Angiotensin II (Ang II) receptors, estimated by the specific binding of the peptide Ang II receptor antagonist [125I] [Sar1,Ile8]Ang II, are localized on multiple ovarian structures, including follicular granulosa cells. Using the Ang II receptor subtype-selective nonpeptide antagonists, DuP 753 [selective for the type 1 Ang II (AT1) receptor] and PD 123319 [selective for the type 2 Ang II (AT2) receptor], we show that follicular granulosa cells, in vivo and in vitro, exclusively express the AT2 receptor. To understand the function of Ang II in ovarian follicles, we compared the biochemical properties and transmembrane signaling pathways of the granulosa cell AT2 receptor with those properties generally associated with Ang II receptors found in the adrenal zona glomerulosa, where the AT1 receptor predominates. The mol wt of the granulosa cell AT2 receptor (approximately 79,000), estimated by affinity cross-linking studies, is similar to that of the adrenal zona glomerulosa Ang II receptor. Like the adrenal zona glomerulosa Ang II receptor, binding inhibition studies show that the granulosa cell AT2 receptor binds Ang II and Ang III with high affinity (IC50, approximately 0.5 nM for both peptides), but not Ang-(1-7) (IC50, approximately 0.5 microM) or Ang-(1-5) (IC50, greater than 10 microM). However, unlike the adrenal zona glomerulosa Ang II receptor, the granulosa cell AT2 receptor does not undergo agonist-induced endocytosis. Further, Ang II does not affect basal or stimulated inositol phosphate production, intracellular Ca2+ mobilization, or adenylyl cyclase or guanylyl cyclase activity in granulosa cells. The granulosa cell AT2 receptor does not appear to directly interact with guanine nucleotide binding regulatory proteins, since agonist dissociation from the AT2 receptor is unaffected by the GTP analog guanosine 5'-O-(3-thiotriphosphate); in contrast, the AT1 receptor appears to directly interact with guanine nucleotide binding regulatory protein, because agonist dissociation from the AT1 receptor is stimulated by guanosine 5'-O-(3-thiotriphosphate). These studies clearly demonstrate that the granulosa cell AT2 receptor is functionally distinct from the well characterized adrenal zona glomerulosa Ang II receptor. The exclusive presence of the AT2 receptor on the granulosa cell makes it an ideal cell type for studying the potential, but as yet unknown, function of this receptor.

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To demonstrate the existence and help clarify the function of renin in the rat ovary, we have characterized rat ovarian renin and examined ovarian renin levels during different stages of the rat estrous cycle. We show that high concentrations of active renin are present in the rat ovary (2.9 ng angiotensin I/h/mg). Ovarian renin activity has a pH optimum of about 7.0 and is due to a glycosylated aspartyl protease with an apparent mol wt of 39,000. These properties of rat ovarian renin are identical to previously characterized rat kidney renin. In PMSG-treated immature and adult 5-day cycling rats, ovarian renin was increased about 2-fold at estrus. At all stages of the estrous cycle in the 5-day cycling rat, the ratio of active to inactive ovarian renin was about 3:1, whereas about 90% of the renin in plasma was inactive. In the hypophysectomized diethylstilbestrol-treated rat ovary, over 90% of active renin remained in the residual ovary after the granulosa cells had been expressed, suggesting a theca-interstitial localization for renin. These studies indicate that active renin exists in the rat ovary, that its levels are cyclically increased at estrus, and that this increase may be due to enhanced local production and activation of the renin precursor. These findings greatly strengthen the concept of a functional renin-angiotensin system in the rat ovary.

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