control were incubated with the cell cultures for 1 h. Cholesteryl oleyl ether was prepared by alcoholysis of cholesterol p-toluene sulfonate [18]. Varying concentrations of this agent in acetone, or acetone control alone were incubated with other cell cultures for 1 h. No cell death was observed by microscopy after treatment with either agent. Cells were then washed three times with media, the media were discarded, and the cells were then scraped with a rubber policeman into 0.15 M Tris-HCl buffer (pH 7.4) containing 0.01 M mer~ptoethanol and 0.5 m g / m l defatted bovine serum albumin. Cells were then completely homogenized using a glass Potter-Elvehjem homogenizer. Assay of NCEH was done using a mixed micellar substrate [19] prepared as follows: 10/tCi of cholesteryl [l"t4C]oleate, (Amersham, 55 mCi/mmol) was freed of contaminating fatty acids by liquid-liquid partitioning using a methanol/water, 1 : 1 phase and a chloroform/ heptane, 5:4 phase [20]. The purified radioactive substrate was added to chloroform containing 3.8 ttmol of phosphatidylcholine and 0.8 /tmol of unlabeled cholesteryl oleate. The solvent was evaporated under N 2 at 37°C and the iipids were suspended in 8.0 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 2.0 trine| sodium taurocholate. This was sonicated at 46 °C in a Branson Sonifier using 100 watts, then centrifuged at 30 000 rpm for 15 min in a Beckman 50 Ti rotor to remove particulate matter. Incubation mixtures were

prepared by adding 50 #1 of the micellar cholesteryl [l-m4C]oleate substrate to 0.8 ml of 0.1 M phosphate buffer (pH 7.0) with 0.05% bovine serum albumin and 0.15 ml cell homogenate (0.20 mg protein) in the same buffer. After incubation for 60 min at 37 ° C, 16.3 ml of methanol/ chloroform/ heptane, 1.4: 1.3 : 1.0, and 5.3 ml of 50 mM potassium carbonate/50 mM potassit, m borate buffer (pH 10.0) was added. The mixture was vortexed for 5 min, shaken for 30 min and centrifuged for 10 min at 2500 rpm to separate the phases. Radioactivities in aliouots of the upper aqueous phases containing the [1J4C]-oleate product were counted in an Aquasol-2 scintillation solution. Micellar [1-14C]oleic acid standards were incubated, processed and counted in an identical manner to monitor recovery. Assay of ACEH was done by a modification of the method of Haley et al. [21] using a micellar cholesteryl [1-14C] oleate substrate prepared as described above for the NCEH assay. After sonication, one part of the prepared phosphatidylcholine dispersion was added to four parts 0.125 M sodium acetate buffer (pH 3.9) containing 5.0 mM sodium taurocholate. Cells were homogenized with a glass mortar and pestle in 0.25 M sucrose containing 1.0 mM EDTA and 0.1~ ethanol (SVE) and centrifuged for 10 win at 1000 rpm. The pellets were rehomogenized in SVE, recentrifuged, and the combined supernatants were used as the enzyme source. These were adjusted to 0.1~ digitonin and incubated for 10 min at 0°C. For ACEH assay 100 pl (0.15 mg protein) of the enzyme preparation and 100/~1 of the sub, irate solution were mixed and incubated at 37°C for 60 min. The reactions were stopped and the substrates and products separated as described above for the NCEH assay. The radioactivities of the [1t4C]oleate in the aqueous phase were counted as described above. Protein content of the cell homogenates was determined by the Lowry method [22]. Specific activities of ACEH in the control rabbit arterial smooth muscle cell cultures were 60.9 + 5.0 pmol/mg protein per min and for NCEH were 4.51 + 1.9 pmols/mg protein per rain. Percentage inhibitions of each of these enzyme activities by varying concentrations of esterastin are shown in Fig. 1. Inhibition was virtually complete at 250 ~M esterastin concentration, and had disappeared at 0.04 ~M. Although esterastin inhibited both enzymes, the inhibition of NCEH was slightly lower at 0.4 and 2.0/~M. The 50~ inhibition of ACEH at 0.4/LM esterastin is in a range similar to that observed by Ecsedi et al. [14] in cultured pig aortic smooth muscle cells. In the latter experiments, esterastin had been presumed to be a specific lysosomal enzyme inhibitor, and had previously been demonstrated to inhibit rabbit fiver lyscsomal acid lipase [23]. When pig aortic ce||s were incubated for 48 h with low-density lipoprotein together with esterastin, this resulted in a

141 13-fold increase in the cellular content of cholesteryl esters [14]. Similarly, cells incubated with cholesleryl oleate liquid crystals and esterasdn showed a 7-fold increase in cholesteryl ester content. The ratio of esterifled to free cholesterol was increased 5-fold in both experiments. These increases cannot be presumed to be a'specific result of inhibition of lysosomal ACEH, however, since our present experiments demonstrate an equal inhibition of NCEH. Results of incubation of rabbit aortic smooth muscle cell cultures with varying concentrations of cholesteryl oleyl ether are shown in Fig. 2. This compound appeared to selectively inhibit NCEH activity, and had no significant effect on ACEH in the concentration range studied. It is possible that cholesteryl oleyl ether did not gain access to the lysosomal ACEH, but it seems likely that since this compound penetrated the cell membrane that it would also penet~ate the lysosome membrane. A concentration of 5 #M cholesteryl oleyl ether resulted in a 50% inhibition of NCEH. Ttfis is somewhat higher than the 50% inhibition by 0.9 #M cholesteryl oleyl ether of partially purified rat liver cholesteryl ester hydrolase assayed at pH 8.0 [15]. The difference was most likely attributable to the differences in enzyme source, preparation, and assay conditions. In the rat liver enzyme study, cholesteryl oleyl ether appeared to be a relatively non-specific inhibitor, since it also showed similar inhibitory effects on retinyl palmitate hydrolase and tdolein lipas¢ [15]. These assays were all done at pH 8.0, and it is therefore possible that the lack of

