Torihashi S, Horowitz B, Pollock J S, Ward S M, Xue C, Kobayashi S, Sanders K M
Department of Anatomy, Nagoya University School of Medicine, Japan.
Histochem Cell Biol. 1996 Jan;105(1):33-41. doi: 10.1007/BF01450876.
The expression of nitric oxide synthase (NOS) in the mucosa of the canine colon was investigated with in situ hybridzation, immunohistochemistry (using isoform specific antibodies), western analysis, and NADPH diaphorase (NADPH-d) histochemistry. In situ hybridization using a common probe for known isoforms of NOS showed that NOS mRNA was strongly expressed in mucosal cells. A gradient in the degree of hybridization was noted from the base of the crypts to the luminal surface. This gradient was also apparent using an endothelial NOS (eNOS)-specific probe. Neural NOS-like immunoreactivity (nNOS-LI) was observed in columnar epithelial cells, and the same population of cells was stained with NADPH-d. Endothelial NOS-like immunoreactivity (eNOS-LI) was also found in mucosal cells; however, this eNOS-LI was confined to mucous cells. These cells were not stained with NADPH-d. The existence of eNOS in mucosal cells was confirmed by in situ hybridization using the probe which specifically hybridized with mRNA of eNOS and by western blots which demonstrated the expression of a 135-kDa protein in mucosal homogenates. The differential expression of NOS isoforms and the gradient in expression along the length of the crypts suggest complex roles for NO in the development of colonic epithelial cells and in secretion and transport functions of the colonic mucosa.
采用原位杂交、免疫组织化学（使用亚型特异性抗体）、蛋白质免疫印迹分析及NADPH黄递酶（NADPH-d）组织化学方法，对犬结肠黏膜中一氧化氮合酶（NOS）的表达进行了研究。使用针对已知NOS亚型的通用探针进行原位杂交，结果显示NOS mRNA在黏膜细胞中强烈表达。从隐窝底部到腔面观察到杂交程度呈梯度变化。使用内皮型NOS（eNOS）特异性探针时，这种梯度变化也很明显。在柱状上皮细胞中观察到神经型NOS样免疫反应性（nNOS-LI），并且同一群细胞被NADPH-d染色。在黏膜细胞中也发现了内皮型NOS样免疫反应性（eNOS-LI）；然而，这种eNOS-LI局限于黏液细胞。这些细胞未被NADPH-d染色。使用与eNOS mRNA特异性杂交的探针进行原位杂交以及蛋白质免疫印迹分析（显示黏膜匀浆中135-kDa蛋白的表达）证实了黏膜细胞中存在eNOS。NOS亚型的差异表达以及沿隐窝长度方向的表达梯度表明，NO在结肠上皮细胞发育以及结肠黏膜的分泌和转运功能中发挥着复杂的作用。
