Addition Of Apo Research Articles

Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4'-Hydroxy-3'-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor.

Apocynin (aPO, 4′-Hydroxy-3′-methoxyacetophenone) is a cell-permeable, anti-inflammatory phenolic compound that acts as an inhibitor of NADPH-dependent oxidase (NOX). However, the mechanisms through which aPO can interact directly with plasmalemmal ionic channels to perturb the amplitude or gating of ionic currents in excitable cells remain incompletely understood. Herein, we aimed to investigate any modifications of aPO on ionic currents in pituitary GH3 cells or murine HL-1 cardiomyocytes. In whole-cell current recordings, GH3-cell exposure to aPO effectively stimulated the peak and late components of voltage-gated Na+ current (INa) with different potencies. The EC50 value of aPO required for its differential increase in peak or late INa in GH3 cells was estimated to be 13.2 or 2.8 μM, respectively, whereas the KD value required for its retardation in the slow component of current inactivation was 3.4 μM. The current–voltage relation of INa was shifted slightly to more negative potential during cell exposure to aPO (10 μM); however, the steady-state inactivation curve of the current was shifted in a rightward direction in its presence. Recovery of peak INa inactivation was increased in the presence of 10 μM aPO. In continued presence of aPO, further application of rufinamide or ranolazine attenuated aPO-stimulated INa. In methylglyoxal- or superoxide dismutase-treated cells, the stimulatory effect of aPO on peak INa remained effective. By using upright isosceles-triangular ramp pulse of varying duration, the amplitude of persistent INa measured at low or high threshold was enhanced by the aPO presence, along with increased hysteretic strength appearing at low or high threshold. The addition of aPO (10 μM) mildly inhibited the amplitude of erg-mediated K+ current. Likewise, in HL-1 murine cardiomyocytes, the aPO presence increased the peak amplitude of INa as well as decreased the inactivation or deactivation rate of the current, and further addition of ranolazine or esaxerenone attenuated aPO-accentuated INa. Altogether, this study provides a distinctive yet unidentified finding that, despite its effectiveness in suppressing NOX activity, aPO may directly and concertedly perturb the amplitude, gating and voltage-dependent hysteresis of INa in electrically excitable cells. The interaction of aPO with ionic currents may, at least in part, contribute to the underlying mechanisms through which it affects neuroendocrine, endocrine or cardiac function.

Mechanism and Physiologic Significance of the Suppression of Cholesterol Esterification in Human Interstitial Fluid

Cholesterol esterification in high density lipoproteins (HDLs) by lecithin:cholesterol acyltransferase (LCAT) promotes unesterified cholesterol (UC) transfer from red cell membranes to plasma in vitro. However, it does not explain the transfer of UC from most peripheral cells to interstitial fluid in vivo, as HDLs in afferent peripheral lymph are enriched in UC. Having already reported that the endogenous cholesterol esterification rate (ECER) in lymph is only 5% of that in plasma, we have now explored the underlying mechanism. In peripheral lymph from 20 healthy men, LCAT concentration, LCAT activity (assayed using an optimized substrate), and LCAT specific activity averaged, respectively, 11.8, 10.3, and 84.9% of plasma values. When recombinant human LCAT was added to lymph, the increments in enzyme activity were similar to those when LCAT was added to plasma. Addition of apolipoprotein AI (apo AI), fatty acid-free albumin, Intralipid, or the d < 1.006 g/ml plasma fraction had no effect on ECER. During incubation of lymph plus plasma, the ECER was similar to that observed with buffer plus plasma. When lymph was added to heat-inactivated plasma, the ECER was 11-fold greater than with lymph plus buffer. Addition of discoidal proteoliposomes of apo AI and phosphatidycholine (PC) to lymph increased ECER 10-fold, while addition of apo AI/PC/UC disks did so by only six-fold. We conclude that the low ECER in lymph is due to a property of the HDLs, seemingly substrate inhibition of LCAT by excess cell-derived UC. This is reversed when lymph enters plasma, consequent upon redistribution of UC from lymph HDLs to plasma lipoproteins.

