Logical properties of angiotensin II receptors in the rat adenohypophysis were analyzed in cultured rat pituitary cells incubated with angiotensin II and known stimuli of pituitary hormone secretion. PRL release during incubation for 3 h with 3 nM angiotensin II was consistently increased by 68 +/- 5%, comparable with that elicited by TRH (63.1 +/- 4%). The ED50 of 0.5 nM for PRL release by angiotensin II was significantly lower than that of TRH (2.9 nM) in the same cell cultures. The antagonist analog [Sar1,Ala8]angiotensin II prevented the angiotensin-induced rise in PRL production but not that evoked by TRH, whereas dopamine and SRIF inhibited basal, angiotensin, and TRH-stimulated PRL release. Angiotensin II also caused a small increase in ACTH release but had no effect on the release of LH, TSH, and GH. Angiotensin II binding and PRL release were measured in partially purified lactotrophs prepared by elutriation, by which the initial cell suspension was separated into seven fractions. Most of the lactotrophs were present in the two fractions eluted at flow rates of 15.7 and 19.8 ml/min, as indicated by their immunoreactive PRL content. The 2.5- to 3.2-fold enrichment of lactotrophs was accompanied by a 2- to 3.5-fold increase in angiotensin II receptor concentration, with no change in binding affinity (Ka = 3.5 x 10(9) M-1). In the same fractions, angiotensin II-induced PRL release was similarly increased by 1.6- to 3.5-fold above basal, compared with values of less than 1 in the initial cell suspension and other fractions. The preferential location of angiotensin II receptors in the lactotroph-containing fractions and the close correlation between angiotensin II binding sites and stimulation of PRL release indicate the functional importance of the pituitary angiotensin II receptor sites. These findings also suggest that angiotensin II could contribute to the physiological regulation of PRL secretion.
The development and morphology of immunocytochemically stained thyrotropes were studied in sections of intact pituitaries and dissociated cell fractions separated by centrifugal elutriation. In the initial cell suspension from six elutriation experiments, adult female rat thyrotropes were 4.8 +/- 0.5% (+/- SE) of the cell population. Correlative morphometric studies of Araldite- or glycol methacrylate-embedded pituitaries showed that there were no changes in the percentage of thyrotropes with the stage of the estrous cycle and that the percentage of thyrotropes (5.14 +/- 0.4%) was not significantly different from the percentages in the initial cell suspension. TSH cell area fraction (a) measurements (with a 10,000-microns 2 ocular grid) showed that adult rat thyrotropes covered 171 +/- 5 microns 2 of the grid area and averaged 15 microns in diameter. In neonatal rats, thyrotropes were half the a of those in the adult for the first 9 days of life. Thereafter, they expanded, and the a reached adult levels by 20-21 days of development. TSH cell percentages remained 2-3 times adult levels throughout postnatal development (2-22 days). This developmental pattern contrasted with that in the male, which showed adult percentages of thyrotropes by 15 days of age. In the elutriation experiments from adult rats, thyrotropes were 2- to 4-fold more concentrated in the fractions eluted at 37.0 ml/min or greater, which contained the largest cells. Fraction 7 (39.5 ml/min) showed a 2-fold enrichment of thyrotropes to 11.6 +/- 1.4%, and fraction 8 showed a 4-fold enrichment to 19.6 +/- 2.7%. A few small TSH cells were found in the fractions 1-3, eluted at 11.8-19.5 ml/min. Electron microscopic studies showed that some of these small TSH cells were poorly granulated and difficult to distinguish from small gonadotropes, whereas the large thyrotropes resembled those described previously in the adult or developing male rat. These studies, thus, combine techniques of immunocytochemistry, morphometrics, and cell separation by elutriation to describe TSH cells in the female rat pituitary. Our findings agree with those reported by Denef et al., who showed that thyrotropes in the adult male rat are among the largest cells in the pituitary. The few small thyrotropes in the female rat may be equivalent to the prolific cells described by Leuschen et al. in the male rat that enrich monolayers from these fractions after 7 days in culture.
The application of centrifugal elutriation to the separation of pituitary cells has produced a fraction that is enriched with LH and FSH gonadotropes. In this study, stains were performed on serial sections of fractions taken from six elutriation experiments with 1:10,000-1:30,000 anti-bLH beta or 1:2,000-1:8,000 anti-hFSH beta and the avidin-biotin-peroxidase complex technique. Over 900 serially sectioned gonadotropes were analyzed. In the initial cell suspensions, 59.6% of the gonadotropes contained both LH and FSH; 18% contained LH, and 23% contained FSH. Fewer than 3% contained ACTH. The elutriation fractions contained several subtypes of gonadotropes. A few small (8 micron), poorly granulated LH or FSH cells were eluted with the smallest cells (10-12%) at flow rates of 15.7 ml/min. Medium sized gonadotropes (10-10.5 micron) that eluted at flow rates of 19.8-30.5 ml/min were infrequent (3-6%) and resembled the larger gonadotrope. An analysis of serially sectioned fields showed that only 15.2%-22% of the small and medium sized gonadotropes contained both LH and FSH, whereas 30-40% contained only LH and 46-50% stored only FSH. The gonadotrope-enriched fraction was eluted at 37-39.5 ml/min; 78% of these gonadotropes (diameter, 14-15 micron) contained both LH and FSH, 10% contained only LH, and 12% contained FSH. Finally, large cells (diameter, 15-16 micron) that contained FSH only were found in the Wash fraction. These studies demonstrate that smaller gonadotropes tend to store only one of the hormones whereas most of the larger cells either store LH and FSH together or FSH alone. These heterogeneous storage patterns may correlate with studies by others who measured different responses to GnRH in gonadotropes separated by size on a unit gravity sedimentation gradient. They may also reflect different secretory phases of gonadotropes from the mixed group of cycling female rats.
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