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Control of adrenocortical cytodifferentiation in vitro by metachromatic extracellular matrix Slavinski, Eva Ann


Methods of propagating homogeneous populations of functional adult rat adrenal cortical cells in monolayer cultures as either differentiated epithelial-like cells or as partially differentiated fibroblast-like cells were developed. Further work has elucidated the local environmental parameters controlling adrenal cortical function and has suggested the mechanism(s) by which these parameters might act. Briefly, cells that were subcultured by mechanical dissociation from confluent primary adrenal cultures and grown in medium containing horse serum, assumed an epithelial-like morphology and resembled adrenal cortical parenchyma in vivo in corticosterone production and steroidogenic, lipolytic response to adrenocorticotrophic hormone. Cells that were subcultured by tryptic dissociation and grown in medium containing fetal calf serum, assumed a fibroblast-like morphology and exhibited: characteristics of both connective tissue and adrenal cortical tissue: cells produced metachromatic extracellular matrix and collagen but funlike fibroblasts of connective tissue origin, they uniformly stained for steroid and pentose shunt dehydrogenases as well as lipid, produced small amounts of corticosterone and responded steroido-genically and morphologically to adrenocorticotrophic hormone. The steroid production of these fibroblast-like cells was lower by 1-2 orders of magnitude than the steroid production of epithelial-like adrenal cells, a degree of cytodifferentiation that is reminiscent of the protodifferentiated state that occurs during the morphogenesis of many embryonic tissues. Several observations suggested that cell morphology and the presence of metachromatic extracellular matrix (MECM) were correlated with cytodifferentiation in terms of steroid production. Addition of adrenocorticotropic hormone (ACTH) to fibroblast-like adrenal cells over 3 days generally caused a disappearance of MECM and the development of an epithelial-like morphology: the magnitude of steroidogenic response to ACTH correlated with the degree of MECM disappearance. Conversely, the addition of medium containing fetal calf serum (FCS- medium) to monolayers of epithelial-like adrenal cells caused modulation to a fibroblast-like form and function within 24 h. A concomitant drop in steroid production and a large increase in MECM production occurred. In order to assess the influence of MECM on adrenal cortical cytodifferentiation more directly, adrenal epithelial-like cells were exposed to (a) 6-diazo-5-oxo-L-norleucine (DON), a drug which inhibits glycosaminoglycan synthesis, together with FCS-medium and (b) hyaluronic acid as an exogenous source of extracellular matrix. Epithelial-like cells exposed to DON in FCS-medium did not produce MECM, did not develop a fibroblast-like morphology, did not migrate away from one another and continued to produce steroids at relatively high levels. Cells exposed to hyaluronic acid (in medium containing horse serum) developed a fibroblast-like form and produced only minute amounts of steroid. The effect of this polyanion on adrenal cell morphology and function could be mimicked by growing cells on surfaces which had been sulphonated to a negative charge density of approximately 170 charges per 100 Ų. These observations implicated components of the extracellular matrix in limiting adrenal cortical cytodifferentiation and suggested that their influence was extracellular and entirely due to charge. The present study demonstrates that cells exist within the adult rat adrenal cortex which are still capable of displaying responses to environmental parameters, such as extracellular matrix, that are characteristic of mesodermal embryonic tissues. Although the physiological counterpart of these adrenal cortical cells within the adult cortex has not been definitively identified, ultrastructural and functional characteristics suggest an origin from capsular tissue, which has been implicated as a stem cell source in vivo during adrenal cortical regeneration. MECM, which surrounds capsular cells in vivo, may well inhibit their overt expression of adult adrenal cortical phenotype and thus provide a store of potential adrenal cortical parenchymal cells capable of expressing their adult phenotype upon dissolution of MECM, an event that could occur in response to very high ACTH levels.

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