Estrogens modulate the inhibitory effect of tumor necrosis factor-alpha on anterior pituitary cell proliferation and prolactin release

Considering that tumor necrosis factor-alpha (TNF-alpha) is involved in normal tissue homeostasis and that its receptors are expressed in the anterior pituitary, we examined the effect of this cytokine on pituitary cell growth. Because anterior pituitary function depends on hormonal environment, we also investigated the influence of gonadal steroids in the effects of TNF-alpha on cell proliferation and the release of PRL from anterior pituitary cells. In addition, the release of TNF-alpha and its action on the release of PRL from anterior pituitary cells of rats at different stages of the estrous cycle was evaluated. In minimum essential medium D-valine, a medium that restricts fibroblastic proliferation, TNF-alpha (10 and 50 ng/mL) reduced 3H-Thymidine incorporation, DNA content, and active cell number. TNF-alpha failed to affect proliferation of cells from ovariectomized (OVX) rats. However, it significantly inhibited growth of cells from OVX rats cultured with 17beta-estradiol (E2) (10(-9) M) and from chronically estrogenized rats. TNF-alpha decreased the release of PRL from cells of intact rats, especially in proestrous, OVX rats cultured with E2 and chronically estrogenized rats. The release of anterior pituitary TNF-alpha was higher in proestrous rats. These results indicate that TNF-alpha plays an inhibitory role in anterior pituitary cell growth and the release of PRL in an estrogen-dependent manner.     

Differential effects of pituitary stalk-section on posterior pituitary and hypothalamic contents of prolactin-releasing factor, oxytocin, dopamine and beta-endorphin

We have recently shown that the posterior pituitary (neurointermediate lobe) contains a potent prolactin (PRL)-releasing factor (PRF) which is distinct from known PRL secretagogues. Posterior pituitary PRF appears to be a small peptide of an unknown cellular origin. Using pituitary stalk-sectioned (SS) male rats, the objectives of this study were: (1) to determine if PRF is transported from the hypothalamus or is synthesized within the pituitary gland, and (2) to compare changes in PRF activity with alterations in the posterior pituitary content of beta-endorphin (beta-END), oxytocin (OXY), and dopamine (DA). One or two weeks following pituitary SS or sham surgery (SHAM), acid extracts of the posterior pituitary and medial basal hypothalamus (MBH) were analyzed for their hormone content. PRF activity was assessed by determining the stimulation of PRL secretion from perifused anterior pituitary cells, DA was measured by HPLC, and OXY and beta-END levels were determined by RIAs. OXY and DA concentrations in the posterior pituitary were reduced more than 95% at both 1 and 2 weeks after SS. PRF activity in the posterior pituitary was significantly reduced by 75 and 90%, 1 and 2 weeks after SS, respectively. In contrast, beta-END levels in the posterior pituitary at these times increased 20 and 60%, as compared to SHAM rats. Unlike the posterior pituitary, OXY levels in the MBH increased 123% 1 week following SS, and 1,260% at 2 weeks. These increases may reflect the accumulation of OXY-containing secretory vesicles in the severed nerves. DA concentrations in the MBH showed a biphasic pattern. DA levels were initially decreased by 70%, and then increased, but remained 30% below SHAM levels. The reason for these alterations in DA levels is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin - releasing hormone (GnRH) content in the medial basal hypothalamus after substance P injection into the 3rd cerebral ventricle in female rats

The effect of substance P (SP) on gonadotropin releasing hormone (GnRH) content in the medial basal hypothalamus (MBH) was studied. To evaluate this effect, 5 microg of SP in saline or saline alone were injected into the 3rd cerebral ventricle in conscious ovariectomized (OVX) and ovariectomized with subcutaneously implanted 17beta-estradiol capsules (OVX+E[_2]) rats. Two hours later the animals were decapitated and GnRH was estimated by radioimmunoassay in the tissue extracts from MBH. SP injected i.c.v. had no effect on the GnRH content in MBH in OVX rats. However, SP significantly decreased GnRH content in OVX+E[_2] rats. These results provide evidence that SP participates in the control of GnRH neurons within MBH. It is suggested that SP may stimulate GnRH release from GnRH neuron terminals and that estrogen may be involved in this process.     

Quantification of vasoactive intestinal peptide immunoreactivity in the anterior pituitary glands of intact male and female, ovariectomized, and estradiol benzoate-treated rats.

