Effects of reserpine on expression of the LDL receptor in liver and on plasma and tissue lipids, low density lipoprotein and fibrinogen in rabbits in vivo

Atherosclerosis 149 (2000) 267 – 275 www.elsevier.com/locate/atherosclerosis

Effects of reserpine on expression of the LDL receptor in liver and on plasma and tissue lipids, low density lipoprotein and fibrinogen in rabbits in vivo Shahida Shafi a,*, Irina P. Stepanova a, C. Fitzsimmons b, D.E. Bowyer b, D. Welzel c, G.V.R. Born a a

Pathopharmacology Unit, The William Har6ey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK b Department of Pathology, Uni6ersity of Cambridge, Cambridge, UK c Department of Pharmacology, Uni6ersity of Regensburg, Germany Received 22 February 1999; received in revised form 12 July 1999; accepted 3 August 1999

Abstract The effects of administering reserpine (0.1 mg/kg) or 17a-ethinyloestradiol (2.5 mg/kg) to New Zealand White rabbits on low density lipoprotein receptors in liver, on plasma low density lipoprotein and fibrinogen and on plasma and tissue lipids were determined. Blood pressure and heart rate were also followed. The drugs were injected subcutaneously into conscious unrestrained rabbits for 5 days. On the 6th day homologous 125I-tyramine cellobiose labelled low density lipoprotein (125I-TC-LDL) was injected intravenously and 24 h later the animals were killed. Compared to controls, reserpine significantly increased LDL receptor expression in the liver by about threefold, and reduced total cholesterol in plasma, aorta and heart, without affecting plasma triglycerides. The reductions in plasma cholesterol and heart were due to decreases in both unesterified and esterified cholesterol. Similar effects were observed with oestrogen, except that there was no change in esterified cholesterol in aorta. In liver, a decrease of 24% in total cholesterol was due mainly to decreased esterified cholesterol. In adrenal glands total cholesterol increased by 25%. Reserpine significantly accelerated the plasma clearance of intravenously injected homologous 125I-TC-LDL and reduced its accumulation in aortic wall. Neither reserpine nor oestradiol affected blood pressure, haematocrit or plasma fibrinogen. The results suggest that reserpine is an affective anti-atherogenic drug capable of decreasing cholesterol in plasma, arteries and heart by increasing high affinity LDL receptors in the liver. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Reserpine; Atherosclerosis; Hepatic LDL receptor; Cholesterol and arterial walls

1. Introduction Atherogenesis is associated with the accumulation of LDL-derived cholesterol in the intima of susceptible arteries, with the development of fatty streaks. The atherosclerotic process is also promoted by high concentrations of fibrinogen [1 – 4]. Decreases in plasma LDL cholesterol by means of drug therapy reduce morbidity and mortality from atherosclerotic disease [5,6].

* Corresponding author. Tel.: +44-171-9826046; fax: + 44-1719826071. E-mail address: [email protected] (S. Shafi)

In previous studies from our laboratory it was shown that infusions of noradrenaline or adrenaline into rabbits and rats increased the uptake of LDL (as well as fibrinogen) by large arteries [7–9], showing that the catecholamines promote atherogenesis. Reserpine in experimental animals depletes catecholamines in tissues [10,11], and decreases atheromatous lesions [12,13]. In rats, single injections of reserpine accelerated the clearance of intravenously injected radiolabelled LDL from the circulation and reduced its accumulation and that of cholesterol in aortic wall and heart, while increasing the amounts in liver (unpublished data, 1999). The liver is the main site for the removal of circulating LDL via the high affinity LDL receptors on hepatic parenchymal cells [14–17], and changes in hepatic receptor activity produce changes in plasma LDL levels [18–20].

0021-9150/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 9 9 ) 0 0 3 2 7 - 5

268

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275

The purpose of this study was, therefore, to investigate the effects of reserpine administration to conscious unrestrained rabbits over a period of 5 days on the expression of LDL receptors in the liver and on plasma and tissue lipids, low density lipoprotein and fibrinogen levels. For positive control, other rabbits were treated with 17a-ethinyloestradiol. This is the first study of the effects of reserpine on the expression of LDL receptors in liver.

2. Materials and methods

2.1. Reagents Reserpine was provided by Sandoz AG in Nu¨rnberg. The cholesterol serum standard (Precinorm L®) and assay reagents were from Boehinger-Mannheim (Sussex, UK) and Genzyme Diagnostics (Kent, UK). 17aEthinyloestradiol and all other chemicals and reagents were purchased from Sigma (Poole, Dorset, UK); carrier-free radioactive iodide (sodium [125I]-iodide in 1 M NaOH) was from Amersham (UK). Monoclonal antibody MAC 188 (rat monoclonal antibody that recognises rabbit LDL receptor) and the relevant reagents were as described previously [21 – 23].

