Mancini P, Santi P A
Department of Otolaryngology, University of Minnesota Medical School, Minneapolis, MN 55455.
Hear Res. 1993 Jan;64(2):151-65. doi: 10.1016/0378-5955(93)90001-h.
Cholera toxin is an ubiquitous activator of intracellular adenylate cyclase and is divided in two major components: A and B. The B-component consists of several subunits that specifically bind to the external cell membrane. The receptor for the toxin, the GM1 ganglioside, is concentrated in nervous tissues. The B subunit of the cholera toxin, conjugated to different molecules (i.e., choleragenoid) is therefore a sensitive anatomical tracer and has been used to detect the presence of GM1 in mammalian tissues. Using choleragenoid, unlabeled and labeled with FITC, we have determined the distribution of the GM1 ganglioside in the vestibular system of the chinchilla. Vestibular tissues were fixed in 4% paraformaldehyde in phosphate buffer, decalcified in 10% EDTA and prepared as either whole-mount, surface-preparations, or for radial cryosections. Positive control tissue consisted of binding to normal brain tissues. Negative controls consisted of several treatments: masking of the GM1 receptors with unlabeled choleragenoid, tissue extraction of GM1 using ethanol, and preabsorbing the choleragenoid with bovine GM1. In addition, to exclude staining of glycoproteins that may have a carbohydrate structure similar to GM1, tissues were digested with trypsin prior to choleragenoid exposure. In the vestibular system, a strongly positive reaction was observed in: the sensory stereocilia and supporting cells of the maculae and cristae, epithelial cells of the planum semilunatum, and polygonal cells of the semicircular canal. Positive but less strong reactivity was observed in the sensory cell body of maculae and cristae, nerve fibers, epithelial cells of utricle and ampulla walls and flattened epithelial cells of the semicircular canals. No reactivity was present in the supporting connective tissue cells and fibrils, blood vessels, gelatinous cupula of the cristae ampullaris and statoconial membranes. Brain tissue showed strong choleragenoid reactivity. The negative controls showed no or greatly reduced reactivity to choleragenoid. Trypsin digestion did not decrease reactivity to choleragenoid.
霍乱毒素是细胞内腺苷酸环化酶的一种普遍存在的激活剂，可分为两个主要成分：A和B。B成分由几个亚基组成，这些亚基特异性结合细胞外膜。毒素的受体GM1神经节苷脂集中在神经组织中。因此，与不同分子（即类霍乱原）结合的霍乱毒素B亚基是一种敏感的解剖学示踪剂，已被用于检测哺乳动物组织中GM1的存在。我们使用未标记和用异硫氰酸荧光素标记的类霍乱原，确定了灰鼠前庭系统中GM1神经节苷脂的分布。前庭组织用磷酸盐缓冲液中的4%多聚甲醛固定，在10%乙二胺四乙酸中脱钙，并制成整装片、表面制剂或放射状冰冻切片。阳性对照组织包括与正常脑组织的结合。阴性对照包括几种处理：用未标记的类霍乱原掩盖GM1受体、用乙醇提取组织中的GM1以及用牛GM1预吸收类霍乱原。此外，为了排除可能具有与GM1相似碳水化合物结构的糖蛋白的染色，在暴露于类霍乱原之前，用胰蛋白酶消化组织。在前庭系统中，在以下部位观察到强烈的阳性反应：黄斑和嵴的感觉静纤毛和支持细胞、半月平面的上皮细胞以及半规管的多边形细胞。在黄斑和嵴的感觉细胞体、神经纤维、椭圆囊和壶腹壁的上皮细胞以及半规管的扁平上皮细胞中观察到阳性但较弱的反应性。支持结缔组织细胞和纤维、血管、壶腹嵴的胶状顶和位砂膜中没有反应性。脑组织显示出强烈的类霍乱原反应性。阴性对照对类霍乱原没有反应或反应性大大降低。胰蛋白酶消化并没有降低对类霍乱原的反应性。
