Biomedical subjects

N Ben-Jonathan

Publications and source records attributed to N Ben-Jonathan.

At least 37 records · Page 2Linked to original sources

PubMed1995

Identification of two classes of prolactin-releasing factors in intermediate lobe tumors from transgenic mice.

Targeted tumorigenesis, using the POMC gene promoter ligated to the simian virus 40 large T antigen, generated transgenic mice with massive tumors of the intermediate lobe (IL) of the pituitary. Inoculation of nude mice with the IL tumor cells resulted in very large secondary tumors. As the IL from several species produces a potent PRL-releasing factor (PRF), it was of interest to determine whether IL tumors from these mice also contain PRF. The objectives were to 1) measure serum PRL levels in mice with IL tumors, 2) determine whether these tumors contain PRF and examine its chromatographic properties, and 3) analyze whether this PRF is related to POMC, its derivatives, or other PRL secretagogues. Serum PRL levels were 5- to 6-fold higher in transgenic than in control mice. Primary and secondary IL tumors were acid extracted and successively fractionated using Sephadex G-100 gel filtration and reverse phase and gel permeation HPLC. PRF activity was determined using short term incubation of tissue extracts or column fractions with GH3 cells. Crude tumor extracts exhibited a strong and dose-dependent PRF activity. Upon chromatography, the PRF activity from either primary or secondary tumors resolved into two classes of compounds: a big PRF with an estimated mol wt of 70-80 kilodaltons and two small, very hydrophobic peptides. The elution profiles of the three PRFs differed from those of beta-endorphin, alpha MSH, beta MSH, ACTH, TRH, oxytocin, angiotensin II, vasoactive intestinal polypeptide, or corticotropin-like intermediate peptide. In summary, we have identified an animal model with IL tumors that has hyperprolactinemia and overproduces PRF. Two classes of PRFs, big and small, were resolved which differ from POMC derivatives and known regulators of PRL release. These data suggest that PRF is produced by melanotrophs, but is not a product of the POMC gene. The IL tumors should provide an excellent source for the purification and structural elucidation of PRFs.

Animals↗

PubMed1995

Prolactin is a local growth factor in rat mammary tumors.

PRL is a mitogenic hormone that shares many characteristics with growth factors. The recent demonstration that rat mammary tissue expresses PRL messenger RNA (mRNA) led us to hypothesize that PRL may act as an autocrine/paracrine growth factor in the mammary gland and may be a determinant in mammary carcinogenesis. To examine this, mammary tumors were induced in rats by injection of the carcinogen nitrosomethylurea (NMU). In vitro studies used a cell line derived from NMU-induced mammary tumors. Expression of PRL and PRL receptor was assessed by reverse transcriptase-polymerase chain reaction. The NMU-induced mammary tumors and the cell line express mRNA for both PRL and PRL receptor (the long and short isoforms); additional hybridizing polymerase chain reaction products were seen in the tumors, but not in lactating mammary tissue. Immunoreactive PRL was detected in the NMU-induced tumors. The effect of PRL on cell proliferation was assessed by culturing NMU cells with PRL antiserum. The PRL antiserum inhibited cell proliferation by up to 70% compared to the effect of normal rabbit serum or GH antiserum. In summary, we showed that NMU-induced mammary tumors express mRNA for PRL and PRL receptor. Addition of PRL antiserum to cultured NMU cells significantly inhibited their growth. We propose that PRL may be acting as a local growth factor that stimulates the proliferation of mammary tumors.

Animals↗

PubMed1994

Effects of aporphine isomers on rat prolactin.

S(+)-aporphines are partial agonists at D2 dopamine receptors. High selectivity of anti-dopaminergic action in limbic vs. extrapyr amidal regions of rat brain and lack of induction of dopaminergic supersensitivity have suggested their potential as atypical antipsychotic drugs. Now, in testing for effects on circulating prolactin, a typical D2 antagonist haloperidol elevated, and potent agonist R(-)-11-hydroxy-N-propylnoraporphine lowered, serum prolactin levels in gentled male rats, while S(+)-N-propylnorapomorphine and its 11-monohydroxy analog had little or no effect, even at high doses. Lack of hyperprolactinemia adds to characteristics of S(+)-aporphines that are desirable in improved antipsychotics.

Animals↗

PubMed1994

Prolactin-releasing activity of neurohypophysial hormones: structure-function relationship.