2.2. Animals, drugs and treatment procedures A total of 15 male New Zealand White rabbits (2.6 –3.0 kg) were housed individually in cages and acclimatised for 5 days on a 12-h light/dark cycle. Their daily water intake and food consumption were determined. Rabbits were weighed and then randomly divided into three experimental groups of five: controls, reserpine and 17a-ethinyloestradiol. All rabbits were maintained on standard chow diet and water was available ad libitum. The procedures used on these animals were approved by the Animal Care and Safety Committee and conformed to the Animal Care Guidelines established by the Home Office. The treatment period was 5 days and drugs or vehicle alone were administrated subcutaneously into conscious unrestrained rabbits. Reserpine was dissolved in 4% citric acid and injected at a dose of 0.1 mg/kg in 1-ml volume on days’ 1, 3 and 5. Control animals received an equal volume of citric acid only. 17a-Ethinyloestradiol was injected daily (11:00 h) at a dose of 2.5 mg/kg.

ples for haematocrit, plasma fibrinogen and cholesterol collected. Haematocrit was determined in heparinised capillary haematocrit tubes. Blood samples for fibrinogen were collected in 6% citrate (1:9) and for cholesterol in EDTA (1.5 mg/ml). Separated plasma samples were stored at − 70°C until assayed.

2.4. Preparation and radiolabelling of low density lipoprotein LDL (1.019B dB1.063 g/ml) was isolated from freshly-drawn rabbit plasma containing disodium ethylenediamine tetra-acetate (EDTA, 1 mg/ml) by sequential ultracentrifugation at 4°C at densities of 1.019 and 1.063 g/ml [24]. The final LDL fraction was dialysed at 4°C against 154 mM NaCl containing 0.3 mM EDTA (5× 5 l) before labelling. The LDL preparation was labelled with tyramine cellobiose (TC) ligand and iodinated with 148 MBq 125I [16,17,25]. Free iodine in the dialysed LDL preparation was less than 4%. Protein concentration was determined with BCA protein assay reagent (Pierce, Cambridge, UK). The specific activity of the preparation was 775 cpm/ng of apo B LDL. The radio-labelled LDL preparation was used within 5 days. Coupled to LDL, the 125 I-TC marker enters the artery wall where for the duration of the experiments it remains trapped intracellularly while LDL undergoes degradation. Therefore, the tissue radioactivity includes both the undegraded and degraded LDL [16,17,25,26].

2.5. Experimental procedures

2.3. Blood samples

2.5.1. Plasma clearance of 125I-TC-LDL After the 5 days of drug treatment period, on the 6th day 125I-TC-LDL (0.5 ×108 cpm/64 mg apo B LDL per animal) was injected into marginal ear vein of all three groups of rabbits under light anaesthesia (Hypnorm 0.1 ml/rabbit). To prevent re-uptake of radio-iodide by the thyroid glands, sodium iodide (3 mg) was added to the drinking water 1 day before the radio-label injection. Then 5 min after the injection of 125I-TC-LDL duplicate blood samples (10 ml) were collected from the marginal vein of contralateral ear and then at 2, 4, 6, 8 and 24 h. The radioactivity of blood over 24 h was expressed as percentage of that determined at 5 min (100%). The results were fitted to a biexponential function and the biological half life (t1/2) was calculated from the mean rate clearance constant (K) obtained from five rabbits (t1/2 = 0.693/K).

All blood samples were collected from the marginal ear vein just before the start and at the end of treatments. Animals were lightly anaesthetised with 0.2 ml Hypnorm (fentyl citrate 0.3 mg/ml and fluanisone 10 mg/ml; Janssen, UK,) intramuscularly and blood sam-

2.5.2. Preparation of aortas and other tissues At 24 h after the final blood samples were collected, the animals were killed by exsanguination via cardiac puncture under pentobarbital (35 mg/kg i.v.) anaesthesia and laparotomy was performed. The cardiovascular

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275

system was perfused at 80 – 100-mmHg pressure for 5 – 10 min via a needle inserted into the left ventricle of the heart with 1–2 l of ice-cold saline (154 mM NaCl) and efflux was through the vena cava. The thoracic aorta was excised intact, from aortic arch to the diaphragm, carefully cleaned of loose adventitia under a stereomicroscope, and opened longitudinally. The aorta was washed four times in ice-cold saline for 5 min each, rinsed once in TCA (12%) and finally with 5% (w/v) KI for 5 min and then cut into two halves longitudinally. The radioactivity of the solutions, counted after rinsing aorta, show that this washing procedure did not lead to loss of any trapped radioactive counts, other than the loosely bound. The whole liver was immediately removed, blotted, weighed, and pieces of 1 – 2 g were cut and frozen in liquid nitrogen and stored at − 70°C for receptor determination. Two samples of each tissue, i.e. liver, heart and adrenal glands, were excised from each rabbit and rinsed in 154 mM ice-cold saline ( × 3). All tissue samples were gently blotted and weighed. One set of samples from each tissue was immediately frozen at − 70°C for total cholesterol and the other set was used for the determination of radioactivity and dry weight. For this, the samples were dried first at 50°C and then in a dessicator to constant weight.

