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  • The requirement for two distinct signalling pathways for cAM


    The requirement for two distinct signalling pathways for cAMP-induced growth arrest likely represents an important mechanism allowing VSMC to finely modulate their cellular responses to cAMP-elevating stimuli. Our data demonstrates for the first time that activation of both pathways in VSMC results in potent and synergistic growth arrest and these observations highlight Epac alongside PKA as a major player in cAMP-dependent growth arrest in VSMC in vitro. The future development of Epac-specific pharmacological antagonists and analysis of Epac-deficient mice will allow validation of the relative roles of PKA and Epac in the regulation of VSMC proliferation during the development of vascular disease in vivo. The following are the supplementary materials related to this article.
    Acknowledgements This work was supported by British Heart Foundation grant FS/07/038 and by the NIHR Bristol BRU in Cardiovascular Medicine. We would like to thank Prof. Patrick Casey and Candice Bailey for the generous gift of the Rap1GAP adenovirus, Prof. David Murphy for the PKAI adenovirus and Prof. Peter Cullen for the GST-Ral-GDS ret pathway vector. We thank Gill Tarlton and Sue Finerty for their expert support. We also thank Prof. Nickolai Dulin and Prof. Donald Maurice for their helpful advice and discussion.
    Empirical data Briefly, 8-pCPT (8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate), an EPAC activator, and 8-Br-cAMP, a selective PKA activator, were injected into hippocampus bilaterally through a guidance cannula for three consecutive days. After behavioral test, the rats’ hippocampi were collected for evaluation of the proteins changes. Total RNA were extracted and cDNA was synthesized through Maniatis protocol [24]. Quantitative real-time PCR experiments were done using QuantiFast SYBR Green PCR Kit, rat’s St8sia2, St8sia4 and Gapdh primers (Qiagen, Germany). Products specificity was confirmed using agarose gel electrophoresis and melting curve analysis. Changes in expressions were calculated using 2−ΔΔ method [25] and were presented as fold changes in expression using REST 2008 software. St8sia4 (PST) mRNA expression levels were decreased in 8-Br-cAMP - treated and were not changed in 8-pCPT treated hippocampi (Fig. 1a). The unexpected results were obtained in expression levels of STX mRNA of the tissues which were received 8-pCPT; as +4 and −2-fold changes in EPAC and PKA activated hippocampi, respectively (Fig. 1b).
    Evaluation of the hypothesis EPAC expression is under constant regulation during CNS development. Microarray data reveal that Pka (Prkaca and Prkacb) expression has an ascending profile in the CNS of the developing mouse [26] while EPAC1, the active isomer, expression shows descending trend during development and postnatal rat brain, spinal cord and dorsal root ganglion [27]. Intracellular signal transduction is dependent to kinases as key regulators of a developing brain and cAMP is known as a major propagator of the kinase activation. Moreover, there are couples of anti-tumor substances which act through inhibition of these enzymes like multi-kinase inhibitors and some new compounds [28], [29]. Due to over-expression of EPAC in human pancreatic ductal adenocarcinoma cells, applying selective inhibitor of EPAC resulted to antitumor properties without involvement in regulatory kinase [30]. On the other hand, stimulating roles of PSA in tumor cell growth, differentiation [31] and invasion [32] have been reported. PST expresses in normal and tumor tissue, in contrary to STX (the embryonic isoform) which only expresses in proliferative tumor cells [33]. It is possible to induce neural sprouting by controlling EPAC function on NCAM modifications [34]. Therefore, administration of EPAC enhancers can stimulate neurogenesis in a clinical approach.
    Conflict of interest statement
    Acknowledgement Experiments of the present article were supported by grant No. 10507 from Tehran University of Medical Sciences. We thank Dr. Bijan Djahanguiri for his kind help and advice.