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Cardiovascular Research 1998 38(2):522-530; doi:10.1016/S0008-6363(98)00040-6
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Downregulation of β-adrenergic receptors by low density lipoproteins and its prevention by β-adrenergic receptor antagonists

Bernhard R Brehma,*, Matthias Meergansa, Dorothea I Axela, Martin Pfohlc, Helmut Heinleb and Karl R Karscha

aDepartment of Cardiology, University of Tübingen, Otfried-Müllerstrasse 10, D-72076 Tübingen, Germany
bInstitute of Physiology, University of Tübingen, Tübingen, Germany
cDepartment of Endocrinology, University of Tübingen, Tübingen, Germany

* Corresponding author. Tel.: +49 (7071) 298-2711; Fax: +49 (7071) 360245; E-mail: 101566.341@compuserve.com

Received 11 August 1997; accepted 7 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Vasodilation by β-adrenergic receptors of smooth muscle cells appears to be impaired early after the onset of hypercholesteremia. The aim of this study was to analyze the modulation of β-adrenergic receptor density and adenylyl cyclase activity in the presence of moderately elevated concentrations of LDL. The effects of β1- and β2-adrenergic receptor antagonists on LDL-induced receptor changes were studied. Methods and results: Media explants of porcine coronary arteries were incubated with moderately elevated LDL concentrations (0.7–3.9 mmol/l). The density of β-adrenergic receptors was determined in plasma membranes using the radioligand [125I]iodocyanopindolol. LDL (3.9 mmol/l) resulted in a decrease of β-adrenergic receptor density (control 137±5 vs. 89±7 fmol/mg protein, P<0.01). After removal of LDL and cultivation for an additional 3 days β-adrenergic receptors increased to 129±5 fmol/mg. In the presence of the β1- or β2-adrenergic receptor antagonists the LDL-mediated decrease was inhibited. Addition of metoprolol after 3 days of LDL incubation caused a restoration of receptor density. The basal, isoproterenol- and forskolin-stimulated adenylyl cyclase activities were increased after LDL incubation by 180, 110 or 80%, respectively. Conclusion: Moderately elevated LDL levels decreased β-adrenergic receptor density while adenylyl cyclase activity was simultaneously increased. β1- or β2-adrenergic receptor antagonists prevented this receptor decrease and might preserve the β-adrenergic receptor density in the presence of moderately elevated LDL levels.

KEYWORDS Moderately elevated LDL; Porcine coronary artery; β-Adrenergic receptor; β-Blockade; Adenylyl cyclase


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In the early phase of the development of coronary heart disease, vasospasms can be observed even before typical histopathological changes are detectable [1–5]. It is clear that a complex cross talk between vasodilating β-adrenergic receptors and LDL receptors occurs since an increase of cAMP levels (cyclic adenosine monophosphate) results in an elevated expression of LDL receptors. In animal studies with maximal cholesterol levels of 25.8±5.4 mmol/l (10-fold above controls) β-adrenergic receptors decreased in heart plasma membranes by about 30% [6]. However, it has been shown that vasoregulation is changed at much lower serum LDL concentrations of about 5.7±1.0 mmol/l [3]and that mild oxidation of LDL leads to a changed vasomotor activity [7, 8].

In this study, we postulate a relationship between modest elevations of LDL (between 0.7 and 3.9 mmol/l) and both the density of β-adrenergic receptors and their ability to activate adenylyl cyclase – the enzyme which brings about vasodilation by increasing cAMP. Furthermore, the effects of selective β1- or β2-adrenergic receptor antagonists on LDL-induced receptor changes were analyzed.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
[125I]Iodocyanopindolol ([125I]ICYP, 81.4 Tbq/mmol) and [{alpha}-32P]adenosine triphosphate ([{alpha}-32P]ATP, 111 Tbq/mmol) were from NEN (Dreieich, Germany). ICI 118.551 and bisoprolol were gifts from ICI (Planckstadt, Germany) and Merck (Darmstadt, Germany) respectively. Dulbecco's modified essential medium was from Life Technologies Gibco (Eggenstein, Germany). All other chemicals used in this trial were from Sigma (Munich, Germany). Human LDL was obtained from one male patient suffering from heterozygous familial hypercholesteremia with a TGC/CGC mutation in codon 113 of exon 4 of the LDL-receptor gene. The presence of any additional familial defective apolipoprotein B-100 was excluded by temperature-gradient gel electrophoresis [9]. The subject's LDL levels were 11.5 mmol/l without any treatment and between 6 and 7 mmol/l immediately before the LDL-apheresis treatment. During the study period the patient was only on pravastatin 40 mg/day, taken in the evening. The LDL was removed from the patients plasma using dextran sulfate apheresis as described previously [9], and was eluted from the dextran sulfate column using physiological (0.9%) sodium chloride solution. The LDL cholesterol concentration in the eluate was 11.4±0.8 mmol/l. A major concern of this study was whether the LDL obtained from our patient was typical for that of healthy individuals. Receptor downregulation studies, however, confirmed that this was the case since LDL obtained from a healthy individual provided identical results. Because large amounts of LDL were necessary for performing the experiments, a healthy person could not be used alone for acquiring the LDL. Pravastatin was taken only in the evenings so that at the time when apheresis was undertaken (mid-afternoon), only negligible levels were present in the plasma (its plasma half-life is only 2 h). The LDL was collected in a sterile bottle and used within 2 days for the experiments.

