The prostacyclin analogue iloprost and prostaglandin E1 suppress sterol synthesis in freshly isolated human mononuclear leukocytes

BBA Report

BBA 50105

The prostacyclin analogue iloprost and prostaglandin E , suppress sterol synthesis in freshly isolated human mononuclear leukocytes Wilhelm Krone, Peter Kaczmarczyk, Unir~ersitBts-Krankenhaus

Eppendorf, Medi:rnrsche Kernklinik

(Revised

Key words:

Dirk Miiller-Wieland und Polrkhnrk,

and Heiner Greten Humburg (I? R. G.)

(Received December 12th. 1984) manuscript received March 6th. 1985)

Sterol synthesis:

Prostaglandin:

(Human

mononuclear

leukoq

te)

The effects of the stable prostacyclin analogue iloprost, prostaglandin E, and prostaglandin F,, on sterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for 6 h in a medium containing lipid-depleted serum led to a 3-fold rise in the rate of sterol synthesis from 1“Clacetate or tritiated water. Iloprost and prostaglandin E, added in increasing concentrations at zero time resulted in an inhibition of the synthesis of sterols, the suppression being 50 and 55% at a concentration of 1 pmol/l, respectively. Both prostaglandins yielded a sigmoidal log dose-effect curve. In contrast, prostaglandin F,, had no influence on sterol synthesis up to a concentration of 1 pmol/ 1. The action of the prostacyclin analogue and prostaglandin E , on the relative rate of sterol synthesis was not immediate, since the prostaglandins had no effect when given at 6 h to the incubation medium, and the incorporation of 1“C]acetate into sterols was measured thereafter. The results suggest that prostacyclin and prostaglandin E, affect cholesterol synthesis and therefore may play a role in the regulation of cellular cholesterol homeostasis and in the development of atherosclerosis.

Raised plasma levels of low-density lipoprotein (LDL) are associated with an increased risk of coronary heart disease [l]. The role of LDL is to provide cholesterol to extrahepatic cells, where it is required for maintaining normal structure and function, and to control endogenous cholesterol synthesis thereby regulating cellular’ cholesterol homeostasis. Cholesterol biosynthesis is modulated by various hormones (e.g., insulin, catecholamines, glucocorticoids, thyroid hormones (for review see Ref. 2)). Although prostaglandins appear to play a role in atherosclerosis [3] little is known about their direct action on cellular cholesterol metabolism. We therefore have examined the effects of the stable prostacyclin analogue iloprost, prosFz, on sterol taglandin E, and prostaglandin synthesis in freshly isolated human mononuclear leukocytes. 000%2760/85/$03.30

12 1985 Elsevier Science Publishers

Peripheral human blood mononuclear leukocytes from healthy subjects were isolated by the method of Boyum [4]. The cells were washed with Krebs-Ringer phosphate buffer (pH 7.4) containing glucose (15 mmol/l). The washed pellet was resuspended and transferred to 50-ml plastic conical tubes (Falcon, Oxnard, CA, U.S.A.). Each tube contained (l-2). lo6 cells suspended in KrebsRinger phosphate buffer (pH 7.4) supplemented with glucose (15 mmol/l), sodium acetate (0.5 mmol/l), minimum essential medium vitamin and minimum essential medium amino acid solution without L-glutamine (Gibco, Glasgow, U.K.), 100 units penicillin/ml, 100 pg streptomycin/ml and 40% lipid-depleted serum (v/v). Outdated ABnegative plasma was obtained from the blood bank, and was converted to serum by the addition of 0.035 ml calcium chloride (0.11 mol/l) and 1.2

B.V. (Biomedical

Division)

155

units thrombin (Behring-Werke, Marburg, F.R.G.) per ml of plasma. Serum complement was inactivated by heating to 56°C for 30 min. Serum prepared in this way was depleted of lipid by the method of McFarlane [5]. The flasks containing mononuclear leukocytes were incubated in a shaking-water bath at 37°C for the indicated time periods, when the volume of the incubation was made up to 2 ml by the addition of 50 ~1 of [2-14C]acetate (10 PCi; 55 mCi/mmol) or tritiated water (10 mCi; 5 Ci/ml) (Amersham International, Amersham, U.K.) and the incubation continued for 2 h. Prostaglandins were added as indicated in the legends to the figures. Incubations were terminated by the addition of 7.5 ml chloroform/methanol (1 : 2, v/v) and 1 . 10’ cpm of [1,2- 3H]cholesterol (Amersham International, Amersham, U.K.) was added as an internal standard. Lipids were extracted by the method of Bligh and Dyer [6], and were saponified with methanolic potassium hydroxide (2 mol/l) for 1 h at 70°C. The nonsaponifiable fraction, which is identical with and precursors such as sterols, i.e., cholesterol lanosterol, desmosterol and squalene [7], was extracted three times with 2 ml hexane, the extracts were combined, and a 5 ml portion was counted after adding 10 ml of Insta-Gel scintillation fluid (Packard Instruments, Downers Grove, IL, U.S.A.) in a Packard Tri-Carb scintillation spectrometer (model 300 C). The recovery of [3H]cholesterol was used to correct for procedural losses of [14C]acetate incorporated into nonsaponifiable lipids. Cell viability assessed by erythrosin B exclusion was routinely determined for each cell preparation and was greater than 95% under all experimental conditions. Purity of cell preparations was routinely assessed after staining smears with Wright-Giemsa. Differential counts before and after incubation showed that 92-95% of the mononuclear leukocytes were morphologically identifiable as lymphocytes. About 5% of the cells were identified as monocytes by their ability to ingest latex particles. Functional integrity of the cells was assessed as follows. Cells were incubated for 6 h with iloprost (1 pmol/l) and prostaglandin E, (1 pmol/i) to inhibit the induction of sterol synthesis caused by lipid-depleted serum. At the end of the incubation period, cells were washed,

