Resta Francesco, Masi Alessio, Sili Maria, Laurino Annunziatina, Moroni Flavio, Mannaioni Guido
Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.
Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy; Azienda Ospedaliero-Universitaria Careggi, Toxicology Unit, Largo Brambilla 1, 50139, Florence, Italy.
Neuropharmacology. 2016 Sep;108:136-43. doi: 10.1016/j.neuropharm.2016.04.038. Epub 2016 Apr 27.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of cellular excitability. HCN2, a subgroup of the HCN family channels, are heavily expressed in small dorsal root ganglia (DRG) neurons and their activation seems to be important in the determination of pain intensity. Intracellular elevation of cAMP levels activates HCN-mediated current (Ih) and small DRG neurons excitability. GPR35, a Gi/o coupled receptor, is highly expressed in small DRG neurons, and we hypothesized that its activation, mediated by endogenous or exogenous ligands, could lead to pain control trough a reduction of Ih current. Patch clamp recordings were carried out in primary cultures of rat DRG neurons and the effects of GPR35 activation on Ih current and neuronal excitability were studied in control conditions and after adenylate cyclase activation with either forskolin or prostaglandin E2 (PGE2). We found that both kynurenic acid (KYNA) and zaprinast, the endogenous and synthetic GPR35 agonist respectively, were able to antagonize the forskolin-induced depolarization of resting membrane potential by reducing Ih-mediated depolarization. Similar results were obtained when PGE2 was used to activate adenylate cyclase and to increase Ih current and the overall neuronal excitability. Finally, we tested the analgesic effect of both GPR35 agonists in an in vivo model of PGE2-induced thermal hyperalgesia. In accord with the hypothesis, both KYNA and zaprinast showed a dose dependent analgesic effect. In conclusion, GPR35 activation leads to a reduced excitability of small DRG neurons in vitro and causes a dose-dependent analgesia in vivo. GPR35 agonists, by reducing adenylate cyclase activity and inhibiting Ih in DRG neurons may represent a promising new group of analgesic drugs.
超极化激活的环核苷酸门控（HCN）通道在控制细胞兴奋性方面起着关键作用。HCN2是HCN家族通道的一个亚组，在小直径背根神经节（DRG）神经元中大量表达，其激活似乎在疼痛强度的决定中起重要作用。细胞内cAMP水平的升高会激活HCN介导的电流（Ih）以及小直径DRG神经元的兴奋性。GPR35是一种与Gi/o偶联的受体，在小直径DRG神经元中高度表达，我们推测其由内源性或外源性配体介导的激活可能通过降低Ih电流来实现疼痛控制。在大鼠DRG神经元的原代培养物中进行膜片钳记录，并在对照条件下以及用福斯可林或前列腺素E2（PGE2）激活腺苷酸环化酶后，研究GPR35激活对Ih电流和神经元兴奋性的影响。我们发现，犬尿喹啉酸（KYNA）和扎普司特，分别为内源性和合成的GPR35激动剂，均能够通过减少Ih介导的去极化来拮抗福斯可林诱导的静息膜电位去极化。当使用PGE2激活腺苷酸环化酶并增加Ih电流和整体神经元兴奋性时，也获得了类似的结果。最后，我们在PGE2诱导的热痛觉过敏的体内模型中测试了两种GPR35激动剂的镇痛作用。与假设一致，KYNA和扎普司特均显示出剂量依赖性的镇痛作用。总之，GPR35激活导致体外小直径DRG神经元的兴奋性降低，并在体内引起剂量依赖性镇痛。GPR35激动剂通过降低腺苷酸环化酶活性并抑制DRG神经元中的Ih，可能代表了一类有前景的新型镇痛药。