There are considerable data suggesting that vasoactive intestinal peptide (VIP) is involved in the regulation of PRL secretion; however, the role and cell of origin of anterior pituitary VIP remain to be determined. Immunocytochemical (ICC) studies have generally failed to detect VIP-immunoreactive (IR) cells in the pituitary of the untreated rat, although VIP-IR cells have been observed in the pituitaries of hypothyroid or estrogen-treated rats. This study was designed to examine the cellular distribution and tissue content of VIP in the anterior pituitary gland of rats under selected endocrine conditions known to alter the rates of PRL and VIP synthesis and secretion. To this end, anterior pituitary VIP and PRL content (ICC and RIA) and serum PRL levels were determined in ovariectomized (OVX) and OVX rats 3 days after treatment with 7 or 70 micrograms estradiol benzoate (EB). For comparison, pituitary VIP and PRL content (ICC and RIA) and serum PRL levels in untreated male and diestrous female rats were determined. Immunostaining for VIP was accomplished using a newly developed primary antiserum. Significant numbers of VIP-IR cells per 5-microns section were found in the anterior pituitary glands of all animals examined (275 +/- 33 in diestrous to 481 +/- 103 cells in male rats). VIP was not colocalized with PRL in any of the pituitaries regardless of steroid treatment or sex. Furthermore, the number of VIP-IR cells per pituitary gland was not significantly correlated with sex or EB treatment. Treatment with 70 micrograms, but not 7 micrograms, EB significantly increased the pituitary content of VIP and serum PRL levels compared to those after ovariectomy. However, both EB treatments resulted in a significant increase in pituitary PRL content compared to that in untreated OVX rats. Pituitaries from male rats had several-fold more VIP and less PRL content than pituitaries from diestrous rats. These data show that 1) in contrast to previous ICC studies, VIP-IR cells are readily detected in the anterior pituitary of intact male and female and OVX as well as EB-treated rats; 2) VIP is localized to cells other than lactotrophs, regardless of the steroid background; and 3) marked changes in anterior pituitary VIP content are not accompanied by changes in VIP-IR cell number.     

Effect of sulpiride-induced hyperprolactinaemia on catecholamine turnover and LRH concentration in the medial basal hypothalamus of rats

In order to clarify the mechanism of hyperprolactinaemic anovulation, the medial basal hypothalamic (MBH) catecholamine (CA) turnover and LRH concentration, and the serum levels and pituitary contents of gonadotrophins and prolactin (Prl) in hyperprolactinaemic female rats were examined. Hyperprolactinaemia (HPrl) was produced by oral administration of sulpiride for 10 consecutive days; each measurement made on the sulpiride-treated rats was compared with that of control dioestrus rats. Prl, LH, FSH and LRH were determined by radioimmunoassay; CA turnover, as assessed by the accumulation of CA following monoamine oxidase inhibition, was assayed by high performance liquid chromatography with electrochemical detection. Sulpiride treatment induced (1) an increase in the serum Prl and a decrease in the serum LH, (2) an increase in the pituitary FSH and LH contents, (3) an increase in the MBH LRH concentration, and (4) an increase in the MBH dopamine (DA) turnover. These results suggest that HPrl may induce anovulation by impaired LH secretion which was caused by the suppression of LRH release due to an increase in DA turnover in the MBH.     

Evidence for prolactin feedback actions on hypothalamic oxytocin, vasoactive intestinal peptide and dopamine secretion

Ovariectomized rats, when transplanted with 4 anterior pituitaries (APs) to the kidney capsule for 2-3 weeks, had elevated plasma prolactin (PRL) levels (3.8-fold) and showed decreased in situ AP weights (0.62-fold) and PRL concentrations (0.63-fold). The concentrations of dopamine (DA) and oxytocin (OT) in pituitary portal plasma of hyperprolactinemic rats were increased 1.7- and 1.9-fold, respectively. However, the levels of vasoactive intestinal peptide (VIP) in pituitary portal plasma of these rats were decreased 0.31-fold. The secretion of DA, dihydroxyphenylalanine (DOPA) and OT from fetal hypothalamic cells in primary culture was increased, whereas VIP secretion from these cells was reduced in a dose-dependent fashion following PRL treatment. These data are the first in vivo and in vitro demonstration of a stimulatory action of PRL on OT release and an inhibitory action of PRL on VIP release. Furthermore, these data suggest that a subtle imbalance between the secretion of the PRL-inhibiting factor (DA) and the PRL-releasing factors (VIP and OT) during elevated systemic levels of PRL is responsible for decreased lactotrophic function.     

