Arterioscler Thromb Vasc Biol Cyclic AMP Raises Intracellular Ca2+ in Human Megakaryocytes Independent of Protein Kinase A by Els den Dekker, Johan W.M. Heemskerk, Gertie Gorter, Hans van der Vuurst, José Donath, Christine Kroner, Katsuhiko Mikoshiba, and Jan-Willem N. Akkerman Arterioscler Thromb Vasc Biol Volume 22(1):179-186 January 1, 2002 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Calcium and cAMP increases in megakaryoblastic cell lines. Figure 1. Calcium and cAMP increases in megakaryoblastic cell lines. Megakaryoblastic cell lines were loaded with fura-2/AM, and Ca2+ increases, in the presence of 1 mmol/L extracellular Ca2+ (A, C), and cAMP formation (B), induced by iloprost (1 μmol/L) or thrombin (5 U/mL), were measured in MEG-01 cells (open bars) and CHRF-288-11 cells (hatched bars). Basal levels of [Ca2+]i were 131±15 and 112±9 nmol/L for MEG-01 and CHRF-288-11 cells, respectively. Basal levels of cAMP were 12±3 and 8.0±3 pmol/106 cells for MEG-01 and CHRF-288-11 cells, respectively. Data are expressed as mean±SD (n=3). Els den Dekker et al. Arterioscler Thromb Vasc Biol. 2002;22:179-186 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Ca2+ responses in stem cells and cultured megakaryocytes. Figure 2. Ca2+ responses in stem cells and cultured megakaryocytes. A (upper panel) shows Ca2+ responses in the presence of 1 mmol/L extracellular Ca2+ induced by iloprost (1 μmol/L) in suspensions of stem cells (1), immature (2), and mature megakaryocytes (3). A representative tracing for 3 observations with similar results is shown. A (lower panel) shows Ca2+ responses of single immobilized cells: (1) stem cells (day 1 cells adhering to anti-CD34); (2) immature megakaryocytes (CD42blow day 7 cells adhering to anti-CD61); and (3) mature megakaryocytes (CD42bhigh day 14 cells adhering to anti-CD61). A representative tracing for at least 7 observations with similar results is shown. B shows similar experiments for cells stimulated by 1 U/mL thrombin. Els den Dekker et al. Arterioscler Thromb Vasc Biol. 2002;22:179-186 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. cAMP-elevating agents induce release of Ca2+ from stores and influx. Figure 3. cAMP-elevating agents induce release of Ca2+ from stores and influx. A and B show Ca2+ responses of immobilized immature megakaryocytes in the presence of 1 mmol/L extracellular Ca2+ after stimulation with (A) carbaprostacylin (1 μmol/L) or (B) forskolin (100 μmol/L). The inserts show Ca2+ responses in nominally Ca2+-free buffer. A representative tracing for at least 10 observations with similar results is shown. C shows Ca2+ responses of suspensions of MEG-01 cells in the presence of 1 mmol/L extracellular Ca2+ after stimulation with forskolin (40 μmol/L, left panel), IBMX (500 μmol/L, middle panel), and iloprost (1 μmol/L) after preincubation with IBMX (500 μmol/L, 5 minutes, 37°C; right panel). Els den Dekker et al. Arterioscler Thromb Vasc Biol. 2002;22:179-186 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Forskolin-induced Ca2+ increases do not involve IP3 receptors or PKA. A, Platelets and MEG-01 cells were preincubated with the IP3 receptor inhibitor 2-APB (100 μmol/L, 2 minutes, 37°C), and Ca2+ increases were measured in nominally Ca2+-free buffer on addition of thrombin (1 U/mL; left and right panels) or forskolin (100 μmol/L; middle panel). Figure 4. Forskolin-induced Ca2+ increases do not involve IP3 receptors or PKA. A, Platelets and MEG-01 cells were preincubated with the IP3 receptor inhibitor 2-APB (100 μmol/L, 2 minutes, 37°C), and Ca2+ increases were measured in