Characterization of adenosine receptors in intact cultured heart cells

Abstract

Adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. We used the hydrophilic A 1 adenosine receptor radioligand [ 3 H]-8-cyclopentyl-1,3-dipropylxanthine ([ 3 H]CPX), to monitor the level of the receptors on intact cardiocytes. The binding showed high affinity (K d = 0.13 nM) and the number of [ 3 H]CPX binding sites (B max ) was 23.1 fmol/dish (21 fmol/mg protein). The K i of the agonists R-N 6 -(2-phenylisopropyl)-adenosine (R-PIA) and S-N 6 -(2-phenylisopropyl)-adenosine (S-PIA), and of the antagonists CPX and theophylline were 3.57, 49.0, 1.63 and 4880 nM, respectively. The number of adenosine receptors was very low during the first days in cultures (5 fmol/dish) and increased gradually until it reached a plateau on days 8-10. Treatment with norepinephrine or isoproterenol which accelerated the rate of contractions, induced up regulation of the receptors. B max increased 2-3-fold by application of norepinephrine for 4 days, while receptor affinity to the radioligand was unaffected. Lactate dehydrogenase (LDH) and creatine kinase (CK) activity increased only by 22 and 38%, respectively. Similarly, 3 days treatment with triiodothyronine (T 3 , 10 −8 M), which also accelerated heart rate, increased the number of adenosine receptors by 56% without a significant change in the affinity of the receptors to [ 3 H]CPX. Carbamylcholine (5 × 10 −6 M), which reduced the rate of heart contractions, caused 26% down regulation while the affinity of the receptors remained unchanged. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, the level of adenosine receptors may respond to drug-induced chronic changes in cardiac contractile activity so as to restore conditions to normal (basal) contractions. KeywordsCPX; heart-rate; norepinephrine; thyroid hormones Adenosine modulates a variety of physiological functions in the heart. These actions are mediated by cell surface adenosine receptors, which can be classified into A 1 and A 2 † Corresponding author. Tel. (972)3-5318265; FAX (972)3-5351824. In conditions of stress like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP [4]. In these conditions adenosine has therapeutic and protective effects on the heart [5,6]. Adenosine causes negative chronotropic, dromotropic and ionotropic effects on the cardiac tissue via A 1 receptors [7,8]. Adenosine also acts as a coronary vasodilator via A 2 receptors [9]. These effects of adenosine occur also without prior catecholamine treatment [10]. Stimulation that enhances the cAMP content (catecholamines, forskolin, amrinone), makes the heart more sensitive to adenosine [11][12][13][14]. In addition, adenosine causes hyperpolarization by activation of K + channels [15] via G proteins [16, 17]. These K + channels are the same channels that are stimulated by acetylcholine in the cardiac tissue [18][19][20]. HHS Public AccessChron...

Regulation of A1 adenosine receptors by amiodarone and electrical stimulation in rat myocardial cells in vitro

“…ES at low temperature or amiodarone, respectively. As in the previous study utilizing adrenergic and cholinergic stimulation [7], the change in contraction rate in rat heart cell cultures was associated with changes in the density of A 1 adenosine receptors. …”

“…The elevation in A 1 adenosine receptors probably increases cardiac sensitivity to adenosine and indicates by which mechanism the heart cells may adapt to electrical stimulation stress. Further support for this mechanism of adaptation to stress was demonstrated by the effect of thyroid hormones and catecholamines that accelerated heart rate and also upregulated the A 1 adenosine receptor [7,15].…”

“…The A 1 adenosine receptor is upregulated in hyperthyroidism [7], an effect accompanied by stimulation of myocardial contractility and a biochemical transition of slow-twitch ("red") ‡ Corresponding author: Prof. Asher Shainberg, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. TEL: 972-3-5318265; FAX: 972-3-5351824; shaina@ashur.cc.biu.ac.il.…”

“…The elevation in A 1 adenosine receptors probably increases cardiac sensitivity to adenosine and indicates by which mechanism the heart cells may adapt to electrical stimulation stress. Further support for this mechanism of adaptation to stress was demonstrated by the effect of thyroid hormones and catecholamines that accelerated heart rate and also upregulated the A 1 adenosine receptor [7,15].The important role of the A 1 adenosine receptor and not A 2 receptors in relation to heart contractility was reported for electrically stimulated cardiocytes that were acutely treated with R-PIA [30,31]. In those cells R-PIA decreased contractility, while CPX, a selective A 1 adenosine antagonist, antagonized the effect of R-PIA on contractile response.…”

Thyroid hormones decrease the affinity of 8-cyclopentyl-1,3-dipropylxanthine (CPX), a competitive antagonist, for the guinea pig atrial A1 adenosine receptor

“…This phenomenon cannot be exclusively explained by the moderately decreased A 1 receptor affinity explored in the present study, so (an)other mechanism(s) should be supposed. Thyroid hormones increased the A 1 receptor number in both the rat atrium (Kaasik et al 1994) and ventricle (El-Ani et al 1994), with unchanged concentration of the relevant G protein subtypes (El-Ani et al 1994;Kaasik et al 1994). Starting from this, the suppressed negative inotropic response cannot be ascribed to a change in the A 1 receptor or G protein density in hyperthyroidism.…”