Evidence to suggest that rat growth hormone is modified when secreted by the pituitary gland.

The relationships were examined between the concentrations of GH determined by bioassay (rat tibia test) and radioimmunoassay in homogenates of rat adenohypophysial tissue and in the incubation medium in which the tissue had been maintained. Adenohypophyses from young male rats were incubated in Medium 199 with or without a putative synthetic GH-releasing factor (GRF) to achieve a dynamic state of GH secretion, but the GRF did not consistently stimulate the release of GH as measured by either bioassay or radioimmunoassay. Both assays gave closely similar values for the concentration of GH in homogenates of the adenohypophyses of undisturbed rats. When the two assays were used to measure the concentrations of GH in incubated pituitary tissue, the values showed no correlation, although the concentrations of GH in the incubation medium were again highly correlated. However, the bioassay : radioimmunoassay ratio for the secreted hormone [2.14 +/- 0.09 (S.E.M.)] was significantly higher than that for the intrapituitary form from the unincubated gland (0.93 +/- 0.06). By radioimmunoassay, secreted rat GH was found to be less stable than purified rat GH when the two forms were incubated in vitro with slices of various rat tissues. Similarly, the rate of clearance of secreted rat GH from the plasma of hypophysectomized rats, as determined by radioimmunoassay, was much faster than that of the purified GH. The biological activity of purified bovine GH was blocked by aprotinin, an inhibitor of proteolysis, but the drug had no inhibitory effect on the bioactivity of rat GH secreted into the incubation medium. Overall, these results indicate that secreted rat GH has properties intermediate between those of the intrapituitary form and the form found in the circulation.     

Evidence that autoregulation of prolactin production does not occur at the pituitary level

PRL production in primary cultures of rat pituitary cells was examined for evidence of autoregulation. The kinetics of PRL accumulation in the culture medium were linear (r = 0.993) over a 20-fold range of PRL concentrations (85-1800 ng/ml). The addition of 0.01-1000 ng/ml biologically active ovine PRL for 2 h had no effect on the rate of secretion or the intracellular levels of PRL. The functional integrity of the cells was verified by the demonstration that estradiol (10(-8) M) caused a 3-fold increase in both PRL synthesis and secretion. Ergocryptine (10(-8) M) inhibited PRL secretion by 57% (P less than 0.001), and this response was modulated by pretreatment with estradiol. Increasing concentrations of endogenously secreted PRL did not detectably alter the rate of incorporation of [35S]methionine into cellular PRL in control or ergocryptine-treated cells for up to 8 h. These results indicate that autoregulation of PRL does not operate directly at the level of the pituitary.     

Dexamethasone inhibits the effects of oestrogen on the pituitary gland in rats

The administration of glucocorticoids has been shown to be effective for the induction of ovulation in patients with anovulation. In the present study, we investigated the effects of a glucocorticoid on oestrogen-induced changes in the pituitary gland. A single ip injection of 10 micrograms oestradiol-17 beta (E2) in ovariectomized and adrenalectomized rats resulted in a significant increase in pituitary weight and progesterone receptor (PgR) concentration. In these animals, serum LH level was initially suppressed and restored to control level 24 h after E2 injection. However, 1 mg of dexamethasone (Dex) injected before E2, but not after E2 administration, completely inhibited both the increases in pituitary weight and PgR concentration. The restoration of serum LH level 24 h after E2 was also prevented. These antioestrogenic effects of Dex were blocked by ip administration of the synthetic antiglucocorticoid, RU486. Dex treatment alone did not have any effect on E2-induced changes in the dynamics of pituitary oestrogen receptor. Finally, E2-pellet implanted sc in ovariectomized and adrenalectomized rats for 7 days caused marked increases in pituitary weight and PgR concentration. A single ip injection of 250 micrograms clomiphene citrate (clomiphene) significantly reduced both the pituitary weight and PgR concentration in these animals, but 1 mg of Dex failed to have a similar effect. These results suggest that glucocorticoids antagonize E2 effects on the pituitary by a mechanism different from antioestrogens such as clomiphene. These antioestrogenic effects of glucocorticoid may be involved in induction of ovulation in anovulatory women.     

