Spatio-temporal propagation of Ca2+ signals by cyclic ADP-ribose in 3T3 cells stimulated via purinergic P2Y receptors

Abstract

The role of cyclic ADP-ribose in the amplification of subcellular and global Ca2+ signaling upon stimulation of P2Y purinergic receptors was studied in 3T3 fibroblasts. Either (1) 3T3 fibroblasts (CD38- cells), (2) 3T3 fibroblasts preloaded by incubation with extracellular cyclic ADP-ribose (cADPR), (3) 3T3 fibroblasts microinjected with ryanodine, or (4) 3T3 fibroblasts transfected to express the ADP-ribosyl cyclase CD38 (CD38+ cells) were used. Both preincubation with cADPR and CD38 expression resulted in comparable intracellular amounts of cyclic ADP-ribose (42.3 +/- 5.2 and 50.5 +/- 8.0 pmol/mg protein). P2Y receptor stimulation of CD38- cells yielded a small increase of intracellular Ca2+ concentration and a much higher Ca2+ signal in CD38-transfected cells, in cADPR-preloaded cells, or in cells microinjected with ryanodine. Confocal Ca2+ imaging revealed that stimulation of ryanodine receptors by cADPR or ryanodine amplified localized pacemaker Ca2+ signals with properties resembling Ca2+ quarks and triggered the propagation of such localized signals from the plasma membrane toward the internal environment, thereby initiating a global Ca2+ wave.

Figure 1.

ATP-induced Ca^{2 +} release in CD38 ⁺ and CD38 ⁻ 3T3 fibroblasts. ATP (100 μM final concentration) was added to FURA-2/AM–loaded CD38⁺ (open rhombus) and to CD38⁻ (open square) 3T3 cells in calcium buffer (A), or in the presence of 2 mM EGTA (B). [Ca²⁺]_i changes were measured using a fluorescence plate reader, as described in the Materials and methods. Characteristic tracings are shown (n = 9 for CD38⁺ in calcium buffer, n = 13 for CD38⁻ in calcium buffer, n = 5 for CD38⁺ in the presence of 2 mM EGTA, n = 7 for CD38⁻ in the presence of 2 mM EGTA).

Figure 2.

ATP-induced Ca^{2 +} release in CD38 ⁻ 3T3 fibroblasts preincubated with cADPR and in CD38 ⁺ 3T3 fibroblasts preincubated with 8-Br-cADPR. (A) CD38⁻ 3T3 cells were preincubated for 10 min in the presence (filled square) or absence (open square) of 50 μM cADPR, as described in the Materials and methods, before ATP addition (100 μM final concentration). (B) CD38⁺ 3T3 cells were preincubated for 2 h with (filled rhombus) or without (open rhombus) 100 μM 8-Br-cADPR, as described in the Materials and methods, before ATP addition (100 μM final concentration). [Ca²⁺]_i was monitored using a fluorescence plate reader, as described in the Materials and methods. Characteristic tracings are shown (n = 13 for CD38⁻ in the presence of 50 μM cADPR, n = 11 for CD38⁻ in the absence of 50 μM cADPR, n = 4 for CD38⁺ in the presence of 100 μM 8-Br-cADPR, n = 4 for CD38⁺ in the absence of 100 μM 8-Br-cADPR).

Figure 3.

Role of IP₃ in the ATP-induced Ca^{2 +} release in CD38 ⁺ 3T3 fibroblasts. (A) CD38⁺ 3T3 cells were incubated for 5 min in the presence of 250 μM 2-APB. (B) CD38⁺ 3T3 cells were incubated for 5 min in the presence of 1 μM U73343 (open rhombus) or U73122 (filled rhombus). ATP (100 μM) was then added to cells, and [Ca²⁺]_i was measured using a plate reader fluorometer, as described in the Materials and methods. Characteristic tracings are shown (n = 6 for CD38⁺ in the presence of 250 μM 2-APB, n = 3 for CD38⁺ in the presence of 1 μM U73343, n = 3 for CD38⁺ in the presence of 1 μM U73122).

Figure 4.

Global Ca^{2 +} wave development in 3T3 fibroblasts. Confocal pseudocolor Ca²⁺ images of selected cells for each condition: CD38⁻ (A), CD38⁻ cells preincubated with 50 μM cADPR (B), CD38⁻ cells microinjected with ryanodine 15 min before addition of ATP (Ry; pipette concentration 100 μM, final intracellular concentration ∼1 μM) (C), and CD38⁺ cells (D) immediately before and after stimulation by ATP are displayed (Bars, 10 μm). (E) Quantitative analysis of Ca²⁺ wave development in the four cells displayed in A–D; [Ca²⁺]_i from different subcellular regions of interest (color coded; the dashed circles indicate nuclei) are plotted against time. Characteristic cells were selected from n = 18 CD38⁻ (A), n = 19 CD38⁻ cells preincubated with cADPR (B), n = 15 CD38⁻ cells microinjected with ryanodine (C), and n = 20 CD38⁺ cells (D) analyzed in total.

Figure 5.

Analysis of subcellular Ca^{2 +} signals in 3T3 fibroblasts. Confocal pseudocolor Ca²⁺ images of the early pacemaker phase upon ATP stimulation of a CD38⁻ cell (A), a CD38⁻ cell preincubated with 50 μM cADPR (B), a CD38⁻ cell microinjected with ryanodine 15 min before addition of ATP (Ry; pipette concentration 100 μM, final intracellular concentration ∼1 μM) (C), and a CD38⁺ cell (D), and magnifications of subcellular regions are displayed (Bars, 2.5 μm). The cells were stimulated at time point 115 s by 100 μM ATP. Right panel, Ca²⁺ tracings of selected ROIs (as indicated and color coded in the middle panel) are displayed. Characteristic cells were selected from n = 18 CD38⁻ cells (A), n = 19 CD38⁻ cells preincubated with cADPR (B), n = 15 CD38⁻ cells microinjected with ryanodine, and n = 20 CD38⁺ cells.