Phosphatidylinositol- and phosphatidylcholine-dependent phospholipases C are involved in the mechanism of action of atrial natriuretic factor in cultured rat aortic smooth muscle cells.

We have investigated the involvement of specific phospholipase systems and their possible mutual relationship with the mechanism by which atrial natriuretic factor (ANF) increases phosphatidate (PA) and diacylglycerol (DAG) in rat aortic smooth muscle cells (RASMC), one of the major targets of this hormone. Our results indicate that ANF initially stimulates a phosphatidylinositol-dependent phospholipase C (PI-PLC) with a significant increase of DAG, enriched in arachidonate, and inositol trisphosphate (IP3) and then a phosphatidylcholine-dependent phospholipase C (PC-PLC) with formation of DAG, enriched in myristate, and phosphocholine (Pcho). Moreover, ANF stimulates PA formation at an intermediate stage between early and late DAG formation. The transphosphatidylation reaction, as well as its labeling ratio, demonstrate that phosphatidylcholine-dependent phospholipase D (PC-PLD) is not involved. Our experiments with R59022, a DAG kinase (DAGK) inhibitor, indicate that such an increase may be due to the phosphorylation of DAG derived from phosphatidylinositol (PI) hydrolysis. Our results show that phorbol 12-myristate 13 acetate (PMA) plays a significant role in late DAG formation and that Pcho is released concomitantly, suggesting there is a relationship between the two phospholipase Cs (PLCs) that occurs through a protein kinase C (PKC) translocation from cytosol to the plasma membrane. These findings are confirmed by the use of PKC inhibitors calphostin, H7, and staurosporine. The involvement of membrane phospholipid hydrolysis and the ensuing production of second messengers might explain the vasorelaxant effect of ANF.

Inhibition of acyl-CoA: cholesterol acyltransferase decreases apolipoprotein B-100-containing lipoprotein secretion from HepG2 cells.

There is evidence that the overproduction of apoB-100-containing lipoproteins by the liver is the underlying event in some forms of dyslipoproteinemia. This metabolic status is associated to an increased risk of developing premature coronary artery disease CAD. The conclusions from previous studies suggested that the availability to the hepatocytes of cholesterol that is readily esterified is an important determinant for VLDL and LDL secretion. In the present study, we set out to investigate the effect of the specific stimulation and inhibition of the rate-limiting enzyme of the cholesterol esterification, acyl-CoA:cholesterol acyltransferase (ACAT, E.C. 2.3.1.26), on the lipid and on the apoB-100 secretion rate from a human hepatoma cell line (HepG2). When the specific ACAT inhibitor FCE 27677 (10-5 M) was added to the cultures, a decrease of the cellular cholesteryl ester content and at the same time a significant reduction of the neutral lipids and of the apoB-100 secretion rate were noticed. The stimulation of ACAT by 25-hydroxycholesterol (20 microgram/ml) caused a 4-fold increase of the cellular cholesteryl ester content and a 2-fold increase of the lipoprotein secretion rate. FCE 27677 (10-5 M to 10-7 M) prevented the effects elicited by the oxysterol. On the contrary, lovastatin (10-6 M) and gemfibrozil (10-6 M) had no effect. The analysis of the lipid and of the apolipoprotein composition of the lipoproteins secreted in the medium revealed that ACAT inhibition had the dual effect of both decreasing the number of apoB-100-containing lipoproteins secreted as well as their cholesteryl ester load. Altogether, these data support the idea of a close relationship between ACAT activation, leading to increased cholesteryl ester availability, and apoB-100-containing lipoprotein secretion. It is speculated that ACAT inhibitors may prove useful for the treatment of human dyslipoproteinemias caused by the hepatic overproduction of apoB-100-containing lipoproteins.

Oxidized lipoproteins induce long-lasting inhibition of nitric oxide synthase from a murine endothelioma cell line (bEnd.4).

