Autoradiographic determination of angiotensin II receptors in prepubertal and postpubertal pig ovarian tissue

Abstract

The aim of this study was to determine whether specific receptors for angiotensin II are present in prepubertal and postpubertal pig ovaries using an autoradiographic technique and computerized densitometry. Frozen sections were cut from prepared samples, and (125)I-labelled angiotensin II and the angiotensin II receptor subtype-specific nonpeptide antagonists for receptor subtype 1, AT(1) (GR117289) and subtype 2, AT(2) (PD123319) were used. In both pre- and postpubertal pig ovarian tissue, specific receptors for angiotensin II were demonstrated. These receptors had a density of 2487.6 (range: 267.5-5177.6, n = 4) and 3703.8 (range: 1819.9-5207.8, n = 4) fmol per mm(2), respectively, and dissociation constants of 130.0 and 26.3 nmol l(-1), respectively (prepubertal ovarian range: 106.0-165.4 nmol l(-1); postpubertal ovarian range: 26.1-100.3 nmol l(-1); P < 0.05, Mann-Whitney U test). AT(1) receptors with a K(i) for (125)I-labelled angiotensin II of 346.9 nmol l(-1) in the prepubertal and 268.1 nmol l(-1) in the postpubertal ovary were located predominantly in follicle wall tissue. Competitive inhibition studies using both angiotensin II antagonists resulted in a decrease in K(i) with prepubertal tissue (283.7 nmol l(-1)) and an increase in postpubertal tissue (293.9 nmol l(-1)). Immunocytochemistry using sections from paraffin wax-embedded prepubertal (n = 4) and postpubertal (n = 4) pig ovaries confirmed the presence of AT(1) receptors on the granulosa cell layer, but not the thecal cell layer, of antral follicles in both pre- and postpubertal pig ovarian tissue, and AT(2) receptors within the granulosa cell layer of prepubertal pig ovarian antral follicles. In summary, these results indicate that angiotensin II receptors are of higher affinity in postpubertal tissue than they are in prepubertal tissue, and indicate an active renin-angiotensin system within the pig ovary.