Cell Total Cholesterol Research Articles

Cellular mechanisms of hypertension and atherosclerosis

The effects of hypoxia on the lipid metabolism of cultured vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats (SHRSP) were examined. The total cholesterol in smooth muscle cells cultured with hyperlipidaemic serum reached higher levels under hypoxic conditions (3.5% O2, 5.0% CO2, 91.5% N2) than under normal conditions (20% O2, 5.0% CO2, 75% N2). In normal serum, cholesterol increased slightly under hypoxic conditions. No difference was noted in cholesterol synthesis from [14C]-acetic acid. Triglyceride levels in smooth muscle cells cultured with hyperlipidaemic serum and normal serum were significantly increased compared with fetal calf serum. The increase in triglyceride levels was inhibited markedly under hypoxic conditions. Triglyceride synthesis induced by normal serum in the hypoxic cells was decreased compared with control cells. The metabolic rate of triglyceride did not differ between the two. These results indicate that hypoxic conditions accelerate the cellular uptake of cholesterol, an initial step in atherogenesis.

Cholesteryl ester handling by RAW264 macrophages: response to native and acetylated low density lipoprotein.

The effects of LDL and Ac-LDL on the growth properties, morphology, and cholesteryl ester (CE) metabolism of the RAW264 macrophage cell line have been characterized. Cells were grown in media supplemented by a defined media (DM) mixture or fetal bovine serum (FBS). The addition of LDL or Ac-LDL to the culture media did not significantly alter cell growth properties. Cytoplasmic deposition of CE was observed by fluorescence microscopy in macrophages treated with LDL or Ac-LDL but not in untreated controls. Dose-response studies have shown that cholesteryl ester (CE) can accumulate in RAW264 treated with LDL. Cellular cholesterol content saturated at 4 hours with 50 micrograms/ml LDL; this effect may be associated with receptor saturation. Dose-response studies conducted with Ac-LDL in DM have shown dramatic increases in total cell cholesterol content. However, deposition of CE was not observed below Ac-LDL concentrations of 100 micrograms/ml. This indicates that a critical concentration of Ac-LDL must be reached to trigger deposition in DM. In contrast, no critical concentration of Ac-LDL was observed in macrophages grown in medium supplemented with 10% FBS. Cholesterol esterification in response to LDL and Ac-LDL was examined by 14C-oleic acid incorporation into CE. These results confirmed the mass cellular cholesterol and CE measurements. Kinetic studies conducted with RAW264 cells treated with 50 or 100 micrograms/ml Ac-LDL resulted in a cholesterol efflux from the cells at 6-12 hours of incubation. Therefore, these studies show that (1) the nature of CE deposition is highly dependent upon the incubation media and (2) CE deposition is very sensitive to Ac-LDL concentration under certain conditions.

Cholesterol accumulation in aortic smooth muscle cells exposed to low density lipoproteins. Contribution of free cholesterol transfer.

Incubation of cultured arterial smooth muscle cells with large concentrations of low density lipoproteins (LDL) resulted in a net increase in cell cholesterol and cholesteryl ester mass that was dependent on LDL concentration and time of incubation. Use of an inhibitor of acyl-CoA:cholesterol acyl-transferase (ACAT) reduced the accumulation of cholesteryl ester mass by 40% (range 25% to 50%), suggesting that a significant proportion of the cholesteryl ester mass that accumulated from LDL did so without being hydrolyzed and re-esterified. Quiescent arterial smooth muscle cells exposed for 48 hours to 0.5 mg/ml of 125I-LDL accumulated 115 nmol total sterol/mg cell protein. However, these cells took up and degraded only 21 micrograms of 125I-LDL protein, which contains 64 nmol total cholesterol. Hence, only about 60% of the increase in cell-associated cholesterol mass was accounted for by LDL particle uptake and degradation. Further, when cells were incubated with 3H cholesteryl linoleyl ether-labeled LDL, the net increase of total cell cholesterol was 81 nmol/mg cell protein. However, only 49 nmol of total cholesterol was taken up by LDL particle uptake, as calculated from the uptake of the 3H cholesteryl linoleyl ether tracer. It thus appears that about 40% of the accumulated cholesterol mass was derived independent of LDL particle uptake, suggesting the possibility of transfer of free cholesterol from the surface of LDL to the cell surface. The occurrence of cholesterol surface transfer was independently verified by the measurement of the uptake and cellular distribution of LDL-derived free 3H-cholesterol. A substantial fraction of the accumulated cell cholesterol mass (approximately 40%) was derived from surface transfer of LDL free cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of low density lipoproteins and mevinolin on cholesterol content and muscarinic cholinergic responsiveness in cultured chick atrial cells. Regulation of levels of muscarinic receptors and guanine nucleotide regulatory proteins.