In-vitro evidence for the autoregulation of prolactin secretion at the level of the pituitary gland in the rat

The autoregulation of rat prolactin secretion at the level of the pituitary gland was investigated, using a static incubation system. The rate of prolactin secretion from the female anterior pituitary gland in vitro was found to be constant when the medium was changed at 20-min intervals. However, when the medium was left unchanged and secretory products were allowed to accumulate, prolactin secretion began to decline within 60 min. This effect was not observed with the male tissue, where the level of accumulated prolactin did not reach that at which the inhibition occurred using female tissue. The nature of the putative secretory product causing the inhibition of prolactin secretion was investigated. Exogenous bovine prolactin (1-4 mg/l) caused an inhibition of endogenous rat prolactin secretion. Inclusion of monoamine oxidase in unchanged medium, to prevent dopamine accumulation in the medium (a possible consequence of co-storage and co-secretion with prolactin granules), did not prevent the inhibition observed in the control incubation. We therefore conclude that in-vitro autoregulation of prolactin secretion can occur at the level of the pituitary gland, probably due to the accumulated prolactin having a feedback action on the lactotroph. This might be of physiological significance if localized concentrations of the hormone within the gland are high.     

Hormonal regulation of prolactin cell development in the fetal pituitary gland of the mouse

The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol (E(2)) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E(2), with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER)alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E(2) action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E(2) on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E(2). The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.     

Possible role of dopamine and noradrenaline in the regulation of prolactin secretion from an ectopic anterior pituitary gland in female rats

It was recently reported that anterior pituitary tissue transplanted to an ectopic site contains measurable amounts of dopamine and noradrenaline. To examine the possibility of local catecholaminergic control of prolactin secretion from ectopic pituitaries, pituitary grafted and sham-operated female rats were submitted to several pharmacological treatments modifying catecholamine synthesis. Administration of a single dose of alpha-methyl-p-tyrosine (alpha-MPT) significantly reduced dopamine content in the graft, while noradrenaline content was not modified. Similar changes in the contents of dopamine and noradrenaline after alpha-MPT administration were observed in the hypothalamus and in the in-situ pituitary in both grafted and sham-operated rats. Plasma concentrations of prolactin were increased in both grafted and sham-operated rats after administration of alpha-MPT. A single injection of L-3,4-dihydroxyphenylalanine (L-DOPA) increased dopamine content in the ectopic pituitary gland without altering the noradrenaline content, and produced similar effects in the hypothalamus and in-situ pituitary of grafted and control rats. Plasma prolactin concentrations were decreased by L-DOPA in both pituitary grafted and control rats. Administration of DL-treo-dihydroxyphenylserine (DOPS) increased noradrenaline content in the ectopic pituitary and reduced plasma prolactin concentrations in pituitary grafted rats. In contrast, injection of DOPS to control rats increased both hypothalamic noradrenaline content and plasma prolactin concentrations. These results suggest that dopamine and noradrenaline present in the ectopic pituitary tissue have a role in mediating prolactin release from pituitary transplants.     

Failure of rat placenta to inhibit prolactin secretion by ectopic pituitary gland

At mid-pregnancy in the rat, episodic secretion of pituitary prolactin ceases when the placenta is sufficiently developed. At this time, sufficient placental lactogen is secreted by the placenta to inhibit prolactin secretion. The present study tested whether the fully developed placenta at mid-pregnancy can inhibit prolactin secreted by a donor pituitary gland implanted under the kidney capsule. Three pituitary glands were implanted in rats on day 7 of pregnancy; muscle fragments were implanted in controls. Blood was collected during the first and second halves of pregnancy. It was found that prolactin concentrations in the animals with the pituitary implants were high on days 9 and 10 of pregnancy and remained raised during the second half of pregnancy while in control animals nocturnal surges were absent in the second half of pregnancy, the last one occurring on day 10. This observation indicates that the placental hormone cannot act directly on the pituitary gland to inhibit prolactin secretion, but presumably exerts its suppressive effect on prolactin secretion through the hypothalamus.     

In vitro studies on the regulation of prolactin secretion in the bullfrog pituitary gland

The effects of dopamine, a dopamine agonist, dopamine antagonists, and serotonin on the incorporation of [³H]leucine into bullfrog pituitary prolactin (PRL) in vitro were studied. CB154, a dopamine agonist, as well as dopamine, inhibited the release of the newly synthesized PRL from the pituitary gland. Incomplete restoration of the dopamine-inhibited PRL release was observed when pimozide or haloperidol, a dopamine antagonist, was added to the incubation medium. Relatively higher doses of pimozide or haloperidol were effective by themselves in enhancing the synthesis and release of PRL in a dose-dependent manner. On the other hand, a lower dose of pimozide or haloperidol acted rather suppressively on the release of PRL. Serotonin was found to be effective in stimulating the synthesis and release of PRL by acting directly on the pituitary gland. The possible regulatory mechanism of PRL secretion in bullfrogs by dopamine and serotonin is discussed.     

Function of prolactin cells in the individual rat pituitary gland is location dependent.