Dopamine transporters participate in the physiological regulation of prolactin

Three populations of hypothalamic neuroendocrine dopaminergic (NEDA) neurons, arising from the arcuate and periventricular nuclei of the hypothalamus release dopamine (DA) that acts at the pituitary gland to regulate the secretion of PRL. It is generally accepted that NEDA neurons lack functional DA transporters (DATs), which are responsible for uptake of DA from the synaptic cleft into the presynaptic axon terminal. This study localized DATs to the hypothalamo-pituitary axis and evaluated the effect of DAT blockade on the hypothalamo-pituitary regulation of PRL. After 7 days of treatment with cocaine (a nonspecific amine transporter blocker) or mazindol (a specific DAT blocker), the relative abundance of PRL messenger RNA (mRNA) in the anterior lobe (AL) of OVX rats was significantly decreased, whereas the relative abundance of tyrosine hydroxylase mRNA in the hypothalamus was significantly increased. The effect of cocaine or mazindol administration on DA turnover and serum PRL concentration was examined in estradiol (E2)-treated OVX rats. E2 administration (i.v.) resulted in a significant increase in serum PRL within 4 h; however, cocaine or mazindol administration abolished the E2-induced increase of PRL. Cocaine or mazindol significantly increased the concentration of DA at the site of the axon terminals within the median eminence (ME), intermediate lobe (IL) and neural lobe (NL), indicating blockade of uptake. Because formation of DOPAC requires uptake of DA, concentrations of DOPAC in the ME, IL and NL decreased following treatment with either cocaine or mazindol. These data, together with the presence of immunopositive DAT in the ME, pituitary stalk, IL, and NL, suggest that a functional DAT system is present within all three populations of NEDA neurons. Moreover, similarity between the effects of cocaine and mazindol treatment indicate that blockade of the DAT, but not other amine transporters, is responsible for suppression of PRL gene expression and secretion. Blockade of DATs prevent uptake of DA into NEDA neurons and consequently increases the amount of DA that diffuses into the portal vasculature and reaches the AL. These data provide evidence that DATs play a physiological role in the regulation of DA release from and TH expression in NEDA neurons and consequently PRL secretion and PRL gene expression and further support our previous observation that the regulation of PRL secretion involves all three populations of NEDA neurons.     

Tumor necrosis factor-alpha-induced nitric oxide restrains the apoptotic response of anterior pituitary cells

We previously reported that tumor necrosis factor-alpha (TNF-alpha) inhibits cell proliferation whereas it stimulates apoptosis of anterior pituitary cells in an estrogen-dependent manner. Also, we showed that nitric oxide (NO) mediates the inhibitory effect of TNF-alpha on prolactin release. Here, we studied the effect of TNF-alpha on nitric oxide synthase (NOS) activity and expression in anterior pituitary cells from cycling and ovariectomized (OVX) rats, and the role of NO in TNF-alpha induced apoptosis of anterior pituitary cells. NOS activity was higher in anterior pituitary cells from rats in proestrus than in diestrus and was stimulated by 17beta-estradiol (10(-9) M, E2). TNF-alpha (50 ng/ml) stimulated NOS activity in anterior pituitary cells from rats at both stages of the estrous cycle and in cells from OVX rats cultured either with or without E2. Inducible NOS (iNOS) gene expression was higher in anterior pituitary cells from rats in proestrus than in diestrus and its expression was enhanced by TNF-alpha. Acute administration of E2 to OVX rats increased endothelial NOS (eNOS) expression in the anterior pituitary gland. Also, E2 increased eNOS mRNA in dispersed anterior pituitary cells from OVX rats, and this effect was blocked by TNF-alpha. nNOS expression in the anterior pituitary gland was higher at proestrus than at diestrus but eNOS expression was similar at both stages. TNF-alpha decreased eNOS mRNA in anterior pituitary cells from rats at proestrus or diestrus. In anterior pituitary cells from OVX rats, TNF-alpha failed to induce apoptosis but was able to induce it when cells were incubated with NAME or NMMA, NOS inhibitors that did not affect cell viability per se. In the presence of E2, NAME induced apoptosis and enhanced the proapoptotic effect of TNF-alpha. In conclusion, our study shows that TNF-alpha upregulates iNOS gene expression whereas it downregulates estrogen-induced eNOS expression in anterior pituitary cells. Endogenous NO may restrain rather than mediate the proapoptotic effect of TNF-alpha in anterior pituitary cells.     

Endotoxin-induced release of interleukin-6 from rat medial basal hypothalami

Interleukin-6 (IL-6) is an inflammatory cytokine that stimulates T-cell activation and B-cell differentiation. We recently reported that picomolar concentrations of IL-6 stimulated PRL, GH, and LH release in vitro. These data suggested that IL-6 may function as a hypothalamic releasing factor for anterior pituitary hormones. Medial basal hypothalami (MBH) were incubated for 60-90 min in Krebs-Ringer bicarbonate buffer supplemented with 0.025% BSA, and the conditioned medium was assayed for IL-6 concentrations by the 7TD1 cell growth factor assay. It was found that MBH released IL-6 in vitro. Although depolarizing concentrations of K+ (56 mM) did not increase IL-6 release, somatostatin release from the MBH was increased significantly. The bacterial endotoxin lipopolysaccharide (LPS; 1-100 ng/ml) induced significant increases in IL-6 release from the MBH. The presence of IL-6 in the hypothalamus suggested a possible role for this cytokine in the regulation of neuropeptide release; however, the release of somatostatin was not affected by 20 ng/ml IL-6. Comparison studies of neural and neuroendocrine tissues revealed that the anterior and posterior pituitaries released larger amounts of bioactive IL-6 than the MBH or parietal cortex during a 4-h incubation; induction of IL-6 release by endotoxin occurred only in the anterior pituitary and hypothalamus. IL-6 mRNA was detectable in the MBH and anterior pituitary tissue after a 4-h incubation; however, no IL-6 mRNA was detectable in freshly isolated tissues. LPS (100 ng/ml) and (Bu)2cAMP (1 mM) increased IL-6 mRNA accumulation in and IL-6 release from the MBH and anterior pituitary. These data suggest that the MBH synthesizes and releases IL-6 via a nonneuronal source in vitro.     

