Clinical and pharmacological significance of α2-adrenoceptor polymorphisms in cardiovascular diseases

International Journal of Cardiology 97 (2004) 367 – 372 www.elsevier.com/locate/ijcard

Review

Clinical and pharmacological significance of a2-adrenoceptor polymorphisms in cardiovascular diseases Christodoulos Flordellis a, Antonios S. Manolis b, Mika Scheinin c, Herve´ Paris d,* a

Department of Pharmacology, School of Medicine, University of Patras, Rio Patras, Greece b Department of Cardiology, School of Medicine, University of Patras, Rio Patras, Greece c Department of Pharmacology and Clinical Pharmacology, University of Turku, Turku, Finland d INSERM Unit 388, Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse cedex 4, France Received 10 February 2003; received in revised form 12 August 2003; accepted 12 October 2003 Available online 4 March 2004

Abstract The a2-adrenoceptors (a2-ARs) are receptors for endogenous catecholamines (norepinephrine and epinephrine) that mediate a number of physiological and pharmacological responses such as hypotension and sedation. Three distinct subtypes, denoted a2A-, a2B- and a2C-AR, have been characterized and cloned. Employment of mutation screening in the study of human populations from various ethnic backgrounds has shown that a2-AR genes are polymorphic. The functional and biochemical consequences of these polymorphisms have been analyzed by expressing the wild-type receptors and their respective genetic variants in heterologous systems such as CHO and COS-7 cells. Changes include alteration in G-protein coupling and in agonist-promoted receptor phosphorylation and desensitization. Case-control and populationbased studies have shown clinical association with cardiovascular risk. Further investigation of the genetic variants in specialized cells and transgenic animals will provide the molecular basis of cardiovascular disease and may reveal a2-AR variants as potential targets for selective pharmacological interventions. D 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Cardiovascular disease; a2-Adrenoceptor; Polymorphism; GPCR; Pharmacogenetic

1. Introduction Genetic polymorphism is defined as the occurrence within a population of two or more allelic variants of a given gene sequence, in such proportions that the rarest cannot be maintained merely by recurrent mutations. For practical purposes, genetic polymorphisms are often defined as common genetic variants [1]. Their importance lies in the fact that in many cases such variations have consequences on the functions of metabolizing enzymes, transporters, receptors and other potential targets of drug interventions, which may therefore provide the basis for observed interindividual variability in clinical phenotypes and pharmacological responses [2]. The G protein-coupled receptors (GPCRs) represent a suitable system for investigating the potential contribution of receptor polymorphisms to the propensity for disease and * Corresponding author. Tel.: +33-561-32-30-90; fax: +33-562-1725-54. E-mail address: [email protected] (H. Paris). 0167-5273/$ - see front matter D 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2003.10.014

variability of response to drug therapy. GPCRs are widely expressed in the organism and are involved in the regulation of numerous physiological functions. Their clinical importance is underlined by the fact that almost one third of all prescription medications are agonists or antagonists of GPCRs [3]. All GPCRs, including the adrenoceptors (ARs), are seven-transmembrane domain proteins with extracellular amino-terminus and intracellular carboxyl-terminus. Of the hydrophilic loops that link the seven hydrophobic a-helix, the third cytoplasmic loop is particularly important for receptor function, because it contains the domains both for coupling to the cognate G-protein and for phosphorylation by GPCR kinases (GRKs) and subsequent desensitization [4]. Mutations of GPCRs resulting in constitutive activity (agonist-independent increased function) or decreased responsivity to agonists (loss of function) are the primary cause of relatively rare diseases [5]. Examples of the former are the familial male precocious puberty and the hyperfunctioning thyroid adenomas that, respectively, result from mutations in the luteinizing hormone receptor [6] and thyrotropin receptor [7]. Loss of function as causal

