Freitas A C N, Silva G C, Pacheco D F, Pimenta A M C, Lemos V S, Duarte I D G, de Lima M E
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, M.G., Brazil.
Departamento Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, M.G., Brazil.
Nitric Oxide. 2017 Apr 1;64:31-38. doi: 10.1016/j.niox.2017.01.004. Epub 2017 Jan 10.
and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway.
Nociceptive thresholds were measured by paw pressure test. PGE (2 μg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and K channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot.
PnPP-19 (5, 10 and 20 μg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of K (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 μg/paw). Tissue nitrite concentration increased after PnPP-19 (10 μg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE injection. Administration of PnPP-19 reverted this PGE-induced effect.
The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-K pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.
源自蜘蛛毒素PnTx2-6（重新命名为δ-CNTX-Pn1c）的肽PnPP-19通过激活氮能系统增强勃起功能。由于一氧化氮（NO）已被作为一种抗伤害感受分子进行研究，且已知PnPP-19可诱导外周抗伤害感受，我们旨在评估PnPP-19是否能通过激活该途径诱导外周抗伤害感受。
通过爪部压力试验测量伤害感受阈值。将前列腺素E（PGE，2μg/爪）与PnPP-19以及一氧化氮合酶（NOS）、鸟苷酸环化酶和钾通道的抑制剂/阻断剂经足底内注射给药。通过格里斯试验测定亚硝酸盐浓度。通过蛋白质免疫印迹法测定内皮型一氧化氮合酶（eNOS）和神经元型一氧化氮合酶（nNOS）的表达及磷酸化情况。
PnPP-19（5、10和20μg/爪）可诱导大鼠外周抗伤害感受。给予NOS抑制剂（L-NOarg）、选择性nNOS抑制剂（L-NPA）、鸟苷酸环化酶抑制剂（ODQ）和钾通道阻断剂（格列本脲）可部分抑制PnPP-19（10μg/爪）的抗伤害感受作用。给予PnPP-19（10μg/爪）后组织亚硝酸盐浓度升高。在所有测试组中，eNOS和nNOS的表达保持不变，然而，注射PGE后，nNOS丝氨酸852（失活位点）的磷酸化增加，eNOS丝氨酸1177（激活位点）的磷酸化减少。给予PnPP-19可逆转这种PGE诱导的效应。
PnPP-19诱导的外周抗伤害感受作用是由NO-环磷酸鸟苷（cGMP）-钾通道途径的激活所致。这种效应可能需要eNOS和nNOS的激活。我们的结果表明PnPP-19是一种治疗疼痛的新药候选物，并强化了nNOS和eNOS激活以及内源性NO释放对于诱导外周抗伤害感受的重要性。
