Vascular endothelial cells (ECs) release a number of biologically-active mediators (PGI2, nitric oxide (NO) and endothelin) that act on adjacent smooth muscle cells to influence vascular tone. In the adrenal cortex, as adrenal arteries get smaller and divide into capillaries and sinusoids, the smooth muscle layer is lost and the abluminal side of the EC comes in close anatomical contact with the steroidogenic cells. Thus, in the capillaries of the adrenal cortex, these mediators may influence the function of the adjacent steroidogenic cells. We have focused our studies on the role of endothelial-derived NO on steroidogenesis. We found that the NO donors inhibit angiotensin II (All)-stimulated aldosterone release. The inhibition by NO is not mediated by cyclic GMP but by NO binding to the cytochrome P450 enzymes involved in aldosterone biosynthesis. We were unable to detect NO synthesis, NO synthase (NOS) activity or NOS protein in ZG cells. However, adrenal capillary ECs contain NOS and release NO. We wilt test the hypothesis that ECs, which are in close anatomical proximity to ZG cells in the adrenal cortex, release NO and regulate or modulate aldosterone release. The proposed studies will investigate the effects of exogenous, endogenous and endothelial-derived NO on aldosterone release. These studies will be conducted in vitro in cultured bovine ZG cells and adrenal capillary ECs. Also, studies will be performed in the perfused rat adrenal gland and in vivo in anesthetized rats. The hypothesis will be tested by addressing four specific aims: (1) We will characterize the effect of exogenous NO on aldosterone release in cultured bovine ZG cells. The effect of the NO donor, deta-nonoate, will be tested on aldosterone release under conditions that vary the oxygen concentration. Additional studies will determine the biosynthetic step inhibited by NO and the effect of chronic NO treatment on aldosterone production and steroidogenic enzymes. (2) Since ZG cells do not have NOS, we will determine the effect of endogenous NO by conferring NOS activity on ZG cells by transducing the cells with an adenovirus containing NOS. (3) We will characterize the influence of endothelial-derived NO on ZG cell aldosterone release. Using co-incubation of ECs and ZG cells, we will determine the effect of agonists on the release of NO from ECs and the action of this NO on ZG cell aldosterone release. (4) We will determine the role of endothelial NO in regulating steroidogenesis and adrenal blood flow in the in situ perfused adrenal gland and in vivo in anesthetized rats.
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