Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture

Summary Semi-thin sections of three-dimensional reaggregates from adult female rat pituitary, cultured in serum-free defined medium, were stained for prolactin, gonadotropin, thyrotropin, growth hormone and S-100, using the double immunolabelling technique. The frequency of juxtaposition between lactotrophs and gonadotrophs was enumerated and compared with the expected frequency at random distribution of polygonal cell profiles in a hexagonal configuration. The proportions of lactotrophs and gonadotrophs in the aggregate sections were determined using stereometrical analysis. The observed frequency of juxtaposition did not differ significantly from the expected frequency. Hence, no reason was found to assume a selective adhesion between lactotrophs and gonadotrophs in adult female rat pituitary reaggregates. A constant proportion of lactotrophs was found to meet the criteria of a cup-shaped morphology, and 70%±9% (mean ±S.D.) of these so-called cupshaped lactotrophs were found to be juxtaposed at their concave side to gonadotrophs. Administration of 0.01 nM 17-oestradiol to the culture medium resulted in a significant reduction of the proportion of cup-shaped lactotrophs but did not affect the selectivity of juxtaposition to gonadotrophs. The selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs may be the morphological correlate of the functional relationship between these cells, which are known to be involved in an intra-pituitary paracrine communication system.     

Trypsin-like activity of rat anterior pituitary in relation to secretory activity.

Biochemical changes in trypsin-like activity were studied in rat anterior pituitary during experimentally induced changes of hormone secretion. While dexamethasone and ACTH treatment of female rats led to a significant decrease of adenohypophyseal trypsin-like activity, elevated enzyme activity was observed after adrenalectomy and ovariectomy. This correlation together with our previous data on the subcellular localisation and some biochemical properties of the trypsin-like proteinases in the anterior pituitary suggests that these enzymes may be involved in the biosynthesis of some polypeptide hormones.     

Somatotrophs and lactotrophs in the anterior pituitary of fetal and neonatal rats

Summary The ultrastructure of immunoreactive somatotrophs and lactotrophs in pituitaries of fetal rats at 19, 20 and 21 days of gestation and on the day of birth was studied. Somatotrophs, first detectable at 19 days of gestation, undergo only minor modifications before reaching the structure described for adults. In particular there is an increase in the endoplasmic reticulum and Golgi apparatus. Lactotrophs, first identifiable in newborn rats, are very different in ultrastructure from adult cells, because the secretory granules are generally small, but variable in shape and size, and the Golgi complex is prominent.     

Discrete early changes in cellular subpopulations of rat uterine and anterior pituitary estrogen receptors in response to acute exposure to exogenous estradiol

Distribution of estrogen receptors among ligand-occupied and unoccupied species in cytosolic and nuclear subcellular compartments has been analyzed as an acute response to administration of 5 micrograms of estradiol in adult female rats. Patterns of anterior pituitary and uterine receptor turnover were monitored at intervals over a 5-h period, using either intact or 2-weeks ovariectomized animals. In terms of total cellular receptor content, initial levels were higher in castrate animals, but rapidly fell to intact levels within an hour following estradiol injection. Cycloheximide given shortly before estradiol had no effect on total pituitary receptor patterns, but appeared to result in an elevation in total uterine receptor content at early intervals. Unoccupied cytosol receptors were rapidly depleted and, with the exception of castrate pituitary samples, showed some replenishment within 5 h, all of which was cycloheximide-sensitive. Initially, occupied cytosol receptors were low in intact rats, but were present at levels approaching those of the unoccupied cytosol receptor forms in the ovariectomized rat tissues. Occupied cytosol receptor levels fluctuated in response to estradiol. Subpopulations of nuclear receptors, especially the unoccupied species, showed significant tissue specificity. In the uterus, unoccupied nuclear forms were initially present in high amounts, and the levels did not change in response to estradiol administration. In the pituitary, the levels of these receptors rose and subsequently fell over the 5-h interval. Cycloheximide conferred a similar biphasic response to estradiol upon the otherwise insensitive unoccupied nuclear forms of the uterus. Occupied nuclear receptors turned over completely during the 5-h study interval, with the kinetics being faster in the castrate than the intact tissues. Cycloheximide affected occupied nuclear forms of the uterus only, dramatically increasing their levels in response to estrogen and causing prolonged retention in the castrate animal model. Collectively, the cycloheximide effects on this system are consistent with early estrogen induction or stimulation of a protein which inhibits accumulation of occupied or unoccupied receptor species within the nucleus. This re-examination of all forms of cellular estrogen receptors as they fluctuate acutely in response to exogenous estrogen has revealed several heretofore undetected responses which must be incorporated into the overall scheme of early estrogen action.     