2.6. Determination of radioacti6ity 125

I-TC-LDL radioactivity in plasma and tissues was determined in a gamma scintillation counter (Cobra 5002 Autogamma). The resultant radioactive counts of tissues were divided by the specific activity of the preparation to express results as ng of apo B LDL/g dry weight.

2.7. Extraction and determination of lipids Frozen samples of tissues were ground into fine powder in liquid nitrogen and cholesterol extracted in isopropanol in a ratio of 1:10. All assays were performed in duplicate. The plasma and tissue total and unesterified cholesterol and plasma triglyceride concentrations were determined by microenzymatic methods [27]. Esterified cholesterol concentration was calculated by difference.

2.8. Determination of the hepatic LDL receptor expression The expression of hepatic LDL receptors in the three groups of rabbits was investigated by Western immunoblotting procedures using monoclonal antibody MAC 188 [21–23]. Briefly, liver samples (1 g) were washed, homogenised, solubilized with buffer containing Triton X100 and then centrifuged twice for 10 min

269

at 4°C. After centrifugation, aliquots (1 ml) of solubilized extract were frozen in liquid nitrogen until used. The proteins in these extracts were then separated by electrophoresis on 7.5% SDS–PAGE [28] under non-reducing conditions and transferred by electroblotting to nitrocellulose. The membranes were finally probed with the monoclonal MAC 188, raised against purified LDL receptor protein [21]. The amount of bound MAC 188 was determined by quantitative Western blotting [22,23] and the amount of LDL protein was expressed in arbitrary units measured under standard densitometry conditions. Each liver was analysed in triplicate.

2.9. Determination of plasma fibrinogen concentration Fibrinogen levels in plasma were determined by the clotting method of Clauss [29], using Amelung KC10 coagulators linked to computers which calculate the fibrinogen concentration of each sample by reference to a standard curve generated at the start of the assay.

2.10. Blood pressure and heart rate measurements The blood pressure and heart rates were monitored in all three groups of rabbits under light anaesthesia (Hypnorm 0.1 ml). A fine Teflon Y-cannula (20 G) filled with saline, connected to a transducer coupled to a Multitrace 2 recorder 5022 (Lectomed, UK), was inserted into the central marginal ear artery and used for the measurements.

2.11. Statistical analysis Results analysis was performed with the statistical package Unistat, and expressed as mean 9 S.E.M. Significance of differences between means of controls and treated animals was evaluated by unpaired Student’s t-test. A probability of 0.05 or less was considered as significant.

3. Results The baseline characteristics of rabbits were compared between groups to determine whether there were differences before the start of treatments (Table 1). There were no statistically significant differences between the three groups of rabbits regarding initial body weight, haematocrit, plasma lipids and fibrinogen concentrations. The pre-treatment (basal) plasma levels of fibrinogen, total cholesterol (TC), unesterified cholesterol (UC), cholesteryl ester (CE) and triglyceride (Table 1) of rabbits were similar to published values [30–33].

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275

270

Table 1 Initial basal characteristics of three groups of rabbits before the start of experimentsa Variables

Control

Reserpine

17a-Ethinyloestradiol

2.88 9 0.07 40.69 0.8 8.919 0.7b

2.87 90.07 40.8 9 1.1 8.9090.9b

1.29 90.1c 0.96 9 0.1c 0.33 9 0.03c

1.159 0.05 0.859 0.04 0.339 0.04

1.03 90.1 0.76 9 0.1 0.27 9 0.03

0.949 0.2

0.9190.2

0.58 9 0.06

Body weight (kg) 2.88 9 0.05 Haematocrit (%) 40.3 9 1.2 Plasma fibrino8.93 9 0.8b gen (mmol/l) Plasma lipid (mmol/l) Total cholesterol Cholesteryl ester Unesterified cholesterol Triglyceride

a Blood samples were collected from the marginal ear vein before the start of treatments for the measurement of above plasma variables. Results are mean 9S.E.M. from five rabbits per group, except where indicated. b n= 4. c n= 6.

3.1. Effects on plasma lipids The effect of treatment with either reserpine or oestradiol for 5 days on plasma TC, UC, CE, and triglyceride concentrations are presented in Table 2. In reserpine treated rabbits, there was a significant change overall in the plasma cholesterol compared to the pretreatment values in the same animals (Table 1). The plasma total cholesterol concentration significantly decreased by 23% (PB0.05), owing to decreases in both the UC and CE fractions of 29 and 19%, respectively. In contrast, there were no changes in plasma triglyceride concentrations. The decreases in TC, UC, CE and triglyceride were greater (47, 63, 41 and 45%, respectively) in rabbits treated with 17a-ethinyloestradiol. These reductions in plasma lipids were highly significant compared to the pre-treatment concentrations in Table 1 (0.003B PB0.05).

