![]() ![]() C, VSMC were stimulated with the forskolin doses indicated for a further 6 h in the presence of 10 μM BrdU and analysed for BrdU incorporation, n = 3 D and E, cell lysates were analysed by western blotting as indicated, n = 5. B, VSMC were incubated for 6 h in the presence of 10 μM BrdU and analysed for BrdU incorporation, n = 9. A, Rap1GAP activity was confirmed by Rap1 activity assay. VSMC were stimulated with the indicated forskolin doses for 18 h. High dose of Ad:Rap1GAP was used unless otherwise indicated. Where indicated, asynchronous VSMC were infected with an adenovirus encoding Rap1GAP at 10 8 pfu/ml (high) or 2 × 10 7 pfu/ml (low) (A–E), or transfected with siRNA specific for Rap1 as described (F, G). Inhibition of Rap1 does not negate the effects of cAMP on cell-cycle progression. # indicates p < 0.05 versus corresponding dose in Ad:Control. * indicates p < 0.05, ** indicates p < 0.01 versus Ad:Control alone. Recombinant PKA catalytic subunit and lysis buffer were included in the assay as positive and negative controls respectively. Upper bands represent PKA phosphorylated kemptide while lower bands are un-phosphorylated kemptide. C, Asynchronous VSMC were stimulated for the indicated times with 200 μM BNZ, 200uM CPT, 200 μM BNZ plus 200 μM CPT, or 100 μM forskolin, and analysed for PKA activity by kemptide phosphorylation assay. BrdU incorporation was quantified by immuno-staining for BrdU and counting of positively stained nuclei, n = 7. B, Asynchronous VSMC were stimulated with forskolin for 24 h with the last 6 h in the presence of 10 μM BrdU. Total cell lysates were analysed for non-phosphorylated (lower band) and phosphorylated (upper band) VASP by western blotting, n = 3. A, 40 h later, cells were rendered quiescent by serum deprivation for a further 6 h followed by stimulation with 200 μM BNZ for 15 min. VSMC were infected with either Ad:Control or Ad:PKAI at 2 × 10 8 pfu/ml. PKA is required but not sufficient to inhibit VSMC cell-cycle progression. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.Ĭopyright © 2010 Elsevier Ltd. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. However, 6-Benzoyl-cAMP and 8-CPT-2'-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Epac activation using the selective cAMP analogue 8-CPT-2'-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. cAMP-mediated growth arrest was PKA-dependent. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. 246, 177–185 (1971).Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. in Colloquium on Smooth Muscle, Pharmacology and Physiology (eds Worcel, M. in Excitation–Contraction Coupling in Smooth Muscle (eds Casteels, R., Godfraind, T. & Brown, B.) (Plenum, New York, 1975).Īndersson, R. in Regulation of Function and Growth of Eukaryotic Cells by Intracellular Cyclic Nucleotides (eds Dumont, J. ![]()
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