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Biomedical subjects

O Stein

Publications and source records attributed to O Stein.

At least 217 records · Page 12Linked to original sources
PubMed1967

Lipid synthesis, intracellular transport, storage, and secretion. I. Electron microscopic radioautographic study of liver after injection of tritiated palmitate or glycerol in fasted and ethanol-treated rats.

A time sequence study of intracellular movement of labeled lipid in the liver was carried out on fasted and ethanol-treated rats injected with either palmitate-(3)H or glycerol-(3)H by electron microscopic radioautography. The elimination of water-soluble lipid precursors during specimen preparation was checked and found to be complete. The labeled lipid product in the tissue was identified as mostly triglyceride. A dehydration procedure was adapted to minimize the loss of lipid during specimen preparation. At 2 min after injection, the earliest time interval studied, both precursors were found to have penetrated the liver cells, and the label was found over both rough and smooth elements of the endoplasmic reticulum, which is the site of glyceride esterification. From 5 min on, in fasted and especially in ethanol-treated rats, the label was seen also over lipid droplets 0.5-2.0 micro in diameter, which represent "storage lipid" (slowly turning over compartment). Mitochondria became labeled mostly at later time intervals after injection. From 10 min on, concentration of label was seen over the Golgi apparatus, containing small osmiophilic particles. Association of label with groups of particles in smooth-surfaced vesicles and vacuoles in and near the Golgi apparatus and in the vicinity of the sinusoidal border was seen, both after palmitate-(3)H and glycerol-(3)H. It is proposed that these particles represent lipoproteins which are formed in the endoplasmic reticulum, "processed" in the Golgi apparatus, and transported in vacuoles to the sinusoid surface to be discharged into the circulation.

Adipose Tissue↗
PubMed1967

Lipid synthesis, intracellular transport, and secretion. II. Electron microscopic radioautographic study of the mouse lactating mammary gland.

In the mammary glands of lactating albino mice injected intravenously with 9, 10-oleic acid-(3)H or 9, 10-palmitic acid-(3)H, it has been shown that the labeled fatty acids are incorporated into mammary gland glycerides. The labeled lipid in the mammary gland 1 min after injection was in esterified form (> 95%), and the radioautographic reaction was seen over the rough endoplasmic reticulum and over lipid droplets, both intracellular and intraluminal. At 10-60 min after injection, the silver grains were concentrated predominantly over lipid droplets. There was no concentration of radioactivity over the granules in the Golgi apparatus, at any time interval studied. These findings were interpreted to indicate that after esterification of the fatty acid into glycerides in the rough endoplasmic reticulum an in situ aggregation of lipid occurs, with acquisition of droplet form. The release of the lipid into the lumen proceeds directly and not through the Golgi apparatus, in contradistinction to the mode of secretion of casein in the mammary gland or of lipoprotein in the liver. The presence of strands of endoplasmic reticulum attached to intraluminal lipid droplets provides a structural counterpart to the milk microsomes described in ruminant milk.

Animals↗
PubMedDate not supplied

Expression of lipoprotein lipase mRNA in rat heart is localized mainly to mesenchymal cells as studied by in situ hybridization.

The expression of lipoprotein lipase mRNA (LPL mRNA) was studied in rat hearts by use of a sulfur-35-labeled antisense mRNA probe. Rats were studied under three conditions: fed, fasted, and injected with cholera toxin (an irreversible agonist of adenylate cyclase) and then fasted. The highest LPL activity was found in the hearts of cholera toxin-injected, fasted rats. After injection of cholera toxin, LPL mRNA levels were 3.5-fold higher than those from fed rats. Using in situ hybridization, we studied the site of expression of LPL mRNA under the same three experimental conditions. In sections of hearts from cholera toxin-injected, fasted rats, concentrations of autoradiographic grains, representing the site of LPL mRNA, were seen over interstitial elements, which comprise capillary and perivascular cells. A more diffuse and sparse reaction was seen over cardiac myocytes and was not always distinguishable from background. A similar but much less definitive localization was seen in sections of hearts from fasted rats. The present results indicate that in the rat heart, the main site of LPL synthesis and processing, especially after stimulation with an irreversible agonist of adenylate cyclase, is localized to interstitial elements rather than to adult cardiac myocytes.