Oxytocin (OT) and arginine vasopressin (AVP) have been reported to release PRL both in vivo and in vitro. The objectives of this study were 1) to compare the potencies of the PRL-releasing activities of OT and TRH using cultured anterior pituitary (AP) cells, and 2) to assess the PRL-releasing activity of naturally occurring neurohypophysial hormones and selected analogs. AP cells were incubated with peptides for 15 min, and medium PRL concentrations were determined by enzyme-linked immunosorbent assay. OT at 25, 100, and 400 nM increased PRL release by 110%, 175%, and 270%, respectively; higher concentrations (1600 and 6400 nM) did not cause a further increase in PRL release. TRH was 5-10 times more potent than OT on a molar basis. GH3 cells, a somatommamotroph tumor cell line, did not respond to OT and related compounds, but showed a similar responsiveness to TRH as AP cells. Twelve neurohypophysial peptides and selected analogs were incubated with AP cells, and their relative PRL-releasing activities were compared. OT and arginine vasotocin (AVT) showed the highest PRL-releasing activity. T4-G7-oxytocin, mesotocin, isotocin, lysine vasotocin, and AVP showed a moderate PRL-releasing activity, whereas, lysine vasopressin, desmopressin, tocinoic acid, pressinoic acid, and oxytocin free acid showed very low or no PRL-releasing activity. Coincubation of OT, AVT, or AVP with a specific OT receptor antagonist abolished their PRL-releasing activity. We conclude that 1) OT and related peptides are capable of stimulating PRL release in vitro, but their potencies are significantly lower than that of TRH; 2) unlike primary AP cells, GH3 cells are unresponsive to OT and related peptides; 3) AVT and AVP probably stimulate PRL release by acting via an OT receptor; and 4) the amino acid residues in positions 3 and 8 in the peptide chain and an amidated C-terminus are critical for the PRL-releasing activity of the neurohypophysial peptides.

Amino Acid Sequence↗

PubMed1994

Activation of the prolactin promoter in transfected GH3 cells by posterior pituitary cells.

UNLABELLED: We previously reported that PRL production is significantly enhanced by a PRL-releasing/regulating factor derived from the posterior pituitary (PP). Specifically, the levels of PRL messenger RNA synthesis and release were dramatically increased in cocultures of GH3 and PP cells. The present objectives were to: 1) determine whether PP cells activate PRL gene transcription in a promoter- and cell-specific manner; 2) compare promoter activation by PP cells with that caused by selected substances that regulate the PRL gene; and 3) examine which region of the promoter (proximal and/or distal) mediates the action of the PP. In Exp 1, GH3 cells, transfected either with luciferase reporter plasmids containing a wild type PRL promoter, a GH promoter, or a glycoprotein alpha-subunit promoter, were cocultured with PP cells. Luciferase activity was used as an index for promoter activation. PP cells induced an 18-fold stimulation of the PRL promoter, as compared with a 2-fold stimulation of the GH promoter and no effect on the glycoprotein alpha-subunit promoter. In Exp 2, GH3 cells transfected with the wild type PRL promoter were cocultured with PP, anterior pituitary, uterine, or PC12 cells for 24 h. PP cells caused a 20-fold stimulation of the PRL promoter, whereas anterior pituitary cells showed a moderate 5-fold stimulation; uterine and PC12 cells caused minimal (< 2-fold) increases in luciferase activity. In Exp 3, GH3 cells were transfected with either the wild type PRL promoter (-2500 PRLluc) or with a truncated promoter (-425PRLluc) containing the proximal region only and were incubated with PP cells, TRH, vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), or estradiol (E2) for 24 h. Compared with the induction of the wild type promoter, PP cells activated the truncated promoter by 30% only. Stimulation of the promoter by relatively high concentrations of TRH, VIP, EGF, or E2, either alone or in combination, was significantly less effective than that caused by PP cells. CONCLUSIONS: 1) PP cells stimulate PRL gene transcription in a tissue- and promoter-specific manner; 2) the magnitude of induction caused by PP cells far exceeds that caused by high concentrations of TRH, VIP, EGF, or E2; and 3) the distal enhancer region of the rat PRL gene is necessary for maximal responsiveness of the PRL promoter to PP cells.

Animals↗

PubMed1994

Peptide V: a VGF-derived neuropeptide purified from bovine posterior pituitary.