resuspended in a medium containing lipid-depleted serum and incubated for a further 2, 4 and 6 h, before labelling. This resulted in a linear induction of sterol synthesis which was the same as in cells incubated at zero time in the presence of lipid-depleted serum without prostaglandins [8] indicating that iloprost and prostaglandin E, did not damage cells under our experimental conditions. Results are expressed as means k S.D. Iloprost, prostaglandin E, and prostaglandin F,, were generously donated by Dr. Schillinger, Research Laboratories of Schering AG, BerlinBergkamen, F.R.G. While prostaglandin E, and prostaglandin F,, were dissolved in very small amounts of ethanol and diluted with Krebs-Ringer phosphate buffer, iloprost was dissolved in small amounts of ethanol and Tris buffer. The solvents per se had no effect on sterol synthesis. The chemical formula of iloprost has been previously published [9]. Sterol synthesis in freshly isolated human mononuclear leukocytes was measured by the incorporation of [14C]acetate and tritiated water into non-saponifiable lipids. It has been demonstrated previously that in mononuclear leukocytes [ 14C]acetate was incorporated mainly into lanosterol and cholesterol and to a minimal extent into squalene [lo]. Because of the possibility that treatment of cells with prostaglandins could alter the pool size of acetate, an intial comparison was made between [14C]acetate and tritiated water as precursor substrates. Parallel results were obtained using either substrate for the stable prostaglandin analogue iloprost (1 pmol/l), prostaglandin E, (1 pmol/l) and prostaglandin F,, (1 pmol/l). Incubation of freshly isolated human mononuclear leukocytes for 6 h in a medium containing lipiddepleted serum resulted in a 3-fold increase in sterol synthesis from [14C]acetate or tritiated water. The rate of sterol synthesis from [14C]acetate was 1700 + 240 and 5300 f 720 cpm/h per 10’ cells and from 3H,0 it was 350 + 85 and 1080 + 210 cpm/h per 10’ cells at 0 and 6 h, respectively (n = 6). The induction of sterol synthesis from tritiated water in human leukocytes caused by lipid-depleted serum was similar to that published by others [ll]. The difference between the rate of [14C]acetate incorporation into sterols at 0 and 6 h was defined as 100%. The prostacyclin analogue

156

iloprost, added in increasing concentrations to the incubation medium at zero-time inhibited this induction of sterol synthesis. Prostaglandin E, led to a similar inhibition of [14C]acetate incorporation into sterols, both prostaglandins yielding a sigmoidal log dose-effect curve (Fig. 1). The suppression of the biosynthesis of sterols by the prostacyclin-analogue was 50% and, by prostaglandin E,, 55% at a concentration of 1 pmol/l. In contrast, prostaglandin FZa had no effect on the relative rate of sterol synthesis up to a concentration of 1 pmol/l (data not shown). While iloprost (1 pmol/l) and prostaglandin E, (1 pmol/l), when added at zero-time, led to a marked suppression of

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Oosis ( - log mol/ I I Fig. 1. Effects of the stable prostacyclin analogue iloprost and prostaglandin E, on sterol synthesis from [t4C]acetate in freshly isolated human mononuclear leukocytes. Cells were incubated at 37°C in a medium containing lipid-depleted serum for either 0 h or 6 h before labelling. Incubation for 6 h resulted in 3-fold induction of sterol synthesis. The control was defined as the difference between sterol synthesis at zero-time and 6 h without agents ( A 0% inhibition). Prostaglandins were added in varying concentrations at the beginning of the incubation. For further details see text. Values are means* S.D. of triplicate incubations of 4-6 experiments.

