Long-term blockade of the angiotensin II receptor in renin transgenic rats, salt-loaded Dahl rats, and stroke-prone spontaneously hypertensive rats.

These studies were designed to investigate the protective effects of the new angiotensin II receptors antagonist BAY 10-6734 (6-n-butyl-4-methoxycarbonyl-2-oxo-1[(2'-(1H-tetrazol-5-yl) -3-fluorobiphenyl-4-yl)methyl] 1,2-dihydropyridine, CAS 156001-18-2) on haemodynamic, hormonal, renal, and structural parameters in renin transgenic rats (TGR(mRen2)27), salt-loaded Dahl S and R rats, and salt-loaded stroke-prone spontaneously hypertensive rats (SHR-SP) in long-term trials. Study 1: In SHR-SP the development of blood pressure, cardiac hypertrophy, and the deleterious effects of salt loading on kidney structure and kidney function was prevented by BAY 10-6734. Study 2: In salt-loaded Dahl S rats with a suppressed plasma renin activity treatment with BAY 10-6734 did not delay the increase in blood pressure but prevented cardiac hypertrophy and the increase in plasma ANP (Atrial natriuretic peptide). Study 3: TGR develop malignant hypertension associated with cardiac hypertrophy, elevated left-ventricular end-diastolic pressure and increased plasma ANP. After 6 weeks of treatment with BAY 10-6734 (30 mg/kg p.o. bid) cardiac pump function was improved and cardiac hypertrophy was reversed in this angiotension dependent form of hypertension. The beneficial effects of BAY 10-6734 in these different animal hypertension models are also emphasized by a reduction in mortality.

Acyl-CoA binding protein (ACBP) regulates acyl-CoA:cholesterol acyltransferase (ACAT) in human mononuclear phagocytes.

It is demonstrated that the acyl-CoA:cholesterol acyltransferase (ACAT) enzyme activity in rough endoplasmatic reticulum membranes is regulated by the acyl-CoA binding protein (ACBP). The ACAT activity is strongly inhibited by different ACBP/oleoyl-CoA complexes depending from the molar ratio of protein and fatty acid-CoA. Other lipid binding proteins such as bovine serum albumin and the liver fatty acid binding protein do not show any effects on ACAT activity. In addition, we can show that cholesterol loading with acetylated low density lipoproteins does not lead to an increase of the ACBP mRNA level. Consequently, the increase of the intracellular concentration of fatty acids because of the cholesteryl ester accumulation renders ACAT more active for cholesterol esterification. In binding studies we have characterized binding sites on microsomal membranes for the ACAT substrate oleoyl-CoA and the ACAT inhibitor diazepam. Diazepam competes with oleoyl-CoA and vice versa for its binding to microsomal membranes. This common binding site is suggested to be responsible for the transfer from ACBP-bound oleoyl-CoA to ACAT and, therefore, to be essential for the microsomal cholesterol esterification.

Dysregulation of lipid metabolism in Tangier monocyte-derived macrophages.

The cellular defect in Tangier mononuclear phagocytes (MNP) was shown to be associated with significant abnormalities in cellular phospholipid, triglyceride, and cholesteryl ester metabolism by using various radiolabeled precursors (32Pi, 3H-serine, 3H-choline, 14C-acetate, and 14C-oleic acid). Tangier MNP expressed increased rates of synthesis for phospholipids (twofold), triglycerides (fivefold), and cholesteryl esters (threefold) as compared to normal MNP when incubated in McCoy's medium containing 0.2% human serum albumin. The turnover rate of cellular phospholipids was also enhanced, while the turnover rates for triglycerides and cholesteryl esters were normal, thus leading to the accumulation of a larger pool of labeled triglycerides and cholesteryl esters in Tangier MNP. The individual phospholipid classes, phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine were similarly affected. Cholesterol loading led to approximately 30% down-regulation of phospholipid synthesis in normal cells, but Tangier MNP showed a smaller response. When nonloaded normal MNP were exposed to high density lipoprotein3 (HDL3), they diminished cellular cholesterol esterification mediated by acyl-CoA:cholesterol acyltransferase (ACAT); in Tangier MNP, ACAT activity increased in the presence of HDL3. When cholesterol-loaded normal and Tangier MNP were treated with HDL3, an up-regulation of phospholipid synthesis was observed in both cell types, but Tangier MNP showed a smaller response. We conclude that the defect in Tangier disease, which we recently described as a "disorder of intracellular traffic" (Schmitz et al. Proc Natl Acad Sci USA 1985;82:6305-6309), is associated with a dysregulation of cellular lipid metabolism, leading to an overproduction of triglycerides and esterified cholesterol and to enhanced synthesis and catabolism of phospholipids.

Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoprotein-mediated cholesterol efflux in macrophages.

We have studied the rate of phospholipid synthesis and turnover in mouse peritoneal macrophages in reaction to cholesterol influx and high density lipoprotein (HDL)-mediated cholesterol efflux, using three different radioactive precursors, 32PO4(3-), [3H]choline, and [14C]oleic acid. The cells were loaded with cholesterol for up to 18 h with acetyl-low density lipoprotein (LDL), and phospholipid synthesis was measured at various time intervals and compared with nonloaded macrophages. In the first 2 h of cholesterol loading, a twofold increase in the rate of synthesis for sphingomyelin, phosphatidylcholine, phosphatidylserine-inositol, and phosphatidylethanolamine was observed. After this initial up-regulation, the rate of phospholipid synthesis continuously declined upon further cholesterol loading, while the turnover rate of cellular phospholipids was not affected under the same conditions. The lysosomal inhibitor chloroquine abolished the down-regulation, revealing a strong correlation between phospholipid synthesis and lysosomal enzyme activity which was presumably dependent on the release of cholesterol from the lysosome. The reduction in phospholipid synthesis induced by cholesterol loading is reversible by the addition of HDL3 to the cells. When HDL3 was added to the culture medium, a two- to threefold increase in phosphatidylcholine synthesis and a twofold increase in sphingomyelin formation was observed after 3 h. Ca2+ antagonists of the dihydropyridine type, which down-regulate HDL-receptor activity and promote the formation and cellular release of lamellar bodies derived from the lysosomal compartment (Schmitz, G., et al. 1988. Arteriosclerosis. 8: 46-56, and Robenek, H., and G. Schmitz. 1988. Arteriosclerosis. 8: 57-67), specifically enhance the synthesis of sphingomyelin in cholesterol-loaded macrophages. Inhibitors of acyl-CoA:cholesterol acyltransferase (Octimibate, progesterone) increase both the synthesis of sphingomyelin and phosphatidylcholine, and enhance HDL-receptor activity. The results indicate that cholesterol and phospholipid metabolism are coordinately regulated in macrophages. Moreover, the formation of phosphatidylcholine and sphingomyelin seems to be an important factor for the promotion of HDL-receptor-mediated cellular cholesterol efflux.

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. I. Characterization of cellular lipid metabolism.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on the cholesterol metabolism of cholesterol-loaded macrophages were compared. We demonstrated that apolipoprotein A-I containing high density lipoproteins (HDL) bind to specific receptor sites on macrophages, are internalized, take up cholesterol, and are then released from the cells as native lipoproteins. The ACAT inhibitor enhances HDL receptor activity and promotes HDL-mediated cholesterol efflux from cultured mouse peritoneal macrophages. In contrast, the Ca++ antagonist increases acetyl LDL-mediated cholesterol influx, abolishes the increase in HDL binding induced by cholesterol accumulation, enhances apo E synthesis, and promotes cholesterol efflux by a mechanism independent of the presence of HDL in the surrounding medium. Concomitantly, a decrease in nucleoside transporter activity, an increase in intracellular ATP hydrolysis, adenosine and cyclic AMP concentration, and a stimulation of the activities of acid and neutral cholesteryl ester hydrolase and ACAT indicated that protein kinase A-catalyzed phosphorylation reactions might be involved in the increase in cholesterol efflux. The Ca++ antagonist-induced efflux occurred only with lysosomal-associated cholesterol, while the ACAT inhibitor acted on the formation of cytoplasmic lipid droplets. The secreted lipoprotein particles contained 68% unesterified cholesterol and 21% phospholipids, 8% esterified cholesterol, and 3% triglycerides. The phospholipid components were: 72% phosphatidylcholine, 22% sphingomyelin, and 6% phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine. We conclude that macrophages release cholesterol in two ways: 1) an HDL-mediated release of unesterified cholesterol increasing upon ACAT inhibition, and 2) an HDL-independent secretion of cholesterol which can be amplified by Ca++ antagonists.