Purification of functional lactotrophs and somatotrophs from female rats using fluorescence-activated cell sorting

As the secretory granules of anterior pituitary cells fuse with the cell surface, there would appear to be sufficient hormone present on the cell surface to be labelled by polyclonal hormone antibodies and thus analysed by flow cytometry. We have therefore applied fluorescence-activated cell sorting to these labelled pituitary cells. Percentage purity and depletion of other cell types was assessed by immunocytochemistry and the reverse haemolytic plaque assay (RHPA). Results demonstrate that fluorescence-activated cell sorting allows almost complete purification of functional lactotrophs and somatotrophs to 96.7 +/- 1.7 (S.E.M.)% and 98 +/- 1.0% respectively by immunocytochemistry, and to 95.8 +/- 1.1% and 97 +/- 0.8% respectively by RHPA. Depletion of other anterior pituitary cell types to less than 2% was demonstrated by both immunocytochemistry and RHPA. Fluorescence-activated cell sorting to this degree of purity was routinely possible with cell yields of 91 +/- 3.4%. To obtain such purity/depletion, it was necessary to use specific antisera of high titre, at concentrations which ensured maximal cell-surface labelling associated with maximal stimulation of hormonal secretion by the appropriate hypothalamic stimulatory factor. Separating cells on the basis of the intensity of prolactin cell-surface labelling demonstrated a low level of binding of the prolactin antibody to gonadotrophs (but not of sufficient fluorescence intensity to be sorted into the prolactin enriched population), raising the possibility of prolactin receptors on gonadotrophs. We were unable to demonstrate the presence of mammosomatotrophs in the normal female rat, since purified lactotrophs did not contain or secrete GH nor did purified somatotrophs contain or secrete prolactin.     

Effects of hypothyroidism on somatotrophs and lactotrophs of rat pituitary

Hypothyroidism was induced in male Wistar rats with propylthiouracil (PTU) in the diet and the adenohypophyses were inimunostained for growth hormone and prolactin. In addition, autoradiography with [³H]thymidine demonstrated cells undergoing DNA synthesis. Feeding of PTU for 18 days reduced the immunostainable somatotrophs from 50% to virtually zero, whereas there was no change in the number of lactotrophs. In hypothyroidism, somatotrophs did not disappear from the gland but due to an almost complete degranulation, they were not recognizable by the immunoperoxidase technique. After withdrawal of PTU, these cells regranulated and became identifiable again as somatotrophs by immunostaining. In the hypothyroid pituitary labeled after 18 days of PTU, the labeling index was markedly decreased, compared to the normal gland and to the hypothyroid gland labeled before PTU was started. Thus it appears that hypothyroidism reduces but does not completely abolish DNA synthesis in somatotrophs, and has no effect on the replication of lactotrophs.     

Endothelin-like immunoreactivity in lactotrophs, gonadotrophs, and somatotrophs of rat anterior pituitary gland are affected differentially by ovarian steroid hormones

It has been previously found that all hormone-producing phenotypes of the anterior lobe of the pituitary gland are capable of producing endothelin (ET)-like substances. The aim of this study was to determine whether the expression of ET-1-like peptides in lactotrophs, gonadotrophs, and somatotrophs is influenced by different in vivo ovarian hormonal conditions. Anterior lobes of the pituitary gland were harvested from ovariectomized and ovarian steroid-replaced adult female rats 10–12 d after surgery. Quantitative immunocytochemistry was performed on enzymatically dispersed pituitary cells. The presence of ET-1-like immunoreactivity in prolactin-, luteinizing hormone-, or growth hormone-producing cells was demonstrated by double-label immunocytochemistry. The incidence of ET-1 immunopositive pituitary cells was unaffected by progesterone treatment alone. Estradiol replacement caused a modest decrease in the number of lactotrophs and somatotrophs expressing ET-1 but increased the incidence of ET-1 immunopositive cells among gonadotrophs. Combined treatment with estradiol and progesterone robustly increased the incidence of ET-1 immunopositive lactotrophs and gonadotrophs but had no effect on somatotrophs. These data reveal that the synthesis of ET-1-like peptides in lactotrophs and gonadotrophs (and, to a lesser extent, in somatotrophs) is sensitive to ovarian steroids. Furthermore, these findings predict that ovarian steroids modulate ET-1 biosynthesis during the estrous cycle, suggesting a possible mechanism by which the ovarian steroid milieu may regulate the responsiveness of lactotrophs and gonadotrophs to their hypothalamic secretagogues.     