Adenylyl Cyclase Inhibitors↗
PubMedDate not supplied

Use of 3H-cholesteryl linoleyl ether for the quantitation of plasma cholesteryl ester influx into the aortic wall in hypercholesterolemic rabbits.

In this study use was made of 3H-cholesteryl linoleyl ether CLE), a nondegradable analogue of cholesteryl ester (CE) to measure plasma lipoprotein CE influx into rabbit aorta. Autologous serum labeled with 3H-CLE was injected into seven hypercholesterolemic rabbits, and more than 90% of the label was recovered in the plasma compartment 10 minutes after injection. Between 4 hours and 3 days the label was cleared from the circulation with a t1/2 of about 24 hours. Between 4 and 24 hours the lipoproteins isolated at d less than 1.006, d less than 1.019, and d less than 1.063 approached similar specific activity, assuming that 3H-CLE had mixed with the lipoprotein CE pool. The rabbits were killed 7 to 14 days after injection when plasma radioactivity decreased to less than 0.03% of injected dose/ml. Total recovery of the CLE ranged from 70% to 95% and 48% to 72% were found in the liver. The minimum influx of plasma CE into the aortic intima was determined by dividing the label found in the artery by the mean specific activity of the labeled compound in the plasma. The minimum influx into regions with atheromatous involvement ranged from 0.8 to 3.4 micrograms CE/cm2/hr. The rate of influx was highly correlated with the amount of CE mass in the intima and media indicating that the bulk of aortic CE is derived from plasma lipoprotein CE. The method described might be useful in distinguishing between possible effects of "antiatherogenic" drugs on plasma CE influx into the aortic wall from an effect on intracellular CE hydrolysis and subsequent efflux of free cholesterol from the artery.

Animals↗
PubMedDate not supplied

In vitro metabolism of LDL labeled with a nondegradable cholesteryl ester analogue.

Low density lipoprotein (LDL) metabolism by human skin fibroblasts was studied using LDL labeled in the neutral lipid fraction with a nonhydrolysable cholesteryl ester analogue 3H-cholesteryl linoleyl ether (3H-CLE-LDL). LDL uptake could be quantitated accurately using 3H-CLE-LDL, since the label accumulated intracellularly due to its resistance to hydrolysis. 3H-CLE-LDL was taken up via apo B,E receptor-mediated endocytosis in a manner similar to 125I-labeled LDL. This was demonstrated by similar rates of uptake of the two differently labeled LDL preparations, saturation kinetics of uptake with respect to 3H-CLE-LDL concentration, regulation of 3H-CLE-LDL uptake by procedures that up-regulate or down-regulate the number of apo B,E receptors, and negligible uptake of 3H-CLE-LDL by receptor-negative cell strains. The major difference between the handling of 125I-LDL and 3H-CLE-LDL was the finding that, while the amount of trypsin-releasable (surface) 3H-CLE increased progressively over a 24-hour experimental period, trypsin-releasable 125I-LDL reached a maximum within 30 minutes. After 24 hours of incubation, the 3H radioactivity released by brief trypsinization was three to four times higher than could be accounted for by 125I radioactivity released by a similar treatment. Possible reasons for this behavior are discussed.

Cholesterol↗
PubMedDate not supplied

Verapamil enhances receptor-mediated endocytosis of low density lipoproteins by aortic cells in culture.

Bovine aortic endothelial and smooth muscle cells (SMC) and human skin fibroblasts (HSF) were used to study the effect of verapamil on cellular interactions with human low density lipoprotein (LDL). Verapamil, 10 to 50 microM, increased 125I-LDL uptake and degradation by 70% to 200% in the various cells after 24 to 48 hours of incubation. The increase in the total amount of LDL endocytosed, labeled with 3H-cholesteryl linoleate (3H-CL), was comparable to that determined with 125I-LDL. In HSF and SMC, a delay in 125I-LDL degradation and hydrolysis of 3H-CL was seen in cells treated for 3 to 24 hours with verapamil. Pretreatment of HSF with 50 microM verapamil for 24 hours and incubation with 2 to 50 micrograms 125I-LDL protein/ml for 1 hour resulted in a 50% to 200% increase in heparin releasable and in a 40% to 130% increase in cellular 125I-LDL. Thus, the increase in 125I-LDL binding and uptake in verapamil-treated cells was apparently due to an increase in receptor number, rather than in receptor affinity. The effect of verapamil on LDL uptake and degradation was also seen in cells that were pretreated for 24 hours and incubated with 125I-LDL in the absence of verapamil. The effect of verapamil was not apparent in LDL receptor-negative cells. Cycloheximide blocked the verapamil effect. The Na+ channel blocker, tetrodotoxin x 10(-6) M, caused a 30% to 50% increase in the total amount of LDL endocytosed, but no delay in LDL degradation; amiloride 2 x 10(-3) M was not effective. If the presently described effect of verapamil also occurs in vivo, this might contribute to the reported beneficial effects of Ca++ channel blockers in experimental atherosclerosis by promoting transfer of LDL cholesteryl ester from the aortic interstitium to a catabolic compartment.