Apolipoprotein B and Cardiovascular Disease Risk: Position Statement from the AACC Lipoproteins and Vascular Diseases Division Working Group on Best Practices

Low-density lipoprotein cholesterol (LDL-C) has been the cornerstone measurement for assessing cardiovascular risk for nearly 20 years. Recent data demonstrate that apolipoprotein B (apo B) is a better measure of circulating LDL particle number (LDL-P) concentration and is a more reliable indicator of risk than LDL-C, and there is growing support for the idea that addition of apo B measurement to the routine lipid panel for assessing and monitoring patients at risk for cardiovascular disease (CVD) would enhance patient management. In this report, we review the studies of apo B and LDL-P reported to date, discuss potential advantages of their measurement over that of LDL-C, and present information related to standardization. In line with recently adopted Canadian guidelines, the addition of apo B represents a logical next step to National Cholesterol Education Program Adult Treatment Panel III (NCEP ATPIII) and other guidelines in the US. Considering that it has taken years to educate physicians and patients regarding the use of LDL-C, changing perceptions and practices will not be easy. Thus, it appears prudent to consider using apo B along with LDL-C to assess LDL-related risk for an interim period until the superiority of apo B is generally recognized.

P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b 5 from the house fly ( Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1–P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo- b 5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo- b 5. The effects of cytochrome b 5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b 5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.

The influence of lipoprotein(a) on fibrinolytic activity

Lipoprotein(a) is a plasma lipoprotein whose structure and composition are similar with low density lipoprotein (LDL) with an addition of apo(a) that is bound to apo B100. The structure of apo(a) is similar with plasminogen, a proenzym in fibrinolytic system. Due to this similarity, it is assumed that Lp(a) can inhibit plasminogen activity and decreases fibrinolytic activity. The purpose of this study is to prove that addition of Lp(a) to normal plasma can inhibit fibrinolytic activity. Four healthy people whose fibrinogen levels, plasminogen activities and euglobulin clot lysis time were within normal range were enrolled in this study. Fibrinolytic activity were assessed by euglobulin clot lysis time (ECLT). In the first experiment, the addition of Lp(a) was done before centrifugation to obtain euglobulin precipitates, while in the second experiment, Lp(a) was added to the euglobulin precipitates. As a control, ECLT was performed in the plasma with the addition of NaCl 0.9% in the same volume with Lp(a). The results of the study showed that in the first experiment, there was no clot formation. It is assumed that Lp(a) can bind fibrinogen and both of them floated in the supernatant, so there was no fibrinogen in the euglobulin precipitate that can be clotted by thrombin. In the second experiment, the clot did not dissolve until the fourth day. In conclusion, the addition of Lp(a) to normal plasma can inhibit the activity of fibrinolytic system. (Med J Indones 2004; 13: 135-9) Keywords: plasminogen, fibrinogen, apo(a), euglobulin clot lysis time (ECLT)

Quantitative Analysis of Apolipoprotein E Secretion by Human Monocyte-Derived Macrophages in Culture

Apolipoprotein E (apo E) has an impact on lipid metabolism and its production by macrophages is considered to play a protective role against atherosclerosis. Apo A-I stimulates secretion of apo E from macrophages. We developed a new method to evaluate the ability of human monocyte-derived macrophages to secrete apo E, and the effects of factors such as apo A-I were examined. Monocytes separated from peripheral venous blood were cultured. The levels of apo E in macrophage-conditioned medium were quantified by immunoblotting with an anti-human apo E antiserum conjugated with alkaline phosphatase. The basal levels of apo E secretion and the response to exogenous apo A-I in macrophages from 10 healthy volunteers were measured. Sufficient accuracy and sensitivity were confirmed and coefficient of variation of the method was 18 +/- 11% (n = 10). It was confirmed that macrophage secreted apo E in a concentration-dependent manner in response to M-CSF and apo A-I. The average apo E concentration in the conditioned medium of macrophages from 10 healthy subjects was 30.9 +/- 14.7 ng/mg cell protein. After the addition of apo A-I, the average apo E concentration increased, by about 60%, to 49.4 +/- 29.7 ng/mg cell protein (p < 0.05). There was a positive correlation between the apo A-I-induced increase and plasma LDL cholesterol levels (r = +0.54, p < 0.05).

