adrenoceptor coupled to guanine nucleotide binding ... - Springer Link

Basic Research in

Cardiology

Basic Res Cardiot 86:117-126 (1991)

Effect of metoprolol on activity of [5-adrenoceptor coupled to guanine nucleotide binding regulatory proteins in adriamycininduced cardiotoxicity L. X. Fu, C-H. Bergh, J. Hoebeke*), Q. M. Liang, K. G. Sj6gren, F. Waagstein, and A~. Hjalmarson Wallenberg Laboratory, Division of Cardiology, Sahlgren's Hospital, University of G6teborg, G6teborg, Sweden *) Laboratoire des Prot6ines des Liquides Biologiques, U R A , CNRS 1334, Tours, France

Summary: Prevention of cardiotoxicity without interfering with the therapeutic efficacy of adriamycin is a very crucial question. We have investigated the activity of [3-adrenoceptor coupled to guanine nucleotide binding regulatory proteins (G-proteins) and CaZ+-ATPase activity in experimental adriamycin-induced cardiotoxicity and the influence of metoprolol treatment on these variables. Adriamycin was administered to rats intravenously as a single dose of 6 mg/kg, and metoprolol was continuously given by means of implanted osmotic pumps, f3-adrenoceptor characteristics were measured by radioligand-binding experiments and by basal and stimulated adenylyl cyclase activity. Northern blot and dot blot analysis was used to quantify G-protein mRNA. It was shown that adriamycin did not induce any change in the total [3-adrenoceptor density, nor did the high affinity agonist binding to 13adrenoceptor change. Adriamycin did not induce any alteration in the amount of mRNA encoding for stimulatory (Gs) or inhibitory (Gi) G-proteins. Also, basal and stimulated adenylyl cyclase activities were identical in the different experimental groups. In contrast, the Ca2+-ATPase was shown to increase in adriamycin-treated rats compared to control rats (45 +_3.8 versus 23 _+1.2 p.mol Pi/mg/h, P < .01). Metoprolol was shown to normalize this increase (29 _+2.1 ~tmol Pi/mg/h). Thus, it may be concluded that in experimental adriamycin-induced cardiotoxicity, despite Ca2+-overloading, the [3-adrenoceptorG protein-adenylyl cyclase system remains intact. Metoprolol seems to prevent Ca2+-overloading independently of the 13-adrenoceptors studied here. Key words: Metoprolol; adriamycin; G-proteins; l}-adrenoceptor; _adenylyl cyclase Ca2+-ATPase; myocardium

Introduction

Adriamycin (ADR), an effective chemotherapeutic agent, is used in the treatment of a variety of human neoplasms. It has been shown that repetitive administration of A D R in patients is associated with cardiotoxic effects and the development of a specific cardiomyopathy (12). Several hypotheses have been presented for the mechanism behind the cardiotoxicity of A D R , including inhibition of D N A synthesis, interference with energy metabolism and modification of Ca 2+ transport (1, 12). Also, membrane lipid damage due to free radical formation which may interfere with the cardiac-[~-adrenoceptor function has been proposed (1, 28, 31). Especially the increase of Ca2+-ATPase activity is a useful marker of adriamycin-induced cardiotoxicity (31) and was used as such in the present investigation. Adriamycin-induced cardiomyopathy has some 657

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characteristics in c o m m o n with other types of cardiomyopathies. Significant alterations in [3a d r e n o c e p t o r - G proteins-adenylyl cyctase system has been shown to occur in subjects with idiopathic dilated cardiomyopathy. These alterations result in subsensitivity of the failing human heart to ~-adrenergic stimulation (11). Long-term [3-blockade has been shown to improve cardiac function in patients with dilated cardiomyopathy (35, 36). This is associated with an up-regulation of cardiac [3-adrenoceptors (15, 35). The present study was undertaken with the purpose of investigating the influence of adriamycin-toxicity on the cardiac [3adrenoceptor-signalling system and calcium handling, and the potential use of metoprolol, a [3-adrenergic receptor antagonist, to prevent these cardiotoxic effects.