DNA synthesis in lactotrophs of primary rat anterior pituitary cell cultures. Effects of thyroliberin, bromocriptine and somatostatin

Prolactin immunostaining in combination with thymidine autoradiography was used to characterize changes in the DNA-synthesizing activity of lactotrophs in primary monolayer cultures of the rat anterior pituitary gland treated for 3 days with thyroliberin (TRH), somatostatin (SRIF) and bromocriptine (CB 154). The number of lactotrophs labelled with 3H-thymidine within the total pool of labeled pituitary cells was used to estimate DNA synthesis in prolactin-producing cells. TRH (10 ng/ml) stimulated DNA synthesis in the whole population of cultured cells but not in lactotrophs. TRH only weakly counteracted the noticeable inhibitory effect of CB 154 (0.75 microM/l) on thymidine incorporation into lactotrophs. SRIF (20 ng/ml) inhibited DNA synthesis in lactotrophs to a lesser extent than CB 154. The combination of methods used in this paper may be useful for studying the selective effects of regulators on the proliferative activity of various pituitary cell types in vivo and in culture.     

Developmental and lactational changes in the rat anterior pituitary VIP receptor

The regulation of female rat anterior pituitary vasoactive intestinal peptide (VIP) receptors was examined during postnatal development and lactation. VIP receptor binding to anterior pituitary membranes from 1- to 60-week-old rats and lactating rats was examined using HPLC purified [Tyr(125I)10]VIP. Nonlinear regression of competitive binding studies indicated the presence of 2 VIP binding sites in 3-week and older animals, whereas only 1 site was identified in 1- and 2-week-old rats. The single site did not differ significantly in affinity or number when compared to the low affinity site of older animals. The guanine nucleotide, GTP-gamma-S, decreased the specific binding of VIP by 60-80% in 3-week and older animals, but not in younger animals. Compared with adult nonlactating animals, the number of high affinity binding sites decreased significantly during lactation, with no change in receptor binding affinity.     

N-Acetylated endorphins in ovine anterior pituitary and neuro-intermediate lobe

We have used an antiserum for immunohistochemistry and RIA/RP-HPLC which recognizes all fragments of N-acetylated endorphin (NacEP). In the rat neurointermediate lobe (N-IL), in addition to the N-acetylated forms of immunoreactive-β-endorphin (ir-βEP) already reported, we have demonstrated NacβEP_1–17 as a minor component. In the sheep pituitary processing of βEP is markedly different. In the anterior pituitary (AP), staining was indistinguishable with βEP and NacEP antisera, in contrast with the rat where many fewer AP cells stained with the NacEP antiserum. Secondly, as in the rat, all N-IL cells stained with both antisera; on RP-HPLC, however, the major forms of NacEP in the sheep N-IL were NacβEP_1–17 (40%), NacβEP_1–27 (25%) and NacβEP_1–16 (20%), with NacβEP_1–31 (2%) as a minor component. A similar profile was seen on RP-HPLC of sheep AP. These data suggest that (1) patterns of processing in sheep AP are similar to those in N-IL, though the extent of acetylation is less and (2) in the sheep pituitary low molecular weight acetylated fragments predominate, in contrast with the rat.     