BACKGROUND: The vascular endothelium produces nitric oxide, which has vasodilatory properties. It has been postulated that some lipoproteins may increase arterial vascular tone by decreasing the availability of endothelium-derived nitric oxide. The mechanism underlying this effect, however, is still poorly understood. METHODS: We investigated the effect of native and oxidized human low- and high-density lipoproteins on the nitric oxide synthetic activity of an endothelioma cell line (bEnd.4). Oxidized lipoproteins were obtained by incubation with CuSO4. The production of nitric oxide by the cells was monitored by quantifying the nitrite concentration in the medium using Greiss reagent. RESULTS: The synthesis of nitric oxide by the bEnd.4 cell line was calcium-dependent and was abolished by a selective inhibitor of the constitutive nitric oxide synthase. Incubation with oxidized lipoproteins caused a time- and dose-dependent inhibition of nitric oxide synthetic activity. At a concentration of 100 micrograms/ml cholesterol, oxidized low- and high-density lipoproteins inhibited the production of nitric oxide by 27 and 51%, respectively, within 6h. The lipid fraction obtained from the native or the oxidized lipoproteins mimicked the effect of the intact lipoproteins. CONCLUSION: These results support the involvement of oxidized lipoproteins in the modulation of endothelial functions relevant to the pathogenesis of cardiovascular disease.

Interaction of oxidized HDLs with J774-A1 macrophages causes intracellular accumulation of unesterified cholesterol.

Uptake of modified lipoproteins by resident arterial monocytes/macrophages is believed to be a key event in the formation of foam cells and thus in the early phases of atherosclerosis. Low-density lipoproteins (LDLs) that undergo oxidative changes become suitable for uptake by macrophages through a specific scavenger receptor that leads to cholesteryl ester accumulation. Because the interaction of other oxidized lipoproteins with macrophages has been poorly investigated, we studied the effect of oxidatively modified high-density lipoproteins (HDLs) on the sterol metabolism of J774-A1 macrophages. Unlike native HDLs, oxidized HDLs caused a concentration-dependent accumulation of unesterified cholesterol and decreased [14C]oleate incorporation into steryl esters. Oxidized HDLs also decreased [14C]acetate incorporation into newly synthesized sterols. Cell surface binding of 125I-oxidized HDLs to the macrophages was saturable, with an apparent dissociation constant (Kd) of 0.96 nmol/mL. Both oxidized and acetylated LDLs but not native lipoproteins could compete for binding of 125I-oxidized HDL. The data support the conclusion that the effects elicited by oxidized HDLs on the sterol metabolism of macrophages are significantly different from those of native HDLs. The binding of oxidized HDLs to macrophages occurs at sites that are likely the same as those for modified LDLs. We speculate that, if occurring in vivo, HDL oxidation would generate modified lipoproteins capable of modulating the cholesterol homeostasis of macrophages.

Decreased cholesterol biosynthesis in fibroblasts from patients with Parkinson disease.

The underlying cause of cellular degeneration in the substantia nigra of patients with Parkinson disease has not been clearly established. With the objective of investigating whether metabolic abnormalities would be detected in peripheral non-neuronal cells, we began assessing key metabolic parameters in skin fibroblasts of these patients. The present report focuses on the finding of a remarkably reduced cholesterol biosynthetic capability of fibroblasts from patients with Parkinson disease. 14C-Acetate incorporation into cholesterol of these fibroblasts was 27.8 +/- 9.4% that observed in normal fibroblasts, and the reduced cholesterol synthesis was confirmed by measuring the activity of the rate-limiting enzyme HMGCoA reductase which averaged 6.64 +/- 2.50 nmol/h/mg protein in the patient's fibroblasts compared to 14.70 +/- 0.69 nmol/h/mg protein in the control fibroblasts. Cholesterol esterifying activity, as cholesteryl oleate formed from 14C-oleate, of the fibroblasts from Parkinson patients, was reduced by an average 43%. Two hypotheses are put forward to link these findings with the current experimental evidences for both increased lipid peroxidation and defective mitochondrial respiratory chain complex I activity in a number of cell types from Parkinson patients. Considering that decreased cholesterol biosynthesis has been detected in all the Parkinson cell lines thus far investigated, it is suggested that this may be a hallmark of the disease.

Oxidatively modified HDLs are potent inhibitors of cholesterol biosynthesis in human skin fibroblasts.