Fig. 1. Effects of reserpine, 17a-ethinyloestradiol and vehicle alone on the liver LDL receptor level in rabbit. Rabbits were injected with reserpine (0.1 mg/kg on days 1, 3, and 5) or 17a-ethinyloestradiol (2.5 mg/kg daily for 5 days) subcutaneously and samples of liver excised on 7th day. Liver homogenates were prepared and LDL receptor quantified as described in Section 2. Each liver was analysed in triplicate. The results are expressed as mean 9 S.E.M. Number of rabbits and significance of differences compared to the controls are indicated above each column. **PB 0.001; ***PB 0.0001.

3.2. Effects on expression of LDL receptors in li6er To determine whether reserpine increases the expression of LDL receptors in the liver, we compared the effect of this drug with 17a-ethinyloestradiol and with vehicle alone (controls). As shown in Fig. 1, the LDL receptor number in liver was increased threefold by reserpine and 4.5-fold by oestradiol compared with control animals and those increases were all highly significant. Reserpine and oestradiol treatments produced 10 and 26% reductions in liver weights compared to controls.

Table 2 Effect of reserpine or 17a-ethinyloestradiol injection over 5 days on plasma cholesterol and triglyceride concentrations in normocholesterolemic rabbitsa Plasma lipids

Control, mmol/l

Reserpine, mmol/l

17a-Ethinyloestradiol, mmol/l

Total cholesterol Cholesteryl ester Unesterified cholesterol Triglyceride

1.239 0.1 0.839 0.1 0.359 0.04 0.899 0.2b

0.95 9 0.06* 0.67 90.05**,b 0.25 9 0.01 0.88 9 0.04

0.55 9 0.01** 0.45 9 0.07** 0.10 90.02*** 0.32 9 0.06*

a

Reserpine (0.1 mg/kg on days 1, 3 and 5) or 17a-ethinyloestradiol (2.5 mg/kg per day) was injected subcutaneously over 5 days. Blood samples were collected from the marginal ear vein at the end of treatment period on the 6th day. Results are mean 9 S.E.M. from five rabbits per group, except where indicated. b n= 4. * PB0.05, significantly different compared to the pre-treatment values in the same animals (Table 1). ** PB0.03, significantly different compared to the pre-treatment values in the same animals (Table 1). *** PB0.003, significantly different compared to the pre-treatment values in the same animals (Table 1).

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275

3.3. Effects on plasma clearance of low density lipoprotein The effect of reserpine treatment on the plasma clearance of 125I-TC-labelled LDL in rabbits over 24 h was compared with controls and 17a-ethinyloestradiol treatment (Fig. 2). Both reserpine and oestradiol treatments increased 125I-TC-LDL clearance from blood (t1/2 = 6.6 h and t1/2 = 6.4 h) compared to controls (t1/2 =8.9 h). The differences in plasma clearance of 125I-TC-LDL were apparent at 6 h after the injection, while 24 h later, the percentage of injected 125I-TC-LDL remaining in the blood was significantly lower for reserpine and oestradiol treated rabbits (15.39 0.7 and 10.49 0.4%, respectively; PB0.05 and P B 0.004, respectively) than in controls (19.49 1.4%).

3.4. Lipid contents of aortic wall, heart, li6er and adrenal gland Table 3 shows the effects of reserpine and oestradiol on total, unesterified and esterified cholesterol in aortic wall, heart, liver and adrenal gland. In aortic wall and

271

heart there were significant reductions in total cholesterol content compared to controls (P B 0.05 and PB 0.01, respectively); these reductions (17 and 21%, respectively) were similar in both reserpine and oestradiol groups (Table 3). The decrease in total cholesterol content of aortic wall was mainly accounted for by a significant decrease in unesterified cholesterol (14–17%; PB 0.05), with no significant changes in cholesteryl ester content. However, in heart the reduction in total cholesterol was due to significant decrease in both the unesterified and esterified cholesterol fractions (P B 0.05 and PB 0.01). In the liver neither total cholesterol nor unesterified cholesterol were reduced significantly by the two drugs compared to control (Table 3). Surprisingly, cholesteryl ester was significantly reduced by 60% (PB 0.01) in the reserpine group, whereas, in the oestradiol group, there was a threefold increase. Compared to reserpine treated animals, the esterified cholesterol content of liver in oestradiol treated animals was increased sixfold. This implies that reserpine either inhibits accumulation of cholesteryl ester or decreases esterification of free cholesterol in liver. In adrenal gland, reserpine had no significant effect on cholesterol content at PB 0.05 level. Oestradiol, however, caused a significant decrease in total cholesterol (30%; PB0.05) which was the result of a 47% decrease in unesterified cholesterol and of 30% in esterified cholesterol.

