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  • The question remains however as to the

    2019-07-11

    The question remains, however as to the downstream signaling by Epacs that contributes to persistent sensitization of sensory neurons. Activation of the Ras family of GTPases by Epac1 (RapGEF3) or Epac2 (RapGEF4) (Bos, 2003, de Rooij et al., 1998) is linked to a number of downstream signaling effectors that may contribute to persistent hypersensitivity, including but not limited to phospholipase Cε (PLCε), phospholipase D (PLD), mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3-kinase (PI3K) (Baviera et al., 2010, Holz et al., 2006, Schmidt et al., 2013, Yano et al., 2007). Although Epacs were initially discovered as GEFs for Rap1, controversy exists, however, as to whether Epacs may also act as GEFs for Ras and whether Ras mediates some of the physiological effects regulated by activation of Epacs. (Li et al., 2006, Lopez De Jesus et al., 2006, Zheng and Quilliam, 2003).
    Methods
    Results
    Discussion Our findings clearly demonstrate that Epac activation sensitizes small-diameter capsaicin-sensitive sensory LY2940680 and that this action is dependent on the small G protein, Ras. Exposing sensory neuronal cultures to the Epac agonist 8CPT-AM increases Ras activity and augments the number of APs generated in response to a ramp of depolarizing current, an effect that is completely blocked by internally perfusing the neurons with GDP-βS or with the Ras neutralizing antibody. Overexpressing DN-Ras also prevents Epac-induced increases in AP firing and attenuates the Epac-induced augmentation of stimulated iCGRP release. There are currently no assays available to measure Ras activity at the single cell level. Consequently, Ras activity cannot be measured directly in the patch clamp experiments. However, the activity assays do offer support for the electrophysiology data as activation of Epacs increases Ras activity and inhibition of Ras activity blocks the Epac agonist from enhancing excitability. These two pieces of data support the conclusion that Epac activation elicits hypersensitivity in a Ras-dependent manner. Our manipulations of Ras did not affect the ability of neurons to generate APs under control conditions or alter the ability of the PKA selective agonist, 6BNZ-AM, to augment ramp-evoked AP firing, i.e., to sensitize sensory neurons. Thus, inhibition of Ras specifically alters the ability of sensory neurons to respond to Epac activation, but does not alter the ability of neurons to be sensitized by activation of PKA, which is a canonical pathway mediating acute sensitization of sensory neurons (Aley and Levine, 1999, England et al., 1996, Hingtgen et al., 1995, Lopshire and Nicol, 1998, Sachs et al., 2009). This is important since activation of Epacs is implicated in long term sensitization of sensory neurons (Eijkelkamp et al., 2013, Hucho et al., 2005, Vasko et al., 2014, Wang et al., 2007). Consequently, inhibition of either Epac or Ras-activation is a useful strategy for blunting chronic hypersensitivity without altering the ability of sensory neurons to be acutely sensitized, i.e., treating chronic inflammatory pain without compromising acute pain perception. 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.