title: Epac activation sensitizes rat sensory neurons through activation of Ras.
abstract: Guanine nucleotide exchange factors directly activated by cAMP (Epacs) have emerged as important signaling molecules mediating persistent hypersensitivity in animal models of inflammation, by augmenting the excitability of sensory neurons. Although Epacs activate numerous downstream signaling cascades, the intracellular signaling which mediates Epac-induced sensitization of capsaicin-sensitive sensory neurons remains unknown. Here, we demonstrate that selective activation of Epacs with 8-CPT-2'-O-Me-cAMP-AM (8CPT-AM) increases the number of action potentials (APs) generated by a ramp of depolarizing current and augments the evoked release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons. Internal perfusion of capsaicin-sensitive sensory neurons with GDP-βS, substituted for GTP, blocks the ability of 8CPT-AM to increase AP firing, demonstrating that Epac-induced sensitization is G-protein dependent. Treatment with 8CPT-AM activates the small G-proteins Rap1 and Ras in cultures of sensory neurons. Inhibition of Rap1, by internal perfusion of a Rap1-neutralizing antibody or through a reduction in the expression of the protein using shRNA does not alter the Epac-induced enhancement of AP generation or CGRP release, despite the fact that in most other cell types, Epacs act as Rap-GEFs. In contrast, inhibition of Ras through expression of a dominant negative Ras (DN-Ras) or through internal perfusion of a Ras-neutralizing antibody blocks the increase in AP firing and attenuates the increase in the evoked release of CGRP induced by Epac activation. Thus, in this subpopulation of nociceptive sensory neurons, it is the novel interplay between Epacs and Ras, rather than the canonical Epacs and Rap1 pathway, that is critical for mediating Epac-induced sensitization.
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Behzad
authors.initials: B
authors.lastname: Shariati
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Thompson
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Grant D
authors.initials: GD
authors.lastname: Nicol
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Anesthesia, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address: vaskom@iupui.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Vasko
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D009619:Nociceptors / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011984:Sensory Receptor Cells / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018631:ras Proteins / Q000378:metabolism*
chemical_list: C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D020662:Guanine Nucleotide Exchange Factors; C526973:Rapgef3 protein, rat; D000242:Cyclic AMP; D018631:ras Proteins; D015740:Calcitonin Gene-Related Peptide
keywords: Calcitonin gene-related peptide (CGRP); Exchange proteins activated by cAMP (Epacs); Excitability; Peripheral sensitization; Rap1; Ras; Sensory neurons