Anterior pituitary glands from individual ovariectomized (ovx) or ovx-estrogen (E2) treated rats were sectioned into 1/8 cubes. Each section was incubated for four consecutive 15 min periods in order to measure the release of immunoreactive and bioactive prolactin (PRL); each individual section was then trypsinized into a single cell suspension for determination of PRL cell numbers in that section. Hormone release (ng PRL/1000 PRL cells) was not uniform throughout the gland; the consistency of the secretory patterns demonstrated that the amount of PRL release from the gland was location-dependent. Statistical analysis of the data showed that the most active cells were in the gland's left lobe, while the least active were in the right lobe. Within these lobes, the dorsal-caudal and ventral-rostral left lobe areas released the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas were least active. Injection of ovx rats with E2 for 2 days altered these secretory patterns. This sectioning procedure should prove useful in future studies addressing issues of cell-cell interaction and geographic location as they relate to pituitary cell function.     

Indirect evidence to suggest that prolactin mediates the adrenal action of haloperidol to stimulate aldosterone and corticosterone secretion in rats.

The effect of dopamine-antagonists on steroid secretion has revealed conflicting results regarding the confirmation of in vivo findings in vitro. In order to discriminate in vivo systemic and local action of the dopamine-antagonist haloperidol (HAL) on aldosterone and corticosterone secretion, microdialysis of the adrenal cortex in conscious, freely moving rats was employed. The effects of 2.5 mg HAL ip or intraadrenal dialysis of 20 micrograms/ml HAL in rats with an intact pituitary gland on PRL, aldosterone, and corticosterone secretion were examined. Systemic HAL application resulted in a 40-fold increase in PRL secretion and stimulated aldosterone and corticosterone production significantly. In contrast, intraadrenal dialysis of HAL had no effect on the secretory pattern of PRL or either steroid hormone, indicating no direct drug action on cells of the rat adrenal cortex. Similarly, ip injection of 2.5 mg HAL in hypophysectomized rats did not alter PRL or steroid hormone levels. We conclude that the dopamine-antagonist HAL stimulates aldosterone and corticosterone secretion in rats through a pituitary factor, probably PRL, but not through direct effects at the adrenal cortex.     

Stimulation by leumorphin of prolactin secretion from the pituitary in rats

The effect of leumorphin (LM), one of big leu-enkephalins derived from preproenkephalin B, on PRL secretion was studied in the rat in vivo and in vitro. Intracerebroventricular injection of synthetic porcine LM (0.06-6 nmol/rat) caused a dose-related increase in plasma PRL levels in urethane-anesthetized male rats and in conscious freely moving rats. Intravenous injection of LM (3 nmol/100 g BW) also raised plasma PRL levels in these animals. The plasma PRL response to intracerebroventricular LM (0.6 nmol/rat) was blunted by naloxone (125 micrograms/100 g BW, iv). The stimulating effect of LM on PRL release was the most potent among the peptides derived from preproenkephalin B. In in vitro studies, PRL release from superfused anterior pituitary cells was stimulated in a dose-related manner by LM (10(-9)-10(-6) M), and the effect was blunted by naloxone (10(-5) M). These results suggest that LM has a potent stimulating effect on PRL secretion from the pituitary in the rat by acting, at least in part, directly at the pituitary through an opiate receptor.     

Effect of prolactin inhibitory agents on the ectopic anterior pituitary and the mammary gland in rats.

The purpose of the present study was to investigate the biological effectiveness of two highly potent prolactin (PRL) inhibitors, lisuride hydrogen maleate (LMH) and 2-Br-alpha-ergocryptine (CB-154), in the absence of hypothalamic factors acting directly at the level of the anterior pituitary. Hypophysectomized female rats bearing 4 transplanted pituitaries beneath the kidney capsules were treated with oestradiol benzoate (OeB) and progesterone (P) with or without simultaneous administration of LHM or CB-154 for 22 days in order stimulate or inhibit lobula-alveolar growth of the mammary glands. In addition to the investigation of the mammary glands by DNA determination and assessment of the histological pictures, the aim of this study was directed towards the influence of the substances tested at the level of the anterior pituitary remote from the hypothalmus. In this connection the changes in the different cells within the ectopic pituitaries as revealed by immunoenzyme-cytochemical studies were investigated. The results obtained support the classical view of a neuroendocrine regulation of mammary gland growth and the importance of oestrogens, P and PRL within this system. Both ergot derivatives LHM and CB-154 were able to antagonize the stimulatory effect of OeB combined with P on the mammary gland. With regard to the mechanism of action of LHM and CB-154 it is concluded that both substances act via a direct action on dopaminergic receptors within the ectopic anterior pituitary.     