Full oestrogenic activity of C19-delta 5 adrenal steroids in rat pituitary lactotrophs and somatotrophs

Oestrogens are known to exert specific stimulatory effects on basal and dopamine (DA)-inhibited prolactin (PRL) release as well as on PRL cell content in rat adenohypophysial cells in primary culture. Recently, we have demonstrated that classical oestrogens increase growth hormone (GH) release and cellular GH content in rat pituitary somatotrophs. Since there is evidence that 5-androstene-3 beta, 17 beta-diol (delta 5-diol), a metabolite of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) can induce typical oestrogenic responses in target tissues, we have investigated the effect of C19 adrenal steroids and compared them to that of 17 beta-oestradiol (E2) on the above-indicated parameters. Following a 72-h incubation, maximal concentrations of E2, delta 5-diol and DHEA caused a similar increase in PRL cell content at respective EC50 values of 0.020, 14 and 115 nM. The sensitivity of lactotrophs to DA action was decreased by approximately 4-fold after a 48-h exposure to maximal concentrations of delta 5-diol or DHEA. The time course of the antidopaminergic effect of delta 5-diol and DHEA was almost superimposable to that of E2. A 72-h incubation with 5 microM DHEA-S, a concentration within the range of blood levels found in women, doubled (P less than or equal to 0.001) cellular PRL accumulation. On the other hand, androstenedione (delta 4-dione) was without effect on any of the parameters measured. All stimulatory effects induced by maximal effective concentrations of delta 5-diol, DHEA or DHEA-S were competitively inhibited by simultaneous incubation with the antioestrogen LY156758. The amplitude of the stimulatory effects of delta 5-diol and DHEA on GH cell content as well as on spontaneous GH-releasing factor (GRF)-induced GH release was superimposable to that observed with E2. The effect of the steroids on GH cell content was exerted at 0.016 nM (E2), 12 nM (delta 5-diol) and 250 nM (DHEA). Simultaneous incubation with LY156758 completely blocked the effect of maximally effective concentrations of E2, delta 5-diol and DHEA in somatotrophs. Furthermore, a physiological concentration of DHEA-S (5 microM) enhanced spontaneous as well as GRF-induced GH release. On the other hand, delta 4-dione as well as testosterone and dihydrotestosterone did not alter GH release. The present data demonstrate that delta 5-diol, DHEA and DHEA-S can exert full oestrogenic effects in lactotrophs and somatotrophs, thus supporting their potential oestrogenic role under both physiological and pathological conditions.     

Functional heterogeneity in somatotrophs isolated from the rat anterior pituitary.

Somatotrophs in suspensions of anterior pituitary cells from adult male rats can be separated into 2 fractions by density gradient centrifugation. In addition to their different densities, somatotrophs in these 2 fractions can be distinguished morphologically by their staining characteristics and ultrastructure. Somatotrophs of lesser density (type I; approximately 1.068 g/cm3) have fewer secretory granules and a more extensive Golgi apparatus than the somatotrophs of greater density (type II; approximately 1.073 g/cm3). Responsiveness of type I and type II cells to secretory agents (i.e., dibutyryl cyclic adenosine monophosphate, somatostatin, thyroxine, and hydrocortisone) was evaluated by GH radioimmunoassay. Type I cells were consistently more responsive (% GH release) than type II cells. During 7 days in culture, type I cells produced more (approximately 200%) GH than they initially contained, whereas type II cells did not show evidence of increased GH production. Hydrocortisone significantly stimulated GH production in type I, but not type II cells. These results support the hypothesis that at least 2 functionally distinct populations of somatotrophs are present in the anterior pituitary gland of the adult male rat.     