2.1 Tissue preparation and incubation
Adult pigs (100–120 days, body weight about 100 kg) of either sex, without medication or treatment, were sacrificed as part of a liver transplantation research program. The heart was immediately excised after the liver had been removed. All further preparations were done at 4°C. The coronary arteries (left descending, right coronary artery and left circumflex branch) were dissected and for further processing the adjacent fat and myocardium were removed. The trimmed arteries were placed in 50 ml ice-cold culture medium (Dulbecco's modified essential medium plus HEPES, pH 7.4) and the adventitia was removed under a microscope (Leica). The remaining vessel was dissected longitudinally and the endothelial layer was scraped off. Parts of the coronary arteries (5 mm) were frozen in isopentane which had previously been cooled in liquid nitrogen [10]. Cross sections were stained with hematoxylin and eosin for histological examination to confirm the absence of endothelial cells and adventitia. The media explants were then cut into small pieces (1–2 mm) and cultivated in serum-free tissue medium containing transferrin (10 µg/ml), insulin (5 µg/ml) and thyroglobin (10 µg/ml) for a maximum period of 14 days. Human LDL (1–2 days after apheresis) was added immediately to the culture medium. At the end of the LDL incubation a low TBARS was shown; this confirmed that the LDL was only minimally oxidized (Table 1).


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  		Table 1 Oxidation of LDL in vitro

 
The incubation was stopped by adding ice-cold phosphate-buffered saline (PBS, pH 7.4) and washing the media explants three times with PBS. Finally, plasma membranes were prepared from these media explants. Cultivation of media explants for 14 days resulted in a migration and proliferation of smooth muscle cells.

Porcine coronary arteries were used because healthy human coronary arteries were not available. Furthermore, β-adrenergic receptor density in human coronary arteries and porcine coronary arteries are nearly identical. The percentages of β1- and β2-adrenergic receptors are 80 and 20%, respectively, figures which are comparable to human coronary arteries [11, 12]. Incubation with metoprolol was carried out at a concentration of 10–6 mol/l since clinical studies have indicated that plasma concentrations of 200 nmol/l result only in a 70% occupation of β1-adrenergic receptors [13]. Bisoprolol shows a 200-fold higher β1/β2-adrenergic receptor selectivity than does metoprolol [14]and ICI 118.551 has a 300-fold higher β2/β1 selectivity [15].

2.2 Oil red O stain
An oil red O stain was used to detect the LDL internalized into smooth muscle cells. Cells were grown on cover slips either with LDL-containing serum-free growth medium or without LDL. After fixation with 1.5% glutaraldehyde, cells were incubated in oil red O solution (10 µg in 1 mol/l acetone diluted 1:100 with phosphate buffered saline, 25°C, 1 h) and washed with phosphate buffered saline.

2.3 Measurement of total LDL and oxidized LDL
Total LDL was measured with a LDL assay from Boehringer Mannheim (Germany) according to the manufacturer's instructions. The amount of oxidized LDL was measured according to the method of Niehaus and Samuelson [16]. Thiobarbituric acid reactive substances (TBARS) of oxidized LDL were measured in this procedure in order to rule out the possibility of marked oxidation. LDL was oxidized with 8 µl H2O2 (0.1%) for 3 days and compared with the LDL containing growth medium. TBARS were measured in order to be comparable to the LDL in the growth medium. The LDL which was used showed at the end of the LDL incubation low TBARS indicating that the LDL was minimally oxidized (Table 1). However, since the formation of TBARS is a dynamic process these values only reflect the end point. Furthermore, it was demonstrated that LDL showed lower susceptibility for oxidation after LDL apheresis [17, 18].