The objective of this study was to purify PRL-releasing factor (PRF) from the bovine posterior pituitary (PP) and determine its structure. Five hundred bovine PPs were acid extracted and fractionated using gel filtration chromatography followed by semipreparative and analytical HPLC. PRF activity was determined by an in vitro bioassay. After six chromatographic steps, a single peak with PRF activity was resolved. As determined by mass spectrometry and microsequencing, this peak contained a major peptide composed of 30 amino acids with a mol wt of 3708K. A synthetic peptide was then produced by solid-phase synthesis. When tested both in vivo and in vitro, the synthetic peptide lacked PRF activity. Further HPLC fractionation under different conditions resolved the synthetic peptide from a highly purified PRF activity. This indicated that the isolated peptide was coincidentally eluted with PRF during the purification. The major isolated peptide has 94% identity with a sequence at the C-terminus of a rat protein named VGF. VGF is a nerve growth factor-inducible protein that has been identified in PC12 cells and is localized in selected sites throughout the central nervous system. The isolated peptide has an Arg-Arg cleavage site at its junction within the VGF protein. Based on this information, we named this substance Peptide V (VGF-derived peptide). We postulate that Peptide V is: 1) a natural cleavage product of the VGF protein; 2) produced and processed either in the hypothalamus or within the pituitary proper, and 3) a releasable peptide that fulfills one or more endocrine functions.

Animals↗

PubMed1993

A compartmentalized chamber for studying dopamine neurons in an hypothalamo-pituitary explant.

UNLABELLED: An in vitro technique was developed which could be used to study whole neurons of the tuberohypophyseal dopaminergic (THDA) tract. Explants containing the medial basal hypothalamus (MBH; THDA cell bodies), pituitary stalk (THDA axons) and posterior pituitary (PP; THDA nerve terminals) were carefully dissected and placed in specially designed chambers. The chambers consist of separate compartments for the incubation of the MBH and PP while maintaining an intact pituitary stalk which traverses through a notched barrier. Dopamine (DA) synthesis in the explants was constant for 3 h and was significantly elevated by electrical stimulation. Electrical stimulation of both the PP and MBH increased endogenous DA release from the PP. DA release in response to potassium depolarization and dopaminergic drugs was significantly lower in explants than in the isolated PP even though spontaneous release was similar. CONCLUSIONS: (1) this method is suitable for studying intact THDA neurons in vitro, and (2) intact THDA neurons respond differently to various stimuli than their excised terminals.

Animals↗

PubMed1993

Coculturing posterior pituitary and GH3 cells: dramatic stimulation of prolactin gene expression.

UNLABELLED: Recent evidence suggests that the posterior pituitary (PP), also called the neurointermediate lobe, regulates PRL release. We previously reported that cocultures of anterior pituitary and PP cells resulted in a 2- to 3-fold increase in PRL content and release. For this study we chose GH3 cells (a somatomammotroph tumor cell line) to determine whether coculturing GH3 with PP cells: 1) stimulates the release and cell content of PRL as compared with GH; 2) increases GH3 cell proliferation; and 3) affects PRL messenger RNA (mRNA) levels. Exp 1. GH3 cells (25,000 cells per well; 25K) were cocultured with PP cells (0K, 12.5K, or 25K) from male rats in serum-free media for 1, 2, 4, and 7 days; hormones were measured by RIA. Coculturing resulted in 5- to 10-fold increases in both media and cell PRL that were linear with time and dependent on the number of PP cells. In contrast, media GH increased only 1.5- to 2-fold, and GH cell content reduced by 75%. Exp 2. GH3, PP, and GH3 + PP cells were cultured for 1, 2, and 4 days and then incubated with [3H]thymidine for 5 h. The incorporation of [3H]thymidine in GH3 cells remained constant over time and showed a small, early increase in cocultures. In contrast, incubation of PP cells alone resulted in a 50- to 60-fold rise in [3H]thymidine incorporation from days 1-4 in culture. Exp 3. Cytoplasmic mRNA was determined by slot blot hybridization with 32P-labeled complementary DNA probes for PRL and GH. After coculturing 25K GH3 cells with 12.5K and 25K PP cells for 4 days, PRL mRNA levels increased 15- and 30-fold, respectively, whereas GH mRNA levels rose less than 2-fold. Neither PRL nor GH mRNA were detected in PP cells. CONCLUSIONS: 1) coculturing GH3 with PP cells dramatically stimulates PRL gene expression, synthesis, and release; 2) this response is specific for PRL, has little effect on GH, and is not due to increased GH3 cell proliferation; and 3) we speculate that a subpopulation of intermediate lobe cells, possibly with a proliferative capacity, is responsible for inducing these effects.