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factors in disease is exemplified by vasopressin-insensitive diabetes insipidus resulting from several point mutations in the gene encoding the vasopressin V2 receptor [8]. GPCR polymorphisms that occur with high frequency in the healthy population are unlikely the direct causes of hereditary diseases. Instead, they may be risk factors for complex multifactorial diseases (cardiovascular diseases, diabetes), modifiers of disease characteristics (onset, progression, outcome), or modulators of responses to drug therapies. 2. Cardiovascular regulation by A2-adrenoceptors The A2-ARs are receptors for norepinephrine and epinephrine that mediate a number of physiological responses to endogenous catecholamines such as hypotension, sedation, inhibition of insulin release, anti-lipolysis and platelet aggregation [9]. These receptors preferentially couple to the Gi/ Go family of heterotrimeric guanine nucleotide binding proteins and thereby regulate a variety of effector systems including adenylyl cyclase, K+-channels, Ca2 +-channels and mitogen-activated protein kinases, Erk1 and Erk2 [10,11]. As for other GPCRs, A2-AR activation by agonists also initiates an interrelated set of processes responsible for their desensitization and internalization. The desensitization process includes uncoupling of the receptors from G-proteins as a result of phosphorylation of Ser and Thr residues in their third intracellular loop by GRKs [4]. Although no distinct consensus sequence motifs have been identified, acidic amino acid residues adjacent to phosphorylated sites seem to favor the kinase action [12]. Receptor phosphorylation promotes the binding of B-arrestins, which hinder receptor coupling to the G-proteins. In addition, they interact with a variety of proteins involved in endocytosis, and as scaffolds transduce and compartmentalize alternative signals [13]. Three distinct A2-AR subtypes denoted A2A, A2B and A2C have been characterized and cloned [14 – 16]. They differ in their pharmacological properties, tissue distributions and sensitivity to phosphorylation and desensitization [9]. The generation of genetically engineered mice has recently permitted to elucidate the specific contribution of each subtype in the regulation of several physiological functions [17 –19]. The mechanisms whereby each subtype affects cardiovascular system are depicted in Fig. 1. The A2A-AR is the principal presynaptic inhibitory autoreceptor regulating norepinephrine release from central and peripheral sympathetic nerves. Activation of this subtype, which is abundantly distributed throughout the central nervous system as well as in peripheral tissues, results in reduction of blood pressure and heart rate. Actually most of the classical effects of A2agonists, including sedation, antinociception, anesthetic sparing, hypothermia are now ascribed to this subtype [20 – 22]. Compared to controls, transgenic mice lacking A2A-AR exhibit higher resting systemic blood pressure and heart rate, which correlate with increased norepinephrine release from cardiac sympathetic nerves [23]. Moreover,

Fig. 1. Regulation of cardiovascular function by a2-adrenoceptor subtypes. a2A-AR plays a crucial role in the mediation of a2-agonist effect on blood pressure by inhibiting norepinephrine release from central and peripheral sympathetic nerves. Reduced sympathetic tone results in decreased cardiac output and inhibition of renin secretion as a consequence of attenuated activation of h1-ARs from heart and juxtraglomerular apparatus. a2C-AR participates to inhibition of norepinephrine release from sympathetic nerve endings and is also responsible for vasoconstriction of cutaneous arterioles. Central a2B-AR is involved in induction of hypertension in response to high-salt diet. This subtype is also responsible for constriction of large arteries. In addition to central and vascular effects, a2B may be involved in the control of blood pressure by regulating water and electrolyte handling in the kidney.

they develop more rapidly hypertension in response to salt loading after subtotal nephrectomy [24]. Expression of the A2B-AR in rat brain is restricted to the thalamus and to the nucleus of the solitary tract [25,26]. This subtype is also found in peripheric tissues including vascular smooth muscle [27], renal cortex [25], fetal liver and placenta [28]. According to results obtained with transgenic mice, A2B-AR plays a critical role in the development of the placental vascular system [29]. It is also responsible for the peripheral vasoconstrictor action of A2-agonists. Thus, contrary to what is observed in controls, intravenous administration of an A2-agonist to A2B-AR knock-out mice fails to elicit the initial transient increase in blood pressure [9]. This receptor subtype has also been found to play a dominant role in salt-induced experimental hypertension. Indeed, mice lacking one copy of the A2BAR gene do not develop hypertension in response to highsalt diet in a DOCA-salt model of hypertension [30]. The physiological role of A2C-AR remained enigmatic for a long time as it did not appear as a major player in cardiovascular regulation [9]. Experiments on transgenic mice with targeted inactivation of the A2C-AR gene or with 3-fold overexpression of A2C-AR in striatum demonstrated that this subtype possibly regulates the dopamine system in the brain [31]. Moreover, examination of these strains using behavioral tests has shown that antagonists of this subtype may be of therapeutic value in stress-related psychiatric disorders [32,33]. However, it was recently shown that both A2A- and A2C-subtype participate to presynaptic inhibition of norepinephrine release. Whereas