2.4 Plasma membrane preparation
For preparing plasma membranes, coronary media explants were homogenized in 40 ml of buffer A (50 mmol/l Tris–HCl, 5 mmol/l EDTA, pH 7.4) using a Polytron homogenizer (Kinematika, Switzerland, 10 000 U/min, probe 10, 3x10 s, at 4°C) [19]. After sedimentation (350xg, 5 min, 4°C) the supernatant was filtered through two layers of gauze. After the second sedimentation step (48 000xg, 10 min, 4°C) the pelleted membranes were resuspended using a tight fitting pestle (Ikra, Germany, 800 U/min, 3x10 sec, 4°C) and washed twice in 40 ml buffer A by centrifugation (48 000xg, 10 min, 4°C). The final pellet was resuspended in 50 mmol/l Tris–HCl (pH 7.4, 4°C) to give a final concentration of 0.2–0.4 mg protein/ml and used immediately for the various assays [19, 20]. To obtain the so-called ‘light vesicles’ the supernatant of the first 48 000xg spin was centrifuged at 160 000xg [19].

2.5 Radioligand binding
β-Adrenergic receptor density was determined on the plasma membranes by radioligand binding using the radiolabeled β-adrenergic receptor antagonist [125I]ICYP [21]. For saturation isotherms increasing concentrations of the radioligand (15–500 pmol/l) in 50 mmol/l Tris–HCl, 12.5 mmol/l MgCl2, pH 7.4, 4°C, were incubated (1 h, 25°C) with plasma membranes (approx. 10–20 µg protein/tube). The incubation was stopped by rapid vacuum filtration on Whatman GF/C filters. The filters were washed (3x4 ml, 50 mmol/l Tris–HCl, pH 7.4) and counted in a {gamma}-counter with a counting efficiency of 80%. Non-specific binding was determined as the residual binding in the presence of alprenolol (10–6 mol/l) [21].

For β-adrenergic subclassification to β1- and β2-adrenoceptor subtypes, [125I]ICYP was incubated at a concentration of 0.05 nmol/l with the β1-adrenergic receptor antagonist bisoprolol or the β2-adrenergic receptor blocker ICI 118.551 (10–10–10–4 mol/l). In competition experiments, displacement of [125I]ICYP at a concentration of 0.05 nmol/l was attempted with increasing concentrations of the β-agonist isoproterenol (10–10–10–4 mol/l). The incubation (45 min, 25°C) was performed in the presence or absence of the non-hydrolyzable GTP-analog guanylimidodiphosphate (Gpp(NH)p, 10–4 mol/l) in order to identify the guanine nucleotide sensitive ‘high affinity state’ of the receptors [22–24]. Control experiments, which were performed with or without LDL (2.6 mmol/l) directly in the binding assay, showed no difference in the number of maximal binding sites and the affinity for the radioligand used (data not shown). In competition experiments, LDL (2.6 mmol/l) was added directly to the reaction mixture. No competition with the radioligand for receptor binding was observed.

2.6 Adenylyl cyclase assay
Adenylyl cyclase activity was determined by using [{alpha}-32P]ATP (approx. 2 µCi/tube final concentration) as the substrate in a final incubation volume of 100 µl (final concentration: 75 mmol/l Tris–HCl (pH 7.4), 6 mmol/l MgCl2, 1 mmol/l DTT, 1 mmol/l EDTA, 10 µmol/l GTP, 0.1 mmol/l ATP, 100 µmol/l cAMP, 20 mmol/l phosphoenolpyruvate, 0.041 mg creatine kinase, 50–100 µg plasma membrane). Isoproterenol (10–6 mol/l) or the diterpene forskolin (50 mmol/l) was added. The incubation (10 min, 37°C) was stopped according to the method of Jakobs et al. through precipitation of the non-reacted ATP by addition of NaHCO3 (120 mmol/l) and zinc acetate (125 mmol/l); the labeled cAMP was separated on alumina columns with a recovery of 80–90% [25].

Protein was measured by the method of Bradford using bovine serum albumin as standard [26].

2.7 Statistical analysis
Saturation curves were analyzed by computer assisted techniques using law of mass action based on linear least square curve fitting techniques originally developed by Munson and Rodbard (software LIGAND) [22, 27]. The proportion of the receptors able to form the β-agonist-promoted ‘high affinity state’ was determined using a ‘two site one ligand fit' according to the law of mass action. The percentage of β1-/β2-adrenergic receptors was calculated using a ‘two site one ligand fit'. Statistical analysis was performed using analysis of variance and Student–Newman–Keuls test. P-values <0.05 were considered as statistically significant. Data are expressed as mean±s.d. of 3–5 sets of experiments.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Decrease of β-adrenergic receptors after LDL incubation
Isolated media explants were incubated either with serum-free growth medium or with serum-free medium supplemented with LDL at a concentration of 2.6 mmol/l for three days. Chronic LDL incubation led to a significant 25% decrease in the β-adrenergic receptor density of plasma membranes obtained from the media of porcine coronary arteries (137±7 to 102±5 fmol/mg, P<0.01, n=5). The affinity of the β-adrenergic receptors for their radioligand was unchanged (Kd, control 36±11 pmol/l, LDL 44±12 pmol/l) (Fig. 1). The so-called ‘light vesicles’ did not bind [125I]ICYP (data not shown). The ratio of β1- to β2-adrenergic receptors after LDL incubation did not change during LDL incubation (80–20%, data not shown).