Materials and methods Experimental protocol and procedure Male Sprague-Dawley rats weighing 200-250 g were used, The rats were divided into three groups: an adriamycin-treated group (ADR group), an adriamycin plus metoprolol-treated group (ADR + MET group), and a control group. A D R group: A D R (Farmitalia Carlo Erba S.A., Spain) was administered as a single dose of 6 mg/kg intravenously (i.v.) to seven rats. Osmotic pumps (Model 2 ml 4, Alzat, USA) containing vehicle (sterilized isotonic saline) were implanted subcutaneously (s.c.). The single dose protocol was chosen and a sufficiently high dose was given. This was done in order to produce typical ADR-indnced cardiac damage and to reduce the inter-animal variation (2, 7, 32). ADR + MET group: in seven rats treated with a single dose of A D R (as above) osmotic pumps containing metoprolol tartrate (in isotonic NaCI) at a concentration of 150 mg/ml were implanted s.c. This pump will deliver metoprolol to the circulation at a constant rate of 1 p~mole/hr for 4 weeks. This dose was chosen to correspond to a pharmacologic effective myocardial fi 1-blockade according to information provided by manufacturer (H~issle AB, M61ndal, Sweden). Control group: Six rats received an equal volume of phosphate-buffered saline i.v. and osmotic pumps containing vehicle by s.c. implantation. After 4 weeks the rats were sacrificed by cervical dislocation, myocardial ventricles were quickly removed and immediately placed in liquid nitrogen. Northern blot and dot blot hybridizations Total RNA was extracted from rat ventricles by the guanidinum thiocyanate-cesium chloride method (8). Plasmids (with insert lengths) containing rat complementary DNAs (cDNAs) encoding the cz subunit of stimulatory G protein (Gs-alpha) and subtypes of the c~subunit of inhibitory G protein (Gialpha 1, 2, 3), as well as a plasmid containing a 911-bp insert encoding frog rRNA were radiotabeled with (c~-3ZP) dCTP (Dupont, 3000 ci/mmol) as previously described (10, 17). Northern blot analysis was performed as previously described (11). The relative amounts of mRNAs were determined by dot blot analysis. Briefly, an equal amount of total RNA (43.75 ug) from this tissue of each rat was denatured and serially diluted before application to a hybridizing membrane (Amersham, UK) mounted on a 96-well manifold (Schleicher & Schuell, USA). The membrane was then hybridized with radiolabeled probles (11). The calculation of the relative levels of specific mRNA was based upon the assumption that each hybridized probe molecule was specifically bound to the appropriate mRNA. The specific radioactivity of the probe was calculated from scintillation counting of radioactivity and spectrophometry of its molarity. All probes were stable as radioactive decay followed the expected half-life of (c~-3~P) dCTP. Membrane preparation Cardiac membrane was prepared by mincing the rat ventricle. The pieces were homogenized in a buffer of the following composition: 20 mM Tris-HC1; 250 mM sucrose; 5 mM MgCI2 (pH 7.5). Nuclei were removed by centrifugation at 900× g (10 rain, 4°C) and the postnuclear fraction was spun at 43 000× g (10 rain, 4 °C). The resulting pellet was resuspended in 600 mM KCI in homogenizing buffer

Fu et al., Metoprolol in adriamycin-induced cardiotoxicity

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and placed on ice for 10 rain. It was then recentrifuged at 43000x g (20 rain, 4°C) and washed twice. This membrane fraction was used for radioligand binding and adenylyI cyclase studies. For Ca 2+ATPase, the membrane fraction was additionally extracted for 30 min with 400 mM LiBr in 10 mM TrisHC1 (pH 7.4). After centrifugation at 1000× g (10 min, 4°C) the pellet was washed twice with buffer (26). The protein content was measured according to the method of Lowry (23).