Several biological properties of lipoproteins are modified by oxidative reactions. Modified lipoproteins are rapidly degraded by macrophages, and this is likely to be a major pathway for the formation of foam cells in the early phases of atherosclerosis. The effect of modification on other aspects of cholesterol homeostasis has, however, received lesser attention. In this study, the influence of copper ion- as well as rat aortic smooth muscle cell-oxidation-modified high density lipoprotein (HDL) on cholesterol biosynthesis in human skin fibroblasts has been investigated. Modified lipoproteins eluted at higher ionic strength than did control HDL on a Mono-Q 5/5 anion-exchange column. However, only copper ion-modified HDLs displayed greater electrophoretic mobility than did control lipoproteins on agarose gel electrophoresis. Both control and modified HDLs decreased cholesterol esterification in fibroblasts. On the other hand, whereas control HDLs were virtually ineffective in modulating cholesterol biosynthesis, modified HDLs had a significant suppressing effect. This was observed in normal as well as low density lipoprotein (LDL) receptor-defective fibroblasts, which are unresponsive to the LDL-mediated downregulation of cholesterol synthesis. These results are consistent with the concept that oxidative modification of HDLs drastically alters their effect on cholesterol homeostasis in fibroblasts. The data furthermore suggest the existence of a lipoprotein pathway for cholesterol biosynthesis regulation that is independent of the LDL receptor-mediated pathway. Downregulation of cholesterol biosynthesis would be a new function for oxidatively modified lipoproteins.

Peritoneal macrophage cholesteryl ester content as a function of plasma cholesterol in rats.

Cholesterol accumulation in macrophages that have migrated in the subintimal space leads to foam cell formation, which is believed to be one of the initiating events in atherosclerosis. In this study we investigated the effect of cholesterol feeding on peritoneal monocyte/macrophage cholesterol content and peritoneal cavity lipoprotein composition in rats. A cholesterol (2%) and cholic acid (1%) diet caused significant hypercholesterolemia in plasma, and at the same time the cholesterol content of peritoneal monocytes/macrophages was increased. At day 7, the cellular cholesteryl ester content had risen to 30.1 micrograms/mg cellular protein from a baseline value of 9.2 micrograms/mg. The unesterified cholesterol content also increased by 56%. At this time, acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity was doubled, whereas neutral and acidic cholesteryl ester hydrolase activities were unchanged. Reversal to the regular chow diet after 7 days of the cholesterol-enriched diet normalized plasma cholesterol levels as well as peritoneal monocyte/macrophage cholesteryl ester content. ACAT activity also decreased toward normal levels. Analysis of the d less than 1.21 g/ml peritoneal lipoproteins isolated by ultracentrifugation revealed the presence, in both normal and hypercholesterolemic rats, of apolipoprotein A-I-rich lipid complexes with pre-beta mobility on agarose gel electrophoresis. The size of the peritoneal lipoproteins was smaller than that of plasmatic high density lipoproteins, and their chemical composition was also different from that of the major plasma lipoproteins. The cholesteryl ester content of peritoneal lipoproteins increased after feeding of the cholesterol-enriched diet. In conclusion, our results show that cholesterol feeding leads to rapid accumulation of cholesteryl esters in monocytes/macrophages. As soon as plasma cholesterol levels are returned to normal, cellular cholesterol content is also normalized.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a detergent-solubilized plasma membrane protein from rat liver recognizing apolipoprotein A-IV.

Apolipoprotein A-IV (apo A-IV) is present in plasma associated to both HDL and as a complex with lipids that cannot be floated by ultracentrifugation at 1.21 g/ml density. Apo A-IV is likely an important molecular determinant in HDL binding to the liver. In this communication, data are presented supporting the view that a specific liver plasma membrane protein of Mr 95,000 is a constituent of the apo A-IV binding site. The protein was solubilized with CHAPS from purified rat liver plasma membranes and subjected to SDS-PAGE. Transblotted to nitrocellulose sheet could be identified as recognizing 125I-apo A-IV-DMPC by autoradiography. 125I-apo A-I-DMPC and radioiodinated rat apo E-poor HDL, also bound to the protein. Apo B-100 (as human LDL) and apo C-III did not bind. The protein identified is likely to be the same that has been previously identified by Graham and Oram [1987) J. Biol. Chem. 262, 7439-7442) as 'HDL receptor protein'.

Metabolism of apolipoprotein A-IV in rat.

