Mouse and monkey adrenal glands were used to study the relationships between gap junction protein expression, intercellular communication and adrenal zonation. Dye communication patterns were determined by incubating freshly excised and hemisected adrenal glands in Lucifer yellow, a gap junction permeable fluorescent dye. Immunohistochemical techniques were used to localize adrenal gap junction proteins. The combination of these two techniques permitted the correlation of gap junction proteins with dye transfer and hormone responses in specialized regions of the adrenal cortex. Lucifer yellow dye communication was most pronounced in the inner glucocorticoid/androgen-producing regions (zona fasciculata/zona reticularis), but was virtually absent in the outer mainly mineralocorticoid-producing region (zona glomerulosa). This pattern of dye communication was coincident with immunohistochemical localization of the gap junction protein, 1 Cx43. The variations in communication and 1 Cx43 expression within the adrenal cortex are thought to be relevant to normal physiological regulation of the adrenal gland.

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.

Gap junctional communication disorders have been implicated in the etiology of benign and malignant tumors. Understanding the type, distribution, and frequency of gap junctions in adrenal disorders should provide insight into the role of gap junctions in adrenal carcinogenesis as well as information that may be useful in developing improved diagnosis and treatment of adrenal diseases. Using immunocytochemical techniques, we have characterized and compared alpha1 connexins 43 gap junction protein levels in normal adrenal glands to those in benign and malignant adrenocortical human tumors. In addition, gap junction protein levels were studied in a human adrenal cancer cell line (H295). In both normal and neoplastic adrenal tissues, only alpha1 connexin 43 could be detected, whereas beta1 connexin 32 and beta2 connexin 26 were not found. In the normal adrenal gland, the zona fasciculata was demonstrated to have the highest number of gap junctions per cell (mean +/- SEM, 13.78 +/- 1.93). In contrast, in benign adrenocortical adenomas, the number of gap junctions per cell compared to that detected in normal adrenal glands was significantly reduced (mean +/- SEM, 4.6 +/- 1.17; P < or = 0.05), and the lowest number was found in malignant adrenocortical tumors (1.42 +/- 0.58; P < or = 0.05). Similarly, there were few or no alpha1 connexin 43 gap junctions in the H295 population. There was a progressive decrease in gap junction plaques in adrenocortical cancer cell populations compared to those in normal cell populations. Therefore, analysis of gap junction protein may be helpful for the differential diagnosis of benign and malignant adrenal tumors. The induction of gap junctions in malignant cells may provide a novel therapeutic strategy for adrenal cancer.