Auranofin modulates human neutrophil superoxide production and protein phosphorylation.

The effect of auranofin (AF) was examined on human neutrophil superoxide production and protein phosphorylation stimulated by phorbol esters. Low concentrations of auranofin (less than or equal to 0.5 microM) enhanced while higher concentrations (0.5-10 microM) inhibited superoxide release stimulated by a suboptimal concentration (0.005 microM) of phorbol myristate acetate (PMA). The enhancing but not the inhibitory effect of AF was lost if a maximal stimulating dose (0.05 microM) of PMA was used. In contrast AF had a biphasic effect on protein phosphorylation regardless of the stimulating concentration of PMA. Comparison of the dose-response curves for these effects of AF suggest that although changes in protein phosphorylation may be partly responsible for altered activity of the NADPH oxidase responsible for superoxide production, it is unlikely that they are mediated by a direct effect of AF on protein kinase C. Also, measurement of 3H-PDBu-binding to neutrophils showed that these actions of AF could not be attributed to altered binding of phorbol esters to their cellular receptor (protein kinase C).

Chloroquine and hydroxychloroquine inhibit multiple sites in metabolic pathways leading to neutrophil superoxide release.

The effect of chloroquine and hydroxychloroquine on human neutrophil polymorph superoxide (O2-) production stimulated either by opsonized zymosan, phorbol myristate acetate or fluoride was examined in vitro. Both drugs inhibited the response to all 3 stimuli in a dose and time dependent fashion. Inhibition of zymosan stimulated O2- release was only attributable in a minor degree to inhibition of expression of surface receptors (MO-1), and appeared to be mainly due to inhibitory effects on more distal components of the activation pathway. Possible mechanisms for the inhibition of fluoride and phorbol ester stimulated O2- release are discussed in the light of current understanding of metabolic pathways of neutrophil polymorph activation.

Studies on the mechanism of inhibition of chemotactic tripeptide stimulated human neutrophil polymorphonuclear leucocyte superoxide production by chloroquine and hydroxychloroquine.

The effect of chloroquine and hydroxychloroquine on neutrophil superoxide release stimulated by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was examined. Both drugs caused time and dose dependent inhibition of superoxide release but had no effect on equilibrium binding of [3H]FMLP to its receptor. Preliminary experiments suggest that these drugs may exert their inhibitory effect on superoxide release by inhibiting the FMLP stimulated hydrolysis of phosphoinositides.

Lipoprotein substrates for plasma cholesterol esterification. Influence of particle size and composition of the high density lipoprotein subfraction 3.

Subpopulations of lipoproteins within the high density lipoprotein subfraction 3 (HDL3) have been isolated from human plasma and characterized in terms of their chemical composition and particle size. Since HDL3 are the major substrates for the esterification of plasma cholesterol, and thus play a central role in the transport of cholesterol through the plasma, these studies also examined the relative capacities of different HDL3 subpopulations to interact with lecithin: cholesterol acyltransferase. Substrate reactivity of a given preparation of lipoproteins was defined in terms of the Vmax of the cholesterol esterification reaction in incubations containing a fixed amount of human lipoprotein-free plasma as a source of the enzyme. Substrate reactivity was found to correlate inversely with the radius of HDL3 particles. This inverse relationship between particle size and substrate reactivity was independent of the particle content of cholesteryl ester.

Competitive inhibition of plasma cholesterol esterification by human high-density lipoprotein-subfraction 2.

Mixtures containing subfractions of human plasma high-density lipoproteins (HDL) and human lipoprotein-free plasma were incubated in vitro at 37 degrees C. Esterification of cholesterol was observed both in incubations containing HDL-subfraction 3 (HDL3) and in those containing HDL-subfraction 2 (HDL2). The implication that the lecithin: cholesterol acyltransferase in lipoprotein-free plasma may therefore interact with lipoproteins in both HDL subfractions was developed further by proposing a simple model in which the two HDL subfractions may compete for interactions with the enzyme. This model was described mathematically and tested in experiments in which a constant amount of the enzyme was incubated with a wide range of concentrations of HDL2 and HDL3 present either alone or in combination. The model was able to predict experimentally observed rates of cholesterol esterification with great accuracy. The best fit was obtained with a Vmax for HDL3 that was 2.4-4-times greater than that for HDL2 and values of the apparent Km for HDL3 free cholesterol and HDL2 free cholesterol of 43-60 nmol/ml and 167-391 nmol/ml, respectively. The model thus predicts that, at physiological concentrations of lipoproteins, HDL2 will function as a competitive inhibitor of the cholesterol esterification reaction by displacing lecithin: cholesterol acyltransferase from a more effective substrate, HDL3, to a less effective substrate, HDL2.

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase.

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Changes in the density distribution of pig high density lipoproteins during incubation in vitro. Influence of esterified cholesterol transfer activity.

