Department of Pharmacy, DiFAR, Pharmacology and Toxicology Section, Viale Cembrano 4, 16148, Genoa, Italy.
Department of Pharmacy, DiFAR, Pharmacology and Toxicology Section, Viale Cembrano 4, 16148, Genoa, Italy; Center of Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV, 16132, Genoa, Italy.
Neuropharmacology. 2018 May 1;133:429-439. doi: 10.1016/j.neuropharm.2018.02.030. Epub 2018 Feb 27.
Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H]D-aspartate ([H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HT heteroreceptors. Actually, the 15 mM KCl-evoked [H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT agonist (±)DOI, an effect reversed by the 5-HT antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT receptors colocalize and cross-talk in these terminals and if 5-HT ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15 mM KCl-evoked [H]D-Asp overflow as well as its inhibition by 100 nM (±)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis.
突触前 mGlu2/3 自身受体存在于大鼠脊髓神经末梢，这一发现表明 LY379268 以 LY341495 敏感的方式抑制 15mM KCl 诱导的 [H]D-天冬氨酸 ([H]D-Asp) 的释放。脊髓谷氨酸能神经末梢还具有突触前释放调节 5-HT 异受体。实际上，5-HT 激动剂（±）DOI 可减少脊髓突触小体 15mM KCl 诱导的 [H]D-Asp 胞吐作用，该作用可被 5-HT 拮抗剂 MDL11,939、MDL100907、酮色林和曲唑酮 (TZD) 逆转。我们研究了 mGlu2/3 和 5-HT 受体是否在这些末梢中存在共定位和串扰，以及 5-HT 配体是否调节 mGlu2/3 介导的谷氨酸胞吐作用的控制。Western blot 分析和共聚焦显微镜强调了 mGlu2/3 和 5-HT 受体蛋白在脊髓 VGLUT1 阳性突触小体中的存在，其中 mGlu2/3 和 5-HT 受体免疫反应性大量共定位。此外，脊髓突触小体的 mGlu2/3 免疫沉淀物也为 5-HT 阳性。有趣的是，当两种激动剂同时添加时，100pM LY379268 诱导的 15mM KCl 诱导的 [H]D-Asp 溢出减少及其被 100nM（±）DOI 抑制变得无法检测到。相反，5-HT 拮抗剂（MDL11,939、MDL100907、酮色林和曲唑酮）增强了 mGlu2/3 自身受体的释放调节活性。突触小体质膜中 mGlu2/3 受体蛋白的表达增加与 5-HT 拮抗剂引起的 mGlu2/3 自身受体功能获得相平行。基于这些结果，我们提出在脊髓谷氨酸能神经末梢中，i）mGlu2/3 和 5-HT 受体共定位并以拮抗剂样方式相互作用，ii）5-HT 拮抗剂是控制谷氨酸胞吐作用的 mGlu2/3 自身受体的间接正变构调节剂。