Figure 1
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  		Fig. 1 Reduction of β-adrenergic receptors in media explants of porcine coronary arteries after persistent LDL incubation. Porcine coronary arteries were isolated and media explants prepared. These were grown with serum-free medium (control) or serum-free medium supplemented with LDL (LDL 2.6 mmol/l, dashed lines) for 3 days. In crude plasma membranes of controls and treated explants the density of β-adrenergic receptors was determined using the radiolabeled β-adrenergic antagonist [125I]ICYP in saturation isotherms. Non-specific binding was the residual binding in the presence of high concentrations of alprenolol (10–6 mol/l, straight line). The average of five different sets of experiments are shown using least-square curve fitting techniques based on the law of mass action. The affinities of the receptor for the radioligand were unchanged after LDL incubation.

 
3.2 Cell viability and cholesterol accumulation
In order to test the viability of the smooth muscle cells, media explants were cultivated for 14 days in the presence of serum-free medium (Fig. 2a) or medium supplemented with various concentrations of LDL (0.7, 1.3, 2.6, 3.9 or 5.1 mmol/l, Fig. 2b). The proliferation and migration of smooth muscle cells served as parameters to indicate cellular viability. After 12–14 days, both migration of cells from the explants and proliferation could be shown; this confirmed the viability of the smooth muscle cells which were identified using immunocytochemical stains incorporating antibodies against {alpha}-actin. No migration or proliferation of cells from media explants could be seen after incubation with LDL at 5.1 mmol/l because these explants were not attached to the culture dishes. Therefore these explants were not included in the experiments.


Figure 2
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  		Fig. 2 Accumulation of lipids in smooth muscle cells of porcine coronary arteries after LDL incubation. The explants of the media of porcine coronary arteries were grown with serum-free medium or in the presence of LDL (2.6 mmol/l). After 14 days, smooth muscle cells were migrating from the media explants. The accumulation of lipids within the cells was shown using oil red O dye. The LDL incubated cells presented a marked accumulation of lipid droplets (red droplets within the cells (a)) within the cytoplasma whereas control cells showed only a few small red spots (b). An oil red O stain of migrating smooth muscle cells is illustrated at 40x magnification. Scale bar: 0.2 mm.

 
Oil red O stain was applied to detect intracellular lipid accumulation. In media explants grown with serum-free medium for 12 days smooth muscle cells migrated from the explants and accumulated small amounts of cholesterol in their cytoplasm. Smooth muscle cells cultured in the presence of 2.6 mol/l LDL showed an increased accumulation of cholesterol. In comparison to controls, smooth muscle cells appeared larger and contained fewer nucleoli.

3.3 Dose–response of LDL-induced decrease in β-adrenergic receptors
Incubation of media explants for 3 days with LDL concentrations varying between 0.7 and 3.9 mmol/l (n=4) showed a dose-dependent decrease in the number of β-adrenergic binding sites (Table 2). LDL at a concentration of 3.9 mmol/l reduced β-adrenergic receptor density by 35%. Thus, all further experiments were continued with a LDL concentration of 2.6 mmol/l.


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  		Table 2 Dose-response of the LDL-induced decrease in β-adrenergic receptors

 
3.4 Time course of β-adrenergic receptor decrease
Incubation of LDL (2.6 mmol/l) for various time periods (1, 6, 12, 24, 36, 48 and 72 h) for the purpose of analysing the initial phase of the β-adrenergic receptor decrease showed (Fig. 3) significant decreases for incubations longer than 24 h. A maximal decrease of β-adrenergic receptors occurred after 48 h.


Figure 3
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  		Fig. 3 Time course of downregulation of β-adrenergic receptors during incubation with moderately elevated LDL. Media explants of coronary arteries were incubated with LDL (2.6 mmol/l) for different periods of time. The experiments were started at varying times and stopped at the same time. The density of β-adrenergic receptors was measured using the radioligand [125I]ICYP. The reduction of β-adrenergic receptors began after 12 h and the maximum effect was reached after 48 h. Additional growth up to 72 h did not reduce the receptors any further. The average±s.d. of three sets of experiments is shown using least square fitting techniques.

