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.     

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.     

Autoregulation of follicle-stimulating hormone secretion in the hamster: evidence for control at the level of the anterior pituitary gland

Pituitary tissue obtained from proestrous, phenobarbital-treated hamsters was placed in organ culture, and the release rates of FSH and LH were monitored. Addition of LHRH to the culture medium increased gonadotropin release rates. Preincubation of pituitaries in medium that contained 1 microgram purified FSH ( NIADDK rat FSH-I-5) resulted in enhanced basal and LHRH-induced FSH release rates. Full expression of FSH hypersecretion by pituitary tissue occurred after 3-h exposure to purified FSH. This phenomenon appears to require adequate tissue calcium concentrations. Further, exposure of pituitaries to purified FSH slightly but significantly enhanced basal but not LHRH-stimulated LH release compared with that in untreated controls. Experiments that employed labeled FSH, reduced exposure time to purified FSH, or decreased calcium concentration in the medium proved that the increased FSH secretory rate was not due to contamination of the medium with the purified FSH used to stimulate the pituitary. These studies suggest that FSH has the ability to influence its own secretion by an action at the level of the anterior pituitary gland.     

Ultrastructural evidence for oxytocin in the rat anterior pituitary gland

OT is synthetized in the hypothalamus. These neurons project to the posterior lobe of the pituitary and the external zone of the median eminence. In order to localize OT in the male rat anterior pituitary we have used immunocytochemistry on ultra-thin sections in target cell(s) obtained by cryoultramicrotomy. OT-like immunoreactivity was observed in lactotropes only. No immunoreactivity was observed in gonadotropes, somatotropes, corticotropes or thyrotropes. In lactotropes, immunoreactivity was localized at the plasma membrane level, in the cytoplasmic matrix and around the secretory granules, but not in the other organelles, and in the nucleus. No reaction was observed by using either non-immune serum or anti-OT serum incubated with OT. No modification of OT-like immunoreactivity was observed by using anti-OT serum incubated with heterologous peptides. These results 1) provide immunocytological evidence for the presence of OT in the anterior pituitary gland; 2) indicate the presence of this peptide in one particular cell type, and 3) support the hypothesis that OT could have a direct participation in the regulation of the PRL release.     

In-vitro secretion of prolactin and growth hormone in the presence of melatonin by pituitary gland from mink kept under long or short days

Mink anterior pituitaries were incubated in Medium 199 for up to 9 or 13 days. Biological activity of prolactin and GH was determined. Daily concentrations of prolactin and GH in the incubation medium were also measured by radioimmunoassay and radioreceptor assay. When females were kept under short days for several weeks before the experiment, a significant decrease in prolactin secretion by the anterior pituitary was observed as compared with that in females maintained under long days. In contrast, secretion of GH was not modified by the photoperiodic history of the animals. Pineal gland denervation by ablation of the superior cervical ganglia a few months before the experiment, or addition of melatonin to the incubation medium of anterior pituitaries from intact or ganglionectomized females, did not modify the secretion of prolactin and GH. The pituitary gland does not therefore seem to be a direct target site for melatonin in transducing the duration of daylength on the hypothalamo-pituitary axis.     

Cleaved prolactin: evidence for its occurrence in human pituitary gland and plasma

A form of PRL that is cleaved in its large disulfide loop has been reported in rat and mouse pituitary glands, but it is not known whether it exists in the human pituitary gland and whether it circulates in blood. Recently, we developed an immunoelectrophoretic technique which is capable of measuring immunoreactive variants of PRL and other hormones in plasma. We describe here detection of cleaved PRL in normal and tumor pituitary tissue from a prolactinoma patient and in the plasma of pregnant women.     