Animals↗
PubMedDate not supplied

Cholesteryl ester efflux from extracellular and cellular elements of the arterial wall. Model systems in culture with cholesteryl linoleyl ether.

Cholesteryl ester (CE) accretion in human atheroma is a slow process during which lipoproteins infiltrate the arterial extracellular space and then gain entry into the cellular components. The present aim was to simulate this process in model systems in culture to learn whether cholesteryl ester transfer protein (CETP) may promote CE efflux at different stages of atheroma formation. To simulate CE efflux from arterial interstitium, cationized LDL labeled with 3H-cholesteryl linoleyl ether (3H-CLE) was added to fixed aortic smooth muscle cells (SMC) or to extracellular matrix. To study efflux of 3H-CLE taken up by cells via receptor-mediated endocytosis of LDL, the SMC cultures were fixed and permeabilized prior to the determination of CE efflux. The cellular model included macrophages, which had ingested acetylated LDL labeled with 3H-CLE. Efflux of 3H-CLE and 14C-CE was studied during postincubation of the labeled cultures with human lipoprotein deficient serum (LPDS) or partially purified CETP. As controls, we used SMC cultures incubated with albumin. In all systems, a 3- to 12-fold increase of 3H-CLE or 14C-CE efflux was found in the postincubation medium containing human LPDS or partially purified CETP when compared to controls. Permeabilization of the cells with saponin enhanced cellular 3H-CLE and 14C-CE efflux in the presence of human LPDS. The findings indicate that CETP may promote CE efflux from aortic interstitium or disintegrating cells. We propose that CETP may play an important role in aortic CE homeostasis under physiological and pathological conditions.

Animals↗
PubMedDate not supplied

Effect of verapamil on cholesteryl ester hydrolysis and reesterification in macrophages.

The macrophage-like cell line, J774, and its variant, CT2, were used to study the effect of verapamil on metabolism of esterified cholesterol. The cells had been labelled with 3H-cholesterol for 24 hours and thereafter acetylated low density lipoprotein (LDL) or d less than 1.019 g/ml fraction of hypercholesterolemic rabbit plasma was added. After an additional 24 hours, 60% or 40% of the label, respectively, was recovered in esterified cholesterol in control dishes, but only 2% to 15%, in the presence of 50 microM verapamil. Enhancement of esterification of cellular 3H-cholesterol was also obtained by addition of liposomes with a cholesterol/phosphatidylcholine molar ratio of 2:1. This reaction was almost completely inhibited by compound 58-035, an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, while addition of verapamil resulted in a dose-dependent partial inhibition which was evident after 4 hours. To determine whether the reduction in cholesterol esterification was due to a direct inhibition of ACAT or to an effect of verapamil on the transport of cholesterol to the site of ACAT activity, the cells were incubated with acetylated LDL labelled with 3H-cholesteryl ester, and the amount of uptake, lysosomal hydrolysis, and cytoplasmic reesterification was determined. In control cells and in cells exposed to the ACAT inhibitor, more than 98% of the 3H-cholesteryl ester taken up had been hydrolyzed. Reesterification of 3H-cholesterol was negligible in the presence of the ACAT inhibitor and amounted to 37.1% in control cells. When verapamil was present, reesterification of 3H-cholesterol to cholesteryl ester was only 4%, while the amount of 3H-cholesteryl ester hydrolyzed was 58% of that taken up.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Long-term effects of verapamil on aortic smooth muscle cells cultured in the presence of hypercholesterolemic serum.

