The fact that Epac induced sensitization of
The fact that Epac-induced sensitization of small diameter sensory neurons is Ras-dependent, but does not require Rap1, is a novel finding since a majority of studies show that Epacs are predominately GEFs for Rap GTPases (de Rooij et al., 1998, Kawasaki et al., 1998). For example, in isolated cerebellar granule cells, activation of Rap1 mediates the Epac-induced increase in conductance of calcium-activated potassium channels (BK) (Ster et al., 2007) and in rat cortical neurons, Epac-induced remodeling of synaptic spines is Rap dependent (Woolfrey et al., 2009). In our sensory neuronal cultures, we demonstrate that Epac activation by 8CPT-AM increases Rap1 activity. This increase in activity, however, is not causally linked to Epac-induced sensitization since inhibiting Rap1 activity by either perfusing a Rap1-neutralizing antibody into sensory neurons or by using shRNA to reduce expression of Rap1 did not prevent the increase in evoked APs and transmitter release caused by the Epac agonist. These manipulations of Rap1 blocked 8CPT-AM and GTP-γS induced increases in Rap1-GTP, confirming that they were effective in inhibiting Rap1. Thus, our findings suggest a specificity of Epac signaling that is likely unique and that could provide a specific target for altering peripheral sensitization without affecting Epac signaling in other neuronal populations. Although our studies show that Ras activation is necessary for sensitization of a subpopulation of sensory neurons, the question remains whether the Epacs produce their effects by acting as Ras GEFs. Some evidence suggests that Epacs can augment exchange of GTP for GDP in Ras (Lopez De Jesus et al., 2006), but other work suggests that the activation of Ras by Epac agonists is dependent on PLC-ε and a subsequent increase in intracellular calcium (Keiper et al., 2004, Metrich et al., 2010). Interestingly, studies in the H1299 lung carcinoma cell line and in insulin-secreting pancreatic β you want to be indicate that Ras is involved in trafficking Epac2 to the plasma membrane, thereby promoting an interaction with Rap1 (Idevall-Hagren et al., 2013, Li et al., 2006), although this is unlikely in sensory neurons since inhibiting Rap1 does not alter Epac-induced sensitization. Further studies are warranted, however, to ascertain whether Epacs serve as Ras GEFs in sensory neurons, or whether the ability of the Epac agonists to increase Ras activity is secondary to other, as yet, undetermined actions of Epacs. 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 cells (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.