Localization of immunoreactive glandular kallikrein in lactotrophs of the rat anterior pituitary

Glandular kallikrein (a trypsin-like serine protease) is an estrogen-induced and dopamine-repressed protein in the rat anterior pituitary which predominantly exists as a latent zymogen (prokallikrein). Its regulation, presence in estrogen-induced pituitary tumors in F344 rats, and expression in GH3 cells has suggested a localization in lactotrophs (prolactin-producing cells). This study examined the cellular origin of glandular kallikrein using immunocytochemical techniques. Anterior pituitaries from estrogen-treated rats were fixed and embedded in paraffin (for preparation of semi thick sections; 5 microns) or methacrylate (for preparation of thin sections; 1 micron). Glandular kallikrein immunostaining was readily detected in the perinuclear (Golgi) region of parenchymal cells of the anterior pituitary in both thin and semi thick sections. Two-color double immunoperoxidase staining of thin and semi thick sections indicated that glandular kallikrein was localized in cells containing prolactin (PRL) but not other pituitary hormones. Immunoperoxidase staining of consecutive serial thin sections with alternating antisera confirmed a localization of glandular kallikrein in lactotrophs. The results establish that glandular kallikrein is colocalized with PRL in lactotrophs of the rat anterior pituitary. This is consistent with the hypothesis that the function of anterior pituitary glandular kallikrein is linked to PRL in some fashion--possibly as a PRL-processing protease.     

Production and partial characterization of a monoclonal antibody to rat anterior pituitary somatotrophs

A cell type-specific monoclonal antibody (Mab) against a cell surface antigen of rat anterior pituitary somatotrophs has been generated by fusion of a nonsecreting mouse myeloma line with spleen cells from a mouse immunized with enzymatically dispersed anterior pituitary cells of adult random cycling female rats. Hybridomas were initially screened for antibodies to cell surface antigens by an enzyme-linked immunosorbant assay using rat anterior pituitary cells and smooth muscle cells of aorta as positive and negative controls, respectively. Positive clones were further checked for cell type specificity by immunofluorescence. Mab WHC-1 is an immunoglobulin M (IgM) with kappa-light chains and is cytotoxic in the presence of complement. Based on double immunofluorescence, this Mab reacted with 22.5 +/- 2.0% (+/- SEM) of the anterior pituitary cells of adult random cycling female rats. Among them, about 93.5 +/- 1.4% were somatotrophs, and only 4.1 +/- 1.2% were mammotrophs. Approximately two thirds of the somatotrophs were Mab WHC-1-positive. The reaction of this Mab with gonadotrophs, thyrotrophs, or corticotrophs were negligible. The percentage of Mab WHC-1-positive cells derived from immunoperoxidase staining was significantly greater than that from immunofluorescence. The cell surface antigen defined by Mab WHC-1 is expressed heavily on GH3 cells, but not on smooth muscle cells. It is resistant to trypsin digestion, but sensitive to ethanol treatment, and exhibits the solubility property of a glycolipid. Mab WHC-1 cross-reacts with the anterior pituitary cell of rabbits, but not mice. These results provide the immunological evidence for heterogeneity among somatotrophs and demonstrate the feasibility of making pituitary cell type-specific Mabs.     

Anti-prolactin cell-surface immunoreactivity identifies a subpopulation of lactotrophs from the rat anterior pituitary

Suspensions of cells dissociated from the anterior pituitary of the adult rat include many that contain intracellular PRL. After fixation, these cells can be identified and the distribution of their PRL determined by immunocytochemistry with anti-PRL antibodies. We have found that approximately 50% of the cells that contain intracellular PRL also have PRL or PRL-like immunoreactive material on the outer cell surface. Cell-surface PRL can be detected on unfixed anterior pituitary cells using anti-PRL antibodies and either fluorescence microscopy or fluorescence-activated cell sorting. Cell surface labeling by anti-PRL antibodies was restricted to PRL-containing cells; 85-97% of the labeled cells contained detectable intracellular PRL, while fewer than 2% contained detectable GH, ACTH, LH, or TSH. Subpopulations of live anterior pituitary cells could also be labeled on the cell surface by antibodies against GH, ACTH, LH, or TSH. This suggests that the presence of hormone at the cell surface may be a characteristic and identifying feature of different anterior pituitary cell types.     