nominally Ca2+-free buffer on addition of thrombin (1 U/mL; left and right panels) or forskolin (100 μmol/L; middle panel). B, MEG-01 cells were preincubated with the PKA inhibitor H89 (15 minutes, 37°C, 10 μmol/L), and phosphorylation of VASP induced by forskolin (100 μmol/L) was determined (left panel). Ca2+ increases were measured in H89-treated cells in nominally Ca2+-free buffer on addition of forskolin (100 μmol/L, middle panel) or thrombin (1 U/mL, right panel). C, Phosphorylation of VASP (left panel) and Ca2+ increases in nominally Ca2+-free buffer (right panel) were measured after stimulation of PKA with the activator Sp-5,6-DCl-cBIMPS (800 μmol/L) and subsequently forskolin (100 μmol/L, right panel). Tracings are representative for 3 observations with similar results. Els den Dekker et al. Arterioscler Thromb Vasc Biol. 2002;22:179-186 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. Rap1 activation in MEG-01 and CHRF-288-11 cells. Figure 5. Rap1 activation in MEG-01 and CHRF-288-11 cells. MEG-01 cells (A, B) and CHRF-288-11 cells (C) were treated with iloprost (1 μmol/L, ○) or thrombin (1 U/mL, ). After lysis, GTP-Rap1 was precipitated with glutathione S-transferase-coupled Rap binding domain of guanine nucleotide dissociation stimulator for Ral-coupled glutathione-agarose beads and identified by Western blotting with a monoclonal antibody against Rap1 (A, C) or polyclonal antibodies specifically recognizing Rap1a or Rap1b (B). To quantify the Rap1 activation induced by iloprost and thrombin, films were scanned and bands were quantified with ImageQuant software. The pixel density for each lane within the same experiment and analyzed on the same film was related to the reference sample present on the same blot. Data represent relative amounts of GTP-Rap1 (mean±SD, n=3). Els den Dekker et al. Arterioscler Thromb Vasc Biol. 2002;22:179-186 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. Forskolin releases Ca2+ from stores and activates Rap1 independent of PKA. MEG-01 and CHRF-288-11 cells were treated with forskolin (100 μmol/L), and Ca2+ increases in nominally Ca2+-free buffer (A) and Rap1 activation (B, left panel) were measured. Figure 6. Forskolin releases Ca2+ from stores and activates Rap1 independent of PKA. MEG-01 and CHRF-288-11 cells were treated with forskolin (100 μmol/L), and Ca2+ increases in nominally Ca2+-free buffer (A) and Rap1 activation (B, left panel) were measured. B, right panel, shows Rap1 activation in MEG-01 cells induced by IBMX (500 μmol/L). Representative examples of 3 experiments with similar results are shown. GTP-Rap1 bands on Western blots were scanned and quantified as described in the Methods section and the legend to Figure 5. Mean±SD (n=3) for activated Rap1 in MEG-01 and CHRF-288-11 cells is shown in C. D, MEG-01 cells were preincubated with H89 (15 minutes, 37°C, 10 μmol/L) and BAPTA-AM (30 minutes, 37°C, 30 μmol/L) or vehicle, and Rap1 activation induced by forskolin (100 μmol/L) was determined after 15 seconds. Data show mean±SD, n=3. GTP-Rap1 was significantly increased after forskolin treatment (P<0.05, n=3, compared with control). Pretreatment with H89 and BAPTA-AM did not significantly change the forskolin-induced Rap1 activation (P>0.05). Rap1 phosphorylation was determined by a gel-shift assay for samples derived from MEG-01 cells treated with forskolin (100 μmol/L; E, upper panel) and platelets treated with iloprost (5 μmol/L; E, lower panel). Els den Dekker et al. Arterioscler Thromb Vasc Biol. 2002;22:179-186 Copyright © American Heart Association, Inc. All rights reserved.