Characterization of adenosine receptors in intact cultured heart cells

Adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. We used the hydrophilic A₁ adenosine receptor radioligand [^{3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]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 [3H]CPX binding sites (B_max) was 23.1 fmol/dish (21 fmol/mg} protein). The K_i, of the agonists. R-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) and S-N⁶-(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 (T₃, 10^-8M), which also accelerated heart rate, increased the number of adenosine receptors by 56% without a significant change in the affinity of the receptors to [³H]CPX. Carbamylcholine (5 × lO^-6M), 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.