Coculture of anterior and posterior pituitary cells: selective stimulation of lactotrophs

There is extensive evidence that the posterior pituitary (PP) participates in the regulation of PRL secretion. We recently reported that a putative PRL-releasing factor is localized, and possibly produced, in the intermediate lobe of the PP. The aim of the present investigation was to determine whether cultured PP cells affect anterior pituitary (AP) function in terms of cell content and cumulative release of PRL. Anterior and posterior pituitaries from adult male rats were dispersed with trypsin and cultured either alone or together for 4 and 8 days in serum-free medium. The concentrations of PRL, GH, and LH in cell extracts and culture media were measured by RIA. Coculturing of AP and PP cells at different plating densities resulted in a 2-fold rise in PRL cell content after 4 days. The cumulative release of PRL in the cocultures was significantly increased only after 8 days. LH and GH were affected slightly, or not at all. Medium conditioned by PP cells mimicked the effects of coculture on the cumulative release of PRL, but not on the cell content. Short-term incubation with TRH induced a much larger release of PRL from AP + PP cocultures than from AP cells cultured alone. In conclusion, these data suggest that 1) PP cells stimulate the production and release of PRL in a hormone-specific manner, and 2) coculturing of AP + PP cells augments the responsiveness of lactotrophs to secretagogues such as TRH. We propose that at least two factors, one of which might be PRL-releasing factor, are involved in these effects.     

Effect of estrogen on the expression of a cell-surface antigen associated with rat anterior pituitary somatotrophs

The long-term in vivo effect of diethylstilbestrol (DES) on the expression of a cell-surface antigen associated with the anterior pituitary somatotroph was studied in two strains of female rats using double immunofluorescence techniques. Mab WHC-1, a recently generated and characterized monoclonal antibody, was used to detect the antigen associated with somatotrophs, whereas rabbit anti-rat prolactin (rPRL) and anti-human growth hormone (hGH) antisera were used to identify mammotrophs and somatotrophs, respectively. In F344 rats, Mab WHC-1-positive cells increased from 13.8 +/- 0.5% of total pituitary cells in normal anterior pituitaries to 34.2 +/- 4.0% in DES-induced pituitary tumors. The number of mammotrophs also increased significantly from 58.0 +/- 3.2% in controls to 75.9 +/- 2.2% in tumors. On the other hand, somatotrophs decreased significantly in number following ovariectomy (OVX) and DES implantation (19.7 +/- 0.5% vs. 6.1 +/- 1.2%). Based on double immunofluorescence, the percentage of Mab WHC-1-positive cells, which were somatotrophs, decreased from 85.5 +/- 2.7% in normal controls to 6.7 +/- 1.5% in DES-induced tumors. On the other hand, the percentage of Mab WHC-1-positive cells which were mammotrophs increased significantly from 14.0 +/- 1.4% to 86.1 +/- 1.8% following OVX and DES implantation. A similar change was found in the number of somatotrophs and mammotrophs following the same treatment in Sprague-Dawley (SD) rats which did not develop pituitary tumors. In contrast to F344 rats, the number of Mab WHC-1-positive cells in SD rats decreased significantly from 32.4 +/- 2.8% in sham-operated controls to 19.3 +/- 2.9% in OVX + DES-implanted rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

A procedure for the purification of somatotrophs isolated from rat anterior pituitary glands using Percoll density gradients