Animals↗

PubMed1992

Endothelin-induced biphasic response of lactotrophs cultured under different conditions.

Endothelin (ET), a recently discovered vasoconstrictor peptide, is widely distributed in different tissues including brain and pituitary. Although evidence regarding the role of ET in neuroendocrine processes is still fragmentary, it appears that the release of several anterior pituitary (AP) hormones can be modulated by peptides of the ET family. In the present study, we compared the effects of ET-1 and ET-3 on the release of PRL from AP cells cultured in serum-free (SFM) or serum-containing (SCM) medium. AP obtained from adult male rats were enzymatically dispersed, and the cells were plated in either SFM or SCM. After 4 days, cells were incubated with ET-1 or ET-3 for designated periods of time, and PRL levels in the incubation media were measured by RIA. When AP cells maintained in SCM were exposed to ET for 20 min, both peptides inhibited PRL release, with ET-1 being a more potent inhibitor than ET-3. In contrast, a biphasic response was observed in cultures grown in SFM: low concentrations of ET peptides inhibited the secretion of PRL, while high concentrations caused a significant stimulation. Further characterization of the effects of ET-1 revealed that the stimulatory phase was relatively short (15-30 min) and was followed by inhibition of PRL release. The addition of either horse serum or fetal bovine serum to SFM on the day of plating resulted in a dose-dependent reduction in the ET-induced stimulation of lactotrophs. These findings indicate that the presence of serum in culture medium alters the expression of cell properties underlying inhibitory and stimulatory responses to ET in terms of PRL secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Effects of reuptake inhibitors on dopamine release from the stalk-median eminence and posterior pituitary in vitro.

Similar to other dopaminergic systems, the dopaminergic neurons innervating the stalk-median eminence (SME) and posterior pituitary (PP) possess an uptake mechanism for dopamine (DA). However, the extent of DA reuptake in these tissues and its physiological significance are debated since much of the released DA is removed by the hypophysial portal vasculature before recapture. The objectives of this study were: (1) to establish in vitro conditions for examining the effects of reuptake inhibitors on DA release from the PP and SME; (2) to compare the effects of nomifensine, diclofensine and amphetamine on DA release from the SME and PP; and (3) to distinguish between reuptake and releasing properties of these drugs. Individual SME and PP were dissected from ovariectomized rats and incubated in either a static or perifusion system. Media DA was extracted with alumina and quantitated by high performance liquid chromatography with electrochemical detection. The reuptake inhibitors, nomifensine, dichlofensine and amphetamine, in the presence of pargyline, a monoamine oxidase inhibitor, stimulated both basal and K(+)-evoked release of DA from the SME and PP under static incubation conditions. The drugs elicited a 2-3-fold higher increase in basal DA release from the SME as compared to the PP. Only amphetamine stimulated DA release in the perifusion system whereas nomifensine and diclofensine were without effects. We concluded that: (1) a mechanism for the reuptake of DA is operable in both the SME and PP; (2) the reuptake of DA appears to be more active in the SME than the PP; and (3) unlike amphetamine, nomifensine and diclofensine are pure reuptake inhibitors devoid of direct DA releasing activities.

Amphetamine↗

PubMed1991

Estradiol rapidly stimulates dopamine release from the posterior pituitary in vitro.

