Van Geldre Lieve A, Fraeyman Norbert H, Peeters Theo L, Timmermans Jean-Pierre, Lefebvre Romain A
Heymans Institute of Pharmacology, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium.
Auton Neurosci. 2004 Jan 30;110(1):8-18. doi: 10.1016/j.autneu.2003.05.001.
Neuronal NO-synthase (nNOS) was investigated in rat longitudinal muscle/myenteric plexus (LM/MP) tissue at the cellular and subcellular level. Using preparations and double immune staining and light and electron microscopy, we concluded that, in these preparations, nNOS is only present in neuronal cells. However, in spite of numerous attempts to morphologically identify the NOS-containing subcellular structure, no firm conclusions were possible. Consequently, the problem was approached by biochemical methods including gradient centrifugation followed by analysis of the fractions. Using a protocol involving gentle homogenisation of the tissue, we found that about 10% of the nNOS immune reactivity was particle-bound confirming previous results (Biochem. Pharmacol. 60 (2000) 145). However, applying a different protocol including strong homogenisation, we now demonstrated that about 50% of the immune reactive nNOS was sedimentable. The results suggested that particulate nNOS is associated with one single subcellular structure, which is different from the plasma membrane, rough and smooth endoplasmic reticulum, mitochondria and lysosomes. The equilibrium sedimentation characteristics of the nNOS containing particles corresponded partly to those containing vasoactive intestinal polypeptide (VIP) or synaptobrevin. Application of non-equilibrium centrifugation conditions, however, demonstrated that almost no co-localisation occurred. We conclude that, in the LM/MP tissue, nNOS is about 50% particle-bound in a subcellular structure, which is different from the VIP-containing particle and from synaptobrevin-containing exocytotic particles.
在细胞和亚细胞水平上研究了大鼠纵行肌/肌间神经丛（LM/MP）组织中的神经元型一氧化氮合酶（nNOS）。通过制备标本、双重免疫染色以及光镜和电镜观察，我们得出结论，在这些标本中，nNOS仅存在于神经细胞中。然而，尽管多次尝试从形态学上鉴定含NOS的亚细胞结构，但仍无法得出确切结论。因此，采用生化方法来解决这个问题，包括梯度离心，随后对各组分进行分析。采用一种温和匀浆组织的方案，我们发现约10%的nNOS免疫反应性与颗粒结合，这证实了先前的结果（《生物化学与药理学》60 (2000) 145）。然而，采用一种不同的方案，包括强力匀浆，我们现在证明约50%的免疫反应性nNOS是可沉淀的。结果表明，颗粒状nNOS与一种单一的亚细胞结构相关，该结构不同于质膜、粗面和滑面内质网、线粒体和溶酶体。含nNOS颗粒的平衡沉降特性部分与含血管活性肠肽（VIP）或突触囊泡蛋白的颗粒的沉降特性相对应。然而，采用非平衡离心条件表明几乎没有共定位现象发生。我们得出结论，在LM/MP组织中，nNOS约50%以颗粒结合的形式存在于一种亚细胞结构中，该结构不同于含VIP的颗粒和含突触囊泡蛋白的胞吐颗粒。