Conformational changes of plastocyanin induced by redox state or metal substitution:Effect on electron transfer to photosystem 1

We are using flash-photolysis studies to elucidate the mechanism of electron transfer (ET) from plastocyanin (Pc) to photosystem I (PSI) in spinach. Two different models can describe the kinetics of the reaction: the CC-model where a rate-limiting Conformational Change occurs in the Pc-PSI complex before the ET or the RE-model where the ET is assumed to be Reversible as a consequence of a higher PSI affinity for reduced Pc compared to oxidized Pc. We will report on kinetic studies with different metal-substituted forms of Pc that can be considered to be redox-inert structural analogs of Cu+-Pc or Cu2+-Pc. Addition of apo- and Ag+-Pc (model of Cu+-Pc) results in a reversible inhibition of the ET from Cu+-Pc to PSI, with affinities for the inhibitors that are comparable to that for Cu+-Pc. However, Zn2+-Pc (model of Cu2+-Pc) is a much weaker inhibitor of the reaction. This indicates that the nature of the metal ion can influence the structure of Pc, which in turn can lead to different affinities to PSI. We will also report on high-resolution X-ray diffraction studies of both the oxidized and reduced states of Cu-Pc. There are small, but significant differences in the structures, in particular in regions that make contact with PSI, which could lead to different affinities to PSI for the two redox states of Pc.

Apolipoprotein E and lipoprotein lipase reduce macrophage degradation of oxidized very-low-density lipoprotein (VLDL), but increase cellular degradation of native VLDL

Oxidized low-density lipoprotein (Ox-LDL) has been shown to be taken up by the macrophage-scavenger receptor at an enhanced rate in comparison to native LDL, with consequent cellular cholesterol accumulation. In the present study, we analyzed macrophage interaction with very-low-density lipoprotein (VLDL) from normolipidemic subjects (N-VLDL) that was oxidized in the presence of 10 μmol/L copper ions. Oxidized VLDL (Ox-VLDL) contained increased conjugated dienes and malondialdehyde (MDA) equivalents and showed increased electrophoretic mobility. Gradual fragmentation of VLDL apolipoproteins (apo) was noted, with apo B-100 being the first to be fragmented, followed by apo E and apo C. Degradation of Ox-VLDL by mouse peritoneal macrophages (MPM) was increased almost twofold in comparison to N-VLDL. Upon incubation of VLDL with lipoprotein lipase (LPL), the LPL-treated lipoprotein demonstrated up to 50% increased degradation by macrophages in comparison to control N-VLDL. However, the degradation of LPL-treated Ox-VLDL was decreased by up to 20% in comparison to control Ox-VLDL. Similarly, the addition of apo E to VLDL enhanced its cellular degradation by 56%, whereas a 20% reduction in the degradation of apo E-treated Ox-VLDL was demonstrated in comparison to nontreated Ox-VLDL. These results showed that LPL and apo E, two important regulatory substances in cellular metabolism of plasma lipoproteins, increased macrophage degradation of native VLDL, but reduced the degradation of Ox-VLDL. These inhibitory effects on macrophage uptake of Ox-VLDL suggest that apo E and LPL may possess antiatherogenic potential.

Cholesterol efflux from adipose cells is coupled to diacylglycerol production and protein kinase C activation

Apolipoprotein A-I (apo A-I)*/DMPC complexes have been previously shown to promote cholesterol efflux from cholesterol-preloaded adipose cells whereas apo A-II/DMPC complexes, which bind to the same cell surface binding sites, were ineffective. Addition of apo A-I/DMPC complexes led to a rapid and transient formation of diacylglycerol. However, in contrast to PGF2 alpha (Doglio et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 1148), no accumulation of inositol phosphates was observed. Apo A-II/DMPC complexes had no effect on diacylglycerol formation. Stimulation by apo A-I/DMPC complexes or native HDL3 of cells prelabelled with (2-palmitoyl 9,10[3H])phosphatidylcholine induced also the formation of labelled diacylglycerol whereas apo A-II/DMPC complexes and HDL3 treated with tetranitromethane showed no effect. Direct activation of protein kinase C(s) by PMA promoted cholesterol efflux providing that DMPC liposomes were present as cholesterol acceptor. It is proposed that lipoprotein particles have two separate effects, i.e. a ligand-induced effect leading to cholesterol translocation from intracellular stores to the cell surface and a bilayer-induced effect allowing cholesterol efflux from the cell surface to the acceptor.

