The effect of cyclic AMP analogues on cholesteryl ester synthesis and hydrolysis in cultured rat peritoneal macrophages.

Abstract

The accumulation of large amounts of cholesteryl ester in macrophages which have invaded the artery wall leads to the formation of foam cells, and is one of the early events in the development of atherosclerosis. The activities of both acyl Coenzyme A: cholesterol acyltransferase (ACAT), which is responsible for cholesterol esterification, and cholesteryl ester hydrolase (CEH) have been shown to be increased in foam cells ( 1 ), so that the cholesteryl ester undergoes a continual cycle of formation and hydrolysis ( l,2). Cyclic AMP (CAMP) has been reported to both stimulate (3) and inhibit (4) ACAT activity in the murine macrophage cell line 5774, while in a recent study we were unable to demonstrate any effect (5). CEH activity, on the other hand, has been shown to be increased by dibutyryl cAMP (Bt2cAMP) in two other murine cell lines, P388DI and WEHl (6.7). In the present work, we have investigated the role of cAMP in the regulation of cholesteryl ester metabolism and foam cell formation in cultured rat peritoneal macrophages using cellpermeable cAMP analogues. Macrophages were collected from male Wistar rats (250300g) by peritoneal washing (8) 2 days after intraperitoneal injection of 3ml thioglycollate (3%). Cells from 3-4 rats were pooled for each preparation. After washing, the cells were cultured as monolayers in supplemented Dulbecco's modified Eagle's medium at 37OC in 95% air l 5% CO?. Rat lipoprotein of density < 1.050glml was isolated from rat serum by ultracentrifugation and acetylated by incubation with acetic anhydride (9). Cholesterol esterification was assayed by the incorporation of radioactivity from potassium ('HI =!ca:c in:o cholesteryl esters during a 5h incubation period. Lipids were extracted with chloroform: methanol (2: 1, v:v) and separated by thin layer chromatography. For the determination of CEH activity, the cells were removed from the plates with a plastic scraper, washed with phosphate-buffered saline and disrupted by sonication. The release of I I-'%]oleate from cholesterol I I '%)oleate was then measured in the resulting homogenate (10). The cAMP analogues used were N6 benzoyl cAMP (BEAMP), B-(4-chlorophenylthio) cAMP (CPTcAMP), 8 chloro cAMP (ClcAMP), N6 monobutyryl cAMP (MBcAMP), 8-(6-amino hexyl) amino cAMP (AHA CAMP), 8-piperidino cAMP (PIP CAMP) and dibutyryl cAMP (BtZcAMP). Significance limits were calculated using Student's t test. Cholesterol esterification in the cultured macrophages was unaffected by a range of cAMP analogues at a concentration of