Regulatory peptides produced in the anterior pituitary

Several peptide families have been detected in the anterior pituitary of several species, including man, and for many of them evidence for local synthesis has been found. Although a paracrine action seems evident for a few, the precise function of most of these peptides remains unknown.     

Subcellular localization of thyroxine 5'-deiodinase activity in bovine anterior pituitary

Bovine anterior pituitary glands were fractionated by differential centrifugation. T4 5'-monodeiodination to T3 was found predominantly in microsomal fractions (M2; 105,000 . g pellet) enriched in glucose-6-phosphatase and 5'-nucleotidase activities. T4 5'-deiodinase activity in M2 fraction was 85.2 fmol T3/min X mg protein and represented an 8.5-fold enrichment over homogenate specific activity (10.6 fmol T3/min . mg protein). Further subcellular localization of the T4 5'-deiodinase was effected by discontinuous sucrose density gradient centrifugation. Maximum T4 5'-deiodinase activity was found in fraction P5 at the interface of densities 1.18/1.20 (200 fmol T3/min . mg protein) and correlated with the profile of glucose-6-phosphatase and not with that of 5'-nucleotidase, the maximum activity of which was recovered in fraction P1 at the interface of densities 1.03/1.12. Electron microscopic examination of the fractions confirmed that P5 contained in excess of 90% rough membranes in contrast to 10% or less in P1. Characterization of T4 5'-deiodinase activity was carried out in M2 preparations. The reaction was thiol dependent, requiring the presence of 50 mM dithiothreitol or more (Km, 38 mM), with a maximum velocity of 55-150 fmol T3/min . mg protein (n = 8). Enzyme activity was substrate dependent, with a Km for T4 between 35-70 nM. 5'-Monodeiodination of T4 was abolished by heating to 70 C for 30 min and was unaffected by EDTA. Propylthiouracil and methimazole did not inhibit T3 generation. Iopanoic acid, on the other hand, was a competitive inhibitor of the 5'-monodeiodination reaction, abolishing T3 production in a dose-dependent manner with a Ki of 3 microM. These data indicate that the bovine anterior pituitary contains significant T4 5'-deiodinase activity, which shares many properties of the type II 5'-deiodinase of the rat. Bovine anterior pituitary T4 5'-deiodinase appears to be predominantly localized in the rough endoplasmic reticulum.     

Effects of thyroxine on oestrogen receptor concentrations in anterior pituitary and hypothalamus of hypothyroid rats

The effects of thyroxine (T4) were studied on the concentration of oestrogen receptors in the anterior pituitary gland and hypothalamus of ovariectomized euthyroid and hypothyroid rats. A group of rats was made hypothyroid by the administration of 131I. Seven days after ovariectomy, animals were separated into five groups: I, euthyroid controls; II, hypothyroid controls; III, hypothyroid and injected with oestradiol benzoate (10 micrograms/day for 10 days); IV, hypothyroid and injected with T4 (4 micrograms/day for 10 days) and V, hypothyroid and injected with both oestradiol and T4 as described above. In group I, oestrogen receptor levels in pituitary cytosol were 44.4 +/- 3.4 (S.D.) fmol/mg protein and in the nucleus 47.7 +/- 4.0 fmol/mg DNA. In group II the respective values were 12.8 +/- 1.7 fmol/mg protein (P less than 0.01) and 12.7 +/- 1.7 fmol/mg DNA (P less than 0.01 compared with group I). In group III, cytosolic receptor concentrations decreased when compared with those in group II (P less than 0.05), whereas nuclear receptor concentrations rose significantly (P less than 0.01). Group IV had both pituitary cytosolic and nuclear receptors increased (P less than 0.01 compared with group II). In group V there were no changes in cytosolic receptor concentrations but a significant (P less than 0.01) rise in nuclear receptors as compared with group II. Hypothalamic oestrogen receptors in untreated hypothyroid rats (group II) were unchanged in the cytosol and diminished (P less than 0.01) in the nucleus in relation to euthyroid controls (group I). Thyroxine, but not oestrogen, was effective in increasing the concentration of cytosolic receptors (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inverse effects of corticosterone and thyroxine on glucocorticoid binding sites in the anterior pituitary gland.