 
3.5 Reversibility of β-adrenergic receptor decrease after LDL incubation
Media explants were grown for 3 days with LDL (1.3 mmol/l and 2.6 mmol/l). The LDL containing growth medium was then removed and media-explants were grown under serum-free conditions for an additional 4 days where the growth medium was changed every other day (n=3).

LDL incubation (1.3 and 2.6 mmol/l, n=3) decreased β-adrenergic receptors dose-dependently (aP<0.05, Table 3). Further cultivation in LDL-free growth medium for 3 days resulted in a normalization of receptor density (bP<0.05).


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  		Table 3 Reversibility of the β-adrenergic receptor decrease after LDL incubation

 
3.6 Effect of β-blockade on the LDL-mediated decrease of β-adrenergic receptors
Media explants were incubated with LDL (2.6 mmol/l, 3 days, n=4) and the β-adrenergic receptor antagonists metoprolol, bisoprolol, or ICI 118.551 (10–6 mol/l) respectively (Table 4).


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  		Table 4 Effect of β-blockade on the LDL-mediated decrease of β-adrenergic receptors

 
Metoprolol or bisoprolol prevented the LDL-mediated downregulation of β-adrenergic receptors (aP<0.05). The β2-adrenergic antagonist ICI 118.551 also counteracted this downregulation, albeit to a lesser extent.

In another experiment, media explants were incubated for three days with 2.6 mmol/l LDL after which metoprolol (10–6 mol/l) was added to the growth medium for another 4 days. Addition of metoprolol increased the β-adrenergic receptor density almost to control levels after 4 days despite the fact that LDL (2.6 mmol/l) was still present in the growth medium (P<0.05, data not shown).

3.7 Functional coupling of β-adrenergic receptors in media explants after incubation with moderately elevated LDL
In order to elucidate the functional significance of the decreased density of the β-adrenergic receptors, the ability of the receptors to form the guanine nucleotide-dependent ‘high affinity state’ and their capacity to activate adenylyl cyclase was determined. In media explant cultures without supplemented LDL about 52% of the receptors bound the β-agonist with high affinity (Kdh 2.5±0.3 nmol/l), while 48% of the receptor population in the plasma membrane bound the β-agonist with low affinity (Kdl 70±15 nmol/l). By addition of the non-hydrolyzable GTP-analog Gpp(NH)p (10–4mol/l) the curve was both shifted to the right and steeper, which indicated that all receptors bound the β-agonist with low affinity (Fig. 4). In LDL-treated explant cultures, 49% of the β-adrenergic receptors were able to form the agonist-promoted ‘high affinity state'. These data suggest that β-adrenergic receptors can form the agonist promoted ‘high affinity state' after LDL incubation and that there is no uncoupling of the receptor from its guanine nucleotide-binding protein.


Figure 4
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  		Fig. 4 Coupling of β-adrenergic receptors. Explant cultures of porcine coronary arteries were incubated with LDL 2.6 mmol/l ({square}—{square}) for 3 days or with serum-free growth medium (bullet—bullet). The agonist-competition curves in the absence or presence of the non-hydrolyzable GTP analog, Gpp(NH)p are shown. The means±s.d. of three series of experiments are plotted with duplicate determination at each concentration of the β-agonist isoproterenol. After LDL incubation, no changes in the functionally coupled β-adrenergic receptors could be shown.

 
3.8 Sensitization of adenylyl cyclase after moderately elevated LDL
The responsiveness of adenylyl cyclase to stimulation was used as a means to assess the functional significance of the decreases in β-adrenergic receptor levels observed (Fig. 5). Since circulating catecholamines bring about vasodilation by activating both β1- and β2-adrenergic receptors, the unselective β-agonist isoproterenol was used.


Figure 5
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  		Fig. 5 Increased activity of the adenylyl cyclase. Media-explants were grown in the presence of LDL at 1.3 or 2.6 mmol/l for 3 days. Afterwards plasma membranes were prepared and the adenylyl cyclase activity was determined according to the method of Jakobs et al. [25]. Under basal conditions without adenylyl cyclase stimulation the LDL incubated media explants showed an increased cAMP production. Stimulation of β-adrenergic receptors with isoproterenol showed a higher stimulation after LDL incubation than under control conditions even though the β-adrenergic receptors decreased in the plasma membranes. Direct activation of the adenylyl cyclase with forskolin resulted again in an increased cAMP concentration after LDL incubation as compared to control conditions. The means±s.d. of three different sets of experiments with triplicate determinations at each point are depicted.

