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 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 (Kd = 0.13 nM) and the number of [3H]CPX binding sites (Bmax) was 23.1 fmol/dish (21 fmol/mg protein). The Ki 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. Bmax 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(-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 [3H]CPX. Carbamylcholine (5 x 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.

Fig. 1

Time-course of [³H]CPX binding to heart cells. Six-day-old cardiocytes were incubated in the presence of 0.6nM [³H]CPX at 25°. Specific binding was determined as the [³H]CPX binding displaceable by 5 mM theophylline. Each value is the mean ± SE of triplicate determinations from a representative experiment of sister cultures. Inset: Pseudo first order kinetic plot of [³H]CPX binding. B = amount of [³H]CPX bound at each time. Be = amount of [³H]CPX bound at equilibrium.

Fig. 2

Dissociation curve of [³H]CPX binding from heart cells. Reverse kinetics of dissociation of [³H]CPX from cardiocytes (6 days old). Specific bindings at 25° were determined at subsequent time intervals. After 60 min of incubation the binding mixture (0.6 nM [³H]CPX in PBS) was removed and PBS containing 5 mM of theophylline was added. Each value is the mean ± SE of triplicate determinations from a representative experiment. Inset: First order kinetic plot of dissociation of [³H]CPX bound at each time after dilution of the binding mixture at 25°. Be = amount of [³H]CPX bound at time 0. B = amount of [³H]CPX bound at the indicated time. The association constant, k_off = 0.099/min.

Fig. 3

Specific binding of [³H]CPX to heart cells. Six-day-old cardiocytes were exposed to the indicated concentrations of the radioligand as described in Materials and Methods. Specific binding was determined as the [³H]CPX binding displaceable by 5 mM theophylline. Each value is the mean ± SE of triplicate determinations from a representative experiment of sister cultures. Inset: Scatchard plot of specific [³H]CPX binding of Fig. 3.The K_d for [³H]CPX was 0.13 ± 0.07 nM and the maximal binding capacity was 23.1 ± 2.9 nM (21 fmol/mg protein).

Fig. 4

Inhibition of [³H]CPX binding to heart cells. Six-day-old cardiocytes were incubated in the presence of [³H]CPX (0.7 nM) and increasing concentration of different drugs. After 60min of incubation at room temperature, specific binding was estimated as described in Materials and Methods. The results are expressed as the percentage of [³H]CPX specifically bound. Data points represent means ± SE of triplicate determinations from a representative experiment. The K_i, values for CPX, R-PIA, S- PIA and theophylhne were 1.63, 3.57, 49 and 4880 nM, respectively.

Fig. 5

Age dependence of specific binding of [³H]CPX to heart cells in comparison to CK activity. The level of [³H]CPX binding and CK activity were determined at the indicated time on sister cultures according to Materials and Methods. The results are expressed (A) per dish, or (B) per protein (means ± SE of triplicate determinations from a representative experiment).

Fig. 6

Effect of NE on [³H]CPX binding. Four-day-old cardiocytes were treated with 20 μm NE. Specific binding of [³H]CPX was determined following 4 days. Inset: Scatchard plot of specific [³H]CPX binding of Fig. 6. k_d = 0.18 and 0.17nM, and B_max = 31.4 and 88.2 fmol/dish in control and NE-treated cells, respectively.

Fig. 7

Time course of NE effect on adenosine receptors. Four-day-old cardiocytes were treated with 20 μM NE for 2, 3 and 4 days and with 10 μM Iso for 2 days. Then, the levels of specific [³H]CPX binding were determined. The results are expressed as means ± SE of triplicate determinations from a representative experiment.

Fig. 8

Dose–response of NE or Iso on [³H]CPX binding. Four-day-old cardiocytes were treated with the indicated concentrations of NE for 60 hr, or Iso for 48 hr. Specific binding of [³H]CPX was determined.

Fig. 9

Effect of TH on [³H]CPX binding. Four-day-old cardiocytes were treated with 10nM T₃. Specific binding of [³H]CPX was determined after 72 hr. (The experiment was carried out in multi wells.) Inset: Scatchard plot of specific [³H]CPX binding of Fig, 9. The K_d for [³H]CPX was 0.11 nM. B_max was 3.2 and 5.0 fmol/dish in control group and T₃-treated cells, respectively.

Fig. 10

Dose–response of TH on [³H]CPX binding. Five-day-old cardiocytes were treated with various concentrations of T₃. [³H]CPX binding was determined 84 hr later.

Fig. 11

Effect of Carb on [³H]CPX binding. Four-day-old cardiocytes were treated with 5 μM Carb for 72 hr before specific binding of [³H]CPX was determined. Inset: Scatchard plot of specific [³H]CPX binding of Fig. 11. K_d = 0.16 and 0.17 nM, and B_max = 25.5 and 18.7 fmol/dish in control and Carb-treated cells, respectively.

Fig. 12

Dose–response of Carb [³H]CPX binding. Five-day-old cardiocytes were treated with various concentrations of Carb and [³H]CPX binding was determined 84 hr later.