Cellular metabolism of human plasma intermediate-density lipoprotein (IDL)

The cellular metabolism of human plasma intermediate-density lipoprotein (IDL) was investigated in cultured human skin fibroblasts and hepG-2 cell in the absence and presence of exogenous recombinant or plasmatic apo E-3. IDL ( d 1.006–1.019 g/ml) and LDL ( d 1.019–1.063 g/ml) were prepared by centrifugation from the plasma of apo E- 3 3 or 4 3 normolipidemic human subjects. Without added apo E-3, IDL binding and cell association are similar or slightly reduced while their degradation is one third to one half. This results in degradation to binding ratios for IDL that are half those for LDL. Exogenous apo E-3 enhances binding, association and degradation of IDL by 50–150%, but the degradation to binding ratio remains low. Exogenous apo E-3 also increased the ability of IDL but not LDL, to down-regulate the incorporation of [ 14C]acetate to sterol by the cells. The optimal concentration of apo E-3 is 4 μg protein/10 μg IDL protein and at that concentration appreciable amounts of the apo E are found associated with the lipoprotein. Apo E-2 has no effect on the cellular metabolism of IDL and apo E-3 is not effective in receptor-negative human fibroblasts. Monoclonal antibodies that block apo E binding to B, E (LDL) receptor (1D7) abolish the cellular metabolism of IDL while antibodies against B-100 (4G3) are ineffective. In competitive binding experiments, IDL is slightly more effective than LDL in displacing 125I-LDL from receptors in hepG-2 cells and appreciably more effective than LDL when tested against 125I-IDL. Apo E-3 increases the capacity of IDL to compete with either 125I-LDL or 125I-IDL. Addition of apo E-3 also increases the binding affinity of IDL to hepG-2 receptors, with K d values of 2.50, 0.93 μg protein/ml, respectively. The study demonstrates the essential role that functional apo E molecules play in the interaction of human IDL with cellular receptors. Yet, in spite of presence of apo E in IDL (2–3 molecules /particle) and enrichment of IDL with apo E-3 (to 4–5 molecules /particle) the proteolytic degradation of the lipoprotein by specific cellular receptor is similar to LDL.

Intravenous infusion of apolipoprotein E accelerates clearance of plasma lipoproteins in rabbits.

Plasma cholesterol levels in cholesterol-fed rabbits were markedly reduced by the intravenous infusion or bolus injection of recombinant human apo E or rabbit plasma apo E. Administration of 6-70 mg of apo E resulted in an approximately 20-40% acute reduction in plasma cholesterol levels within 2-3 h. Plasma cholesterol levels remained reduced for 4-8 h after the administration of apo E. Furthermore, the intravenous injection of apo E reduced the plasma cholesterol levels in Watanabe heritable hyperlipidemic rabbits. The addition of apo E to [14C]cholesterol-labeled canine thoracic duct lymph or [14C]cholesterol-labeled chylomicrons resulted in accelerated plasma clearance of these diet-induced lipoproteins in normal rabbits, with the uptake occurring primarily in the liver. This study suggests that the amount or availability of apo E in the plasma of cholesterol-fed rabbits may be rate limiting for the normal clearance of diet-induced remnant lipoproteins.