We have used fractionation on density gradients of Percoll to separate the cell types in the rat anterior pituitary gland and to produce a purified preparation of somatotrophs. The method differs from those described previously which used, for example, albumin or Ficoll gradients, in being more rapid and avoiding low temperatures, and therefore gives cells with improved viability. Anterior pituitary glands from male rats were dispersed with trypsin to produce 1.5 x 10 (6) -2.0 x 10 (6) cells/gland. These were fractionated on hyperbolic density gradients of Percoll. Two bands of cells containing somatotrophs were detected, one of which (band A; density 1.075-1.082 g/cm3) contained approximately 90% somatotrophs, whereas the other (band B; density 1.055-1.068 g/cm3) contained about 70% somatotrophs mixed with other cells, especially lactotrophs. Cells in band A appeared more responsive to secretagogues than those in band B; growth hormone secretion was stimulated markedly by cyclic AMP derivatives and prostaglandin E2, and inhibited by somatostatin. Such purified somatotrophs are well suited to biochemical studies on the mechanism of the control of growth hormone secretion.     

Somatostatin receptors on pituitary somatotrophs, thyrotrophs, and lactotrophs: pharmacological evidence for loose coupling to adenylate cyclase

Pharmacological characterization of somatostatin (SRIF) receptors located on somatotrophs, thyrotrophs, and lactotrophs was attempted by measuring the effects of 14 structural agonists of somatostatin (SRIF) on the inhibition of basal and GRF-stimulated GH and basal and TRH-stimulated PRL and TSH secretion. We also checked the abilities of the analogs to displace [125I]N-Tyr-SRIF binding to pituitary cell membranes and their potency to inhibit adenylate cyclase activity. There was a very good correlation (r = 0.975) between the displacement of [125I]N-Tyr-SRIF and the inhibition of adenylate cyclase activity by the analogs. The effects of the analogs on secretion of the three hormones followed the same rank order of potency. However, the active analogs displayed 2-6 times lower affinities in inhibiting PRL than GH or TSH secretions. The shift in affinity was even more pronounced in the case of the lower potency of the analogs as inhibitors of adenylate cyclase activity compared to hormone secretions. Pretreatment of the cells with pertussis toxin (100 ng/ml; 24 h) blocked SRIF inhibition of basal and GRF-stimulated adenylate cyclase activity and decreased by 83% [125I]N-Tyr-SRIF binding. It also blocked the ability of SRIF to inhibit GRF-induced GH and TRH-induced PRL and TSH secretion. However, pertussis toxin also increased GRF stimulation of GH secretion and decreased TRH stimulation of both TSH and PRL secretion. We conclude from our data that SRIF-binding sites located on the three target cells of the adenohypophysis are of a single class. These binding sites are negatively coupled to adenylate cyclase, but the inhibition of hormone secretions by SRIF cannot be explained solely through adenylate cyclase inhibition. Another mechanism of transduction must be involved in the actions of SRIF on its three pituitary target cells.     

The number of lactotrophs is reduced in the anterior pituitary of streptozotocin-induced diabetic rats

AIMS/HYPOTHESIS: Prolactin secretion is often reduced in insulin dependent diabetes mellitus, but little is known about the mechanism involved. Since changes in the hormonal environment modulate cell proliferation, death and cellular makeup of the anterior pituitary, we have analysed whether the number of lactotrophs is reduced in diabetic rats. METHODS: Streptozotocin induced diabetic rats were maintained hyperglycaemic for 2 months. Pituitary prolactin, growth hormone, Bcl-2, Bax and PCNA concentrations were analysed by western blot analysis. In situ hybridisation was used for quantification of prolactin and growth hormone mRNA containing cells. Cell death was detected by TUNEL labelling, alone and in combination with immunocytochemistry for prolactin or growth hormone. RESULTS: Diabetic rats had fewer lactotrophs ( p<0.01). This was coincident with a decrease in overall protein and prolactin content. An increase in pituitary cell death was found and some of the TUNEL labelling co-localised with prolactin immunostaining. No change in the concentration of Bcl-2 or Bax, proteins implicated in apoptosis, was detected. PCNA content was higher in the pituitaries of diabetic rats, suggesting increased proliferation. CONCLUSION/INTERPRETATION: Anterior pituitary cell turnover is affected in poorly controlled diabetes mellitus. A decrease in the number of lactotrophs, as a result of increased cell death, could underlie, at least in part, the reduction in prolactin secretion observed in diabetic animals.     

