Immunocytochemical localization of gamma-glutamyl transpeptidase in porcine ciliary epithelium

Abstract

Our previous work on cultured bovine ciliary epithelial cells indicated that γ-glutamyl transpeptidase (GGTP) activity is primarily associated with the non-pigmented epithelial cells and is virtually absent in the pigmented epithelial cells [Ng and Shichi, (1987) J. Ocular Pharmacol, 3, 341–348]. We have now investigated the localization of GGTP in porcine ciliary processes by immunocytochemical methods using goat anti-rat GGTP serum detected by rabbit anti-goat IgG secondary antibodies conjugated to fluorescein isothiocyanate or horseradish peroxidase, or biotin-conjugated secondary antibodies and streptavidin (Auroprobe). The non-pigmented cells were labeled uniformly and intensely by fluorescent antibodies, while only the plasma membrane (on the stroma side) was labeled in the pigmented cells. Immunoperoxidase reactions demonstrated that only the plasma membrane was labeled also in non-pigmented cells. The apparent uniform labeling of the cytoplasm of non-pigmented cells by fluorescent antibodies was attributed to the extensively infolded plasma membrane. The peroxidase reaction product was not found in the mitochondria, nucleus or endoplasmic reticulum of non-pigmented or pigmented cells. Immunogold labeling indicated that antigenic sites are associated with the plasma membrane infoldings (vitreous side) of non-pigmented cells, and the plasma membrane (stroma side) of pigmented cells. For enzymatic localization of GGTP activity in ciliary epithelial cells, non-pigmented and pigmented epithelial cells were carefully separated from fresh porcine ciliary body and homogenized to isolate microsomal fractions containing vesicles of plasma membrane and endoplasmic reticulum. The specific activity of microsomes from non-pigmented cells was 23 times higher than that of microsomes from pigmented cells. We conclude that GGTP is localized to the plasma membrane of both non-pigmented and pigmented cells and that the markedly high GGTP activity of non-pigmented cells is due to massive plasma membrane infoldings.