Smooth muscle cells derived from rabbit and bovine aorta were cultured for up to 5 weeks in the presence of d less than 1.019 g/ml fraction of hypercholesterolemic rabbit serum. When this fraction was added to serum containing culture medium, there was a significant increase in DNA, protein, and cholesteryl ester per dish. Addition of 50 microM verapamil markedly reduced the stimulatory effect of the d less than 1.019 g/ml fraction on both DNA and protein content per dish. The effect of verapamil on cholesteryl ester content was more complex: there was an increase within the first week, but later the net accumulation of cholesteryl ester per dish was lower than in untreated dishes. The recovery of less DNA in verapamil-treated dishes was not due to increased cell loss, as evidenced by retention of a residualizing marker, 3H-cholesteryl linoleyl ether. Moreover, verapamil did reduce incorporation of 3H-thymidine into DNA. In verapamil-treated dishes, there was flattening and a cobblestone appearance of the cells. A hypothesis is proposed to explain the inhibitory effect of verapamil on the development of atheroma formation in cholesterol-fed rabbits: Assuming that macrophages play an active role in cholesteryl ester removal from atheroma, verapamil, which reduces lysosomal cholesteryl ester hydrolysis in macrophages, would permit the lipid-laden macrophage to remove more cholesteryl ester per cell from the arterial wall. In addition, the presently reported results support the possibility that verapamil may impede the development of atheroma formation by reduction of smooth muscle cell proliferation.

Animals↗
PubMedDate not supplied

Use of 3H-cholesteryl linoleyl ether as a quantitative marker for loss of cholesteryl ester during regression of cholesterol-induced aortic atheromas in rabbits.

In this study, use was made of 3H-cholesteryl linoleyl ether (3H-CLE) to follow regression of aortic atheromatosis induced by feeding cholesterol to rabbits. After a 3-month induction period, the rabbits were divided into two groups with an attempt to match them by plasma cholesterol levels. They were injected with rabbit plasma labeled with 3H-CLE, and the baseline group rabbits were killed 10 to 12 days after injection. The experimental (regression) group rabbits were given rabbit chow containing 3% cholestyramine and were killed up to 330 days thereafter. Aortic 3H-CLE of both the baseline and the regression groups correlated highly with the plasma cholesterol levels at the time of injection of label. The radioactivity recovered in the aortas of the baseline and regression groups was not significantly different, indicating retention of label between day 12 and 330 days after injection. During that time, the mean aortic cholesteryl ester content decreased from 7.6 +/- 1.3 mg to 3.1 +/- 0.7 mg (p less than 0.01). The specific activity of 3H-CLE/cholesteryl ester determined in the aortic arch and the thoracic and abdominal aorta was significantly increased in all three regions examined in the regression group as compared to the baseline group. The present data show that 3H-CLE is retained in the atheromatous aorta for at least 330 days and that its use may add another dimension to the quantitative evaluation of regression of atherosclerotic lesions.

Animals↗
PubMedDate not supplied

The effect of acute and chronic administration of nicotine on lipoprotein lipase activity.

Nicotine was injected intraperitoneally to rats and 2 h later lipoprotein lipase was determined in isolated perfused hearts. There was a significant increase in heparin releasable lipoprotein lipase activity which represents the functional pool without increase in total enzyme activity. When 1 microliter/h of a solution of nicotine (120 mg/ml) was delivered for 3 days from subcutaneously implanted miniosmotic pumps, total lipoprotein lipase activity in the heart increased 1.5-3.0 fold. Endogenous lipoprotein lipase activity in plasma doubled and there was a significant fall in plasma triglyceride levels. The effect of nicotine on heart lipoprotein lipase activity was evident also after 6 days of continuous delivery and was accompanied by a fall in adipose tissue lipoprotein lipase activity. No effect was seen when the dose of nicotine was halved. A positive correlation was seen between plasma nicotine levels and heart lipoprotein lipase activity, while adipose tissue lipoprotein lipase correlated negatively with plasma nicotine levels. Chronic administration of nicotine was accompanied by either weight loss or diminished weight gain. It is concluded that in the rat the acute effect of nicotine on the shift of lipoprotein lipase to the functional pool could be related to enhanced beta-adrenergic stimulation. The chronic effect of nicotine could have been mediated by the loss in body weight, due to reduced caloric intake.

Animals↗