Dopamine (DA) from both the posterior pituitary (PP) and stalk-median eminence (SME) inhibits prolactin (PRL) secretion from the anterior pituitary. Estradiol participates in the regulation of PRL release, in part by modulating DA release from the SME. However, little is known concerning the effects of estradiol on the release of DA from the PP. The objective of this study was to examine whether estradiol rapidly affects the potassium-evoked release of endogenous DA from the PP and SME in vitro. Tissues were dissected from ovariectomized rats and allowed to equilibrate in media for 30 min. Two pulses of 28 mM K+, 3 min each, were then given 30 min apart. Test substances were added 20 min before the second stimulus. DA in the media was determined by HPLC. Estradiol, at a concentration of 1 and 10 nM, significantly stimulated the potassium-evoked DA release from the PP by 34 and 47%, respectively. This stimulation was specific since 17 alpha-estradiol, a biologically inactive isomer, and testosterone, were without effects. Estradiol did not alter DA release from either the SME or isolated neural lobes of the PP. Naloxone, an opioid receptor antagonist, abolished the estradiol-induced stimulation of DA release from the PP. In contrast, amphetamine, a DA-releasing agent, significantly increased DA release in the presence of naloxone. In conclusion, (1) estradiol stimulates DA release from the PP but not the SME or neural lobe; this effect is rapid and stereospecific, and (2) the effects of estradiol appear to be mediated via an opioid(s) peptide(s) from the intermediate lobe.

Amphetamine↗

PubMed1991

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.

Animals↗

PubMed1991

Effects of coculture of anterior and posterior pituitary cells on the responsiveness of lactotrophs to different secretagogues.

The synthesis and release of PRL are regulated by a variety of factors that originate in the hypothalamus, peripheral tissues, or posterior pituitary (PP). We recently reported that coculture of anterior pituitary (AP) and PP cells induced an increase in both PRL cell content and the responsiveness of lactotrophs to TRH. The aim of the present study was to determine whether the augmented response to TRH is due to increased lactotroph sensitivity to this particular secretagogue or to enhancement of the releasable pool of PRL. Cells obtained from anterior pituitaries of adult male rats were plated either alone or together with PP cells at the same total density. Cells cultures were maintained in serum-free medium for 4 days and then incubated for 20 min with the designated substances. Angiotensin-II and TRH evoked a significantly larger release of PRL in AP + PP cocultures than in AP cells cultured alone; the greatest difference between the culture types was observed at the highest concentrations of both secretagogues. The stimulation of PRL release by KCl, the calcium ionophore A23187, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate was higher in the presence of PP cells than in cultures of AP cells alone, although the magnitude of this effect was lower than that seen with PRL secretagogues. The concomitant application of A23187 and 12-O-tetradecanoylphorbol-13-acetate resulted in an increased response in both types of culture and a greater relative effect of PP cells on the evoked PRL release. In contrast to other secretagogues, oxytocin (OT) elicited a smaller response in AP + PP cocultures than in AP cultures. OT was present in significant amounts in medium from cocultures, apparently after being released from the severed neuronal terminals. When AP cultures were pretreated for 4 days with comparable concentrations of OT, the acute OT-evoked PRL release was greatly diminished. These findings suggest that coculture with PP cells increases the releasable pool of PRL in lactotrophs. The stored PRL is accessible for release by secretagogues known to act via the Ca2+ second messenger system, involving both Ca2+/calmodulin and protein kinase-C pathways. The diminished response of cocultures to OT is probably due to desensitization of lactotrophs by the residual amounts of this peptide present in the disrupted nerve endings.

Animals↗

PubMed1990

Estradiol-induced prolactinomas: differential effects on dopamine in posterior pituitary and median eminence.

Prolactin release is inhibited by dopamine and stimulated by estradiol. Dopamine is released from nerve terminals in the median eminence and posterior pituitary. Estradiol may act directly on the anterior pituitary or by modulating the two dopaminergic systems. Estradiol treatment induces the formation of prolactinomas in Fischer 334 rats. Therefore, this strain was chosen as the experimental model. The first objective was to determine whether estradiol differentially regulates the two dopaminergic systems. The second objective was to explore whether the anterior pituitary in estradiol-treated rats acquires the capability for de novo synthesis of dopamine. Rats were ovariectomized and implanted with estradiol capsules (OVEX + E2). Controls were untreated ovariectomized rats (OVEX). Three weeks thereafter, rats were killed. Anterior and posterior pituitaries and medial basal hypothalami (MBH) were removed and individually incubated for 60 min in Hank's balanced salt solution containing 10 microCi [3H-]tyrosine. The median eminence was then dissected from the MBH. Tissues were homogenized in perchloric acid and the supernatant fluids were extracted with alumina. Both endogenous and tritiated dopamine were simultaneously quantitated by HPLC. Prolactinoma formation in OVEX + E2 rats was confirmed by dramatic rise (50-fold) in plasma prolactin levels and marked enlargement (3-fold) of the anterior pituitary. Estradiol treatment caused a significant 60% reduction in both dopamine content and synthesis in the median eminence. In contrast, estradiol treatment affected neither dopamine content nor synthesis in the posterior pituitary. There was no evidence for de novo synthesis of dopamine in anterior pituitaries from either OVEX or OVEX + E2 rats. We conclude that the two dopaminergic systems which regulate prolactin secretion, exhibit a differential response to estradiol.

