The effect of aging on prolactin secretion in rat pituitary monolayer culture.

A high basal rate of prolactin (PRL) secretion (.16±.03 μg/well/hr) was produced for over four weeks by pre-confluent male rat pituitary monolayer cell cultures. When the media was changed, a rapid release of microgram quantities of PRL occurred followed by a return to the basal PRL secretory rate by seven hours. Theophylline (3.8×10^?3M), but not dibutyrl cAMP (1×10^?3M), produced a significant (p<.02) increase in PRL secretion, and simultaneous addition of these agents potentiated the PRL secretory rate. TRH (2×10^?8M) had no effect on PRL release by six hours, whereas dopamine (4.9×10^?5M) produced a significant suppression (p<.002) of PRL secretion. In addition, the effects of theophylline, TRH, and dopamine on PRL secretion were similar in cultures of various ages. Ovine prolactin in concentrations up to 50 μg per ml produced no change in PRL secretion during 72 hours of incubation suggesting that PRL feedback control of its own secretion may be transmitted via the hypothalamus. These studies show that a high rate of PRL secretion can be maintained by pre-confluent monolayer cultures for extended periods of time, permitting repeated experimentation on the same wells.     

Inhibition of prolactin secretion by a direct effect of methysergide on the pituitary lactotrophs in the rat

Methysergide administered i.p. caused a dose dependent decrease of serum prolactin levels in rats of both sexes bearing large bilateral electrolytic lesions in the median eminence. This prolactin release inhibiting action of methysergide was prevented by pretreatment of the animals with dopamine receptor blockers pimozide or spiroperidol, which by themselves had no effect on serum prolactin levels. Similar results were observed when the dopamine receptor agonist piribedil was used instead of methysergide. It is concluded that methysergide is capable of inhibiting prolactin secretion by activation of dopamine receptors of the pituitary lactotrophs.     

Differential effects of dopamine antagonists on prolactin secretion from cultured rat pituitary cells.

Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing $\text{in vitro}$ H³-haloperidol binding in rat striatum reported by others (10). Concentration-effect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above 25nM. When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an $\text{in vitro}$ test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.     

Muscarinic inhibition of prolactin production in cultures of rat pituitary cells

Acetylcholine inhibits prolactin production from cell cultures of rat pituitary glands with a half-maximal effect at about 0.2 μM, and from GH-cells, clonal strains of rat pituitary cells, with a half-maximal effect at about 1 μM. The inhibition ranges between 80 and 40 % of control values. Inhibition is detectable at 2 hours, and continues for days in the presence of the anticholinesterase, eserine. Muscarinic agonists mimic the cholinergic inhibition and nicotinic agonists do not. The inhibition is blocked by atropine, a muscarinic antagonist, and not by hexamethonium, a nicotinic antagonist.     

Inhibition of pituitary bioactive prolactin secretion in the male rat by the glucocorticoid agonist decadron phosphate.

In the present studies, the soluble glucocorticoid agonist, decadron phosphate (DEC), was administered i.v. to intact adult male rats in order to evaluate the effects of glucocorticoid receptor stimulation on circulating levels of immunoreactive (ir-) and bioactive (bio-) prolactin (PRL). In light of reports that glucocorticoid-specific receptors exist within the rat brain, additional experiments investigated the effects of intracerebroventricular (i.c.v.) administration of graded doses of the same drug on pituitary ir- and bioPRL secretion. Concentrations of ir- and bioPRL in samples obtained before and after drug treatments were determined by standard PRL radioimmunoassay and the Nb2 rat node lymphoma bioassay, respectively. Rats injected i.v. with 0.5 mg DEC/kg body weight, but not those treated with a tenfold lesser dose, exhibited decreased plasma irPRL concentrations. However, both doses promoted a decline in circulating levels of bioPRL compared to vehicle-treated controls, along with an overall reduction in the plasma bio/irPRL ratio. The magnitude and duration of this drug-induced decline in biopotency of secreted hormone was dose-dependent. While the plasma bio/irPRL ratio was diminished only transiently in rats injected with 0.05 mg DEC/kg, treatment with the higher dose led to a sustained decrease in the plasma bio/irPRL ratio for the duration of the experiment. The current studies also show that intracerebral administration of DEC resulted in dose-dependent alterations in pituitary PRL release. Circulating levels of ir- and bioPRL were not altered in rats injected i.c.v. with 10 ng of DEC, the lowest dose tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of [D-Trp6]LHRH infusion on prolactin secretion by perifused rat pituitary cells