Ultrastructural evidence for endogenous substance-P-like immunoreactivity in the rat pituitary gland

Substance P (SP) is known to act on pituitary hormone release either at the hypothalamic level or directly at the pituitary level. In order to investigate whether SP is present in the pituitary gland and to localize the peptide at the cellular and subcellular levels, the immunocytological method was used. Rat pituitaries were fixed and frozen. Ultrathin sections, obtained by cryoultramicrotomy, were incubated with anti-SP serum. The antigen-antibody reaction was detected by 4-chloro-1-naphtol. SP immunoreactivity was observed both in the prolactin and in the gonadotropic cells, but not in the somatotropic, corticotropic and thyrotropic cells. In reactive cells, SP immunoreactivity was observed in the secretory granules, in the cytoplasm, and in the nucleus distributed all over the euchromatin near to the heterochromatin regions. No immunoreactivity was observed when nonimmune serum or anti-SP serum incubated with SP was used. No modification of the immunocytochemical reaction was observed when anti-SP serum incubated with somatostatin, gonado- or thyroliberin was used. These data (1) provide immunocytological evidence for the presence of SP in the pituitary gland; (2) indicate the presence of SP peptide in the gonadotropic and prolactin cells only. They support previous findings indicating that SP could have a direct effect at the pituitary level.     

Mammary gland prolactin receptor and pituitary prolactin secretion in lactating mice with different lactational performance

SHN female mice, a high mammary tumour strain, are superior to SLN, a low mammary tumour strain, in lactational performance. Mammary gland prolactin receptor and pituitary prolactin secretion during lactation were compared between these strains. The binding activity, the number of receptor sites per mg tissue and the association constant were measured by the in vitro incubation of mammary gland slices with 125I-labelled bovine prolactin, and the pituitary and plasma levels of prolactin were assayed by homologous radioimmunoassay. There was only a slight difference between strains in any of the parameters for prolactin receptor and for prolactin secretion on either day 4 or day 9 of the first lactation. Almost all the correlation coefficients between each parameter for prolactin receptor and the pituitary or plasma level of prolactin were not statistically significant. These findings suggest that any parameter for prolactin examined in this study is not always directly indicative of lactational performance and further show that the individual variation in the pituitary prolactin secretion during lactation is not so great as to alter the prolactin receptor.     

Pharmacologic evidence that a D2 receptor subtype mediates dopaminergic stimulation of prolactin secretion from the anterior pituitary gland.

The ability of low concentrations of dopamine (DA) to stimulate the secretion of prolactin (PRL) was examined in perifused or monolayer cultures of anterior pituitary cells. In cultures perifused with media containing 100 nM DA, changing the DA concentration to either 1 or 100 pM caused a significant dose-dependent stimulatory PRL secretory response within 6 min when compared to the PRL secretory response to removal of DA altogether. Picomolar concentrations of DA caused a biphasic PRL secretory response. This response is characterized by an immediate increase in the rate of PRL secretion similar to that seen when the cells were treated with 100 nM thyrotropin-releasing hormone followed by a decrease in the rate of PRL secretion to levels comparable to cells receiving media alone. In a monolayer culture system DA, at concentrations between 10 nM and 1.0 pM, caused significant stimulation of PRL secretion relative to media alone. Maximal stimulation occurred at nanomolar concentrations of DA (approximately 60% greater than control). Although the D2 agonists, bromocriptine and 2-(N-phenethyl-N-propyl)-amino-5-hydroxytetralin hydrochloride (PPHT) caused significant (p less than 0.05) inhibition of PRL secretion at nanomolar concentrations and above, neither had stimulatory activity. The D1 agonists, SKF 38393 and SKF 82958, had no effect on PRL secretion when tested at 0.1 pM to 1 microM. These data suggest that DA not only inhibits PRL secretion in vitro, but also stimulates PRL secretion at relatively low concentrations. Stimulation is mediated by a DA receptor which is neither recognized by D2 nor D1 agonists, suggesting a possible third DA receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses to prolactin secretagogues in oestrogen-treated rats suggest that the defect in prolactin regulation produced by oestrogen is at the level of the pituitary gland