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A2A-AR acts at high stimulation frequencies, A2C-AR is particularly efficient at low stimulation frequencies [34]. Regulation in both frequency ranges is physiologically important. It is moreover remarkable that strains of mice lacking A2A- or A2C-AR subtype exhibit reduced survival following cardiac pressure overload by aortic banding [35]. Increased mortality was due to heart failure. It correlated with elevated level of plasma catecholamines and was associated with remodeling of the left ventricule with cardiomyocyte hypertrophy and intense fibrosis.

3. Identification and biochemical characterization of A2-adrenoceptor polymorphisms Employment of mutation screening methods in the study of cohorts of hypertensive and normotensive individuals from various ethnic backgrounds has indicated that all three A2-AR genes are polymorphic (see Table 1). The functional and biochemical consequences of these polymorphisms have been analyzed by expressing the wild-type receptors and their respective genetic variants in heterologous systems such as CHO and COS-7 cells [36]. Genetic variations in the promoter, 5V- and 3V-untranslated regions have been reported only for A2A-AR [37 – 42]. Several genetic variations were found in the coding region [41]. However, given the importance of the third intracellular loop for G-protein coupling [43], only those located in this region have been analyzed in detail (Fig. 2). A single nucleotide genetic variant of A2A-AR (C to G transversion at nucleotide 753) that results in an Asn to Lys substitution at amino acid 251 (N251K) in the third cytoplasmic loop of the receptor has been identified. Compared to wild-type, the Lys251 variant has higher propensity to

Fig. 2. Topology of a2-AR subtypes and location of polymorphisms in their third cytoplasmic loop. In the upper part of the figure, the topology and the location of the polymorphisms are indicated. In the lower part, the amino acid sequences of the wild-type a2-AR subtypes (WT) and of their corresponding variants (Variant) are aligned and compared.

couple to Gi. It exhibits also a gain of agonist-stimulated function since it displays enhanced potency to inhibit adenylyl cyclase, to activate MAPK and to cause inositol phosphate accumulation [44]. A polymorphic variant of the A2B-AR was identified using PCR-based screening methods. It consists in the deletion of three Glu residues (Del301 –303) in an acidic motif located in the third cytoplasmic loop of the receptor [45]. This polymorphism is more frequent in Caucasians than in African-Americans (allele frequency 0.31 – 0.45 vs. 0.12). The acidic stretch is uniquely present in the A2B-AR and is conserved in mammalian, suggesting it is important for the functionality of this subtype. Consistent with this view, employment of site-directed mutagenesis has demonstrated that substitution or deletion of this motif leads to impairment of agonist-dependent phosphorylation by GRKs

Table 1 Currently known polymorphisms in the human a2-AR genes Gene

Position

a2A-AR

5V-flanking 5V-flanking 5V-flanking 5V-flanking 5V-flanking 5V-flanking ORFb ORF ORF ORF 3V-flanking

region region region region region region

region

Variationa

Allele frequency

Reference

C-1291G (MspI: 174/121 kb) HhaI (299/277 bp) Bsu361 (12/5.8 kb) G-1773T C-1284G G-261A A25G N251K R368L K370N DraI (6.7/6.3 kb)

0.35/0.65 0.89/0.11 0.66/0.34 0.95/0.05 0.73/0.27 0.89/0.11 rare rare rare rare 0.81/0.19 0.73/0.27 0.8/0.2 0.56/0.44 0.8/0.2 0.62/0.38