Regression of redundant lactotrophs in rat pituitary gland after cessation of lactation

Regressive changes occurring in the pituitary gland of the rat after removal of litters were studied. Pituitary glands of lactating rats were characterized by the presence of numerous hypertrophied lactotrophs. Interruption of lactation caused a blockade of prolactin synthesis and secretion, followed by degeneration of lactotrophs. Morphometric analysis of pituitary glands revealed that lactotrophs accounted for about 50% of the total hypophysial cell count in lactating rats. This percentage decreased progressively and reached pre-pregnant levels 7 days after removal of litters; the decrease was inversely correlated with an increase in the number of degenerating lactotrophs which comprised 30% of all lactotrophs 72 h after removal of litters. The morphological changes found in lactotrophs were closely related to changes in the prolactin content of serum and the pituitary gland. Regression of lactotrophs appeared to be the most important cause inducing the reversal of hypophysial lactotrophic activity to pre-pregnant conditions.     

Evidence for paracrine interaction between gonadotrophs and lactotrophs in pituitary cell aggregates

Pituitary cell aggregates prepared from 14-day-old male or female rats and maintained for 4-5 days in culture were superfused with LHRH during periods of 20 or 90 min. LHRH provoked a rapid and sustained rise of PRL release at concentrations similar to those stimulating LH release (10(-11)-10(-8) M). Dopamine, at a concentration inhibiting PRL release for 90%, weakened but did not prevent this stimulation. LHRH also stimulated PRL release in aggregates prepared from adult male rat pituitary cells, but the effect was weaker and seen only after a more prolonged period in culture. There was no PRL response to LHRH in aggregates of lactotroph-enriched populations, obtained by gradient sedimentation at unit gravity, in which only few and small gonadotrophs are present. When a lactotroph-enriched/gonadotroph-poor population was coaggregated with a highly enriched population of large gonadotrophs, LHRH very effectively stimulated PRL release, the extent of stimulation being dependent on the proportional number of gonadotrophs in the coculture. Superfusion of lactotroph-enriched/gonadotroph-poor aggregates with medium in which the gonadotroph-enriched aggregates had previously been incubated for 3 h with 1 nM LHRH (gonadotroph-conditioned medium) also provoked a clear-cut rise in PRL release. This effect was not due to LH, FSH, or the small amounts of PRL present in the gonadotroph-conditioned medium. The LHRH antagonist [D-Phe2-D-Ala6]LHRH was capable of blocking the PRL response to LHRH but not that to the gonadotroph-conditioned medium. In the lactotroph-gonadotroph coaggregates TRH stimulated PRL release but had no effect on LH release. TRH was also ineffective in releasing LH or FSH in populations containing both gonadotrophs and thyrotrophs. The present data suggest that gonadotrophs can activate the secretory activity of the lacotrophs through the release of a paracrine humoral factor.     

Calcium-dependent control of corticotropin release in rat anterior pituitary cell cultures

The role of calcium in the stimulation of ACTH secretion by CRF and other regulators was studied in rat anterior pituitary cells. Incubation of cultured pituitary cells in normal calcium with CRF, vasopressin, angiotensin II, or norepinephrine increased the rate of ACTH release for up to 45 min and then became constant for up to 3 h. In the absence of extracellular calcium, the initial rate of stimulated secretion was unaffected, but after 45 min the secretion rate decreased by 40% for CRF and to a greater extent for the other stimuli. Addition of calcium after 90 min in calcium-free medium restored the CRF-stimulated ACTH release rate to the control value. The absence of extracellular calcium had no effect on CRF-stimulated cAMP accumulation, but intracellular calcium depletion by preincubation of the cells with EGTA completely inhibited CRF-stimulated cAMP production and ACTH release. The voltage-dependent calcium channel antagonist nitrendipine and the calcium channel agonist BK 8644 had little effect on the CRF-stimulated ACTH release rate, while they, respectively, inhibited and enhanced the stimulation by vasopressin and high potassium. In calcium-depleted cells incubated with the calcium ionophore A23187, CRF stimulation of cAMP production and ACTH release were dependent upon extracellular calcium concentrations from 0.1-100 microM. These findings have defined two phases in the stimulation of ACTH release by CRF and cAMP-independent stimuli in cultured pituitary cells: an early phase with a rapid increase in the ACTH release rate which is independent of extracellular calcium, and a late phase of constant secretion rate, with partial extracellular calcium dependence for the stimulation by CRF and complete calcium dependence for the stimulation by non-cAMP-mediated stimuli.     