3.5. LDL content in aortic wall, li6er, heart and adrenal gland

Fig. 2. Plasma clearance of homologous 125I-TC-LDL in control, reserpine or 17a-ethinyloestradiol treated rabbits. 125I-TC-LDL was injected intravenously 5 days after the drug treatments. Biological half life (t1/2) was calculated from the mean rate clearance constant obtained for four or five rabbits. Control (n=4), reserpine (n = 5) and oestradiol (n = 5). Values are mean 9S.E.M. as a percent of initial injected 125I-TC-LDL at zero time. Differences between means for control and treated rabbits: *PB 0.05; **PB0.0002; ***PB 0.0001.

Table 4 shows the effects of reserpine and oestradiol on accumulation of radioactivities derived from 125ITC-LDL (expressed as ng apo B LDL per g dry weight) 24 h later in aortic wall, liver, heart and adrenal gland. The term ‘accumulation’ in this context refers to the sum of radioactivity from both degraded and intact LDL in tissues. In aortic wall reserpine and oestradiol markedly reduced 125I-TC-LDL accumulations by  50% compared to controls (Table 4); these reduction were highly significant (PB 0.01). In heart, reserpine had no effect, whereas oestradiol significantly decreased accumulation by 33% (PB 0.01). In liver and adrenal gland, reserpine and oestradiol had no significant effect on accumulation of 125I-TC-LDL radioactivity, although in the liver there was a trend towards an increase in accumulation with both drugs.

3.6. Blood pressure and heart rate Table 5 shows the effects of reserpine and oestradiol treatment on blood pressure and heart rate of conscious rabbits. Compared to controls, the heart rate of reserpine treated animals was significantly reduced by 

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275

272

Table 3 Tissue concentration of total, unesterified and esterified cholesterol (mmol/g dry wt) of rabbits treated with reserpine, 17a-ethinyloestradiol and controlsa Tissues

Lipids (mmol/g dry wt)

Controlb(mmol/g dry wt)

Reserpine (mmol/g dry wt)

17a-Ethinyloestradiol (mmol/g dry wt)

Aortic

Total cholesterol Unesterified cholesterol Cholesteryl ester Total cholesterol Unesterified cholesterol Cholesteryl ester Total cholesterol Unesterified cholesterol Cholesteryl ester Total cholesterol Unesterified cholesterol Cholesteryl ester

10.990.1 8.6 9 0.3 2.39 0.2 25.39 3.1 19.29 2.3 6.2 9 0.9 456.29 18.0 10.1 92.0 445.99 19.0 15.69 0.6 13.99 0.5 1.79 0.3

9.1 90.5*,b 7.1 9 0.4*,b 2.0 9 0.7b 19.19 0.7 16.09 7.0 3.1 9 0.3** 499 9 13.2 12.4 9 1.4 462.8 9 18.0 12.19 0.5*** 11.39 0.6** 0.99 0.1*

9.3 9 0.2** 7.4 90.3* 1.9 9 0.2 32.7 93.5 13.9 91.7 18.8 93.3** 319.3 946.0* 5.4 91.4 313.9 932.1* 12.5 90.3*** 12.0 90.3** 0.6 90.1**

Liver

Adrenal

Heart

a Rabbits were injected with reserpine or oestradiol and vehicle alone (control) for 5 days and on the 7th day, tissues were excised washed, blotted, weighed and homogenised for cholesterol determinations. Results are expressed as mean 9 S.E.M. of five rabbits, except where indicated. b n= 4. * PB0.05, significantly different compared to the control cholesterol concentrations. ** PB0.01, significantly different compared to the control cholesterol concentrations. *** PB0.003, significantly different compared to the control cholesterol concentrations.

27%, from 197916 to 14397 beats per minute (PB 0.01), and the blood pressure remained unchanged. Oestradiol treatment had no effects on the heart rate or blood pressure (Table 5).

3.7. Haematocrit and plasma fibrinogen le6els Neither reserpine nor oestradiol had significant effects on plasma fibrinogen or haematocrit (Fig. 3).