Pig plasma, depleted of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) by ultracentrifugation, was incubated in vitro at 37 degrees C in the presence of 0.002 M parachlormercuriphenyl sulfonate, a chemical inhibitor of lecithin: cholesterol acyltransferase. Incubation resulted in a consistent and significant shift in the density distribution of esterified cholesterol within the high density lipoprotein (HDL) fraction. After 24 h of incubation the esterified cholesterol in HDL of density greater than 1.125 g/ml was reduced by a mean of 28%, while that in HDL of density less than 1.125 g/ml was increased by a mean of 77%. Both the rate and the magnitude of this redistribution were markedly enhanced when the incubation mixture contained rabbit lipoprotein-free plasma, a rich source of the esterified cholesterol transfer protein. When pig plasma, depleted of VLDL and LDL, was subjected to rate zonal ultracentrifugation, the HDL in non-incubated samples eluted as a single symmetrical peak in a position comparable to that of human HDL3. After 24 h of incubation at 37 degrees C the density of the HDL was reduced, with a peak midway between the positions of human HDL3 and HDL2. When VLDL- and LDL-depleted pig plasma were mixed with human lipoprotein-free plasma (also a source of esterified cholesterol transfer activity) and incubated for 24 h at 37 degrees C, there was an even greater reduction in the density of pig HDL, which now eluted in a position comparable to that of human HDL2. It was concluded that the capacity of the esterified transfer protein to redistribute esterified cholesterol between different lipoprotein particles may be of importance in the process of HDL interconversion.

A unified model of esterified cholesterol exchanges between human plasma lipoproteins.

Exchanges of esterified cholesterol between human high density lipoproteins (HDL) and very low density lipoproteins (VLDL) and between VLDL and low density lipoproteins (LDL) have been fitted to a mathematical model previously developed to describe exchanges between human HDL and LDL. In all cases the fit of the model predicted exchanges to those measured experimentally was extremely good, thus greatly increasing confidence in the validity of the model. The model assumes that esterified cholesterol exchanges are achieved by means of a transfer protein which interacts with lipoprotein particles from which it picks up and deposits esterified cholesterol. The values generated for the model constants indicated that, given equal concentrations of esterified cholesterol in each fraction, the relative probability that the transfer protein will pick up a molecule of esterified cholesterol in HDL vs. VLDL vs. LDL is in the ratio 28.9:4.65:1. According to the model the transfer protein may 'bind' to lipoproteins. The model predicts that, at physiological lipoprotein concentrations, the proportion of transfer protein bound to HDL will be more than double that which is unbound to lipoprotein and that bound to VLDL will be about one tenth that unbound. The model was unable to detect evidence of the transfer protein binding to LDL.

Comparison of different methods of isotopically labelling the esterified cholesterol in human high density lipoproteins.

Different methods of incorporating esterified [3H]cholesterol into human high density lipoproteins (HDL) have been assessed in terms of their influence on the subsequent rate at which esterified [3H]cholesterol was transferred from HDL to other plasma lipoproteins in 37 degrees C incubations containing tracer amounts of the labelled preparations added to aliquots of a common pool of unlabelled human plasma. Two basic methods were used to label the HDL esterified cholesterol: (a) by the action of lecithin: cholesterol acyltransferase in incubations of plasma containing exogenous [3H]cholesterol and (b) by exchange in incubations of plasma containing preparations of prelabelled LDL. The exchange labelling approach was also used to examine the effects of various HDL compositional changes on the subsequent behaviour of its esterified [3H]cholesterol. It was found that the source of the label, per se, whether from the esterification of exogenous [3H]cholesterol or from exchange with labeled LDL, had no influence on the subsequent behaviour of HDL esterified [3H]cholesterol. However, modification of the HDL composition during the labelling process had profound effects. For example, in preparations in which the esterified cholesterol content of the labelled HDL was increased by the action of lecithin: cholesterol acyltransferase, there was a reduced rate of transfer of esterified [3H]cholesterol out of the HDL fraction in subsequent incubations of the labelled HDL with fresh, unlabelled plasma. Conversely, when the esterified cholesterol content of the labelled HDL had been decreased by pre-incubation with Intralipid or very low density lipoproteins (VLDL), the subsequent rate of transfer of esterified [3H]cholesterol out of the HDL fraction was increased markedly. It has been concluded that to obtain a biologically valid, labelled tracer of human HDL esterified cholesterol, the labelling should be achieved with minimal modification to the HDL composition. This means labelling by exchange in incubations in which lecithin: cholesterol acyltransferase is inhibited in plasma from which VLDL has been removed.

Net mass transfer of esterified cholesterol from human low density lipoproteins to very low density lipoproteins incubated in vitro.

In 37 degrees C incubations of either human or rabbit very low density lipoproteins (VLDL), low density lipoproteins (LDL) and lipoprotein-free plasma thee was a net mass transfer of esterified cholesterol from LDL to VLDL which in the human studies resulted in an approximate doubling of the VLDL esterified cholesterol concentration after 24 h. There was also a net mass transfer of triacylglycerol in the reverse direction, from VLDL to LDL.