Cultures of myocytes from embryonic chick atria grown in medium supplemented with fetal calf serum from which lipoproteins had been removed demonstrated a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared to cells grown with control serum. This effect was reversed by growth of cells in medium supplemented with lipoprotein-depleted serum (LPDS) reconstituted with the low density lipoprotein fraction from fetal calf serum. In cells grown in LPDS, total cell cholesterol was increased 32% over control levels and returned to control levels in cells grown with LPDS reconstituted with low density lipoprotein. Growth of cells in LPDS plus mevinolin, an inhibitor of endogenous cholesterol synthesis, also reversed the effects of LPDS on cholesterol content and sensitivity of beating to carbamylcholine. The ability of mevinolin (30 microM) to reverse the effect of LPDS on sensitivity of beating to carbamylcholine was inhibited by mevalonic acid, a metabolic precursor to cholesterol, with an IC50 of 7 x 10(-5) M. These data suggest that mevinolin reverses the effects of LPDS by altering cellular cholesterol levels. Enhanced responsiveness of embryonic chick heart cells to muscarinic stimulation was associated with a 2-fold increase in the number of muscarinic receptors with high affinity for agonist from 82 +/- 10 fmol/mg protein in media containing fetal calf serum to 175 +/- 12 fmol/mg protein in cells grown in the presence of LPDS. The distribution of receptors between high affinity (RH) and low affinity (RL) forms changed from 41% RH and 59% RL in cells grown in control serum to 66.5% RH and 33.5% RL in cells grown in LPDS. Quantitation of the effect of growth in LPDS on the levels of guanine nucleotide regulatory proteins No and Ni which couple the muscarinic receptor to a physiologic response, demonstrated that the relative levels of the 39-kDa alpha subunits of No and 41-kDa alpha subunits of Ni determined by ADP ribosylation with pertussis toxin and immunoblotting increased 2-fold compared to control cells grown with fetal calf serum. Growth of cells with medium supplemented with LPDS plus mevinolin reduced the levels of alpha 39 and alpha 41 to below the levels in control cells. Levels of the beta subunit of No and Ni were unaffected by growth with LPDS.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts.

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.

Uptake of [ 3h]cholesterol from low density lipoprotein by cultured human fibroblasts

The uptake of [ 3H]cholesterol from low density lipoprotein (LDL) was studied in LDL receptor-positive and receptor-negative human fibroblasts. In both cell lines the uptake depended upon temperature, time of incubation and the concentration of LDL in the medium. Although the incorporation of 125I-labeled LDL was minimal after 2 h of incubation in the receptor-negative (homozygous familial hypercholesterolemia, FH) cells, the uptake of [ 3H]cholesterol was only slightly less than that of the receptor-positive (WI-38) cells. With longer periods of incubation, a larger difference in labeled cholesterol incorporation was observed; this appeared to be due to a continued accumulation of the steroid in the WI-38 cells. After 8 and 24 h of incubation, some of the [ 3H]cholesterol was present as the ester in the WI-38 cells, but not the FH cells. Modified (reduced and methylated) LDL did not enter WI-38 cells by the receptor-mediated pathway during 2 h of incubation, as indicated by 125I uptake. [ 3H]Cholesterol uptake, however, was not significantly different from modified and unmodified LDL. While experiments indicated that significant amounts of cholesterol moved rapidly from LDL to cultured cells with a dependence on time and LDL concentration, no increase in total cell cholesterol was detected in either cell line. FH cells contained less total cholesterol and had a higher 3H specific activity than the WI-38 cells. These data suggest that there may be important mechanisms in addition to the LDL pathway for the movement of lipids into cells.