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Fig. 2. Semi-logarithmic plot of effects of cholesteryl oleyl ether on acid cholesteryl ester hydrolase (ACEH) and neutral cholesteryl ester hydrolase (NCEH) in cultured rabbit aortic smooth muscle cells. Mean percentage inhibitions of quadruplicate experiments :LS.D. are shown.

inhibition of ACEH in our experiments was related to the acid incubation conditions. Inhibition of NCEH by cholesteryl oleyl ether reached a maximum of 60%. In a previous study [9], rabbit aortic cholesteryl ester hydrolase activity assayed using the relatively specific ACEH phospholipid-digitonin substrate dispersion showed a small percentage of activity at pH 7.0. Since ACEH activity in the oresent study was 13.5 times l'figher than NCEH, it is possible that the apparent non-irfffibitable portion of NCEH activity may represent that fraction of ACEH activity occurring at pH 7.0. The selective action of cholesteryl oleyl ether can be employed to establish whether prolonged specific inhibition of NCEH by this compound results in accumulation of cellular cholesteryl esters comparable to that observed with esterastin-induced simultaneous inhibition of ACEH and NCEH. These studies were supported by NIH Grant HL 32527, grant awards from the Paul Glenn Foundation, the Candle Foundation, and Research Grant Award 895G-1 from the American Heart Association, Greater Los Angeles Affiliate. The excellent technical assistance of Nora Riley is gratefully acknowledged. We also thank Dr. Hamao Umezawa of the Institute of Micr,~bial Chemistry, Tokyo, Japan, for his generous gift ~f esterastin. References 1 Goldstein, J.L. and Brown, M.S. (1982) in Metabolic Risk Factors in lschemic Cardiovascular Disease. (Carlson, L.A. and Pernow, B., eds.), pp. 17-34, Raven Press, NY. 2 Takano, T., Black, W.J., Peters, T.J. and De Duve, C. (1974) J. Biol. Chem. 249, 6732-6737.

142 3 Shinomiya, M., Saito, Y. and Kumagai, A. (1983) Atherosclerosis 46, 233-238. 4 Severson, D.L. and Fletcher, T. (1978) Atherosclerosis 31, 21-32. 5 Dousset, J.C., Dousset, N., Foglietfi, M.J. and Douste-Biazy, L. (1981) Bioching Biophys. Aeta 664, 273-277. 6 Smith, A.G., Brooks, CJ.W. and Harland, W.A. (197.~) Steroids Lipids Res. 5, 150-161. 7 Kothari, H.V., Bonner, M.J. and Miller, B.F. (1970) Biochim. Biophys. Acta 202, 325-331. 8 Kothari, H.V., Miller, B.F. and Kritchevsky, D. (1972) Biochim. Biophys. Acta 296, 446-458. 9 Hajjar, D.P., Mim'ck, C.R. and Fowler, S. (1983) J. Biol. Chem. 258, 192-198. 10 Brecher, P., Pyan, H.Y. and Chobanian, A.V. (1977) J. Lipid. Res. 18, 154-162. 11 Kritchevsky, D., Tepper, S.A., Genzano, J.C. and Kothari, H.V. (1974) Atherosclerosis 19, 459-462. 12 Haley, N.J., Fowler, S. and De Duve, C. (1980) J. Lipid Res. 21, 961-969.

13 St. Clair, R.W. (1976) Atherosclerosis Rev. 1, 61-106. 14 Escedi, G.G., Amanuma, K., Iman~a, T., Aoyagi, T., Ohkuma, S. and Takano, T. (1985) Biochem. Internat. 10, 337-342. 15 Blaner, W.S., Halperin, G., Stein, O., Stein, Y. and Goodman, D.S. (1984) Biochim. Biophys. Acta 428-434. 16 Brown, M.S., Dana, S.E. and Goldstein, J.L. (1975) J. Biol. Chem. 250, 4025-4027. 17 Umezawa, H. and Aoyagi, T. (1978) J. Antibiotics 31,639-641. 18 Halperin, G. and Gait, S. (1980) Steroids 35, 39-42. 19 Hajjar, D.P. and Weksler, B.B. (1983) J. Lipid Res. 24, 1176-1185. 20 Belfrage, P. and Vaughan, M. (1969) J. Lipid Res. 10, 341-344. 21 Hale)', N.J., Folwer, S. and DeDuve, C. (1980) J. Lipid Res. 21, 961-969. 22 Lowry, D.H., Rosenbrough, N.J. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 23 imanaka, T., Moriyama, Y., Ecsedi, G.G., Aoyagi, T., AmanumaMuto, K., Ohkuman, S. and Takano, T. (1983) J. Bioci:em. 94, 1017-1020.