 
The unstimulated basal activity of adenylyl cyclase was enhanced after LDL incubation (2.6 mmol/l, 3 days) by 180% (control, 280±20; LDL, 500±35 pmol/mg protein/10 min). The isoproterenol-stimulated adenylyl cyclase activity was increased by about 110% after LDL incubation, although the number of functionally coupled β-adrenergic receptors decreased (control, 390±45; LDL, 600±40 pmol/mg protein/10 min). Adenylyl cyclase activated by forskolin was increased by 80% after LDL incubation for 3 days (control, 477±28; LDL, 696±34 pmol/mg protein/10 min).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Vasodilation is already decreased in the presence of moderately elevated LDL although no atherosclerotic lesions can be detected [3]. Changes in β-adrenergic receptors and second messenger pathways have not been fully characterized for the onset of hypercholesteremia with moderately elevated levels of LDL.

The data in this study show that moderately elevated LDL levels result in a subsequent decrease of functionally coupled β-adrenergic receptor density without changing the ratio of β1- to β2-adrenergic receptors. Interestingly, adenylyl cyclase activity is concomitantly increased. A functional relationship between the downregulation of the β-adrenergic receptors and moderately elevated LDL concentrations is supported by the finding that removal of the LDL from the growth medium is associated with an increase in β-adrenergic receptor density. β1- as well as β2-adrenergic receptor antagonists prevented the downregulation of β-adrenergic receptors. The LDL mediated decrease in β-adrenergic receptor density in coronary artery media plasma membranes can be reversed by adding the β1-adrenergic receptor antagonist metoprolol to the medium after the LDL mediated downregulation of the β-adrenergic receptors.

Downregulation of β-adrenergic receptors could also be demonstrated in plasma membranes of marmoset monkey hearts after a dietary cholesterol supplementation for 22 weeks [6, 28]. It is important to note that in that study cholesterol levels were 10-fold higher than those used in our study. The higher membrane cholesterol concentrations led to a decrease in Na+/K+-ATPase activity and other membrane bound enzymes [29]. Marmosets maintained on a high fat and cholesterol diet exhibited a 3-fold increase in endothelin B receptor mRNA levels [30]. Nanda and Henry found increased {alpha}-adrenergic and serotonergic receptors in aortas of rabbits fed with a high cholesterol diet for ten weeks [31].

As such, a specific LDL-modulating effect on these different receptor types appears to exist.

Lurie et al. assessed the effects of hypercholesteremia on β-adrenergic receptor concentration after a six week hypercholesteremic diet regime in a quail model [32]. Erythrocytes showed no significant change in β-adrenergic receptor density. The difference from our study may lie in the fact different cholesterol concentrations were used and that animal models were investigated.

Another explanation for the decreased binding of the radioligand to the membranes of media explants grown in the presence of LDL could be that the β-adrenergic receptors became sequestered in light vesicles. Such an effect has already been shown for agonist-mediated downregulation of β-adrenergic receptors [33, 34]. However, this effect was ruled out because the light vesicles showed no [125I]ICYP binding at all. Another explanation could be that the LDL exerted a cytotoxic effect and thereby reduced the number of smooth muscle cells and the receptor density. However, this was ruled out by the fact that in the presence of LDL and metoprolol, the β-adrenergic receptor concentration actually increased. These results suggest that increased degradation and/or decreased receptor synthesis may be responsible for this form of β-adrenergic receptor downregulation.

Since the antagonist competition curves showed that the ratio of β1- to β2-adrenergic receptors did not change another experimental approach was carried out. Downregulation of β-adrenergic receptors could be prevented in the presence of the specific β1-adrenergic receptor antagonists metoprolol or bisoprolol. The β2-adrenergic receptor antagonist ICI 118.551 also prevented β-adrenoceptor downregulation, although to a lesser extent. This was probably due to the high proportion (80%) of β1-adrenergic receptors in smooth muscle cells of porcine coronary arteries. Thus although downregulation occurred predominantly amongst the β1-adrenergic receptors, this study provided no strong evidence that any one of the two receptors was preferentially downregulated. As well as ruling out a cytotoxic effect for LDL, the fact that the LDL-mediated β-adrenergic receptor decrease was reversed by subsequent metoprolol addition indicates that an ‘empty’ receptor and an antagonist occupied receptor have different effects on cellular signalling and thereby alter regulatory mechanisms in different ways [19]. Such a situation has already been confirmed for endothelin-binding sites in smooth muscle cells whereby incubation with an endothelin receptor antagonist brought about an increase in endothelin receptor density [35].

After LDL incubation, β-adrenergic receptors were able to form the agonist promoted ‘high affinity state’ and thus activate adenylyl cyclase. This implies that changes in the fluidity and rigidity of plasma membranes uncoupled the receptor from its corresponding G-protein and that β-adrenergic receptors maintained their function.