リポ蛋白リパーゼによるVLDL-triglycerideの水解に及ぼすHDLとd&gt;1.25画分の効果

Hypertriacylglycerolemia is often accompanied by low level of plasma high density lipoprotein (HDL). In order to determine whether or not HDL can be a regulartory factor of very low density lipoprotein (VLDL) metabolism, we studied the effect of HDL on VLDL-triacylglycerol (TG) hydrolysis by lipoprotein lipase (LpL) in the presence and absence of d>1.25 fraction. HDL enhanced VLDL-TG hydrolysis by bovine milk LpL; low density lipoprotein (LDL) did not promote the hydrolysis. The increased hydrolysis of VLDL-TG by HDL further enhanced by d>1.25 fraction. Though d>1.25 fraction also had a stimulatory effect on VLDL-TG hydrolysis apart from the effect of HDL, the VLDL-TG was doubly increased by simultaneous existence of HDL and d>1.25 fraction. HDL did not show any effect on the hydrolysis of Triton X-100-emulsified TG. d>1.25 fraction had a weak stimulatory effect on the detergent-emulsified TG. This enhanced VLDL-TG hydrolysis by HDL was not due to additional apoprotein C-II (apo C-II) contained in HDL, because the VLDL hydrolysis system used in the experiment contained a sufficient amount of apo C-II in VLDL and further addition of apo C-II did not increase the hydrolysis. Phosphatidylcholine (PC) liposome increased VLDL-TG hydrolysis by LpL. On phenyl-Sepharose chromatography of d>1.25 fraction showed that VLDL-TG hydrolysis stimulatory fraction was coeluted with cholesteryl ester (CE) transfer activity. Triton X-100-emulsified TG hydrolysis by LpL was inhibited as the content of CE in the TG emulsion was increased.These results suggest that low HDL level in plasma may retard VLDL hydrolysis and cause hypertriacylglycerolemia. PC liposome also activated LpL in VLDL-TG hydrolysis. HDL is a phospholipid rich lipoprotein, so it might act as a liposome in plasma. d>1.25 fraction seemed to play an important role in VLDL hydrolysis. The fact that LpL was inhibited by CE and VLDL hydrolysis stimulatory fraction was coeluted with CE transfer activity on the hydrophobic chromatography implied that d>1.25 fraction may transfer CE from VLDL or VLDL remnant to HDL during the hydrolysis by LpL to promote TG hydrolysis of VLDL and VLDL remnant. The mechanism by which HDL and d>1.25 fraction promoted VLDL-TG hydrolysis by LpL remains to be clarified.

Uptake and degradation of human chylomicrons by macrophages in culture. Role of lipoprotein lipase.

Because macrophages secrete lipoprotein lipase (LPL), we sought to determine if LPL activity would influence the metabolism of chylomicrons by macrophages. In initial studies, we showed that normal chylomicrons were a substrate for the macrophage's LPL activity. Uptake of normal chylomicrons occurred in a saturable fashion and was effectively competed for by human chylomicrons, very low density lipoproteins (VLDL), and rabbit beta-VLDL, but not by acetyl-low density lipoprotein (LDL), and only modestly by native LDL. When apoprotein C-II-deficient chylomicrons were incubated with macrophages, no hydrolysis of triglyceride occurred, yet saturable uptake of chylomicron protein and lipid occurred, demonstrating that LPL activity is not a prerequisite for saturable uptake. However, addition of apo C-II led to marked hydrolysis and enhanced uptake of protein and lipid moieties. When albumin was present in the medium, there was approximately equal enhancement of cellular content of triglyceride and cholesteryl ester, despite the fact that chylomicrons are triglyceride-rich. This was due to uptake of a triglyceride-depleted particle produced by LPL, as well as a preferential re-release of triglyceride. These studies suggest potential pathways by which triglyceride-rich lipoproteins could contribute to accumulation of cholesteryl esters in macrophages, even while only small amounts of triglyceride accumulate.

Prolactin studies in normals: Implications for clinical research

Dopaminergic (DA) transmission is a major regulator of pituitary prolactin (PRL) secretion. Strategies to assess abnormalities of DA regulation in mental illness have thus included comparisons of patients' and normals' serum PRL levels before and after the administration of DA agonists and antagonists. These clinical research strategies suffer from a number of shortcomings. There is a wide interindividual variability of normal basal PRL levels, and intraindividual variability has been little studied. Large interindividual variability of PRL responses to DA antagonist challenges has also been observed in normals and reported to be strongly correlated to variation in serum levels of the challenge drug. Assessment of DA agonist challenges is hampered by the fact that low basal levels of serum PRL make suppression difficult to measure; a further problem is the confounding effect of nausea when these drugs are given in high doses. In this study of normals, individual basal serum PRL levels were found to be stable over a mean period of 10 months, with interindividual variance vastly greater than intraindividual variance. Thus, state alterations in mental illness may best be studied using a longitudinal design for measurements of PRL levels in patients, thereby avoiding confounding interindividual variability. Moreover, it appears that alterations of PRL levels between groups or within patients, even though within the normal range, may have individual physiological significance. A study of the PRL responses to haloperidol (hal) and hal + apomorphine (apo) challenges in normal revealed a strong correlation despite a highly significant 51% reduction in PRL response with the addition of apo. Because this correlation is dependent upon a normal or limited range of DA regulation, the study of these two responses in abnormal populations may be more revealing of DA abnormalities than the study of PRL responses to single DA agonist or antagonist challenges.