The metabolism of apolipoprotein A-IV (apo-IV) has been investigated in the rat. In this animal species, apoA-IV is a major protein constituent of plasma HDL and lymph chylomicron. The apolipoprotein is also present in the lipoprotein-deficient fraction (LDF) of plasma and lymph. In vivo studies with the radioiodinated protein showed the apoA-IV does not exchange freely between HDL and LDF and that LDF apoA-IV had a faster catabolism than HDL apoA-IV. ApoA-IV in chylomicrons is a direct precursor of apoA-IV in plasma HDL but not of that in LDF. On the other hand lymph LDF apoA-IV is an important precursor of plasma LDF apoA-IV. Transfer of apoA-IV from plasma to lymph is negligible, and since most of apoA-IV in lymph is present in LDF, we speculate that LDF apoA-IV is the major apoA-IV secretory product of the intestine. Studies aimed at identifying the site of catabolism of apoA-IV utilizing either radioiodinated or [14C]sucrose labelled apoA-IV, gave results consistent with the view that the liver plays a major role. When tested, human apoA-IV behaved in vivo in rat as the autologous protein. These findings, together with others previously published (Ghiselli, G. et al. (1987) J. Lipid Res. 27, 813-827), support the conclusion that the plasma metabolism of apoA-IV is remarkably similar in rat and human. We speculate that in mammals the rapid plasma catabolism of apoA-IV is mediated by an efficient uptake by the liver.

Lipophilic beta-adrenoceptor antagonists stimulate low density lipoprotein receptor activity in human skin fibroblasts.

The effect of beta-adrenoceptor antagonists on the receptor-mediated low density lipoprotein (LDL) binding and internalization was studied in vitro in human skin fibroblasts. The cellular uptake of 125I-labeled human LDL was dose dependently elevated by some, but not all, of the drugs used. This effect of beta-adrenoceptor antagonists was positively related to their lipophilicity, and was prevented by cycloheximide and by alpha-amanitin. Scatchard analysis of the saturable LDL binding indicates an increased number of LDL binding sites. Our studies show that the stimulating effect of beta-adrenoceptor antagonists on the high affinity LDL binding and internalization in human skin fibroblasts involves DNA transcription and new protein synthesis, and identify drug lipophilicity as a major determinant of this action. This effect could be relevant in vivo in adipose tissue which accumulates lipophilic drugs and derives its cholesterol mainly from circulating LDL.

In vitro inhibition of rat arterial smooth muscle cell growth by extractive sulfated mucopolysaccharides.

The effect of a sulfated mucopolysaccharide mixture of known composition, extracted from pig duodenum, was studied on the proliferation of rat arterial smooth muscle cells cultured from rat aorta. Cell growth, stimulated by fetal calf serum, was monitored by direct cell count and by determination of the mitotic index. The extractive mixture was studied in comparison with commercial heparin, with heparin with different electrophoretic mobilities in barium acetate and with dermatan and heparan sulfates. Heparins and the extractive mucopolysaccharide mixture inhibited cell growth measured at various time intervals, and in their presence the proliferation of smooth muscle cells plateaued at lower cell densities. Dermatan and heparan sulfates were either inactive or significantly less effective than the other mucopolysaccharides. A short preincubation (3 h) of smooth muscle cells with the extractive mixture, followed by incubation with the growing medium with no mucopolysaccharides added, slowed the cell growing rate, suggesting an interaction of the mixture components with the cell surface.

Experimental studies on the hypolipidemic activity of chloridarol.

The efficacy of chloridarol (2-benzofuryl-p-chlorophenyl carbinol) as hypolipidemic agent was evaluated in rats and rabbits. In normolipidemic rats chloridarol, at doses ranging from 50 to 200 mg/kg/day, decreased plasma triglycerides without affecting cholesterolemia and fast- or norepinephrine-induced lipolysis. The drug proved effective in reducing fructose-induced hypertriglyceridemia and dietary hypercholesterolemia in rats; in the latter model chloridarol significantly raised both the HDL cholesterol and the HDL/VLDL + LDL cholesterol ratio. In hyperlipidemic rabbits the drug had no effect on plasma cholesterol, but it lowered triglyceridemia. The action of chloridarol on rat liver ultrastructure was also investigated. Treatment for one month induced peroxisome proliferation, less marked, however, than that elicited by clofibrate; after a prolonged chloridarol treatment (9 months), this effect had almost completely disappeared and the ultrastructure of the hepatocytes was close to that of controls.

