Immunoelectron microscopic localization of the opioid growth factor receptor (OGFr) and OGF in the cornea

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.