The aim of this study was to determine whether pituitary glucocorticoid binding sites are under hormonal control. It was shown that corticosterone and thyroxine exerted antagonistic effects on both the transcortin-like component and true receptor present in the hypophysis: thyroid hormones, in contrast to glucocorticoids which exhibited opposite influence, increased maximum binding without affecting significantly the apparent association constant. Thus, it seems that the concentration of glucocorticoid binding sites is regulated by the glucocorticoid ligands, as well as by a different hormone. Moreover, a striking parallelism was found between plasma transcortin and pituitary transcotin-like capacity, argueing in favour of a plasma origin for this pituitary binder.     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

gamma-Aminobutyric acid- and benzodiazepine-binding sites in human anterior pituitary tissue

The existence of a gamma-aminobutyric acid (GABA) system in human anterior pituitary tissue was examined. Crude membrane fractions prepared from human anterior pituitary tissue bound tritiated GABA. The binding was saturable, and Scatchard analysis indicated a single binding site of high affinity (Kd = 40 nM) and a maximum binding of 1.2 pmol/mg protein. Binding was displaced in a dose-related manner by the GABA agonists muscimol (KI = 1 X 10(-8) M), isoguvacine (KI = 6 X 10(-7) M), THIP (4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol); KI = 5 X 10(-6) M), and the antagonist (+)bicuculline (KI = 5 X 10(-5) M) but not its inactive stereoisomer (-)bicuculline (KI greater than 10(-3) M). In anterior pituitary tissue, a significant concentration of GABA was found (mean, 2.5 +/- 0.5 nmol/mg protein) but no glutamic acid decarboxylase activity, the enzyme synthesizing GABA, was detected using a highly sensitive assay. In addition, benzodiazepine binding was present. An affinity of approximately 15 nM and a Bmax of approximately 0.75 pmol/mg protein were observed when using [3H]diazepam as the ligand. No saturable clonazepam binding occurred, and only slight GABA stimulation of diazepam binding was observed (mean, 18%; range, 6-38%). The ability of GABA and benzodiazepine to alter PRL secretion in rats suggests that the human pituitary GABA-binding sites described here might also mediate effects on PRL release.     

Cell type-specific metabolism of peptidylglycine alpha-amidating monooxygenase in anterior pituitary

Peptidylglycine alpha-amidating monooxygenase (PAM) is a bifunctional enzyme expressed in each major anterior pituitary cell type. We used primary cultures of adult male rat anterior pituitary to examine PAM expression, processing, and secretion in the different pituitary cell types and to compare these patterns to those observed in transfected AtT-20 corticotrope tumor cells. Immunostaining and subcellular fractionation identified PAM in pituitary secretory granules and additional vesicular compartments; in contrast, in AtT-20 cells, transfected PAM was primarily localized to the trans-Golgi network. PAM expression was highest in gonadotropes, with moderate levels in somatotropes and thyrotropes and lower levels in corticotropes and lactotropes. Under basal conditions, less than 1% of the cell content of monooxygenase activity was secreted per h, a rate comparable to the basal rate of release of individual pituitary hormones. General secretagogues stimulated PAM secretion 3- to 5-fold. Stimulation with specific hypothalamic releasing hormones demonstrated that different pituitary cell types secrete characteristic sets of PAM proteins. Gonadotropes and thyrotropes release primarily monofunctional monooxygenase. Somatotropes secrete primarily bifunctional PAM, whereas corticotropes secrete a mixture of mono- and bifunctional proteins. As observed in transfected AtT-20 cells, pituitary cells rapidly internalize the PAM/PAM-antibody complex from the cell surface. The distinctly different steady-state localizations of endogenous PAM in primary pituitary cells and transfected PAM in AtT-20 cell lines may simply reflect the increased storage capacity of primary pituitary cells.     

Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue

Previous studies have suggested that vasoactive intestinal polypeptide (VIP) is involved in regulation of PRL secretion within the pituitary gland. In order to determine whether VIP is synthesized in anterior pituitary tissue, we performed three experiments. In all experiments, anterior pituitaries were obtained from male rats. The tissue was then labeled by incubation in leucine-free minimum essential medium containing [3H]leucine, 14 microCi/ml. In Exp I, the labeled tissue was homogenized, centrifuged, and the supernatant was chromatographed on Sephadex G-50F. The fractions indicated a large peak of counts near the void volume and another peak coeluting with VIP. These latter fractions were pooled and subjected to reverse phase HPLC. Fractions from the HPLC indicated: a protein peak, VIP immunoreactivity, and maximum counts immunoprecipitated by anti-VIP serum at the retention time of synthetic porcine VIP. Exp II consisted of perifusion of labeled pituitary quarters over a 120-min period followed by an additional 60 min in the presence of 56 mM KCl. During this latter period of KCl depolarization, a large amount of 3H-labeled material was secreted. These fractions were then chromatographed on Sephadex G-50F, and the fractions coeluting with [125I]porcine VIP were subjected to immunoprecipitation with anti-VIP serum. In addition, all fractions from the Sephadex column were assayed for VIP, and the only activity was at the elution volume of [125I]porcine VIP. In Exp III, the pituitary labeling procedure included 3.6 X 10(-5) M cycloheximide. Subsequently, the tissue was perifused and the perifusate collected during the 60-min 56 mM KCl perifusion phase was pooled and immunoprecipitated with anti-VIP serum. No immunoprecipitable counts were obtained. These experiments indicate that anterior pituitary tissue synthesizes VIP on the basis of the HPLC profile and immunoprecipitation with specific anti-VIP antiserum. These results, in addition to other studies by our laboratory and others, suggest that intrapituitary VIP may be an important regulator of anterior pituitary hormone secretion, particularly PRL.     

