Epac activation sensitizes rat sensory neurons through activation of Ras

Behzad Shariati, Eric L. Thompson, Grant Nicol, Michael Vasko

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)54-67
Number of pages14
JournalMolecular and Cellular Neuroscience
Volume70
DOIs
StatePublished - Jan 1 2016

Fingerprint

Sensory Receptor Cells
Action Potentials
Calcitonin Gene-Related Peptide
Perfusion
Capsaicin
Neutralizing Antibodies
Guanine Nucleotide Exchange Factors
Nociceptors
Architectural Accessibility
Critical Pathways
Monomeric GTP-Binding Proteins
Guanosine Triphosphate
GTP-Binding Proteins
Small Interfering RNA
Hypersensitivity
Animal Models
Inflammation
2'-O-methyl-8-(4-chlorophenylthio)cAMP
Proteins

Keywords

  • Calcitonin gene-related peptide (CGRP)
  • Exchange proteins activated by cAMP (Epacs)
  • Excitability
  • Peripheral sensitization
  • Rap1
  • Ras
  • Sensory neurons

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience
  • Cell Biology

Cite this

Epac activation sensitizes rat sensory neurons through activation of Ras. / Shariati, Behzad; Thompson, Eric L.; Nicol, Grant; Vasko, Michael.

In: Molecular and Cellular Neuroscience, Vol. 70, 01.01.2016, p. 54-67.

Research output: Contribution to journalArticle

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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.",
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