FIGURE 6.
8-pCPT-modified cAMP/ado analogs elicit activation of adenylyl cyclase, PKA, and anti-apoptotic effect by an ENT1- and A2AR-dependent mechanism in PC12 cells. A, PC12 cells were incubated with 8-pCPT-cAMP or 8-pCPT-ado (100 μm each) in the presence or absence of ADA (2 units/ml) for 10 or 20 min followed by the determination of [cAMP]i. Values are means ± S.D. (error bars) of three independent determinations. B, PC12 cells were stimulated with 8-pCPT-ado (100 μm) or vehicle (DMSO) in the presence of absence of SCH 58261 (100 nm) for the indicated time followed by immunoblotting of the cell lysates with an anti-PKA substrate motif or anti-β-actin. C, PC12 cells were stimulated with adenosine (10 μm) or 8-pCPT-ado (100 μm) in the presence or absence of ADA (2 units/ml) for the indicated time followed by immunoblotting of cell lysates with anti-PKA substrate motif or anti-ERK1/2. D, the determination of [3H]thymidine uptake by PC12 cells in the presence and absence of 8-pCPT-ado (100 μm) or ADA (2 units/ml). Values are means ± S.D. (error bars) of three independent determinations. E, PC12 cells were stimulated with 8-pCPT-cAMP (100 μm), CGS 21680 (1 μm), or vehicle (DMSO) in the presence of absence of SCH 58261 (100 nm) for the indicated time followed by immunoblotting of the cell lysates with an anti-PKA substrate motif or anti-ERK1. F, PC12 cells were incubated with 8-pCPT-cAMP, 8-pCPT-ado, 8-pCPT-2′-O-methyl-cAMP, or 8-pCPT-2′-O-methyl-ado (10 μm each) in the presence or absence of ADA in serum-free DMEM for 24 h followed by the determination of apoptotic cells by Hoechst staining. Values are means ± S.D. (error bars) of three different wells from one representative experiment. Asterisks indicate a significant difference; n.s., not significant.