Pharmacological characterization of beta2-adrenoceptor in PGT-beta mouse pineal gland tumour cells

Abstract

1. The adrenoceptor in a mouse pineal gland tumour cell line (PGT-beta) was identified and characterized using pharmacological and physiological approaches. 2. Adrenaline and noradrenaline, adrenoceptor agonists, stimulated cyclic AMP generation in a concentration-dependent manner, but had no effect on inositol 1,4,5-trisphosphate production. Adrenaline was a more potent activator of cyclic AMP generation than noradrenaline, with half maximal-effective concentrations (EC50) seen at 175+/-22 nM and 18+/-2 microM for adrenaline and noradrenaline, respectively. 3. The addition of forskolin synergistically stimulated the adrenaline-mediated cyclic AMP generation in a concentration-dependent manner. 4. The pA2 value for the specific beta2-adrenoceptor antagonist ICI-118,551 (8.7+/-0.4) as an antagonist of the adrenaline-stimulated cyclic AMP generation were 3 units higher than the value for the betaI-adrenoceptor antagonist atenolol (5.6+/-0.3). 5. Treatment of the cells with adrenaline and forskolin evoked a 3 fold increase in the activity of serotonin N-acetyltransferase with the peak occurring 6 h after stimulation. 6. These results suggest the presence of beta2-adrenoceptors in mouse pineal cells and a functional relationship between the adenylyl cyclase system and the regulation of N-acetyltransferase expression.

Figure 1

Adrenaline- and noradrenaline-induced cyclic AMP production in PGT-β cells. (A) [³H]adenine-loaded cells were preincubated with IBMX (1 mM) for 20 min and then stimulated with variable concentrations of adrenaline (Ad) and noradrenaline (NA) for 20 min. (B) The cells were stimulated with 1 μM adrenaline (Ad) or 300 μM noradrenaline (NA) for the indicated times (0, 1, 3, 5, 10, 20, 30 min) and cyclic AMP generation was measured as described in Methods. The inset shows the time-dependent changes in cyclic AMP accumulation induced by 1 μM adrenaline in the presence of 1 mM IBMX. The experiments were done three times and each point is the mean±s.e.mean.

Figure 2

Effect of α-adrenoceptor agonist and antagonist in PGT-β cells. (A) Cells were stimulated with 1 μM adrenaline, 10 μM phenylephrine, or 300 μM ATP for 30 s, and the reactions were stopped by addition of 15% (w/v) trichloroacetic acid containing 10 mM EGTA. The inositol 1,4,5-trisphosphate generation was measured as described in Methods. (B) [³H]adenine-loaded cells were preincubated with IBMX (1 mM) for 20 min and then stimulated with 1 μM adrenaline (Ad) in the absence or presence of 10 μM clonidine (Cl), 30 μM yohimbine (Yh), and 1 μM propranolol (Pr) for 20 min. The cyclic AMP generation was measured as described in Methods. The experiments were done three times and each point is the mean±s.e.mean. ^*P<0.05, compared to control.

Figure 3

Enhancement of forskolin-stimulated cyclic AMP generation by adrenaline. (A) [³H]adenine-loaded PGT-β cells were preincubated with IBMX (1 mM) for 20 min and then stimulated with various concentrations of forskolin plus 1 μM adrenaline (•) for 20 min. The inset shows the concentration-dependent changes in forskolin-stimulated (○) cyclic AMP generation. (B) The time-dependent changes in cyclic AMP accumulation induced by 10 μM forskolin plus 1 μM adrenaline (•) or forskolin alone (○) for the indicated times (0, 1, 3, 5, 10, 20, 30 min) in the absence of IBMX. The cyclic AMP levels were measured as described in Methods. The experiments were done three times and each point is the mean±s.e.mean.

Figure 4

Effects of ionomycin on cyclic AMP generation. (A) Time-dependent changes in intracellular cyclic AMP levels stimulated by 1 μM adrenaline in the presence or absence of 1 μM ionomycin. (B) Concentration-dependent inhibition of adrenaline-stimulated cyclic AMP generation by ionomycin. [³H]adenine-loaded cells were treated with 1 μM adrenaline and variable concentrations of ionomycin for 3 min. The cyclic AMP generation was presented as percentage of the maximal response induced by treatment with 1 μM adrenaline. (C) Inhibition of forskolin-stimulated cyclic AMP generation by ionomycin. [³H]adenine-loaded cells were treated with 10 μM forskolin and ionomycin for 3 min. The cyclic AMP levels were measured as described in Methods. The experiments were done three times and the results were reproducible. Data are the means±s.e.mean. ^*P<0.05, compared to control.

Figure 5

Effect of isoprenaline and BRL 37344 on cyclic AMP production in PGT-β cells. [³H]adenine-loaded cells were preincubated with IBMX (1 mM) for 20 min and then stimulated with variable concentrations of isoproterenol and BRL 37344 for 20 min. The cyclic AMP generation was measured as described in Methods and presented as percentage of the maximal response induced by treatment with 1 μM isoproterenol. The experiments were done three times and each point is the mean±s.e.mean.

Figure 6

The antagonism by β-adrenoceptor antagonists of adrenaline-stimulated cyclic AMP generation. Concentration-response curves for the antagonistic effect of increasing concentrations of ICI 118,551 (A) and atenolol (C) on adrenaline-stimulated cyclic AMP generation are shown. The pA₂ values of ICI 118,551 (B) and atenolol (D) were calculated for each antagonist by plotting: log (dose ration⁻¹) vs −log(antagonist) concentration. The concentrations of ICI 118,551 used to counteract the effects of adrenaline were 0 (○), 10 nM (•), 100 nM (Δ), 1 μM (▴), and 10 μM (▪). The concentrations of atenolol were 0 (○), 3 μM (•), 30 μM (Δ), and 300 μM (▴). The concentration of adrenaline ranged from 10⁻⁹–10⁻³ M. The cyclic AMP generation was presented as percentage of the maximal response induced by treatment with adrenaline. One typical experiment out of three independent experiments in duplicate is shown.

Figure 7

Time-dependent effect of β-adrenoceptor activation on N-acetyltransferase activity in PGT-β cells. The cells were treated with 1 μM adrenaline and 10 μM forskolin in the absence (○) or presence (•) of ICI-118,551 (10 μM) for the indicated times (0, 1, 3, 6, 12, 24 h) and N-acetyltransferase activity was assayed as described in Methods. Results were confirmed by two independent experiments and expressed as the means±s.e.mean.