Differential regulation of P2Y(11) receptor-mediated signalling to phospholipase C and adenylyl cyclase by protein kinase C in HL-60 promyelocytes

Abstract

The regulatory mode of the P2Y(11) purinoceptor-mediated signalling cascades towards phospholipase C and adenylyl cyclase was studied in HL-60 promyelocytes. Treatment with the potent P2Y(11) receptor activator dATP evoked an elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) and inositol 1,4,5-trisphosphate (IP(3)) production that was sustained for longer than 30 min. However, the dATP-induced responses were significantly inhibited by the activation of protein kinase C after a short exposure to phorbol 12-myristate 13-acetate (PMA). dATP also potently stimulated cyclic AMP production with half maximum effect seen at 23+/-7 microM dATP. In addition, a 5-min pretreatment with PMA enhanced the dATP-stimulated cyclic AMP accumulation. PMA potentiated the cyclic AMP production when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment. dATP also enhanced the forskolin-mediated cyclic AMP generation. Treatment of the cells with 10 microM U-73122, which almost completely blocked the dATP-stimulated IP(3) production and [Ca(2+)](i) rise, had no effect on cyclic AMP accumulation, while 10 microM 9-(tetrahydro-2-furyl)adenine (SQ 22536), which inhibited the adenylyl cyclase activation, did not effect the dATP-stimulated phosphoinositide turnover. Taken together, the results indicate that P2Y(11) receptor-mediated activation of phospholipase C and adenylyl cyclase occurs through independent pathways and is differentially regulated by protein kinase C in HL-60 cells.

Figure 1

Effects of dATP and UTP on the [Ca²⁺]_i rise in HL-60 cells. Typical patterns of [Ca²⁺]_i rise in cells pretreated with vehicle (control) or 30 nM PMA for 5 min and obtained upon treatment with dATP (30 μM) and UTP (30 μM) in the presence (A) or absence (B) of 2.2 mM extracellular Ca²⁺. (C) Concentration-dependent effect of dATP on [Ca²⁺]_i rise. Fura-2/AM-loaded cells were treated with vehicle (control) or 30 nM PMA for 5 min and then stimulated with various concentrations of dATP. Net increases in [Ca²⁺]_i were measured as described under Methods. (D) Concentration-dependent effect of PMA on dATP- and UTP-induced [Ca²⁺]_i rise. The cells, pretreated with various concentrations of PMA for 5 min, were stimulated with 30 μM dATP or UTP, and net increases in [Ca²⁺]_i were measured. The experiments were done three to five times and each point is the mean±s.e.mean.

Figure 2

Effect of PPADS on dATP- and UTP-stimulated intracellular [Ca²⁺]_i rise. (A) Fura-2-loaded cells were incubated with vehicle (control) or 10 μM PPADS for 5 min and then treated with 100 μM dATP or UTP. Typical patterns of [Ca²⁺]_i rise were presented. (B) Concentration-dependent effect of PPADS on dATP- and UTP-stimulated [Ca²⁺]_i rise. Fura-2/AM-loaded cells were treated with various concentrations of PPADS, and then stimulated with 100 μM dATP or UTP. Net increases in [Ca²⁺]_i were presented as percentage of control (dATP or UTP alone). The experiments were done three times and each point is the mean±s.e.mean.

Figure 3

Effect of dATP and UTP on IP₃ production in HL-60 cells. (A) Time course of IP₃ generation evoked by dATP or UTP. The cells were stimulated with 100 μM dATP or UTP for the designated times (0, 0.25, 0.5, 1, 3, 5, 10, 20, 40, 60 min), and the reactions were stopped by addition of 15% (w v⁻¹) TCA containing 10 mM EGTA. (B) Concentration-dependent stimulation of IP₃ formation by dATP. The cells, pretreated with vehicle (control) or 30 nM PMA for 5 min, were treated with various concentrations of dATP for 15 s, and the IP₃ production was measured by competition assay as described under Methods. (C) Concentration-dependent effect of PMA on dATP- and UTP-stimulated IP₃ generation. The cells were pretreated with various concentrations of PMA for 5 min and then stimulated with 30 μM dATP or UTP for 15 s. IP₃ generation was measured as described under Methods. The experiments were done three times and each point is the mean±s.e.mean.

Figure 4

Effects of dATP and UTP on cyclic AMP generation in HL-60 cells. (A) Time-dependent changes in intracellular cyclic AMP levels stimulated by 100 μM dATP or UTP. The cells were incubated in 50 μM Ro 20-1724 for 20 min and then stimulated with dATP or UTP for the indicated times. The cyclic AMP accumulation was measured as described under Methods. (B) Concentration-dependence of dATP-stimulated cyclic AMP generation. The cells were treated with vehicle (control) or 30 nM PMA for 5 min and then stimulated with various concentrations of dATP for 20 min. (Inset) Cells were simultaneously stimulated with dATP (100 μM) and PMA (30 nM) or pretreated with PMA for 5 min and then stimulated with dATP, and then cyclic AMP production was measured. (C) Concentration-dependent effect of PMA on dATP-stimulated cyclic AMP generation. The cells were treated with 100 μM dATP and various concentrations of PMA or 4-α-PMA for 20 min. (D) PMA effect on cyclic AMP generation in HL-60 cells stimulated by various nucleotide analogues. The cells were treated with the analogues for 20 min in the presence or absence of 30 nM PMA, and the cyclic AMP levels were measured as described under Methods. Each point is the mean±s.e.mean of 3–5 experiments.

Figure 5

Effect of U-73122 on P2Y₁₁ receptor-mediated signalling in HL-60 cells. (A) Concentration-dependent inhibition of dATP-induced [Ca²⁺]_i rise by U-73122. Fura-2/AM-loaded cells were treated with various concentrations of U-73122 for 10 min and then stimulated with 100 μM dATP, and the peak [Ca²⁺]_i level was measured. (Inset) Typical pattern of [Ca²⁺]_i rise after treatment with dATP in the absence or presence of 10 μM U-73122. (B) Effect of U-73122 on dATP-stimulated IP₃ generation. The cells, pretreated with vehicle (control) or 10 μM U-73122 for 10 min, were stimulated with 100 μM dATP for 15 s, and the IP₃ generation was measured as described under Methods. (C) Effect of U-73122 on dATP-induced cyclic AMP generation. The cells, pretreated with vehicle (control) or 10 μM U-73122 for 10 min, were stimulated with 100 μM dATP for 20 min, and the cyclic AMP accumulation was measured as described under Methods. The experiments were done three times and each point is the mean±s.e.mean.

Figure 6

Effect of SQ 22536 on dATP-stimulated signalling pathways in HL-60 cells. (A) Concentration-dependent inhibition of the dATP-induced cyclic AMP production by SQ 22536. Fura-2/AM-loaded cells were treated with various concentrations of SQ 22536 for 10 min and then stimulated with 100 μM dATP, and cyclic AMP accumulation was measured. (B) Effect of SQ 22536 on dATP-stimulated IP₃ generation. The cells, pretreated with vehicle (control) or 100 μM SQ 22536 for 10 min, were stimulated with 100 μM dATP for 15 s, and the IP₃ generation was measured as described under Methods. The experiments were done three times and each point is the mean±s.e.mean.