Nongenomic actions of testosterone on a subset of lactotrophs in the male rat pituitary

Rapid, nongenomic effects of testosterone on PRL release in vitro were investigated. Anterior pituitary tissue from adult male rats was stimulated in vitro for 5 or 20 min with testosterone (T; 1 or 100 nM) or testosterone-BSA (T-BSA; 1 or 100 nM) with or without 1.2 mM tannic acid, which enables visualization of secretory granule exocytosis. Within 5 min, both concentrations of T and T-BSA stimulated exocytosis from type 2 lactotrophs (characterized by small spherical granules), but not from type 1 lactotrophs (characterized by large polymorphic granules). The effects of T on type 2 lactotrophs could be blocked by preincubation with dopamine (500 nM), but were not time or concentration dependent, and could not be inhibited by 1) removal of extracellular Ca2+, 2) the L-type Ca2+ channel blocker nifedipine (100 nM), 3) the Ca2+-adenosine triphosphatase inhibitor thapsigargin (150 nM), 4) the PKC inhibitor retinal (10 microM), or 5) the gamma-aminobutyric acidA chloride channel blocker picrotoxin (100 microM). T-BSA (0.1 nM to 1 microM) for 5 or 20 min also caused an increased release of immunoreactive PRL into the medium compared with control incubations. T and T-BSA did not stimulate exocytosis from gonadotrophs or cause LH release. In conclusion, we report for the first time a rapid, nongenomic effect of T on PRL secretion.     

Immunocytochemical and morphometric studies of mammotrophs, somatotrophs and somatomammotrophs in sheep pituitary cell cultures

Alterations occurring when sheep anterior pituitary cells are placed in culture for 4 days were studied using electron microscopy and immunogold labelling. The majority of cells present showed marked morphological changes during culture, with degranulation and development of extensive rough endoplasmic reticulum. The proportions of somatotrophs and mammotrophs, as identified by immunogold labelling, fell during culture. The majority (70%) of somatotrophs showed relatively little degranulation but the remainder were extensively degranulated after 4 days in culture, suggesting two subpopulations of this cell type. Conversely, most (80%) of the mammotrophs showed extensive degranulation after culture, but one-fifth remained heavily granulated, suggesting that mammotrophs too are heterogeneous. The proportion of cells labelling for both GH and prolactin (somatomammotrophs) increased during culture to about 3% of the cells present, compared with less than 0.2% of all cells before culture.     

alpha 2u-Globulin is present in the rat anterior pituitary.

The possibility that the pituitary gland may contain as yet undiscovered regulatory factors is intriguing. Recent reports have suggested the presence, in the anterior pituitary, or a number of proteins of extrapituitary origin. alpha 2u-Globulin, a rat serum and urinary protein, previously shown to be synthesized in the submaxillary gland and in the liver under anterior pituitary control, has now been localized by immunocytochemistry in the cytoplasm of some cells of the anterior pituitary. No alpha 2u-globulin could be detected in either the intermediate or posterior pituitary. The presence of alpha 2u-globulin was confirmed and quantitated by radioimmunoassay. Using RNA blot analysis and cloned alpha 2u-globulin cDNA probes, we could not detect alpha 2u-globulin mRNA sequences in pituitary RNA, indicating that alpha 2u-globulin is not synthesized therein. The presence of alpha 2u-globulin, presumably of circulatory origin, in certain anterior pituitary cells suggests that it may play a role in anterior pituitary function.