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  • It is well established that a number of intracellular signal


    It is well established that a number of intracellular signaling pathways mediate sensitization of sensory neurons (Gold and Gebhart, 2010, Richardson and Vasko, 2002). This redundancy could be advantageous since it provides diversity in initiating and maintaining hypersensitivity in response to injury, but can negatively impact the development of potential therapies for chronic pain conditions. For example, in large-diameter capsaicin-insensitive sensory neurons (Drew et al., 2002), activation of Epac1 augments mechanically-evoked currents (Eijkelkamp et al., 2013, Lolignier et al., 2015) whereas our previous work demonstrated that Epac2 is necessary for the increase in evoked transmitter release caused by PGE2 (Vasko et al., 2014) in cultures of capsaicin-sensitive sensory neurons maintained in NGF. It therefore seems plausible that these two Epac isoforms could selectively alter different physiological or pathological responses in different sensory neurons using different downstream signaling effectors. Indeed, Epac-mediated signaling is quite diverse and varies in different cell types (Gloerich and Bos, 2010, Grandoch et al., 2010, Holz et al., 2006). For example, Epac2 mediates the cAMP-dependent potentiation of neurotransmitter release in the hippocampus (Fernandes et al., 2015), whereas Epac1 mediates the cAMP-activated release of chloride from human T84 intestinal inhibitor of catalase (Hoque et al., 2010), a mechanism that is important in maintaining hydration of the gastrointestinal tract. Thus, delineating the pathways linked to hypersensitivity in different subpopulations of neurons is critical for understanding mechanisms of different pain modalities. Our previous work demonstrated that in sensory neurons maintained in the presence of NGF, PGE2 enhances evoked AP firing and neurotransmitter release in an Epac-dependent mechanism. In the current study, we demonstrate that direct activation of Epacs in sensory neurons activates both Ras and Rap1 small GTPase proteins; however, in capsaicin-sensitive sensory neurons, the ability of the Epac agonist to enhance AP firing and neurotransmitter release is blocked by inhibition Ras and not Rap1 (Fig. 9). Our demonstration that sensitization of capsaicin-sensitive sensory neurons grown in NGF occurs through activation of the Epac-Ras signaling cascade provides a novel signaling pathway that could serve as an ideal therapeutic target in the treatment of chronic pathological pain without altering the ability to respond to normal physiological sensations.
    Conflict of interest
    Acknowledgments M.R.V. conceived the overall design of the project. B.S., G.D.N., and M.R.V. conceived and designed the experiments. B.S. performed the experiments and analyzed the data. B.S., G.D.N., and M.R.V. interpreted results of experiments. E.L.T. generously provided the dominant-negative Ras lentiviral particles and offered critical feedback. B.S. and M.R.V prepared figures. B.S. drafted the manuscript. G.D.N. and M.R.V. edited and revised inhibitor of catalase the manuscript. We would like to thank Dr. Jill C. Fehrenbacher for valuable suggestions regarding experimental design with dominant negative Ras and Drs. Ruizong Wang and Yihong Zhang for technical assistance. This work was supported by NIH Grant NS069915 to MRV. These studies were conducted, in part, in a facility constructed with the support from the Research Facilities Improvement Program Grant Number C06 RR015481-01 from the National Center for Research Resources, National Institutes of Health.
    Introduction Whereas the physiological and pathological effects of the universal second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) were believed to be mediated exclusively by protein kinase A (PKA) and cyclic nucleotide-regulated ion channels, it has been also shown that the existence of a PKA-independent mechanism of cAMP action is mediated by a family of guanine nucleotide exchange factors, the Exchange Protein directly Activated by Cyclic AMP (EPAC) [1]. EPAC isoforms have important roles in several human diseases notably in heart failure [2], [3], [4]. Sustained EPAC activation is involved in cardiac hypertrophy [5] by initiating the Ca2+ dependent excitation-transcription coupling [6], [7], [8]. Several pathways can serve to increase [Ca2+]i in the cardiac myocyte, e.g. the L-type Ca2+ channels (LTCC), the Na+/Ca2 exchanger (NCX), the ryanodine receptor (RyR) and the SERCA pump [9]. However, it has been shown that, when over-expressed, Transient Receptor Potential Canonical (TRPC) channels can initiate cardiac hypertrophy through increased Ca2+ influx and calcineurin/NFAT activation [2], [10].