[42] [39] [39] http://genecanvas.idf.inserm.fr http://genecanvas.idf.inserm.fr http://genecanvas.idf.inserm.fr [41] [41,44] [41] [41] [37] [38] [40] [45] [58] [48]

a2B-AR

ORF

Ins/Del of 3 glutamic acids

a2C-AR

ORF

Ins/Del of 4 amino acids

a

The position and the kind of single nucleotide mutations (transversion or transition) in the 5V- and 3V-flanking regions or amino acid substitution in the ORF are indicated. Restriction enzyme fragment length polymorphisms are indicated with the restriction enzyme site, whereas the length of fragments generated is depicted in base pairs in brackets. Ins/Del stands for Insertion/Deletion. b ORF stands for open reading frame and indicates genetic variation in the coding region of the receptor gene.

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and abolishment of A2B-AR desensitization [46]. Quite recently, the naturally occurring deleted A2B-AR variant was reported to be similarly refractory to desensitization [47]. The biochemical consequence of the A2B-AR polymorphism is therefore the generation of a receptor with sustained signaling in spite of continued activation by endogenous or exogenous agonists. Finally, an in-frame gene deletion of 12 nucleotides yielding a receptor with a four amino acid deletion (Gly – Ala – Gly –Pro) in the third intracellular loop of the A2C-AR was recently discovered [48]. This Del322 –325 A2C-AR, which is f 10 times more frequent in African-Americans than in Caucasians (allele frequency 0.381 vs. 0.040), displays impaired coupling to Gi proteins and to effector systems including adenylyl cyclase and MAPK. This defect is believed to derive from the fact that the deletion is at proximity of the sequence RRGGRR, a motif identified in a number of receptors as a major Gi coupling domain [49].

4. Physiological consequences and clinical associations of the A2-adrenoceptor polymorphisms The possible associations between clinical diseases and the identified A2-AR polymorphisms have already been investigated in some case-control and population-based studies. The key role of A2A-AR in the mediation of the hypotensive effect of A2-agonist has first prompted the examination of cohorts of individuals suffering essential hypertension in terms of A2A-AR polymorphism. Genotyping using the restriction enzyme DraI, which allows the detection of polymorphism in the 3V-flanking region of the a2A-AR gene, generated inconsistent results. Indeed, association between this polymorphism and hypertension was found in a white population in one study [40] but not in another [50]. On the other hand, a third report has demonstrated the presence of association in black American population [38]. Of note, none of these studies was based on a random population sample. Possible effects of unevenly distributed parameters, such as sex and age, have not been rigorously addressed. More rationally planned clinical genetic studies will be necessary to clarify the so far observed discrepancies. A study, in a Finnish population including 166 obese subjects (BMI>27 kg/m2), investigated the possible association of the deletion a2B-AR genotype with metabolic rate. Basal metabolic rate was determined by indirect calorimetry and adjusted for fat-free body mass, fat mass, sex and age [45]. This analysis showed that the D/D genotype was associated with decreased metabolic rate of 94 cal/day in comparison to subjects with the I/I genotype. Reduction in basal metabolism may be the consequence of a change in vascular resistance and subsequent redistribution of blood flow away from metabolically active tissues, skeletal muscle in particular. Alternatively, it may derive through reflex modulation of the activity of autonomic nervous system.