Effect of 17 beta-estradiol on phosphoinositide metabolism and prolactin secretion in anterior pituitary cells

The present study was undertaken to investigate the effect of 17 beta-estradiol (E2) administration on in vitro prolactin (PRL) release and intracellular phosphoinositide metabolism. The incorporation of [3H]inositol (Ins) into phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphophate [PtdIns(4,5)P2], and the generation of inositol phosphate (InsPx) following thyrotropin-releasing hormone (TRH) stimulation were studied in primary cultures of anterior pituitary cells obtained from ovariectomized rats. Administration of polyestradiol phosphate (PEP) to ovariectomized rats produced a significant increase (p less than 0.05) in serum PRL levels. This treatment also enhanced significantly (p less than 0.01) the in vitro release of PRL in a progressive manner during 24, 48 and 72 h of culture of dispersed anterior pituitary cells. The radioisotopic labeling by [3H]Ins of all species of phosphoinositides was progressive throughout 72 h of culture, and a good correlation was observed between intracellular phosphoinositide synthesis and PRL release from these cells. PEP treatment enhanced significantly (p less than 0.05-0.01) [3H]Ins incorporation into PtdIns and PtdIns(4)P after 48 and 72 h of culture, although it did not alter [3H]Ins incorporation into PtdIns(4,5)P2. Furthermore, this treatment caused a small, but significant increase (p less than 0.01) in InsPx generation following TRH stimulation. However, the increased [3H]Ins incorporation into phosphoinositide and InsPx generation that we observed after TRH stimulation was significantly (p less than 0.01) less than the increased amount of in vitro PRL release following PEP treatment. There was no significant correlation between the percentage increases in PRL release and phosphoinositide metabolism following the same treatment. These data suggest that phosphoinositide metabolism is enhanced in the anterior pituitary cells of ovariectomized rats by treatment with PEP, but this system does not appear to be tightly coupled or causally related to the much greater production of PRL release.     

Regulation of thyroxine 5'-deiodinase activity by 3,5,3'-triiodothyronine in cultured rat anterior pituitary cells

The influence of the medium T3 concentration on iodothyronine 5'-deiodinase activity was studied in cultured anterior pituitary cells derived from chronically hypothyroid rats. Type II (propylthiouracil-insensitive) enzyme activity, measured with T4 as substrate, was reduced by T3 in a dose-dependent manner, with an ED50 of approximately 1.4 X 10(-10) M free T3. Density gradient centrifugation was used to obtain populations of pituitary cells relatively enriched in thyrotrophs, somatotrophs, mammotrophs, or gonadotrophs, and the effect of T3 on type II 5'-deiodinase activity was evaluated in each of these four populations. In the absence of T3, the enzyme activity was 1.5- to 2-fold greater in the somatotroph- and mammotroph-enriched cell pools than in the thyrotroph- and gonadotroph-enriched pools. In contrast, when the cells were cultured in the presence of T3, enzyme activity was reduced to the same low level in all four enriched pools. The results suggest that the increase in whole pituitary type II 5'-deiodinase activity associated with hypothyroidism is due largely or totally to increases occurring within somatotrophs and mammotrophs. The data also suggest that the intrinsic responsiveness of the deiodinase to hypothyroidism is greater in somatotrophs and mammotrophs than in other anterior pituitary cells.     

Bradykinin stimulates phosphoinositide metabolism and prolactin secretion in rat anterior pituitary cells

Bradykinin stimulated prolactin secretion from monolayer cultures of rat anterior pituitary cells, the stimulation being greater from the cells of male rats. This stimulated secretion was accompanied by a rise in total inositol phosphate accumulation, suggesting that the action of bradykinin is mediated by phosphoinositide hydrolysis. The increase in inositol phosphate accumulation was biphasic; a further sharp rise occurred when the concentration of bradykinin exceeded 1 mumol/l. This may indicate that bradykinin acts on other cell types in the pituitary gland. Bradykinin had no effect on growth hormone secretion from cells of normal pituitary glands, or on prolactin secretion and phosphoinositide metabolism in GH3 rat pituitary tumour cells. Bradykinin receptor antagonists (both B1 and B2) had no effect on either bradykinin-stimulated inositol phosphate accumulation or prolactin secretion. Kallikreins, the enzymes responsible for the generation of kinins, are known to be present in the adenohypophysis. Therefore, the results presented here would suggest that kinins may have a role as paracrine agents in the pituitary gland.