4. Discussion This is the first study to demonstrate effects of reserpine on hepatic LDL receptor expression, plasma and tissue lipids and low density lipoprotein in conscious unrestrained New Zealand White rabbits in vivo. 17aEthinyloestradiol, which is known to increase LDL receptors in the liver, was used in parallel as a positive control. The results show that reserpine administration over 5 days significantly increased hepatic LDL receptor numbers and reduced total cholesterol in plasma, aorta and heart, without any effect on plasma triglycerides. The reductions were the result of decreases in both the unesterified and esterified cholesterol fractions, except in aorta, where there was no change in cholesteryl ester. 125 I-TC-LDL clearance from the plasma was increased and the accumulation of radioactivity in the artery wall was significantly reduced by both reserpine and 17aethinyloestradiol. Neither reserpine nor 17a-ethinyloestradiol affected haematocrit, plasma fibrinogen or blood pressure, although in reserpine-treated rabbits the heart rate was decreased.

The increased clearance from the blood of 125I-TCLDL by liver and decreased total plasma cholesterol was associated with up-regulation of hepatic LDL receptors. In vivo, the rate of receptor-mediated removal of LDL-cholesterol from plasma is proportional to the number of hepatic receptors [34]. It has been calculated that the number of receptors in rabbit liver is 1.6×1015 receptors/kg body weight [23]. Thus in the present experiment this number was increased to 4.8× 1015 by reserpine and to 7.7× 1015 by 17a-ethinyloestradiol. Based on certain assumptions about the stoichiometry of binding of LDL particles to the LDL receptor, and Table 4 Effect of reserpine or 17a-ethinyloestradiol treatment on 125I-TCLDL radioacitivities in rabbit organs derived from homologous low density lipoprotein 24 h after the radiolabel injectiona Tissue

Aorta (×10−1) Heart Liver Adrenal gland (×102)

125

I-TC-LDL (ng apo B/g dry wt)

Control

Reserpine

17a-Ethinyloestradiol

4.4 90.6 25.4 91.5 403.6 9 46b 17.0 92.5b

2.2 9 0.2** 23.6 92.1 443.1 943 18.7 9 1.4

2.290.2** 17.1 91.6** 534.5 957 17.8 92.8b

a Rabbits were treated for 5 days and on 7th day, the aortic wall, heart, liver and adrenal gland radioactivity was determined as described in Section 2. Results are from five rabbits except where indicated, expressed as mean 9S.E.M. The results are expressed as equivalent to ng of apo B/g dry weight. Specific activity was 775 cpm/ng apo B LDL. b n =4 per group. ** PB0.01, significantly different compared to the control organs.

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275 Table 5 The effect of reserpine or 17a-ethinyloestradiol injection on mean arterial blood pressure (mmHg) and heart rate (bpm) of rabbita

Control Reserpine 17a-Ethinyloestradiol

Arterial blood pressure (mmHg)

Heart rate (bpm)

72 9 4 71 95 62 9 3

1979 16 14397** 194 99

a

Results expressed as mean9 S.E.M. from five rabbits per group. ** PB0.02, significantly different compared to the control.

that cholesterol represents 30% by weight of the LDL particle and apo B 21%, then in reserpine treated animals the LDL receptors in liver remove 4.8×1015 LDL particles/h per kg body weight, and 8.2 × 1015 particles/h per kg body weight in oestrogen-treated animals. The mechanism by which reserpine increases LDL receptor is unknown, but could occur either as a direct effect of the drug, e.g. by up-regulating the expression of the gene for the receptor, or by depleting catecholamines, which decrease LDL receptor activity in cultured lymphocytes and fibroblasts [35,36]. So far there is little evidence concerning the effect of catecholamines on receptor expression in liver, except that LDL receptor number is increased in human hep-

Fig. 3. Effect of reserpine or 17a-ethinyloesteradiol treatments on plasma fibrinogen levels in rabbit. Rabbits were injected subcutaneously with reserpine (0.1 mg/kg on days 1, 3, and 5) or oestradiol (2.5 mg/kg daily) for 5 days. Blood samples were collected before and on the 6th day after the treatments for measurement of plasma fibrinogen levels. Control animals received vehicle alone. The results are expressed as mean 9S.E.M. Number of rabbits are indicated above each column.