sterol synthesis after 6 h, both prostaglandins had no effect when given at 6 h and [14C]acetate incorporation into sterols was measured thereafter, indicating that both prostaglandins require some time to exert their inhibitory action on sterol synthesis (Table 1). Freshly isolated human mononuclear leukocytes can synthesise cholesterol from acetate and have been useful cells for studying the regulation of cholesterol synthesis by lipoproteins [12], hormones [8] and drugs [13] and for demonstrating LDL receptor defects in familial hypercholesterolaemia [lo]. Accumulation of cholesterol is considered the hallmark of the pathogenesis of atherosclerosis [14]. Therefore, considerable interest has been focussed on factors modulating cellular cholesterol homeostasis. Recently, it has been postulated that prostacyclin and other prostaglandins may alter cholesterol accumulation in intact cultured aortic smooth muscle cells [15]. Accordingly, prostacyclin stimulates the activity of the cholesterol ester hydrolase [16] and may reduce the intracellular levels of cholesterol esters in cultured intimal cells of atherosclerotic human aorta [17]. This study demonstrates a direct effect of the stable prostacyclin analogue, iloprost, and prostaglandin E, on sterol synthesis, both prostaglandins leading to a marked inhibition of the pathway. This action may be mediated by cyclic AMP,

TABLE I FAILURE OF THE STABLE PROSTACYCLIN ANALOGUE ILOPROST AND PROSTAGLANDIN E, TO SUPPRESS STEROL SYNTHESIS FROM [14C]ACETATE WHEN ACTING ONLY FOR A SHORT-TIME PERIOD Cells were incubated at 37°C in a medium containing lipid-depleted serum for either 0 or 6 h before labelling. Incubation for 6 h resulted in a 3-fold induction of sterol synthesis. The control was defined as the difference between sterol synthesis at zero-time and 6 h without agents ( ; 0% inhibition). Prostaglandins were added either at the beginning of the incubation or added at 6 h. For further details see text. Values are means f S.D. of triplicate incubations of four experiments. Agents (1 amol/l)

Inhibition

None Iloprost at 0 h Iloprost at 6 h Prostaglandin E, at 0 h Prostaglandin E, at 6 h

0 50*11 6+ 5 52& 9 2flO

of sterol synthesis

(W)

157

since prostacyclin and prostaglandin E, increase the levels of the cyclic nucleotide in lymphocytes [18]. Accordingly, we have recently shown that dibutyryl cyclic AMP suppressed sterol synthesis in human mononuclear leukocytes [12]. In contrast to the prostacyclin analogue and prostaglandin E,, prostaglandin F,, did not affect sterol synthesis. In accordance to that, E-type prostaglandins appear to act via specific receptors which are coupled to adenylate cyclase, whereas F-type prostaglandins, by stimulating different receptors, might increase the intracellular levels of cyclic GMP (for review see Refs. 19, 20). This work was supported by the Fritz-ThyssenFoundation. The authors thank Mrs. N. Meyer for excellent technical assistance. References Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara, P.M. (1971) Ann. Intern. Med. 74, l-12. Krone, W., Miiller-Wieland, D. and Greten, H. (1984) in Adrenergic control of cholesterol synthesis (Schettler, G., Assman, G., Diehm, C. and Moerschel, J., eds.), pp. 81-89, Springer-Verlag, Berlin Kuo, P.T. (1981) Am. Heart J. 102, 949-953. Boyurn, A. (1968) Stand. J. Clin. Lab. Invest. 97 (Suppl. 21). 77-89.

10 11 12 13 14 15

16 17

18 19 20

McFarlane, A.S. (1968) Nature 149, 439. Bligh, E.G. and Dyer, W.J. (1959) Can. J. B&hem. Physiol. 3, 911-917. Fogelman, A.M., Edmond, J., Saeger, J. and Popjak, G. (1975) J. Biol. Chem. 250, 2045-2055. Krone, W., Hildebrandt, F. and Greten, H. (1982) Eur. J. Clin. Invest. 12, 467-471. Skuballa, W. and Vorbrtiggen, H. (1981) Angew. Chem. Int. Ed. Engl. 20, 1046-1048. Ho, Y.K., Faust, J.R., Bilheimer, D.W., Brown, MS. and Goldstein, J.L. (1977) J. Exp. Med. 145, 1531-1549. Williams, CD. and Avigan, J. (1972) B&him. Biophys. Acta 260, 413-423. Krone, W., Betteridge, D.J. and Galton, D.J. (1979) Eur. J. Clin. Invest. 9, 405-410. Betteridge, D.J., Krone, W., Reckless, J.P.D. and Galton, D.J. (1978) Lancet ii, 1342-1343. Portman, O.W. (1970) Adv. Lipid Res. 8, 41-114. Hajjar, D.P., Weksler, B.B., Falcone, D.J., Hefton, I.M., Tack-Goldman, K. and Minick, CR. (1982) J. Clin. Invest. 70, 479-488. Hajjar, D.P. and Weksler, B.B. (1983) J. Lipid Res. 24, 1176-1185. Tertov, V.V., Orekhov, A.N., Repin, V.S. and Smirnov, V.N. (1982) B&hem. Biophys. Res. Commun. 109, 1228-1233. Polgar, P., Vera, J.C., Kelley, P.R. and Rutenburg, A.M. (1973) B&him. Biophys. Acta 297, 378-383. Samuelson, B., Granstrom, E., Green, K., Hamberg, M. and Hammarstrom, S. (1975) Annu. Rev. B&hem. 44, 669-697. Kuehl, F.A., Jr. (1974) Prostaglandins 5, 325-341.