ZONE-SPECIFIC ADRENOCORTICAL CELL LINES

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Hypertension is a multifactorial disease, and abnormalities in the renal, cardiovascular, and endocrine systems all produce blood pressure elevation. The complex interactions of these systems have hindered efforts to identify the causes of primary hypertension. Disordered adrenal steroidogenesis clearly induces hypertension in certain clinical settings and contributes to the pathogenesis of more complex hypertensive states. To study the normal regulation of steroid hormone biosynthesis and to define the specific contributions of corticosteroids to hypertension, it would be advantageous to develop adrenocortical cell lines that maintain differentiated function. The rat is the logical source of these cell lines because of its preeminent role in whole-animal studies of steroid hormone biosynthesis and genetic models of hypertension. Transgenic targeting of SV40 large T antigen (TAg) has been used by others to produce immortalized cell lines that retained differentiated function. Targeted expression of TAg to specific zones of the adrenal cortex in transgenic rats will generate immortalized cell lines derived from discrete zones. The promoter regions of three steroidogenic enzymes will be used: steroid 11 beta-hydroxylase, which is selectively expressed in the inner zones, aldosterone synthase, which is selectively expressed in the outer zona glomerulosa, and steroid 21 -hydroxylase, which is expressed in all cortical zones. These promoters are expressed in vivo only in adrenocortical cells, and they may ensure that the cells maintain a differentiated phenotype. Two approaches will be used to increase the chances of obtaining differentiated cell lines. First, adrenocortical tumors will be induced in transgenic rats by targeted expression of TAg. Second, transgenic animals will be prepared that express a temperature-sensitive TAg in specific adrenocortical zones. Individual clones will be derived from either the tumors (wild-type TAg) or primary cultures of adrenocortical cells (temperature- sensitive TAg) and various parameters of adrenocortical function will be studied. The role of the adrenal cortex in a novel genetic model of hypertension, the Ren-2 transgenic rat, will also be investigated. Expression of mouse Ren-2 in specific adrenocortical zones will identify essential site(s) for the hypertension. Additional experiments will establish if over-expression of the rat renin gene in specific cortical zones also induces hypertension. These studies will extend previous results with the mouse Ren-2 gene, and will thus increase our understanding of the effects of renin in this provocative genetic model of hypertension.