Cytochemical localization of adenylate cyclase activity in the rat anterior pituitary

Summary The cytochemical localization of adenylate cyclase was studied in relation to the secretory function of the anterior pituitary glands of male rats. The reaction product of adenylate cyclase was localized on the outside of plasma membranes, but was not detected intracellularly. High activity of adenylate cyclase was detected on somatotrophs and microvilli of follicular cells, whereas no activity was found on thyrotrophs or corticotrophs. Although most of the gonadotrophs showed little or no adenylate-cyclase activity, some was detected in a small number of gonadotrophs in the central portion of the gland. In somatotrophs, activity was not detected on the plasma membranes facing perivascular spaces where exocytotic extrusion of secretory granules was frequently observed, although the remaining areas of plasma membranes of the same somatotrophs were associated with high levels of adenylate-cyclase activity. These findings indicate that the association of a high level of adenylate-cyclase activity is not directly related to the ability of the plasma membranes to fuse with secretory granule membranes.     

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.     

Estrogenic activity of phenol red in rat anterior pituitary cells in culture

The estrogenic activity of phenol red, a pH indicator widely used in cell culture media, was studied in rat anterior pituitary cells. After 72 hours of incubation with 40 microM phenol red, a 40-50% increase in prolactin cell content and a 100% stimulation of luteinizing hormone-releasing hormone induced luteinizing hormone release was observed. Both effects could be completely reversed by simultaneous incubation with the antiestrogen LY156758. In the rat uterine [3H] estradiol binding assay, phenol red showed a significant displacement at concentrations above 10 microM while its concentration in the commonly used culture media is about 40 microM. From the present results, we conclude that phenol red acts as a weak estrogen in normal tissues and that its estrogenic activity should be taken into account in studies using estrogen-sensitive cell or tissue cultures.     

Inhibition of prolactin release from anterior pituitary lactotrophs in culture by sulfur-containing analogs of dopamine

The effects of permanently charged and uncharged analogs of dopamine were examined for their ability to inhibit basal prolactin release from primary cultures of rat pituitary lactotrophs. The charged quaternary trimethyldopamine and the charged dimethylsulfonium analogs were active (IC50's were 4.3 and 31 microM, respectively) while the permanently uncharged monoethylsulfide was devoid of significant activity. Dopamine and dimethyldopamine, which are able to exist in both charged and uncharged forms, are more potent (IC50's were 36 and 44 nM, respectively) but all compounds were capable of approaching the same maximum degree of prolactin release inhibition. Haloperidol, a dopamine receptor antagonist, was able to block the actions of each of the agonists. The data suggest that (a) dopamine agonists do not have to be in the uncharged form in order to activate the dopamine receptor that regulates prolactin release, (b) the uncharged monomethylsulfide analog of dopamine is incapable of activating the dopamine receptor, and (c) the nitrogen on the side chain of dopamine can be replaced by another atom and still retain prolactin release inhibiting activity.     

Rat atrial natriuretic factor suppresses proopiomelanocortin-derived peptides secretion from both anterior and intermediate lobe cells and growth hormone release from anterior lobe cells of rat pituitary in vitro

Synthetic rat atrial natriuretic factor (ANF) was found to attenuate, in a dose-dependent manner, basal and corticotropin-releasing factor-induced secretion of proopiomelanocortin-derived peptides from cultured anterior and intermediate lobe cells of rat pituitary. ANF was also found to suppress basal and growth hormone-releasing factor-stimulated secretion of growth hormone from anterior lobe cells of rat pituitary. These results, together with reports of the existence of ANF-positive neurons in the hypothalamus and ANF-positive fibers in the median eminence, suggest that hypothalamic ANF is probably involved in the regulation of pituitary hormone secretion, especially that of proopiomelanocortin-derived peptides and growth hormone.