CHARACTERIZATION OF ANGIOTENSIN-II RECEPTOR SUBTYPE ON BOVINE THECAL CELLS AND ITS REGULATION BY LUTEINIZING-HORMONE

In this study the primary objectives were to localize angiotensin-II (AII) receptors on specific ovarian cells and determine whether, thee receptors are regulated by LH. AII receptor analysis, carried out using membrane fractions prepared from isolated thecal, granulosa, and luteal cells from bovine ovary, revealed that [I-125]AII-binding sites were present only on thecal cells. The K(d) and binding capacity were determined to be 0.29 +/- 0.08 nm and 66.9 +/- 8.1 fmol/mg membrane protein (mean +/- SEM from three experiments, each with duplicate determinations), respectively. None of the peptides unrelated to AII affected the binding of [I-125]AII. Unlabeled AII, saralasin, and AIII were equipotent (IC50, approximately 5 nm for all three peptides) in competing with the radioligand. However, the binding affinity for AI was less by almost 2 log units. Using AII receptor subtype-specific nonpeptide antagonists, Losartan [a selective antagonist for the type 1 AII (AT1) receptor] and PD 123319 [a selective antagonist for the type 2 AII (AT2) receptor], AII receptors on thecal cells could be classified pharmacologically as AT2-type receptors. The IC50 determined from the competitive binding inhibition experiments for the various unlabeled competing substances were 5 nM, 20 nM, 200 nM, and 200-mu-M with respect to AII, p-amino-phenylalanine-AII, PD123319, and Losartan, respectively. Thecal cells cultured in a serum-free medium also expressed AII receptors, which could be up-regulated by LH or 8-bromo-cAMP in a dose-dependent manner. The number of AII receptors on thecal cells nearly doubled when the cells were cultured in the presence of 100 ng/ml LH, with little change in their K(d) value. This increase in the number of AII receptors was inhibitable by a protein synthesis inhibitor, cycloheximide. In summary, we have demonstrated that in the bovine ovary, AII receptors belonging to AT2 subclass are predominantly expressed on thecal cells, and these receptors can be up-regulated by LH via a cAMP-dependent mechanism. Thus, the bovine thecal cells in primary culture can potentially become a useful in vitro system to study the mechanism of regulation of AII receptor induction as well as the so far unknown function of this class of receptor.