The effect of a superactive agonistic analog of luteinizing hormone-releasing hormone (LHRH), [D-Trp6]LHRH on prolactin (PRL) secretion by perifused rat pituitary cells was investigated. Constant infusion of [D-Trp6]LHRH (0.5 ng/min) for 2-3 h elicited a significant decrease in PRL secretion by these cells. This decrease in PRL release started ca. 30 min after the beginning of the infusion with the LHRH analog and lasted up to 1.5-2 h. [D-Trp6]LHRH significantly stimulated luteinizing hormone (LH) secretion during the first 30 min of peptide infusion; thereafter, LH levels began to return to control values. In animals pretreated in vivo with 50 micrograms of [D-Trp6]LHRH (s.c.) 1 h before sacrifice, PRL secretion by the rat pituitary cell perifusion system was significantly lower than vehicle-injected controls throughout the entire [D-Trp6]LHRH infusion period. On the other hand, thyrotropin-releasing hormone (TRH)-stimulated PRL secretion was slightly, but significantly imparied by [D-Trp6]LHRH infusion, while dopamine (DA) inhibition of PRL release was unaffected by this same treatment. These results reinforce previous observations of a modulatory effect of [D-Trp6]LHRH, probably mediated by pituitary gonadotrophs, on PRL secretion by the anterior pituitary. In addition, our findings suggest that basal PRL secretion by the lactotroph may be dependent on a normal function of the gonadotroph. The collected data from this and previous reports support the existence of a functional link between gonadotrophs and lactotrophs in the rat pituitary gland.     

Direct action of ethanol on pituitary prolactin secretion in vitro.

The effect of ethanol on prolactin release in vitro has been studied in order to investigate the direct action of ethanol on pituitary gland of the female rats. Animals were sacrificed in diestrus 2 and pituitary glands were incubated in TC-199 medium containing dopamine, noradrenaline, serotonin, TRH or cycloheximide with or without ethanol. The total amount of prolactin after the incubation period was calculated. Alcohol significantly increased the prolactin release in all groups. Cycloheximide and dopamine decreased the prolactin synthesis, but ethanol reduced the effect of dopamine. It is concluded that part of ethanol-induced hyperprolactinaemia, is due to a direct action of the alcohol on pituitary, affecting release and/or synthesis of prolactin.     

Modulatory effect of steroid hormones on GnRH-induced LH secretion by cultured rat pituitary cells.

The purpose of the present experiments was to examine the short- and long-term effects of estradiol-17 beta (E2), progesterone (P), and 5 alpha-dihydrotestosterone (DHT), alone and in combination, on the gonadotrophin-releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, using an ovariectomized rat pituitary cells culture model. After 72 h in steroid-free medium, pituitary cells were further cultured for 24 h in medium with or without E2 (1 nM), P (100 nM), or DHT (10 nM). Cultures were then incubated for 5 h in the absence or presence of 1 nM GnRH with or without steroids. LH was measured in the medium and cell extract by radioimmunoassay. The results show that the steroid hormones exert opposite effects on the release of LH induced by GnRH, which seems to be dependent upon the length of time the pituitary cells have been exposed to the steroids. In fact, short-term (5 h) action of E2 resulted in a partial inhibition (64% of control) of LH release in response to GnRH, while long-term (24 h) exposure enhanced (158%) GnRH-induced LH release. Similar results were obtained with DHT, although the magnitude of the effect was lower than with E2. Conversely, P caused an acute stimulatory action (118%) on the LH released in response to GnRH and a slightly inhibitory effect (90%) after chronic treatment. GnRH-stimulated LH biosynthesis was also influenced by steroid treatment. Significant increases in total (cells plus medium) LH were observed in pituitary cells treated with E2 or DHT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a coupling of prolactin secretion and synthesis by rat pituitary cells in culture

The relationship between the release and the synthesis of prolactin by rat pituitary cells in culture was studied using a microtubule-disrupting drug, vinblastine. (1) Prolactin secretion was inhibited by vinblastine in short-term incubations. Vinblastine did not act via the dopamine pathway, since a potent anti-dopaminergic drug, fluphenazine, was unable to reverse the inhibiting action of the antimicrotubular agent. (2) Continuous treatment by vinblastine induced a progressive decrease of the rate of incorporation of [3H]leucine in prolactin. The half-inhibition time was about 2 days. This inhibition of prolactin synthesis was selective, since total protein synthesis remained unaffected. (3) Measurements of radioimmunoassayable prolactin showed that the inhibition of hormone release by vinblastine led to a transient increase of the intracellular content of prolactin. The phase of over-accumulation was followed by a progressive reduction of the total (cell + medium) prolactin. This result is in agreement with the observed inhibition of de novo synthesis of prolactin and indicates that a degradation process takes place in pituitary cells in culture. In conclusion, the use of vinblastine allows us to demonstrate that the rate of prolactin synthesis is dependent upon the secretory status of the cell.     

