Shariati Behzad, Thompson Eric L, Nicol Grant D, Vasko Michael R
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA; Department of Anesthesia, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Mol Cell Neurosci. 2016 Jan;70:54-67. doi: 10.1016/j.mcn.2015.11.005. Epub 2015 Nov 18.
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.
环磷酸腺苷直接激活的鸟嘌呤核苷酸交换因子（Epacs）已成为重要的信号分子，通过增强感觉神经元的兴奋性，介导炎症动物模型中的持续超敏反应。尽管Epacs激活众多下游信号级联反应，但介导Epac诱导的辣椒素敏感感觉神经元致敏的细胞内信号仍不清楚。在这里，我们证明用8-CPT-2'-O-Me-cAMP-AM（8CPT-AM）选择性激活Epacs可增加去极化电流斜坡产生的动作电位（APs）数量，并增强离体大鼠感觉神经元中降钙素基因相关肽（CGRP）的诱发释放。用GDP-βS（代替GTP）对辣椒素敏感感觉神经元进行内部灌注，可阻断8CPT-AM增加AP发放的能力，表明Epac诱导的致敏是G蛋白依赖性的。用8CPT-AM处理可激活感觉神经元培养物中的小G蛋白Rap1和Ras。通过内部灌注Rap1中和抗体或使用shRNA降低蛋白质表达来抑制Rap1，并不会改变Epac诱导的AP生成增强或CGRP释放，尽管在大多数其他细胞类型中，Epacs作为Rap鸟嘌呤核苷酸交换因子（Rap-GEFs）发挥作用。相反，通过表达显性负性Ras（DN-Ras）或通过内部灌注Ras中和抗体来抑制Ras，可阻断AP发放的增加，并减弱Epac激活诱导的CGRP诱发释放的增加。因此，在这个伤害性感觉神经元亚群中，对于介导Epac诱导的致敏至关重要的是Epacs与Ras之间的新型相互作用，而不是经典的Epacs与Rap1途径。