Radioligand binding studies and adenylyI cyclase assay The binding characteristics of ~-adrenoceptor was determined by use of (12sI) iodocyanopindolol (ICYP). Briefly, binding assays were carried out in a buffer of the following composition: 25 mM TrisHCI, 75 mM MgC12 (pH 7.4). An aliquot of the membrane fraction was incubated for 2 h in a total volume of 200 ~tl at 37 °C with 100 pM ICYP and different concentrations of isoproterenol (100 pM-100 ~tM). The reaction was terminated by dilution with 5 ml of ice-cold buffer. The sample was then poured over a GF/C 2.5 cm glass microfiber filter (Whatman Int. Ltd., UK) under reduced pressure, followed by a wash with 15 ml of the same buffer. Sample counting was performed in a gamma counter (1282 Compugamma, LKB, Sweden). Saturation-binding isotherms were obtained by incubating an aliquot of the membrane fraction for 2 h at 37 °C with increasing concentrations of ICYP (12.5 pM-400 pM) in the same buffer as above. Non-specific binding was determined in the presence of 2 ~tM 1-propranolol and amounted to less than 5 % of total binding. Specific binding was obtained by subtracting non-specific binding from total binding. All binding data are given as specific binding, Bmax and KD we:re calculated according to the method of Scatchard (30). Agonist binding characteristics were calculated by computer-aided, non-linear least square fitting as previously described (3, 4). Adenylyl cyclase activity was measured according to the method of Salomon et al. (29), Maxilnal adenylyl cyclase activity was determined in the presence of 10 mM sodium fluoride (NaF), and 1 vM isoproterenol (ISO) plus 50 ~uM guanosine triphosphate (GTP). cAMP was eluted by sequential Dowex and alumina chromatography. The yield was calculated by previous addition of (3H) cAMP.

Ca2+-A'IYase assay Membrane Ca2+-ATPase was measured as previously described (26, 18). Briefly, the membrane preparation (100 ~tg protein) was preincubated at 37 °C for 5 rain in 1 ml of a buffer containing 50 mM Tris-HC1 (pH 7.4) and 4 mM CaCI2. The reaction was started by the addition of 4 mM Tris-ATP (pH 7.4) and terminated i0 min later by the addition of 1 ml of 12 % cold trichloroacetic acid. The amount of phosphate liberated (Pi) was assayed according to the method of Taussky and Shorr (34). The activity of Caa+-ATPase was expressed in %tmol Pi/mg/h".

Mater&Is (lasI) ICYP (100 p,Ci), (3H) cyclic AMP (1.0 mCi/ml), (alpha-32P) ATP (10.0 mCi/ml) and (o~-32P) dCTP (3000 Ci/ml) were obtained from New England Nuclear. Adriamycin was a generous gift from Prof. Karl Landys, University of G6teborg, Sweden. Plasmids containing rat cDNAs encoding e~ subunits of Gs and Gi were kindly supplied by R. R. Reed, Johns Hopkins University School of Medicine, Baltimore, USA. Metoprolol and osmotic pumps were kindly provided by Hfissle Cardiovascular Research Laboratories, M61ndal, Sweden. All other chemicals were of reagent grade, and were obtained from Sigma Chemical Co, St. Louis, USA.

Statistical analj~is Student's t-test was used for comparisons when data were normally distributed. The results are reported as the mean + S.D. p < 0.05 in a two-tailed test is considered statistically significant.

Results N o m o r t a l i t y o c c u r r e d t h r o u g h o u t t h e study period. R a t s in e i t h e r A D R g r o u p o r A D R + M E T g r o u p did n o t h a v e any a p p r e c i a b l e fluid a c c u m u l a t i o n in the b o d y cavity. 3[~e activity of Ca2 ~-ATPase was s h o w n to increase dramatically in t h e A D R g r o u p c o m p a r e d to t h e

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Fig. 1+ mRNAs encoding c~ subunits of Gs and subtypes of Gi. Northern blot analysis of sizefractionated total RNA (40 ~g) was sequentially probed under high stringency condition with radiolabeled cDNAs encoding Gs