In our study we found an increased activity of adenylyl cyclase, although in hypercholesteremic quails adenylyl cyclase activity was found to be decreased [32]. These contrasting observations may have arisen either because hypercholesteremia was induced in a different fashion, or that the quail study was in fact an in vivo study.

As this study showed, adenylyl cyclase activity in media explants of coronary arteries could be activated by 180% compared to control levels. However, stimulation by isoproterenol or forskolin amounted to only 110 or 80% of control levels respectively. These data imply an upregulation of adenylyl cyclase activity towards an increased basal activity in conjunction with a lower responsiveness to isoproterenol or forskolin. This suggests that adenylyl cyclase is maximally activated when LDL concentrations are moderately elevated. In the early phase of atherosclerosis development, an increased vasoconstriction due to increased endothelin or {alpha}-adrenergic receptors is well known [5, 30, 31]. An increased basal adenylyl cyclase activity might be useful for enforcing vasodilation in the presence of only low concentrations of epinephrine. An increased vasodilatory activity would therefore be useful for counteracting augmented vasoconstrictor mechanisms.

Time for primary review 35 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Wayne F, Alan P, Curry MD, et al. Plasma apoprotein B and VLDL-, LDL-, and HDL-cholesterol as risk factors in the development of cornary artery disease in male patients examined by angiography. Atherosclerosis (1981) 39:411–424.[CrossRef][Web of Science][Medline]
  2. Carlson LA, Bottiger LE, Ahlfeldt PE. Risk factors for myocardial infarction in the Stockholm Perspective Study. Acta Med Scand (1979) 206:351–360.[Web of Science][Medline]
  3. Merkel LA, Rivera LM, Bilder GE, Perrone MH. Differential alteration of vascular reactivity in rabbit aorta with modest elevation of serum cholesterol. Circ Res (1990) 67:550–555.[Abstract/Free Full Text]
  4. Heistad DD, Armstrong ML, Marcus ML, Piegors DJ, Mark AL. Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys. Circ Res (1984) 454:711–718.
  5. Henry PD, Yokoyama M. Supersensivity of arteriosclerotic rabbit aorta to ergonovine. J Clin Invest (1980) 66:306–313.[Web of Science][Medline]
  6. McMurchie EJ, Patten GS. Dietary cholesterol influences cardiac β-adrenergic receptor adenylate cyclase activity in the marmoset monkey by changes in membrane cholesterol status. Biochim Biophys Acta (1988) 942:324–332.[Medline]
  7. Middleton A, Middleton B. Agents which increase cyclic AMP have diverse effects on low-density-lipoprotein-receptor function in human vascular smooth-muscle cells and skin fibroblasts. Biochem J (1990) 267:607–614.[Web of Science][Medline]
  8. Auwerx JH, Chait A, Wolfbauer G, Deeb SS. Involvement of second messengers in regulation of the low density lipoprotein receptor gene. Mol Cell Biol (1989) 9:2298–2302.[Abstract/Free Full Text]
  9. Pfohl M, Naoumova RP, Klass C, et al. Acute and chronic effects on cholesterol biosynthesis of LDL-apheresis with or without concomitant HMG-CoA reductase inhibitor therapy. J Lipid Res (1994) 35:1946–1955.[Abstract]
  10. Molenaar P, Jones CR, McMartin LR, Summers RJ. Autoradiographic localisation and densitometric analysis of beta-1 and beta-2 adrenoceptors in the canine left anterior descending coronary artery. J Pharm Pharmacol (1988) 246:384–393.
  11. Horwitz J. Muscarinic receptor stimulation increases inositol-phospholipid metabolism and inhibits cyclic AMP accumulation in PC12 cells. J Neurochem (1989) 53:197–204.[CrossRef][Web of Science][Medline]
  12. Schwartz J, Velley J. The β-adrenoceptor of pig coronary arteries: determination of β1 and β2 subtypes by radioligand binding. Br J Pharmacol (1983) 79:409–414.[Web of Science][Medline]
  13. Renaud JF, Scanu AM, Kazazoglou T, et al. Normal serum and lipoprotein-deficient serum give different expressions of excitability, corresponding to different stages of differentiation, in chicken cardiac cells in culture. Proc Natl Acad Sci USA (1982) 79:7768–7772.[Abstract/Free Full Text]