Evidence for a coupling of prolactin secretion and synthesis by rat pituitary cells in culture

The relationship between the release and the synthesis of prolactin by rat pituitary cells in culture was studied using a microtubule-disrupting drug, vinblastine. (1) Prolactin secretion was inhibited by vinblastine in short-term incubations. Vinblastine did not act via the dopamine pathway, since a potent anti-dopaminergic drug, fluphenazine, was unable to reverse the inhibiting action of the antimicrotubular agent. (2) Continuous treatment by vinblastine induced a progressive decrease of the rate of incorporation of [³H]leucine in prolactin. The half-inhibition time was about 2 days. This inhibition of prolactin synthesis was selective, since total protein synthesis remained unaffected. (3) Measurements of radioimmunoassayable prolactin showed that the inhibition of hormone release by vinblastine led to a transient increase of the intracellular content of prolactin. The phase of over-accumulation was followed by a progressive reduction of the total (cell + medium) prolactin. This result is in agreement with the observed inhibition of de novo synthesis of prolactin and indicates that a degradation process takes place in pituitary cells in culture. In conclusion, the use of vinblastine allows us to demonstrate that the rate of prolactin synthesis is dependent upon the secretory status of the cell.     

Paradoxical effects of oxytocin and vasopressin on basal prolactin secretion and the estrogen-induced prolactin surge

The roles of oxytocin (OT) and vasopressin (AVP) on both basal and estrogen-induced prolactin (PRL) secretion were examined. Adult female Sprague-Dawley rats that were ovariectomized for 3 weeks and received estrogen treatment for 1 week were used. Intravenous administration of hormones and serial blood sampling were accomplished through indwelling intraatrial catheters which were implanted two days before. Plasma PRL levels were measured by radioimmunoassay. Oxytocin at a dose of 20 micrograms/rat stimulated a moderate PRL release in the morning and lower doses (5 and 10 micrograms) were without effect. Vasopressin was most effective at a dose of 5 micrograms/rat in stimulating PRL release, while consecutive injections of higher doses (10 and 20 micrograms) were less effective. In contrast, TRH, ranging from 1 to 8 micrograms/rat, induced a dose-dependent increases in PRL secretion. Using the effective dosages determined from the morning studies, repeated injections of either OT, AVP or their specific antagonists MPOMeOVT [( 1-(beta-mercapto-beta, beta-cyclopentamethylene propanoic acid), 2-(O-methyl)tyrosine, 8-ornithine]-vasotocin) and d (CH2)5Tyr(Me)AVP ([1-(beta-mercapto-beta, beta-cyclo-pentamethylene propionic acid), 2-(O-methyl)tyrosine, 8-arginine]-vasopressin), were given hourly between 1300 to 1800 h and blood samples were obtained hourly from 1100 to 1900 h. It was found that either OT or AVP significantly reduced the afternoon PRL surge, while their antagonists were not as effective. When OT or AVP were administered together with their specific antagonists, the inhibitory effects of either hormone on PRL surge were reversed. Thus it is concluded that both OT and AVP assume a non-specific stress-like effect on PRL release, in which basal secretion is stimulated and surge secretion is inhibited.     

Basal and thyroliberin-stimulated prolactin synthesis in single-cell cultures and in populations of rat pituitary cells.

1. Newly synthesized prolactin was obtained from cultures of rat pituitary tumour cells (GH4C1 cells) after incubation with [35S]methionine. 2. Radioactive synthesized and secreted prolactin was quantified by an immunoprecipitation method by using disc-gel electrophoresis of the dissolved immunoprecipitate in the presence of sodium dodecyl sulphate. By using a microanalytical modification, hormone synthesis and secretion could also be studied in single-cell cultures. This technique was combined with a cytoimmunofluorescence method in which rhodamine-conjugated antibodies were used for studying intracellular prolactin. 3. The presence of radioactive synthesized and secreted prolactin was demonstrated in nine out of 13 single-cell cultures. Cell cultures containing 10 cells or more and clonal populations originating from one cell always secreted radioactive prolactin. 4. Thyroliberin treatment (2 muM) for 24h increased the extracellular accumulation of radioactive prolactin in five out of seven single-cell cultures and always in populations of cells. 5. The number of cells showing prolactin specific fluorescence increased from 20 to 50% and the intensity of this fluorescence became greater after thyroliberin treatment. 6. Studies of [35S]prolactin secretion from single cells and immunochemical detection of intracellular prolactin showed that some cells in an unsynchronized population did not secret radioactive prolactin or show prolactin specific fluorescence. 7. The quantitative effect of thyroliberin as studied in single-cell cultures suggested that the main if not the only effect was to increase prolactin synthesis in cells already producing hormone.     

