Hofer Stefanie C, Ralvenius William T, Gachet M Salomé, Fritschy Jean-Marc, Zeilhofer Hanns Ulrich, Gertsch Jürg
Institute of Biochemistry and Molecular Medicine, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Neuropharmacology. 2015 Nov;98:78-89. doi: 10.1016/j.neuropharm.2015.03.021. Epub 2015 Mar 31.
The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼ 40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissues. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.
内源性大麻素系统（ECS）由大麻素受体CB1和CB2及其内源性花生四烯酸衍生的激动剂2-花生四烯酸甘油酯和花生四烯乙醇胺组成，它们发挥着重要的神经调节作用。最近，已经描述了一类新型的CB1受体负变构肽配体，即源自α血红蛋白的类血压素肽，其在中枢神经系统中的起源和功能尚不清楚。我们现在使用单克隆抗体确定了RVD-血压素（pepcan-12）和N端延伸的肽类内源性大麻素（pepcans）在中枢神经系统中的定位，并确定了它们的神经元起源。对啮齿动物的免疫组织化学分析显示，在主要的去甲肾上腺素能神经元群中，包括蓝斑（LC）、A1、A5和A7神经元，有独特而特异性的染色，这些神经元似乎是中枢神经系统中产生/释放的主要部位。在多巴胺能神经元中未检测到染色。在整个大脑（尤其是海马体和大脑皮层）和脊髓中都可见肽能轴突，这表明pepcans存在顺行轴突运输。有趣的是，肾上腺髓质中的嗜铬细胞也被pepcans强烈染色。我们发现pepcans与甘丙肽在LC和肾上腺中都有特异性共表达。使用液相色谱-串联质谱法（LC-MS/MS），仅在未灌注的大脑中检测到pepcan-12（约40 pmol/g），这表明在中枢神经系统中它是分泌型的，存在于细胞外区室。在肾上腺中，未灌注和灌注组织中测得的pepcan-12明显更多（400 - 700 pmol/g）。因此，嗜铬细胞可能是血液中发现的pepcan-12的主要产生部位。这些数据揭示了肽类内源性大麻素出现的重要区域，具有独特的去甲肾上腺素能免疫组织化学染色，为研究它们在ECS中的潜在生理功能打开了新的大门。本文是名为“神经药理学中的荧光工具”的特刊的一部分。