  14. Kaumann AJ, Lemoine H. Direct labelling of myocardial β1-adrenoceptors comparison of binding affinity of 3H-(–)-bisoprolol with its blocking potency. Naunyn-Schmiedeberg's Arch Pharmacol (1985) 331:27–39.[CrossRef][Web of Science][Medline]
  15. Bilski AJ, Halliday SE, Fitzgerald JD, Wale JL. The Pharmacology of a β2-selective adrenceptor antagonist (ICI 118,551). J Cardiovasc Pharmacol (1983) 5:430–437.[Web of Science][Medline]
  16. Niehaus WG, Samuelson B. Formation of malonaldehyde from phospholipid arachidonate during microsomal lipid peroxidation. Eur J Biochem (1968) 6:126–130.[Web of Science][Medline]
  17. Napoli C, Ambrosio G, Scarpato N, et al. Decreased low-density lipoprotein oxidation after repeated selective apheresis in homozygous familial hypercholesterolemia. Am Heart J (1997) 133:585–595.[CrossRef][Web of Science][Medline]
  18. Leitinger N, Pirich C, Blazek I, Endler G, Sinzinger H. Decreased susceptibility of low-density lipoproteins to in-vitro oxidation after dextran-sulfate LDL-apheresis treatment. Atherosclerosis (1996) 126:305–312.[CrossRef][Web of Science][Medline]
  19. Marquetant R, Brehm B, Strasser RH. Chronic beta-blockade transregulates inhibitory A1 adenosine and muscarinic M2 receptors of the adenylyl cyclase system. J Mol Cell Cardiol (1992) 24:535–548.[CrossRef][Web of Science][Medline]
  20. Strasser RH, Krimmer J, Marquetant R, Kübler W. Regulation of β-adrenergic receptors: impaired desensitization in myocardial ischemia. J Cardiovasc Pharmacol (1988) 12:S15–S24.[Web of Science][Medline]
  21. Engel G, Hoyer D, Berthold R, Wagner H. [125I]Iodocyanopindolol, a new ligand for beta-adrenoceptors: identification and quantitation of subclasses of beta-adrenoceptors in guinea pig. Naunyn-Schmiedeberg's Arch Pharmacol (1981) 317:277–285.[CrossRef][Web of Science][Medline]
  22. De Lean A, Munson PJ, Rodbard D. Simultaneous analysis of families of sigmoidal curves: application to bioassay and physiological dose–response curves. Am J Physiol (1978) 235:E102–E978.
  23. Munson PJ. A review of nonlinear least-squares curve fitting techniques for data analysis. Comput Endocrinol Recent Adv (1990) 72:33–44.
  24. Munson PJ, Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding system. Anal Biochem (1980) 107:220–239.[CrossRef][Web of Science][Medline]
  25. Jakobs KH, Saur W, Schultz G. Reduction of adenylate cyclase activity in lysates of human platelets by the {alpha}-adrenergic component of epinephrine. J Cyclic Nucleot Res (1976) 2:381–392.
  26. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem (1976) 72:248–254.[CrossRef][Web of Science][Medline]
  27. De Lean A, Stadel JM, Lefkowitz RJ. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor. J Biol Chem (1980) 255:7108–7117.[Abstract/Free Full Text]
  28. Patten GS, Rinaldi JA, McMurchie EJ. Effects of dietary eicosapentaenoate (20:5 n-3) on cardiac beta-adrenergic receptor activity in the marmoset monkey. Biochem Biophys Res Commun (1989) 162:686–693.[CrossRef][Web of Science][Medline]
  29. McMurchie EJ. Dietary lipids and the regulation of membrane fluidity and function. In: Liss AL, editor. Physiologic regulation of membrane fluidity. Saunders, Philadelphia, 1988, pp. 189-237.
  30. Elshourbagy NA, Korman DR, Wu HL, et al. Molecular characterization and regulation of the human endothelin receptors. J Biol Chem (1993) 268:3873–3879.[Abstract/Free Full Text]
  31. Nanda V, Henry PD. Increased serotonergic and alpha adrenergic receptors in aortas from rabbits fed a high cholesterol diet. Clin Res (1982) 30:209. Abstract.[Medline]
  32. Lurie KG, Chin JH, Hoffman BB. Decreased membrane fluidity and beta-adrenergic responsiveness in atherosclerotic quail. Am J Physiol (1985) 249:H380–H385.[Web of Science][Medline]
  33. Sibley DR, Lefkowitz RJ. Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature (1985) 317:124–129.[CrossRef][Medline]
  34. Collins S, Bouvier M, Lohse MJ, et al. Mechanisms involved in adrenergic receptor desensitization. Biochem Soc Trans (1990) 18:541–544.[Web of Science][Medline]
  35. Yu JC, Davenport AP. Regulation of endothelin receptor expression in vascular smooth-muscle cells. J Cardiovasc Pharmacol (1995) 26(Suppl_3):S348–S350.[Web of Science][Medline]

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