Ether as an anesthetic for decapitation in the rat: gonadotropin secretion by subsequently established anterior pituitary cell cultures

Historically, for the establishment of dispersed anterior pituitary cell cultures, rodents have been killed by decapitation without anesthesia. Because decapitation fails to induce immediate unconsciousness, the American Veterinary Medical Association Panel on Euthanasia has recently recommended that rodents should not be decapitated without previous anesthesia or sedation. Investigators are therefore confronted with the dilemma of wishing to euthanize rodents humanely yet not wishing to potentially compromise experimental data through the use of anesthetics. We present our observations on the effects of diethyl ether anesthesia administered prior to decapitation on the gonadotropin secretory characteristics exhibited in vitro by cultured rat anterior pituitary cells. Neither light nor complete (surgical level) ether anesthesia had any statistically significant effect on either luteinizing hormone or follicle-stimulating hormone responsiveness or cell content of luteinizing hormone and follicle-stimulating hormone or DNA content. The data indicate that ether anesthesia would appear to be acceptable for those studies involving subsequent in vitro luteinizing hormone and follicle-stimulating hormone secretion.     

Absence of pituitary prolactin epitopes in immunoreactive prolactin of rat brain.

Immunoreactive prolactin (ir-PRL) in rat brain has been consistently documented. However, the identity of this ir-PRL is controversial. Ir-PRL is defined by its ability to bind to PRL antibodies. All previous studies of brain ir-PRL have used polyclonal antibodies, at least one of which apparently crossreacts with a portion of the proopiomelanocortin molecule. To begin to define the epitopes comprising ir-PRL in the brain, we utilized two monoclonal antibodies (MAb) that recognize pituitary PRL in a variety of species, including rat. Immunocytochemistry was performed on rat brains and pituitary glands using two monoclonal and one polyclonal PRL antibody. Although both MAb immunostained lactotrophs of the rat pituitary gland, neither antibody immunostained cell bodies or neuronal processes in the brain. However, the polyclonal antiserum immunostained lactotrophs and a system of neuronal cell bodies and processes in the brain. Thus, epitopes found in pituitary PRL from several species are not found in ir-PRL in rat brain.     

Laminin induces formation of neurite-like processes and potentiates prolactin secretion by GH3 rat pituitary cells

Tumor-derived GH3 rat pituitary cell lines are widely utilized to study mechanisms of prolactin secretion and responsiveness to secretagogues. These cells served here as a model with which to study relationships between shape and function. When GH3 cells were routinely grown in serum-supplemented medium, they exhibited the polygonal phenotype of epithelial cells, with scarce secretory granules. In contrast, when seeded in a serum-free medium, they attached loosely and contained more secretory granules. In both cases, they released prolactin in a nonpolarized manner. We show in the present work that laminin extracted from Englebreth-Holm-Swarm (EHS) tumors was a potent attachment and spreading factor for GH3/B6 cells seeded in serum-free medium. Moreover, it induced the formation of neurite-like processes, which were increased in number and length by chronic treatment with a specific secretagogue, thyroliberin (TRH). These changes in cell shape were correlated with a potentiation of prolactin secretion, both basal and TRH-stimulated. Furthermore, using immunocytochemistry and electron microscopy, we revealed--at the dilated tip of processes--an accumulation not only of prolactin, but also of synaptophysin, a vesicle membrane marker, and of several organelles, such as secretory granules, smooth vesicles, dense bodies and mitochondria. The cytoplasmic processes contained long parallel bundles of microtubules and showed a strong immunoreactivity for beta 2-tubulin. In addition, we found immunocyto-chemical evidence for the presence of 200-k Da neurofilament protein in GH3/B6 cell processes as well as in neurites of cultured hypothalamic neurons. We conclude that, in GH3/B6 cells, laminin induced the differentiation of neurite-like processes, which were the site of polarized organelle transport and exhibited some neuronal markers.