Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

In order to efficiently and rapidly label lipoproteins in plasma with [3H]cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and [3H]cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, [3H]cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to [3H]cholesterol from an albumin-stabilized emulsion. The distributions of both [3H]cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.     

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts.

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.     

Relations between particle size of HDL and LDL lipoproteins and cholesterol esterification rate

Particle size of low density (LDL) and high density (HDL) lipoproteins and cholesterol esterification rate in HDL plasma (FER(HDL)) are important independent predictors of coronary artery diseases (CAD). In this study we assessed the interrelations between these indicators and routinely examined plasma lipid parameters and plasma glucose concentrations. In 141 men, healthy volunteers, we examined plasma total cholesterol (TC), triglycerides (TG), HDL and LDL cholesterol (HDL-C, LDL-C) and HDL unesterified cholesterol (HDL-UC). Particle size distribution in HDL and LDL was assessed by gradient gel electrophoresis and FER(HDL) was estimated by radioassay. An effect of particle size and FER(HDL) on atherogenic indexes as the Log(TG/HDL-C) and TC/HDL-C was evaluated. Subjects in the study had plasma concentrations (mean +/- S.D.) of TC 5.2+/-0.9 mmol/l, HDL-C 1.2+/-0.3 mmol/l, TG 2.1+/-1.7 mmol/l, glucose 5+/-0.8 mmol/l. Relative concentration of HDL(2b) was 17.6+/-11.5 % and 14.6+/-11.8 % of HDL(3b,c). The mean diameter of LDL particles was 25.8+/-1.5 nm. The increase in FER(HDL) significantly correlated with the decrease in HDL(2b) and LDL particle size (r = -0.537 and -0.583, respectively, P<0.01) and the increase in HDL(3b,c) (0.473, P<0.01). Strong interrelations among TG and HDL-C or HDL-UC and FER(HDL) and particle size were found, but TC or LDL-C did not have such an effect. Atherogenic indexes Log(TG/HDL-C) and TC/HDL-C correlated with FER(HDL) (0.827 and 0.750, respectively, P<0.0001) and with HDL and LDL particle size.     

Influence of progesterone on the initial rate of cholesterol esterification in rat plasma

The effect of progesterone on the initial rate of cholesterol esterification in rat plasma was measured after daily injections of the hormone or following addition of progesterone $\text{in}$$\text{vitro}$. The administration of progesterone did not modify the lecithin:cholesterol acyltransferase (LCAT) activity nor the progress of the enzyme reaction with time. When increasing concentrations of progesterone were added to the medium the percentage of cholesterol esterified per minute decreased progressively. The addition of progesterone also decreased the slope of the time-course reaction. It is suggested that the inhibition of the LCAT activity due to the presence of the hormone would be masked by an increased hepatic production of the enzyme and/or by the alterations that the hormonal treatments produced in the plama lipid levels.     

Effect of apolipoprotein E-free high density lipoproteins on cholesterol metabolism in cultured pig hepatocytes

We studied cholesterol synthesis from [14C]acetate, cholesterol esterification from [14C]oleate, and cellular cholesterol and cholesteryl ester levels after incubating cells with apoE-free high density lipoproteins (HDL) or low density lipoproteins (LDL). LDL suppressed synthesis by up to 60%, stimulated esterification by up to 280%, and increased cell cholesteryl ester content about 4-fold. Esterification increased within 2 h, but synthesis was not suppressed until after 6 h. ApoE-free HDL suppressed esterification by about 50% within 2 h. Cholesterol synthesis was changed very little within 6 h, unless esterification was maximally suppressed; synthesis was then stimulated about 4-fold. HDL lowered cellular unesterified cholesterol by 13-20% within 2 h and promoted the removal of newly synthesized cholesterol and cholesteryl esters. These changes were transient; by 24 h, both esterification and cellular unesterified cholesterol returned to control levels, and cholesteryl esters increased 2-3-fold. HDL core lipid was taken up selectively from 125I-labeled [3H]cholesteryl ester- and ether-labeled HDL. LDL core lipid uptake was proportional to LDL apoprotein uptake. The findings suggest that 1) the cells respond initially to HDL or LDL with changes in esterification, and 2) HDL mediates both the removal of free cholesterol from the cell and the delivery of HDL cholesteryl esters to the cell.     

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A-I mRNA in Hep G2 cells

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A-I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA-I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA-I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA-I gene expression and regulation.     

