For the current studies we used the selective Epac
For the current studies, we used the selective Epac agonist 8CPT-AM to directly activate the proteins. We chose this pharmacological approach since it allows a direct activation of Epacs rather than using receptor agonists such as PGE2 which could introduce confounding variables. One potential concern, however, is whether 8CPT-AM has off-target actions that could account for its ability to sensitize sensory neurons. For example, at approximately 10-fold higher concentrations than we used in the current work, 8CPT-AM can increase PKA activity. The EC50 of 8CPT-AM for activating PKA is 30μM, whereas the EC50 for Epacs is just 0.9μM (Rehmann et al., 2003). Because the EC50 for 8CPT-AM to augment evoked transmitter release was 2μM (see Fig. 1C), it is unlikely that the effect is mediated by PKA. 8CPT-AM also has been reported to have antagonist actions on the P2Y12 receptor (Herfindal et al., 2013). Since inhibition of Gβγ or adenylyl cyclase did not block the sensitizing actions of 8CPT-AM, it seems unlikely that the effect is mediated by heterotrimeric G proteins. For release studies, we used overexpression of DN-Ras as our means of blocking Ras activity. One potential limitation of DN-Ras (17N) is that the molecule forms complexes with Ras GEFs (Quilliam et al., 1994) and thus could nonspecifically inactivate GEFs for other small G proteins. This seems unlikely, since internal perfusion of the Y13-259 Ras-neutralizing antibody into sensory neurons blocked AP firing in a manner analogous to DN-Ras. Indeed, the Y13-259 Ras-neutralizing antibody blocks Ras activity (Furth et al., 1982, Sigal et al., 1986) without inhibiting the activity of Rap1 (von Lintig et al., 2000). The ability of the DN-Ras to attenuate the Epac-induced increase in potassium-evoked release of iCGRP at lower concentrations of 8CPT-AM, but not at higher concentrations could be explained by the fact that not all the neurons in the cultures expressed DN-Ras. We observed a ~ 70% infection efficiency as measured by the TCS 2002 of green fluorescence in sensory neuronal cultures. In uninfected cells, higher concentrations of 8CPT-AM could presumably cause sufficient activation of Epacs to overcome the inhibitory effects of DN-Ras. Consistent with this notion, when we recorded APs from non-fluorescent sensory neurons in cultures infected with DN-Ras LV, we observed that the Epac agonist, 8CPT-AM, enhanced AP firing in response to a ramp of depolarizing current (data not shown), thus demonstrating that suppression of the Epac agonist-induced enhancement of AP firing only occurs in cells that express DN-Ras. It is also possible that activation of Epacs could engage divergent signaling pathways that separately regulate transmitter release and AP firing. For example, it is well established that the small GTPase, Rab3a is localized on synaptic vesicles and is involved in exocytosis (Geppert et al., 1994, Sudhof, 2013). Furthermore, AP firing is largely dependent on voltage-gated sodium and potassium channels, whereas transmitter release is dependent on free calcium levels which could increase through voltage-gated calcium channels, TRP channels, or intracellular release. Thus, modulation of transmitter release involves an integrated response which includes, but is not limited to, alterations in the number of APs. 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 cells 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.