Unfortunately, no relevant information is available to privilege either one of these possibilities. Another investigation was carried out in a non-selected population-based cohort of male subjects at relatively high risk for cardiovascular diseases [51]. The study hypothesis was based on the peripheral vasoconstrictive property of the a2B-AR subtype in mice [52], the coronary vasoconstrictive property of a2AR agonists in humans [53], and the importance of the acidic region present in the third loop of a2B-AR for receptor desensitization [46]. In this population-based prospective study of 912 Finnish middle-aged men, the D/D genotype was associated with a 2.5-fold increased risk for acute coronary events, i.e. myocardial infarction, as compared with the I/D and I/I genotypes [51]. The association between D/D genotype was very recently confirmed by the analysis of unselected autopsy material collected from out-of-hospital sudden deaths [54]. Analysis of a cohort of 683 middle-aged white men demonstrated that carriers of the D/D genotype had an increased risk for sudden cardiac death (odds ratio, 2.0) and acute myocardial infarction (odds ratio, 2.1). The association between the risk of fatal cardiac accident and D/D genotype was even higher in subjects who died before the age of 55 years, but it was independent of other known major risk factors for coronary heart disease. As suggested by a study of hemodynamic response to acute adrenaline infusion in healthy young men [55], the most plausible mechanism explaining the association is a blunted coronary blood flow increase due to exaggerated a2B-AR mediated vasoconstriction. This alteration could be involved in the incidence of myocardial infarction either directly by increasing coronary spasm or indirectly as the stimulus for plaque fissuring [56,57]. Two additional mechanisms have also been considered as leading to acute coronary events in the D/D group. They include augmented vasoconstriction of small coronary arteries and increased cardiac oxygen demand due to increased peripheral resistance [51]. This polymorphism was not associated with essential hypertension in Finnish [51]. Such a lack of association has already been confirmed in the USA [58] and it is not unexpected as blood pressure is under the control of several different factors interacting in a complex way. Finally, considering its loss of function and the phenotype of a2C-AR knock-out mice the Del322– 325 variant of a2CAR may in the future prove to be pharmacogenetically significant. The clinical importance of this polymorphism was very recently demonstrated by two independent studies in which genomic DNAs prepared from patients with chronic heart failure (NYHA class II through IV) were genotyped for AR polymorphisms [35,59]. In the first of these studies, the frequency of Del322 – 325 a2C-AR did not significantly differ between heart failure patients (11%) and healthy control population (11.4%). However, patients carrying the deletion polymorphism appeared to have a worse clinical status and a poorer outcome. In the second study, the association of the a2C-AR and h1-AR polymorphisms with heart failure was investigated in a cohort of 162 African-

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American subjects. No increase in risk was observed among persons who are homozygous for h1Arg389, a h1-AR variant exhibiting increased activity [60]. The Del322 –325 a2C-AR genotype was found more frequent in heart failure patients than in controls and represented per se a factor of risk for heart failure (adjusted odds ratio, 5.65). Overall, there was a synergistic effect of the two genes so that a clear increase in risk of heart failure was observed among whose homozygous for both variants (adjusted odds ratio, 10.11). In addition to bring further support for a link between unfunctional a2C-AR and heart failure and the latter observation underlines the importance of analyzing the association of multiple candidate genes.

5. Prospectives The studies so far performed to characterize altered functions or differences in the regulation process of a2-AR variants have been carried out in vitro employing heterologous expression systems, such as COS-7 and CHO cells. Different cell-lines or even distinct batches from the same cell-line differentially express effectors or intracellular complements of receptors (such as G-protein subunits, adenylyl cyclase, ion channels, GRK and h-arrestin isoforms), which determine GPCR second messenger responses, desensitization, internalization and phosphorylation-dependent mitogenic signaling. Furthermore, these transformed cells do not accurately reflect the physiological settings where a2AR subtypes are expressed, like vascular smooth muscle cells or specialized neuronal cells. It is thus important that the biochemical properties of the a2-AR variants are in the future analyzed in vitro in such differentiated cellular systems as well as ex vivo in cells or tissues collected from subjects with defined genotypes. In this regard, it will be instrumental to also generate transgenic mice expressing the polymorphic a2-ARs, preferably in a tissue-specific manner. Such mouse strains will hopefully allow the detailed understanding of the consequences of genetic variation of a2-ARs in cardiovascular pathophysiology. In addition, they may serve as models to develop and test new pharmacological therapeutic strategies. Acknowledgements This work was supported by the BIOMED 2 program CEE BMH4 CT98-3373 (European Commission, Brussels, Belgium). Authors thank Mrs. F. Kozatapani for secretarial assistance. References [1] Buscher R, Herrmann V, Insel PA. Human adrenoceptor polymorphisms: evolving recognition of clinical importance. Trends Pharmacol Sci 1999;20:94 – 9.

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