Effects of theophylline and dibutyryl-cAMP on adenosine receptors and heart rate in cultured cardiocytes

Abstract

The effects of chronic exposure to the adenosine antagonist theophylline (Theo) and dibutyryl cyclic-AMP, a membrane-permeant derivative of the second messenger 3', 5'-cyclic-AMP (cAMP), on contractions and adenosine receptor levels in cultured cardiocytes were studied. Binding of the A1-adenosine receptor antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) was used to monitor the level of the receptors in intact cardiocytes. Both Theo and cAMP stimulated the rate of contraction and also increased the density of adenosine receptors. The Bmax value for [3H]CPX binding to intact cardiocytes was increased by 45-47% following 4 days of exposure to either 50 microM Theo or 100 microM cAMP. Scatchard analysis indicated that the affinity of the A1 receptors for [3H]CPX remained unchanged (Kd 0.1-0.2 nM). No significant differences were observed in protein content or in cell number. A linear correlation was achieved between the level of A1-adenosine receptors and heart rate at various Theo and dibutyryl-cAMP concentrations, although Theo was more efficient in elevation of the receptor density. Increases of 82, 78, 138 and 235% in A1 receptor density and increases of 63, 59, 66 and 150% in heart rate were obtained following 5 days of treatment with 1, 10, and 1000 microM of Theo, respectively. It is concluded that there is a linkage between the rate of cardiac contractions and the level of adenosine receptors. Thus, changes in the density of adenosine receptors may compensate for chronic drug-induced changes in cardiac contractile activity so as to restore conditions to the normal state.

Fig. 1

Effect of theophylline on [³H]CPX binding. A. Five day-old cardiocytes were treated with 50 μM Theo. Specific binding of [³H]CPX was determined following 84 hours. The cardiocytes were exposed to the indicated concentrations of the radioligand as described in Materials and Methods. Each value is the mean ± S.D. of triplicate determinations from a representative experiment of sister cultures. B. Scatchard plot of specific [³H]CPX binding of Fig. 1. K_d = 0.19 and 0.18 nM, and B_max = 20.5 and 28 fmol/dish in control and Theo treated cells, respectively.

Fig. 2

Dose response of theophylline on [³H]CPX binding and heart rate. A. Four day-old cardiocytes were treated with the indicated concentrations of Theo for 5 days. Specific binding of [³H]CPX (●) and heart rate (□) were then determined. The results are expressed as means ± S.D. of at least triplicate ([³H]CPX binding) or 5 determinations (heart rate) from a representative experiment. B. The relationship between [³H]CPX binding and heart rate of Fig. 2A. The correlation coefficient, r = 0.941, was obtained using Spearman’s test.

Fig. 3

Contractile responses to theophylline. Four day-old cardiocytes were treated with the following concentrations of Theo for 5 days: a. control, b. 0.1 μM, c. 1.0 μM, d. 100 μM. Contractility (beats/min) measured under a phase contrast microscope was 42±5, 67±7, 71 ±7 and 72±6. respectively. The results are from a representative experiment that was measured during 10 sec from one cardiocyte per dish. For every treatment at least 5 cells from different culture-dishes were monitored.

Fig. 4

Effect of cAMP on [³H]CPX binding. A. Four day-old cardiocytes were treated with 100 μM dibutyryl-cAMP. Specific binding of [³H]CPX was determined following 96 hours. The cardiocytes were exposed to the indicated concentrations of the radioligand as described in Materials and Methods. Each value is the mean ± S.D. of triplicate determinations from a representative experiment of sister cultures. B. Scatchard plot of specific [³H]CPX binding of Fig. 4A. K_d = 0.12 and 0.13 nM, and B_max = 24.4 and 36.0 fmol/dish in control and cAMP treated cells, respectively.

Fig. 5

Dose response of cAMP on [³H]CPX binding and heart rate. Five day-old cardiocytes were treated with the indicated concentrations of dibutyryl-cAMP for 3 days. Specific binding of [³H]CPX (A) and myocyte contractility following 100 μM dibutyryl-cAMP (B) were determined. The results are expressed as means ± S.D. of at least triplicate ([³H]CPX binding) or 5 determinations (heart rate) from a representative experiment.