Skip to Content
MilliporeSigma
  • Epac activation sensitizes rat sensory neurons through activation of Ras.

Epac activation sensitizes rat sensory neurons through activation of Ras.

Molecular and cellular neurosciences (2015-11-26)
Behzad Shariati, Eric L Thompson, Grant D Nicol, Michael R Vasko
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Benzoyladenosine, 96%
Sigma-Aldrich
Phosphatase Inhibitor Cocktail 3, DMSO solution
Sigma-Aldrich
Rap1 Activation Assay Kit, Non-radioactive Rap1 Activation Assay Kit that uses Ral GDS RBD, agarose (Catalog # 14-455) to precipitate Rap1-GTP from cell lysates & detection by a Rap1 specific antibody.
Sigma-Aldrich
Ras Activation Assay Kit
Sigma-Aldrich
Anti-Rap1 Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-v-H-Ras (Ab-1) Rat mAb (Y13-259) Agarose Conjugate, suspension (Liquid), clone Y13-259, Calbiochem®