273

atoma cells following treatment with the a-1adrenoreceptor antagonist doxazosin [37]. Total cholesterol in the liver of reserpine-treated animals was reduced marginally despite a 50% reduction in cholesteryl ester, whereas in oestradiol-treated animals there was a small increase due to threefold increase in esterified cholesterol. This increase in oestradiol treated liver was due to increases in LDL receptor expression and plasma clearance. The unexpected reduction in liver of reserpine animals, suggests that reserpine inhibits either accumulation of cholesteryl ester or the esterification of newly synthesised cholesterol. Even in the plasma there were reductions in both cholesteryl ester and unesterified cholesterol concentrations. One possibility is that the liver ACAT activity is reduced, presumably by reduction in the free cholesterol pool available for esterification. In contrast, estradiol increases ACAT activity in liver [38]. Inhibition of esterification affects apo B production [39]. If reserpine does reduce cholesterol in apo B-containing lipoproteins, the atherosclerotic risk would be reduced. If ACAT inhibition is responsible for the observed reduction in plasma total cholesterol, that should result in an increase in liver unesterified cholesterol. This was not the case, because unesterified cholesterol actually decreased by 17 and 28% in the livers of both reserpine and oestradiol-treated rabbits. This suggests that the decrease in hepatic unesterified cholesterol may have been due to increased bile acid production or decreased de novo cholesterol synthesis. Biliary lipids and bile acid secretions were not measured in these experiments. The results indicate that both drugs had no significant effects on the accumulation of radiolabelled LDL in liver, although there was a tendency towards an increase in uptake. The most likely explanation for this could be due to the short-term administration of the drugs. If these had been administered over longterm period then the increases might have become more apparent. Dietary lipids suppress expression of hepatic LDL receptors [19,40]. Our results indicate that reserpine up-regulates these receptors. Therefore, it will be interesting to know whether in cholesterol-fed rabbits reserpine can reverse the suppression of the hepatic receptors. The reduction in total cholesterol in arterial wall and heart by reserpine is presumably a result of the decrease in plasma LDL cholesterol, as happens with 17a-ethinyloestradiol. This work has shown that reserpine raises LDL receptor levels in liver and lowers LDL in plasma. These findings suggest that reserpine could well find a new indication as a cholesterol-lowering drug for the prevention of atherosclerosis.

274

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275

Acknowledgements For supporting this study we wish to thank the British Heart and the Garfield Weston Foundations.

References [1] Smith EB. Fibrinogen, fibrin and fibrin degradation products in relation to atherosclerosis. Clin Haematol 1986;15:355–70. [2] Handa K, Kono S, Saku K, Sasaki J, Kawano T, Hiroki Y, et al. Plasma fibrinogen levels as an independent indicator of severity of coronary atherosclerosis. Atherosclerosis 1989;77:209 – 13. [3] Smith EB, Crosbie L. Fibrinogen and fibrin in atherogenesis. In: Ernst E, Koening W, Lowe GDO, Meade TW, editors. Fibrinogen: a ‘new cardiovascular risk factor’. Vienna: Blackwell-MZW, 1992:4 – 10. [4] Folsom AR, Wu KK, Shahar E, Davis CE. Association of haemostatic variables with prevalent cardiovascular disease and asymptomatic carotid artery atherosclerosis. Arterioscler Thromb 1993;13:1829–36. [5] Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383–9. [6] Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med 1995;333(20):1301– 7. [7] Cardona-Sanclemente LE, Born GVR. Adrenaline increases the uptake of low-density lipoproteins in carotid arteries of rabbits. Atheroscelerosis 1992;96:215–8. [8] Cardona-Sanclemente LE, Born GVR. Increased by adrenaline or angiotensin II of the accumulation of low density lipoprotein and fibrinogen by aortic walls in unrestrained conscious rats. Br J Pharmacol 1996;117:1089–94. [9] Shafi S, Cusack NJ, Born GVR. Increased uptake of methylated low density lipoprotein induced by noradrenaline in carotid arteries in anaesthetised rabbits. Proc R Soc 1989;235:289 – 98. [10] Okada K, Shinozuka K, Shimoura K, Kobayashi Y, Hattori K, Nakase A. Effects of reserpine on the contents and uptake of dopamine and noradrenaline in rabbit arteries. Clin Exp Pharmacol Physiol 1993;20:261–7. [11] Dahlstro¨m A, Ha¨ggendal J. Recovery of noradrenaline levels after reserpine compared with the life-span of amine storage granules in rat and rabbit. J Pharm Pharmacol 1966;18:751 – 2. [12] Smith THF, Rossi GV. The effects of reserpine and mecamylamine on experimental atheromatosis in the normotensive and hypertensive rats. J Pharmacol 1962;135:367–73. [13] Carrier O Jr, Clower BR, Whittington PJ. Inhibition of cholesterol-induced vascular lesions by dietary reserpine. J Atheroscler Res 1968;8:229–36. [14] Nenseter MS, Blomhoff R, Drevon CA, Kindberg GM, Norum KR, Berg T. Uptake of LDL in parenchymal and non-parenchymal rabbit liver cells in vivo. Biochem J 1988;254:443 – 8. [15] Ma PTS, Yamamoto T, Goldstein JL, Brown MS. Increased mRNA for low density lipoprotein receptor in livers of rabbits treated with 17alpha-ethinyl estradiol. Proc Natl Acad Sci USA 1986;83:792 – 6. [16] Pittman RC, Carew TE, Glass CK, Taylor CA, Attie AD. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem J 1983;212:791 – 800.