Cysteamine causes reduction of prolactin monomers followed by aggregation in the rat pituitary gland

Storage forms of PRL were studied in control and cysteamine-treated cultures of estradiol-induced tumors in Fischer 344 rats and in secretory granules isolated from these tumors to further investigate the mechanism of action of cysteamine on PRL. The two major bands visible when protein is stained after electrophoresis of isolated granules migrate to the position of PRL and GH monomers. Electrophoresis under reducing conditions changes the position, but does not noticeably increase the amount of each band. [3H]PRL in cells labeled for 8 h with [3H]leucine also exists predominantly as monomer. Immunoreactivity of PRL in cell lysates or isolated granules is not affected by incubation with reducing agents beta-mercaptoethanol or glutathione at concentrations up to 5 mM, but cysteamine decreases PRL immunoreactivity in isolated granules at concentrations of 3 mM and higher. Electrophoresis of isolated granules after incubation with 25 mM cysteamine for 1 h demonstrates that cysteamine converts PRL to the reduced form. After 4 h, or after dilution of the granules before solubilization, the amount of reduced monomer is decreased, and larger molecular weight species appear. The reduced monomer can be recovered by electrophoresis under reducing conditions. The fully immunoreactive form can be recovered by incubation for 1 h with dithiothreitol at concentrations of 0.3 mM-3 mM. These data indicate that: PRL exists predominantly in monomeric form in the rat pituitary gland, and cysteamine reduces PRL, and formation of disulfide-linked aggregates of PRL occurs subsequently under some conditions.     

Indomethacin inhibits the effects of oestrogen in the anterior pituitary gland of the rat

Two inhibitors of prostaglandin synthesis, indomethacin and aspirin, blocked the increase of oestrogen-binding sites in the nuclear subcellular fraction, an increase which occurs after the administration of oestradiol. Consequently the biological effects of oestrogens in the anterior pituitary gland of the rat (prolactin synthesis, concentration of progesterone-binding sites and cell proliferation) are diminished. The anterior pituitary gland synthesized prostaglandin F2 alpha (PGF2 alpha), PGE2 and PGD2 from arachidonic acid. This synthesis was blocked when indomethacin was added to the culture media. Oestrogen increased the concentration of PGE2: an increase that was partially prevented by indomethacin. Prostaglandins may have an important role on the effects of oestrogen in the anterior pituitary gland of the rat.     

Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth

PR1 cells are a prolactin (PRL)-secreting cell line derived from a pituitary lactotroph tumor found in 17β-estradiol-treated Fischer 344 rats. We examined the effect of estrogen on cell proliferation and PRL synthesis under various culture conditions. Estrogen, at extremely low concentrations, induces cell proliferation in this cell line, whereas antiestrogen inhibits proliferation. Interestingly, the proliferation response is much more sensitive than the PRL response because 0.01 pM estradiol or diethylstilbestrol induces half-maximal growth induction [≈0.1% estrogen receptor (ER) occupancy is required], whereas 0.01 nM concentration is required for half-maximal PRL induction (≈50% ER occupancy is required). The proliferation response is not as sensitive to antiestrogen as the PRL response, because 10 nM concentration of the pure antiestrogen ICI 182,780 could not inhibit 1 nM estradiol- or diethylstilbestrol-induced proliferation. The same concentration of ICI 182,780 decreased PRL secretion to 1% of estradiol- or diethylstilbestrol-induced prolactin secretion suggesting a possible dichotomy of ER control of proliferation and PRL synthesis. The K_d of ER binding in these cells is about 3 × 10⁻¹¹ M. These results with the PR1 cells extend previous studies in other estrogen- regulated systems and suggest that only a small pool of ER is required for cell proliferation in contrast with the regulation of expression of specific genes. They also raise questions as to how a dimeric receptor functions when only one ligand site is occupied or when both an estrogen and an antiestrogen occupy one dimer.     

Role of the pituitary gland in the sex differentiation of the level of corticosteroid-binding globulin in rats

A study was made of the role of hypophyseal hormones in the regulation of the sex-associated level of corticosteroid-binding globulin (CBG) in the serum of female and male rats. Pituitary body removal did not change the level of CBG in males but caused its decrease in females to the level in intact males. Ectopic transplantation of the homologous pituitary body under the renal capsule increased the level of CBG in animals of both sexes. Intermittent infusion of STH did not change CBG content in hypophysectomized female rats and lowered it in intact females. A negative effect of androgens on the studied sign did not manifest itself either against a background of hypophysectomy or hypophyseal ectopy. Corticosteroids and thyroid hormones preserved their effects with regard to the level of CBG in hypophysectomized rats.