Prolactin responses to pharmacological agents were used to characterize the defect in prolactin regulation which occurs after administration of high doses of oestrogen to rats. Animals with chronically implanted venous cannulae were injected with 2 mg oestradiol benzoate in oil and 2-3 days later prolactin concentrations were measured after injections of saline, thyrotrophin-releasing hormone (TRH), fenfluramine, apomorphine and butaclamol. The responses were compared with those in oil-injected animals. Hyperprolactinaemia in oestrogen-treated animals was unresponsive to apomorphine, but was even more sensitive to dopamine receptor blockade than controls. These results suggest that the lactotrophs in oestrogen-treated animals are already maximally suppressed by endogenous dopamine, though ineffectively. Although there was an increased prolactin response to TRH in oestrogen-treated animals, there was an impaired response to fenfluramine, indicating suppressed serotonergic prolactin-releasing factor mechanisms. Maximal endogenous dopaminergic activity and suppressed prolactin-releasing factor mechanisms are appropriate hypothalamic responses to hyperprolactinaemia. The operation of these responses in the earliest stages of the development of pituitary hyperplasia indicates that oestrogen induces a disturbance of prolactin regulation in the lactotroph, independent of hypothalamic control.     

Kallidin stimulates prolactin secretion from individual rat anterior pituitary cells

Kinins are localized within the adenohypophysis where they have been shown to stimulate the release of pituitary hormones. In the present study we have investigated the effect of [Lys]-bradykinin (kallidin) on prolactin secretion at the single cell level from cultured male rat anterior pituitary cells. This was assessed by use of a reverse haemolytic plaque assay which permits quantitative evaluation of the proportion of all pituitary cells which are secreting prolactin, and the amount of prolactin secreted per lactotroph (plaque area). The rate of plaque development was used as an index of the rate of hormone secretion in time-course studies. Kallidin induced a dose-dependent increase in both the percentage of plaque-forming cells and the median plaque area during the first 2 and 3 h of incubation respectively. The threshold concentration of kallidin was 10 nmol/l. After 4 h of kallidin stimulation there was no difference between treated and control monolayers with respect to median plaque area and the total secretion index. Although recruitment of additional cells into the secretory pool cannot be excluded, this seems unlikely since at 3 and 4 h little or no difference was observed in the number of plaque-forming cells. The data suggest that initially kallidin accelerated the rate of prolactin secretion primarily by inducing an increase in the number of cells secreting prolactin, and subsequently by increasing the amount of hormone secreted per lactotroph. The results presented here are consistent with the proposed role of the kallikrein-kinin system in the paracrine or autocrine control of prolactin release from the pituitary gland.     

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.     

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.     

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.     

Regulation of prolactin secretion in patients with Cushing's disease. A comparative study on the effects of dexamethasone, lysine vasopressin and ACTH on prolactin secretion by the rat pituitary gland in vitro

In 15 untreated patients with Cushing's disease the regulation of prolactin (PRL) was evaluated. Plasma PRL was 11.5 +/- 4.8 vs. 5.3 +/- 3.6 ng/ml (patients with Cushing's disease vs. control; mean +/- S.D.; p less than 0.001). The maximal increment of plasma PRL in response to TRH was 32.3 +/- 17.3 vs. 27.9 +/- 17.2 ng/ml (NS); the maximal increment of plasma PRL in response to an insulin-induced hypoglycemia was 3.8 +/- 4.6 vs. 22.7 +/- 12.4 ng/ml (p less than 0.001). Additionally the effect of dexamethasone, lysine vasopressin and ACTH on the secretion of PRL by rat pituitary glands in vitro was studied. Dexamethasone (1.25--10 microM) inhibited the secretion of PRL. However, in the presence of dexamethasone modulation of PRL release by TRH and dopamine remained unaltered. Lysine vasopressin (5 nM - 5 microM) and ACTH (0.5--12.5 microM) did not have a direct effect on PRL release by normal rat pituitary glands in vitro and these substances also did not interfere with dopamine-mediated inhibition of PRL release. Conclusions: In Cushing's disease the PRL responses to TRH (normal) and to insulin-induced hypoglycemia (blunted) are differentially affected. Therefore, hypercortisolism probably selectively interferes with the regulation of PRL secretion at a suprahypophyseal level. It is concluded that TRH and dopamine regulate PRL release at sites which are not under corticosteroid regulation, while corticosteroids modulate PRL secretion in response to stress.