Animals↗

PubMed1990

Regulation of dopamine release in vitro from the posterior pituitary by opioid peptides.

UNLABELLED: Opioid peptides are present in both the posterior pituitary (PP) and stalk-median eminence (SME). Their effects on the dopaminergic neurons in the SME are well documented, but little is known concerning their role in the regulation of dopamine (DA) release from the PP. The objectives of this study were (1) to develop an in vitro method suitable for examining the regulation of endogenous DA release from PP and SME, and (2) to describe and compare the effects of selected opioid peptides on potassium-evoked DA release from these tissues. Tissues were dissected from ovariectomized rats and incubated in media. After equilibration, two pulses of 28 mM potassium (K+), 3 min each, were delivered 30 min apart. Test substances were administered 20 min before the second K+ stimulus. DA in the media was determined by high-performance liquid chromatography. Potassium at 28 and 56 mM elicited a marked increase in DA release from the PP and SME; this was abolished by the removal of calcium. The opioid receptor antagonist, naloxone, significantly increased the release of DA from both PP and SME by 55%. Dynorphin A elicited a significant inhibition of DA release from PP and SME by 33 and 50%, respectively. In contrast, methionine enkephalinamide decreased DA release from the SME by 50%, but was without effect in the PP. The release of DA from both PP and SME was significantly inhibited by beta-endorphin, and this was reversed by naloxone. However, beta-endorphin was fourfold more effective in the SME. N-acetyl-beta-endorphin did not alter DA release. CONCLUSIONS: (1) we have developed a simple and sensitive in vitro method for studying the effects of hormones and drugs on the release of endogenous DA from PP and SME; (2) tuberoinfundibular dopaminergic and tuberohypophyseal dopaminergic nerve terminals are subjected to a similar inhibitory control by endogenous opioid peptides, and (3) exogenously applied opioid peptides exert differential effects on the release of DA from SME and PP which could be attributable to a dissimilar distribution of opioid receptor subtypes in these two tissues.

Animals↗

PubMed1990

The preovulatory prolactin surge is prolonged by a progesterone-dependent dopaminergic mechanism.

The preovulatory PRL surge consists of a sharp peak, a prolonged plateau, and a termination phase. This study examined the role of progesterone in maintaining elevated PRL release during the plateau phase and its effect on dopaminergic (DA) neuronal activity. Immature rats were injected with PMSG on day 28, and blood was collected during the periovulatory period. Plasma estradiol levels were elevated before and during the PRL peak and declined during the plateau. Plasma progesterone levels were low before and during the peak, rose 4- to 5-fold during the plateau, and decreased to basal levels at the termination phase. In a second experiment rats were subjected to acute ovariectomy (OVEX) or sham surgery (SHAM) just before the onset of the PRL surge. Some OVEX rats were either immediately implanted with an estradiol-containing capsule or given three injections of progesterone during the time of the plateau phase. Blood PRL levels in SHAM rats showed the typical peak, plateau, and termination phases. The PRL peak was evident, but the plateau was missing in OVEX rats with or without estradiol treatment. Replacement with progesterone restored the plateau. In a third experiment, the stalk-median eminence, posterior pituitary, and striatum were removed during the time of the midplateau phase. Tyrosine hydroxylase activity was determined in tissue homogenates by a coupled hydroxylation-decarboxylation assay. Tyrosine hydroxylase activity in the stalk-median eminence of SHAM and progesterone-treated OVEX rats was similar, but was significantly lower than that in OVEX rats with or without estradiol. Tyrosine hydroxylase activity in the posterior pituitary and striatum was unchanged. To assess the functional DA input to the anterior pituitary, haloperidol, a DA antagonist, was injected during the midplateau phase. It induced a 12- to 17-fold rise in plasma PRL in both untreated and estradiol-treated OVEX rats, but failed to increase PRL above the plateau levels in SHAM and progesterone-treated OVEX rats. We conclude that the plateau phase of the preovulatory PRL surge is dependent on the concomitant rise in progesterone. Progesterone probably acts by reducing the DA neuronal activity in the SME, resulting in an absence of functional DA input to anterior pituitary lactotrophs.

