背景:和目的:肽PnPP-19,源自蜘蛛毒素PnTx2-6(更名为δ-CNTX-Pn1c),通过激活硝化系统增强勃起功能。由于NO已被研究为一种抗伤害感受分子,并且PnPP-19已知可诱导外周抗伤害感受,我们旨在评估PnPP-19是否可以通过激活该途径诱导外周抗伤害感受.
方法:通过爪压试验测量伤害性阈值。PGE2(2μg/paw)与PnPP-19和NOS抑制剂/阻断剂一起,鸟苷酸环化酶和KATP通道。通过Griess试验获得亚硝酸盐浓度。通过westernblot测定eNOS和nNOS的表达和磷酸化。
结果:PnPP-19(5、10和20μg/爪)在大鼠中诱导外周抗伤害感受。给药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KATP阻断剂(格列本脲)部分抑制了PnPP-19(10μg/paw)的镇痛作用。PnPP-19(10μg/爪)给药后组织亚硝酸盐浓度增加。各试验组eNOS和nNOS的表达均相同,然而,注射PGE2后,nNOSSer852(失活位点)的磷酸化增加,而eNOSSer1177(活化位点)的磷酸化降低.PnPP-19的施用恢复了这种PGE2诱导的作用。
结论:PnPP-19诱导的外周抗伤害作用是由NO-cGMP-KATP途径的激活引起的。这种作用可能需要激活eNOS和nNOS。我们的结果表明,PnPP-19作为治疗疼痛和加强nNOS和eNOS激活的重要性的新候选药物,以及内源性NO释放,用于诱导外周抗伤害感受。
BACKGROUND: 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.
METHODS: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 μg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot.
RESULTS: 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 KATP (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 PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect.
CONCLUSIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP 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.