[On the growth of cultured bovine aortic endothelial cells. Preliminary observations on ultrastructural morphology by freeze-etching and quantitation of growth rate by phase contrast microscopy: effect of glycosaminoglycans]. / Sull'accrescimento in coltura di cellule endoteliali di aorta bovina. Osservazioni preliminari di morfologia ultrastrutturale al crio-decappaggio e quantizzazione della velocita' di crescita in microscopia a contrasto di fase: effetto di glicosaminoglicani.

Bovine aortic endothelial cells (BAEC), detached from thoracic aorta by collagenase treatment, have been studied after adding a mixture of glycosaminoglycans (GAGs) (the composition of which has been studied by Casu et al. -6), at different concentrations. GAGs at the concentration of 90 mg/100 ml of medium (Dulbecco's modification of Eagle's MEM with 10% foetal calf serum) produced an increase in the growth speed. We have quantitized these data both by counting the number of cells 24 h after trypsinization in phase-contrast-microscopy, and by measuring the time necessary to reach a certain number of influences. Ultrastructural observation at transmission electron microscope after freeze-etching preparation, didn't show any relevant modification either in the organization of cell membrane or in the cytoplasmatic structures. In some observations a conspicuous increase of "vesicular openings" ("pits") on the plasmatic membrane was evident. Statistical evaluation of this phenomenon is under consideration.

Fecal cholesterol excretion studies in type II hypercholesterolemic patients treated with the soybean protein diet.

The fecal steroid elimination profile was studied in 7 type II hyperlipoproteinemic patients given a low-lipid diet with textured soybean proteins, in order to define the mechanism of the hypocholesterolemic activity of this new dietary regimen. Four of the patients followed a 3- + 3-week cross-over protocol, comparing the soybean diet with a reference low-lipid diet with animal proteins. In these, fecal neutral steroids and bile acids were analyzed by chromatography during the two dietary periods. In spite of the clear hypocholesterolemic effect, no significant differences in steroid output were noted between the two dietary periods. In the 3 remaining patients, a chromatographic + isotopic method (by injecting 14C-labelled cholesterol i.v. 4-6 weeks prior to the dietary study) was employed. Again, no marked changes were noted in the fecal neutral steroid and bile acid outputs and the slope of the decay curve of the plasma cholesterol-specific activity was not changed by the experimental diet, in spite of the remarkable decrease in plasma cholesterol. The reported results do not provide a definitive contribution to the mode of action of the soybean protein diet. They suggest, however, that it is not an effect mediated by undigestible dietary components. The possibility of a cholesterol redistribution from plasma to tissue pools should be considered.

Gastric antisecretory, antiulcer and cytoprotective properties of 9-hydroxy-19,20-bis-nor-prostanoic acid in experimental animals.

9-Hydroxy-19,20-bis-nor-prostanoic acid (IBI-C83) was evaluated on gastric acid secretion and gastric lesions induced in laboratory animals by a variety of experimental conditions: compound IBI-C83 is proved effective in decreasing basal, histamine- and pentagastrin-stimulated total acid output in rats and in pentagastrin perfused dogs. The concentration of N-acetylneuraminic acid in the gastric fluid, a marker of mucus secretion, is enhanced in rats by IBI-C83. This drug prevents gastric damage induced by non-steroidal antiinflammatory compounds such as acetylsalicylic acid, indometacin and phenylbutazone, gastric ulcers following pylorus ligation, and facilitates healing of the gastric ulcers evoked by subserosal injection of acetic acid. A prominent feature of IBI-C83 is its capacity to protect the rat from gastric damage elicited by necrotizing agents such as absolute ethanol, hydrochloric acid and hypertonic saline. This property, called "cytoprotection" and common to naturally occuring prostaglandins, is independent on the antisecretory activity of IBI-C83 and is not shared, at least in the reported experimental models, by the H2-receptor antagonist cimetidine. In spite of the prostaglandin-like properties displayed in its cytoprotective activity, compound IBI-C83 does not affect cardiovascular functions, gastrointestinal transit and uterine motility.