[17] Carew TE, Pittman RC, Marchand ER, Steinberg D. Measurements in vivo of irreversible degradation of low density lipoprotein in the rabbit aorta: predominance on intima degradation. Arteriosclerosis 1994;4(3):214 – 24. [18] Brown MS, Kovanen PT, Goldstein JL. Regulation of plasma cholesterol by lipoprotein receptors. Science 1981;212:628–35. [19] Kovanen PT, Brown MS, Basu SK, Billheimer DW, Goldstein JL. Saturation and suppression of hepatic lipoprotein receptors: a mechanism for the hypercholesterolemia of cholesterol-fed rabbits. Proc Natl Acad Sci USA 1981;78:1396 – 400. [20] Chao Y-S, Windler EE, Chen GC, Havel RJ. Hepatic catabolism of rat and human lipoproteins in rat treated with 17a-ethinylestradiol. J Biol Chem 1979;254:11360– 6. [21] Gherardi E, Brugni N, Bowyer DE. Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies. Biochem J 1988;253:409–15. [22] Gherardi E, Bowyer DE, Fitzsimmons C, Le Cras T, Hutchings A, Butcher G. Probing of the expression of the low-density lipoprotein receptor in vivo using an anti-receptor monoclonal antibody. Biochem J 1991;280:1 – 7. [23] Fitzsimmons C, Bush R, Hele D, Goodman E, Gherardi E, Bowyer DE. Measurements of the absolute number of functioning low-density lipoprotein receptors in vivo using monoclonal antibody. Biochem J 1995;305:897 – 904. [24] Havel RJ, Edner HA, Bragdon J. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 1955;34:1345 – 53. [25] Wiklund O, Bjo¨nheden T, Olofsson SO, Bondjers G. Influx and cellular degradation of low density lipoproteins in rabbit aorta determined in an in vitro perfusion system. Arteriosclerosis 1985;5:135 – 41. [26] Schwenke DC, Claire RW. Accumulation of 125I-tyramine cellobiose-labelled low density lipoprotein is greater in atherosclerosis susceptible region of white Careau pigeon aorta and further enhanced once atherosclerosis develops. Arterioscler Thromb 1992;12:446 – 60. [27] Nanjee NM, Miller NE. Sequential microenzymatic assay of cholesterol, triglycerides, and phospholipids in a single aliquot. Clin Chem 1996;42:915 – 26. [28] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(259):680– 5. [29] Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des fibrinogens. Acta Haematol 1957;7:237 – 46. [30] O’Donnell L, Owens D, McGee C, Devery R, Hession P, Collins P, et al. Effects of reserpine on serum lipoproteins of normally fed and cholesterol-fed rabbits. Metabolism 1988;37(10):910– 5. [31] Hong MK, Vossoughi J, Mintz GS, Kauffman RD, Hoyt RF Jr, Cornhill JF, et al. Altered compliance and residual strain precede angiographically detectable early atherosclerosis in lowdensity lipoprotein receptor deficiency. Arterioscler Thromb Vasc Biol 1997;17:2209 – 17. [32] Fujioka T, Nara F, Tsujita Y, Fukushige J, Fukami M, Kuroda M. The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochim Biophys Acta 1995;1254:7 – 12. [33] Tvedskov TF, Vad H, Bendix-Hansen K, Albrechsten K, Gram J. Studies of initial anticoagulant response in tissue-thromboplastin induced intravascular coagulation. Blood Coagul Fibrinolysis 1996;7:595 – 601. [34] Dietschy J, Turley SD, Spady DK. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animals species. J Lipid Res 1993;34(10):1637–57.

S. Shafi et al. / Atherosclerosis 149 (2000) 267–275 [35] Krone W, Naegele H, Behnke B, Greten H. Opposite effects of insulin and catecholamines on LDL-receptor activity in human mononuclear leucocytes. Diabetes 1988;37:1386–91. [36] Maziere C, Maziere JC, Mora L, Gardette J, Polonovski J. Epinephrine decreases low density lipoprotein processing and lipid synthesis in cultured human fibroblasts. Biochem Biophys Res Commun 1985;133:958–63. [37] D’Eletto R, Javitt NB. Effect of doxazosin on cholesterol synthesis in cell culture. J Cardiovasc Pharmacol 1989;13(2):S1 – 4. [38] Davies RA, Showalter R, Kern F Jr. Reversal by triton WR-

.

275

1339 of ethinyloestradiol induced hepatic cholesterol esterification. Biochem J 1978;174:45 – 51. [39] Carr TP, Hamiliton RL, Rudel LL. ACAT inhibitors decrease secretion of cholesteryl esters and apolipoprotein B by perfused livers of African green monkeys. J Lipid Res 1995; 36:25 – 30. [40] Wilkinson J, Higgins JA, Fitzsimmons C, Bowyer DE. Dietary fish oils modify the assembly of VLDL and expression of the LDL receptor in rabbit liver. Arterioscler Thromb Vasc Biol 1998;18:1490 – 7.