Interaction of apolipoprotein(a) with apolipoprotein B-containing lipoproteins

Recombinant DNA-derived apolipoprotein(a) was used to demonstrate that the apo(a) moiety of lipoprotein(a) (Lp(a)) is responsible for the binding of Lp(a) to other apolipoprotein B-containing lipoproteins (apoB-Lp) including LDL2, a subclass of low density lipoproteins (d = 1.030-1.063 g/ml). The r-apo(a).LDL2 complexes exhibited the same binding constant as Lp(a).LDL2 (10(-8) M). Treatment of either recombinant apo(a) or Lp(a) with a reducing agent destroyed binding activity. A synthetic polypeptide corresponding to a portion of apo(a)'s kringle-4 inhibited the binding (K1 = 1.9 x 10(-4) M) of LDL2 to Lp(a). Therefore, we concluded that binding to apoB-Lp was mediated by the kringle-4-like domains on apo(a). Using ligand chromatography which can detect complexes having a KD as low as 10(-2) M, we demonstrated the binding of plasminogen to apoB-Lp. Like Lp(a), binding of plasminogen to apoB-Lp was mediated by the kringle domain(s). The differences in binding affinity may be due to amino acid substitutions in the kringle-4-like domain. In most of the kringle-4-like domains of apo(a), the aspartic residue critical for binding to lysine was substituted by valine. Consistent with this substitution, we found that L-proline and hydroxyproline, but not L-lysine, inhibited the binding of LDL2 to apo(a). Inhibition by L-proline could be reversed in the binding studies by increasing the amount of apo(a); and L-proline-Sepharose bound plasma Lp(a), suggesting that L-proline acted as a ligand for the kringle-4-like domain(s) of apo(a) involved in the binding of apoB-Lp. The binding of apo(a) to proline and hydroxyproline could be responsible for the binding of apo(a) to the subendothelial extracellular matrix, i.e. domains of proteins rich in proline or hydroxyproline (e.g. collagen and elastin).     

In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.     

Uptake of [(3)H]glycine by synaptosomes of channel

It is known that channel catfish erythrocytes can take up glycine by several distinct transport systems. Further, glycine is an inhibitory neurotransmitter in mammalian brain and spinal cord. Consequently, the uptake of [(3)H]glycine by catfish brain was investigated and found to be a saturable process, dependent on the presence of Na(++) and Cl(--) and sensitive to temperature. A kinetic analysis of transport was performed at 22C. This showed that a high-affinity system existed which exhibited a K(m) of 5.1 (+/- 2. 1) microM. Several structural analogues of glycine were capable of inhibiting uptake in a competitive manner. The most effective inhibitor was sarcosine (IC(50) 5 36 microM). Uptake was also able to be inhibited by harmaline, a drug known to interfere with Na(+)-dependent transport processes. It is concluded that glycine transport by channel catfish brain has much in common with transport by mammalian nervous tissue which is carried out by the membrane carriers GLYT1 and GLYT2. On the other hand, synaptosomal transport differs somewhat from glycine transport by channel catfish erythrocytes.     

Characterization of [(3)H]ryanodine binding sites in mammalian lung

The ryanodine-sensitive calcium channels, also called ryanodine receptors, are intracellular Ca(2+)-release channels that have been shown to bind the neutral plant alkaloid ryanodine with nanomolar affinity. The activity of the skeletal muscle (RyR1), cardiac muscle (RyR2), and brain (RyR3) ryanodine receptor isoforms have been shown to be highly regulated by physiological factors including pH, temperature, and ionic strength; endogenous compounds including Ca(2+), Mg(2+), and adenosine triphosphate (ATP); and pharmacological agents including caffeine, ruthenium red, and neomycin. RyR3 is reportedly expressed in diverse tissues including lung; however, specific [(3)H]ryanodine binding sites in mammalian lung tissue have not been characterized. In this study, hamster lung ryanodine binding proteins were shown to specifically bind [(3)H]ryanodine with an affinity similar to that of RyR isoforms found in other tissues and this binding was shown to be sensitive to Ca(2+) concentration, stimulation by caffeine and spermine, and inhibition by Mg(2+), ruthenium red, and neomycin. The solubilized, intact ryanodine binding protein from hamster lung demonstrated approximately the same 30S sedimentation coefficient as RyR1 and RyR2, but a putative ryanodine receptor subunit from hamster lung was not found to cross-react with antibodies specific for the three known isoforms. We conclude that the hamster lung ryanodine binding protein demonstrates sedimentation and binding characteristics that are similar to those of the known RyR isoforms, but may exhibit antigenic dissimilarity from the typical RyR isoforms found in muscle and brain.     