Is cholesterol the receptor for polyene antibiotic-induced B-lymphocyte activation

Stimulation induced by polyene antibiotics has previously been considered to be mediated via binding to cell membrane cholesterol. However, stimulation by polyenes such as nystatin was not affected differently than other polyclonal B-cell activators by the addition of cholesteral-blocking drugs such as digitonin, tomatin, or pimaricin, nor did cholesterol-containing serum diminish its mitogenic properties. Incubation of cells with lecithin-cholesterol vesicles, thereby increasing total cell cholesterol, greatly reduces cell reactivity toward polyenes to the same extent as toward other B- or T-cell activators. We therefore, tentatively suggest that polyene-induced mitogenesis is not mediated via binding to cell membrane cholesterol but via interaction with a distinct mitogen receptor on the cell surface.

Regulation of rat ovarian cell growth and steroid secretion.

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.

Effect of contact inhibition on the regulation of cholesterol metabolism in cultured vascular endothelial cells.

Cholesterol synthesis in actively growing bovine vascular endothelial cells is regulated by low density lipoprotein (LDL) at a step prior to mevalonate formation, in a manner comparable to that found in aortic smooth muscle cells. LDL uptake by these cells is associated with induction of cholesterol esterification, an increase in total cell cholesterol, and an inhibition of endogenous sterol synthesis. In contrast, cholesterol metabolism in confluent contact-inhibited endothelial cultures was not significantly affected by LDL even though the cells bind the lipoprotein at high affinity receptor sites. Lysosomal degradation and subsequent regulatory effects on cellular cholesterol metabolism, however, were observed in contact-inhibited endothelial cells incubated with cationized rather than native LDL. Cationized LDL enter the cells independently of the high affinity sites. Therefore, the primary regulation of cholesterol metabolism in these cells is neither through the appropriate intracellular enzymes nor through the high affinity surface receptors, but via an inhibition of LDL internalization. It is suggested that this inhibition is due to a strict contact-inhibited morphology which enables the endothelium of the larger arteries to function as a selective barrier to the high circulating levels of plasma LDL.

The picomole determination of free and total cholesterol in cells in culture.

An enzymatic, fluorometric method for the determination of free and total cholesterol in cells in culture is presented. The method is simple, requiring one reagent that includes all of the enzymes and a second reagent that increases the pH, which enhances the fluorescence of the product. The method is based on the enzymatic hydrolysis of cholesteryl esters to free cholesterol, the oxidation of cholesterol with the liberation of hydrogen peroxide, and the reaction of this peroxide with a fluorogen to form a fluoresencet product in the presence of a peroxidase. It is rapid, in that free cholesterol can be read in 5 minutes and total cholesterol after 20 minutes. The precision of the method is greater than that obtained from gas-liquid chromatography.

Cholesterol metabolism in the macrophage. I. The regulation of cholesterol exchange.

The cholesterol metabolism of homogeneous populations of mouse peritoneal macrophages was evaluated under in vitro conditions. Macrophages are rich in free cholesterol and maintain a constant cholesterol to protein ratio (12 microg cholesterol/mg protein). No detectable cholesterol ester was present within the cell. More than 95% of total cholesterol was membrane associated and the majority was present in subcellular fractions containing lysosomes and plasma membrane. Less than 0.1% of cell cholesterol was synthesized from acetate-1-(14)C. During in vitro cultivation, macrophages rapidly exchanged their membrane cholesterol with that of lipoproteins of calf serum. About 30% of the cell cholesterol was exchanged per hour in 20% serum medium, and exchange was nearly complete by 5 hr. Exchange proceeded in a rapid exponential phase followed by a slower phase. Calculations based on a two compartment model indicated that the rapidly exchanging cholesterol compartment represented 60-70% of the total cell cholesterol, and the slowly exchanging compartment accounted for 30-40%. The relationship between serum lipoprotein concentration and exchange rate exhibited first-order kinetics. The rate was determined by thermal energy, in keeping with a Q(10) of 2, and an activation energy of 12 kcal/mole. Exchange was independent of bulk transport of lipoproteins by pinocytosis and phagocytosis, and was not linked to energy metabolism. The alpha-lipoproteins were the major class of proteins of calf serum participating in exchange.

