Zagon Ian S, Ruth Torre B, Leure-duPree Alphonse E, Sassani Joseph W, McLaughlin Patricia J
Department of Neuroscience and Anatomy, H109, The Milton S. Hershey Medical Center, 500 University Drive, PA 17033, USA.
Brain Res. 2003 Mar 28;967(1-2):37-47. doi: 10.1016/s0006-8993(02)04172-0.
This study was conducted to determine the cellular and subcellular location(s) of the opioid growth factor receptor (OGFr), and the opioid growth factor (OGF), [Met(5)]-enkephalin, in the corneal epithelium. Laser scanning confocal microscopy analysis revealed that both OGFr and OGF were colocalized in the paranuclear cytoplasm and cell nuclei in basal, as well as suprabasal, cells of adult rat corneal epithelium. Using a postembedding immunogold procedure for immunoelectron microscopy that included embedding in Unicryl, both single- and double-face labeling studies were performed. Immunogold labeling of OGFr was detected on the outer nuclear envelope, in the paranuclear cytoplasm proximal to the nuclear envelope, perpendicular to the nuclear envelope in a putative nuclear pore complex, and within the nucleus adjacent to heterochromatin. Immunoreactivity for OGF was noted in locations similar to that for OGFr. In addition, aggregates of staining for OGF were found throughout the cytoplasm, including subjacent to the plasma membrane. Double labeling experiments revealed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, extending across the nuclear pore complex, and in the nucleus. Anti-OGFr IgG by itself, but not anti-OGF IgG alone, was associated with the outer nuclear envelope, and uncomplexed OGF immunoreactivity was detected in the cytoplasm in dual labeling experiments. These results based on complementary approaches of confocal microscopy and immunoelectron microscopy, suggest that: (i) OGFr resides on the outer nuclear envelope, (ii) OGF interacts with OGFr at the outer nuclear envelope, (iii) the colocalized receptor and peptide translocates between the cytoplasm and the nucleus at the nuclear pore, and (iv) signal transduction for modulation of cell proliferation necessitates a peptide-receptor complex that interfaces with chromatin in the nucleus.
本研究旨在确定阿片样生长因子受体（OGFr）以及阿片样生长因子（OGF）、[Met(5)]-脑啡肽在角膜上皮细胞中的细胞及亚细胞定位。激光扫描共聚焦显微镜分析显示，OGFr和OGF在成年大鼠角膜上皮基底细胞以及基底上层细胞的核周细胞质和细胞核中均有共定位。采用包埋后免疫金法进行免疫电子显微镜检查，包括用Unicryl包埋，进行了单面和双面标记研究。在核外膜、靠近核膜的核周细胞质、假定核孔复合体中垂直于核膜的位置以及与异染色质相邻的细胞核内均检测到OGFr的免疫金标记。OGF的免疫反应性在与OGFr相似的位置被发现。此外，在整个细胞质中，包括质膜下方，均发现了OGF染色聚集物。双重标记实验表明，OGF-OGFr复合物在核外膜、核周细胞质、穿过核孔复合体以及细胞核中均有共定位。单独的抗OGFr IgG与核外膜相关，而单独的抗OGF IgG则不然，在双重标记实验中，未结合的OGF免疫反应性在细胞质中被检测到。基于共聚焦显微镜和免疫电子显微镜互补方法的这些结果表明：（i）OGFr位于核外膜上；（ii）OGF在核外膜处与OGFr相互作用；（iii）共定位的受体和肽在核孔处的细胞质和细胞核之间转运；（iv）调节细胞增殖的信号转导需要一种与细胞核中的染色质相互作用的肽-受体复合物。