Isolation and identification of two [(3)H]norharman- ([(3)H]beta-carboline)-binding proteins from rat liver

Norharman (9H-pyrido-[3,4-b]indol) represents a member of the mammalian alkaloids with the group name beta-carbolines. In mammals, it exhibits psychotropic and co-mutagenic actions. Highly specific [(3)H]norharman binding sites have been detected in the liver of rats (B(max): 11 pmol mg(-1) protein; K(D): lower nanomolar range). Two [(3)H]norharman binding proteins with apparent molecular masses of 60 and 80 kDa (SDS-PAGE) were isolated from rat liver crude membrane fraction and identified as the enzyme carboxylesterase (EC 3.1.1.1; 60 kDa) and the stress protein glucose-regulated protein 78 (GRP78; 78 kDa). Possible functional consequences of the interaction of norharman with these two proteins are discussed.     

An enzymatic method for the determination of the initial rate of cholesterol esterification in human plasma.

A method is described for the determination of the initial rate of cholesterol esterification in human plasma, based on the enzymatic determination of free cholesterol in the plasma before and after incubation at 37 degrees C. The cholesterol esterification rate was linear up to 40 minutes. In 18 normal male and 10 normal female subjects the cholesterol esterification rate was 91 +/- 15 (mean +/- SD) and 62 +/- 12 nmoles/hr/ml of plasma, respectively.     

Evidence for non-equilibrating pools of apolipoprotein C-III in plasma lipoproteins

Using immunoaffinity chromatography to isolate apoC-III from radiolabeled lipoproteins for direct determination of specific radioactivity, we have studied the metabolism of human apoC-III in VLDL and in HDL following the bolus injection of 125I-labeled VLDL. Transfer of apoC-III radioactivity from VLDL to HDL was detected in the plasma sample drawn 5 min after injection of the tracer. However, the specific radioactivity of apoC-III in VLDL was found to be higher than that in HDL, with this difference being maintained throughout the sampling period (48-72 hr). The ratios of the respective specific activities ranged from 1.2 to 1.9 in six subjects studied (two normolipidemics and four hypertriglyceridemics). When 125I-labeled HDL was injected as the tracer, however, the higher apoC-III specific radioactivity was associated with the HDL fraction. This lack of complete equilibration of apoC-III between VLDL and HDL in vivo was further characterized by in vitro studies using either 125I-labeled VLDL or 125I-labeled HDL. All incubations were carried out for 3 hr at 37 degrees C followed by 16 hr at 4 degrees C and the apoC-III specific activity in each lipoprotein fraction was directly determined after immunoaffinity chromatography. In a study of plasma from a mildly hypertriglyceridemic subject in which 125I-labeled VLDL was incubated with unlabeled HDL, apoC-III specific activities in VLDL remained 30% greater than that in HDL. When 125I-labeled HDL (from the same subject) was incubated with unlabeled VLDL of apoC-III, final specific activity in VLDL was less than 10% of that of HDL apoC-III. Differences in specific activities were also demonstrated when radiolabeled purified apoC-III was exchanged onto VLDL prior to its incubation with HDL. A consistent difference in apoC-III specific activities in VLDL and HDL was observed after isolation of the particles either by molecular sieve chromatography or by ultracentrifugation. These studies demonstrated that, while the exchange of apoC-III between VLDL and HDL may be very rapid, this equilibration is not complete. Pools of apoC-III that do not participate in the equilibration process are present in both the VLDL and HDL fractions and could account for 30-60% of the total apoC-III mass in each lipoprotein fraction.     

Cold labelled substrate and estimation of cholesterol esterification rate in lecithin cholesterol acyltransferase radioassay

A simple and reproducible radioassay of lecithin cholesterol acyltransferase (LCAT E.C. 2.3.1.43) activity is described. The method is based on cold labelling of the serum, plasma or other LCAT and lipoproteins containing fluids with a trace of 14C-cholesterol spread in ready made sorbent discs. Since the procedure minimizes the chemical, heating and time consuming steps, it may be particularly suitable for routine investigation of the defects of cholesterol kinetics and also for experimental studies.