Quantitation of the in vitro free cholesterol exchange of human red cells and lipoproteins

The exchange of free cholesterol in vitro between human red blood cells and low density lipoproteins (LDL) was quantified. The flux of sterol between LDL and red cells was relatively constant over a wide range of concentrations of free cholesterol in lipoproteins. In a system containing a suspension of red blood cells in a mixed solution of high density lipoproteins (HDL) and LDL, the fractional rate of exchange of HDL cholesterol was most rapid followed by LDL and lastly, by red cells. Increasing the ionic strength of the incubation media had no effect on the exchange of cholesterol between LDL and red cells. However, when both HDL and LDL were incubated with red cells in a buffer of increased ionic strength, total red cell cholesterol exchange was unaltered, but proportionately more exchange occurred with HDL and less with LDL. Addition of acetone to the buffer increased the exchange of cholesterol between LDL and red cells but produced no increment in red cell-HDL exchange.

Effects of Certain Variables on Hematological Characteristics of Rainbow Trout

Abstract Rainbow trout of two strains (Shasta and Idaho) were fed two diets (Colorado and Santa Monica) under experimental conditions. The effects of diet, strain, sex, and age on total blood cell count, hematocrit, hemoglobin, sedimentation rate, bilirubin, alkaline phosphatase, glutamic-oxalacetic transaminase, total protein, glucose, cholesterol, creatinine, and uric acid were measured. No sex-related differences were detected in hematological values. Variations due to time appeared to be random. Variations due to diet, strain, and/or diet-strain interactions were found for many of the blood components. There were differences in several tests between hematological characteristics of “wild” and cultured trout. Hematological effects of diet, strain, age, water temperature, sampling techniques, and test methods were important, and should be considered by researchers. The range of mean values obtained for the blood components measured can be used as “normal” levels for comparison if investigators employ si...

The composition of red cell lipids and the activity of two enzymes of the erythrocyte membrane (ATPase and acetylcholinesterase) in primary refractory anemias

In a group of patients with primary refractory anemia, the content of total red cell cholesterol, total red cell phospholipids and individual phospholipidic fractions and the activity of two enzymes of the erythrocyte membrane—ATPase and acetyl-cholinesterase—were studied. Patients with primary refractory anemia differ from the control group of healthy individuals by a marked decrease in the activity of red cell acetylcholinesterase. This decrease was significantly higher in patients with primary refractory anemia with aplastic bone marrow than in patients with normal or hyperplastic bone marrow. The mechanism underlying this decrease is discussed. On the other hand, other biochemical examinations, i.e. the examination of red cell lipids and the activity of ATPase of erythrocyte ghosts, did not reveal any deviations from normal values in the studied group of patients.

Plasma and Red Cell Cholesterol

Abstract The experimental production of hypercholesteremia in rabbits by three independent methods—namely, high-fat feeding, cortisone injection, and Tween-80 injection—results in an elevated plasma total cholesterol, whereas the total RBC cholesterol remains constant. Previous work on plasma and red cell cholesterol in humans has been substantiated. Normal total plasma and red cell cholesterol values are 187 ± 32 and 129 ± 20 mg.%, respectively. Elevated plasma total cholesterol occurring in coronary arteriosclerosis, diabetes, nephritis, hypothyroid, Hodgkin's disease, and obesity does not affect the erythrocyte levels. Elevated erythrocyte total cholesterol levels occur in sickle cell and pernicious anemia accompanied by slightly sub-normal plasma levels. A hypothesis is advanced to explain the variability of plasma cholesterol and the relative constancy of red cell cholesterol.