Kachi S, Nishizawa Y, Olshevskaya E, Yamazaki A, Miyake Y, Wakabayashi T, Dizhoor A, Usukura J
Department of Ophthalmology, Nagoya University, School of Medicine, Nagoya 466, Japan.
Exp Eye Res. 1999 Apr;68(4):465-73. doi: 10.1006/exer.1998.0629.
Guanylate cyclase activating proteins, GCAP-1 and GCAP-2, have a pivotal role in the activation of guanylate cyclase in phototransduction. Previous studies on the localization of GCAP-1 and GCAP-2 are contradictory. In this study, we tried to avoid possible artifacts accompanied by immunocytochemistry. Immunolabeling of a GCAP was carried out using antibodies pre-adsorbed with a different type of GCAP. In addition, immunolabeling was performed using three different animal species under different fixation and embedding. Electron microscopic immunocytochemistry was also performed to reveal subcellular localization of GCAPs as well as confirming data obtained by light microscopy. All data indicate that anti-GCAP-1 antibody binding sites were found predominantly in cone outer segments, in particular, in disk membrane regions. Sparse labeling was observed in rod outer segments, but the labeling was much lower than that seen in cone outer segments. Less labeling is also found in synaptic regions and inner segments of cones. No labeling was detected in connecting cilia and its cytoplasmic extensions. Such labeling patterns were similar among human, monkey and bovine retinas. The localization of GCAP-1 is consistent with the pattern of a recently reported human cone-specific degeneration. Anti-GCAP-2 antibody binding sites were detected in both inner and outer segments of rods and cones of all three animals although the labeling density was slightly different among species. Cryo-immuno-labeling of GCAP-2 in bovine retinas revealed that labeling sites were more concentrated in rods than those of cones, and that synaptic regions were also labeled. The different localization of GCAPs suggest that roles of GCAP-1 and GCAP-2 may be different.
鸟苷酸环化酶激活蛋白GCAP-1和GCAP-2在光转导中鸟苷酸环化酶的激活过程中起关键作用。先前关于GCAP-1和GCAP-2定位的研究结果相互矛盾。在本研究中，我们试图避免免疫细胞化学可能带来的假象。使用预先吸附有不同类型GCAP的抗体对GCAP进行免疫标记。此外，在不同的固定和包埋条件下，使用三种不同的动物物种进行免疫标记。还进行了电子显微镜免疫细胞化学，以揭示GCAPs的亚细胞定位，并确认光学显微镜获得的数据。所有数据表明，抗GCAP-1抗体结合位点主要位于视锥细胞外段，特别是在盘膜区域。在视杆细胞外段观察到稀疏标记，但标记程度远低于视锥细胞外段。在视锥细胞的突触区域和内段也发现较少的标记。在连接纤毛及其细胞质延伸部分未检测到标记。在人类、猴子和牛的视网膜中，这种标记模式相似。GCAP-1的定位与最近报道的人类视锥细胞特异性变性模式一致。在所有三种动物的视杆细胞和视锥细胞的内段和外段均检测到抗GCAP-2抗体结合位点，尽管不同物种之间的标记密度略有不同。牛视网膜中GCAP-2的冷冻免疫标记显示，标记位点在视杆细胞中比视锥细胞中更集中，并且突触区域也有标记。GCAPs的不同定位表明GCAP-1和GCAP-2的作用可能不同。