Animals↗

PubMed1990

Acute stimulation of prolactin release by estradiol: mediation by the posterior pituitary.

We have previously shown that the posterior pituitary contains a potent PRL-releasing factor (PRF). Estradiol stimulates PRL release by acting at three possible sites: the hypothalamus, the anterior pituitary, and the posterior pituitary. The objectives were 1) to document the profiles of PRL and LH release in response to an acute administration of estradiol, and 2) to identify the site of action of estradiol by employing two surgical approaches, pituitary stalk section (SS) and posterior pituitary lobectomy (LOBEX). Ovariectomized rats were used throughout. In Exp 1, rats were injected iv with 5 micrograms/kg 17 beta-estradiol, and blood was collected at 30-min intervals for 4 h. Estradiol induced a rapid and profound decline in plasma LH levels and a delayed, 5- to 6-fold rise in PRL. The purpose of the second experiment was to determine whether estradiol stimulates PRL release by acting at the anterior pituitary. Injection of estradiol to SS rats failed to stimulate a rise in PRL. We have previously reported that lactotrophs of SS rats are responsive to PRL secretagogues such as TRH. The objective of the third experiment was to differentiate between hypothalamic and posterior pituitary sites of estradiol action. Estradiol induced only a small rise in PRL when injected into LOBEX rats. However, LOBEX and control rats showed similar large rises in PRL in response to injection of alpha-methyl-para-tyrosine, an inhibitor of tyrosine hydroxylase. The latter indicates that the hypothalamic dopaminergic system as well as anterior pituitary lactotrophs are functionally intact in LOBEX rats. We conclude that estradiol administration to ovariectomized rats induces a rapid decline in LH and a delayed marked increase in PRL. The posterior pituitary, probably via PRF, is the primary site that mediates the acute effects of estradiol on PRL release. Estradiol does not stimulate PRL release directly from the anterior pituitary. The role of the hypothalamus is unclear. Estradiol could act directly on PRF-containing cells in the posterior pituitary or indirectly, via hypothalamic neurons terminating in the posterior pituitary. The hypothalamus also has a minor component that responds to estradiol and is independent of the posterior pituitary.

Animals↗

PubMed1990

Prolactin-releasing factor: cellular origin in the intermediate lobe of the pituitary.

Our laboratory has provided substantial evidence for the presence of PRL-releasing factor (PRF) in the posterior pituitary. The objectives of this study were 1) to determine the distribution of PRF activity between the neural and intermediate lobes, and 2) to assess the PRF activity of cultured posterior pituitary cells. Posterior pituitaries from adult male rats were dispersed with trypsin and cultured for 1-7 days. Cultured cells or intact posterior pituitaries were extracted with acid and lyophilized. PRF activity was determined by the ability of reconstituted extracts to increase PRL release from cultured anterior pituitary cells. Upon dissection of the posterior pituitary, PRF activity was primarily present in the intermediate lobe. There was minimal contamination between the two lobes, as indicated by the localization of 90% of the total oxytocin in the neural lobe and 95% of alpha MSH in the intermediate lobe. Extracts from intact posterior pituitaries and posterior pituitary cells cultured for 4 days stimulated PRL secretion in a similar dose-dependent manner. Cultured liver and cerebral cortex cells had very low PRF activity. Both oxytocin and dopamine, two neuronal markers, were reduced to less than 5% of their original values within 1 week of cell culture. There was also a significant reduction in the cell content of alpha MSH. On the other hand, PRF activity was relatively stable during culture. Incubation of posterior pituitary cells for 4 days with either cycloheximide or PRL caused a 55-60% reduction of the PRF activity of the cells. We conclude the following. 1) PRF is localized, almost exclusively, in the intermediate lobe of the pituitary. 2) PRF activity is present within nonneuronal cells, either melanotrophs or a small subpopulation of nonopioid-producing cells. 3) PRF is tissue specific, and its presence in cultured posterior pituitary cells depends at least in part on de novo synthesis. 4) The synthesis and/or release of PRF may be subjected to short loop negative feedback regulation by PRL.

Animals↗