Forskolin-associated luteinizing hormone release by rat anterior pituitary glands: effects of castration and estradiol replacement.

We have previously shown that the diterpene forskolin, a compound which increases intracellular cyclic AMP (cAMP), causes a concentration-dependent release of luteinizing hormone (LH) by continuously perifused anterior pituitary cells from female rats. To test the hypothesis that cAMP-associated LH release is an estrogen-dependent process, we first compared concentration-response relationships between forskolin and both cAMP production and LH secretion by tissue obtained from intact female, intact male and ovariectomized (OVX) rats. Anterior pituitary fragments were perifused with medium alone or medium plus several concentrations of forskolin for 4 h. All three groups demonstrated concentration-dependent cAMP production in response to forskolin. However, while a concentration-dependent release of LH by forskolin was confirmed in pituitary fragments from intact female rats, no such relationship could be identified for tissue from OVX or male rats. Pituitary fragments from OVX rats administered estradiol via silastic capsule for 48 h were next challenged with 3 microM forskolin. In response to this submaximal concentration of the diterpene, a brisk increase in LH secretion was observed. These results demonstrate that, although forskolin stimulates the production of cAMP in intact male, intact female and OVX animals, an associated release of LH can be documented only in the intact female group. These findings, together with the observation that administration of estrogen appears to restore forskolin-associated LH secretion in OVX animals, suggest that estrogen may play a key role in the stimulation of LH release by cAMP.     

Time-dependent increase in plasma prolactin after pituitary stalk section: role of posterior pituitary dopamine

PRL secretion is inhibited by dopamine (DA) input from two systems: the tuberoinfundibular (TIDA) with terminals in the median eminence, and the tuberohypophyseal (THDA) with terminals in the posterior pituitary. The aims of this study were 1) to determine the effects of pituitary stalk section (SS), which prevents DA input from the TIDA neurons, on PRL release, and 2) to assess if the anterior pituitary receives any DA input after SS. Ovariectomized rats were subjected to SS or sham surgery. Jugular blood was collected on the day of surgery (day 0) and for 6 days thereafter and was analyzed for PRL by RIA. DA concentration in the posterior pituitary was determined by HPLC. Unexpectedly, SS caused only a 2- to 3-fold initial rise in plasma PRL on day 0. This was followed by a gradual rise to 4-, 6-, and 8-fold above control levels on days 2, 4, and 6, respectively, without a further increase by 2 weeks. During this time, DA concentrations in the posterior pituitary progressively declined to 66%, 28%, and 6% of control values on days 1, 2, and 6 after SS, respectively. In the second experiment, intact and SS rats were treated with the DA receptor antagonist haloperidol. Haloperidol induced a dramatic 30- to 40-fold increase in plasma PRL in intact rats. Haloperidol induced a 3-fold rise in plasma PRL on day 1 after SS and a transient 2.5-fold rise on day 2. On day 6 after SS, when DA in the posterior pituitary was barely detectable, haloperidol failed to increase PRL secretion. The DA agonist apomorphine caused similar inhibitions of PRL release on days 1 and 6 after SS. Injections of TRH stimulated PRL secretion equally well in intact and SS rats. We conclude that SS does not induce refractoriness to PRL secretagogues or a dysfunction of the anterior pituitary DA receptors. The immediate rise in PRL after SS is modest because the anterior pituitary still receives DA input from the posterior pituitary. A gradual exhaustion of posterior pituitary DA, caused by the disconnection of the THDA terminals from their perikarya, leads to the progressive rise in plasma PRL levels. The DA input affecting PRL release is derived exclusively from the TIDA and THDA neurons, but their relative contributions are yet unknown.     

Interactions of delta 9-tetrahydrocannabinol (THC) with hypothalamic neurotransmitters controlling luteinizing hormone and prolactin release

The effects of delta 9-tetrahydrocannabinol (THC) on hypothalamic norepinephrine (NE) and dopamine (DA) turnover and hypothalamic serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA) and LHRH content preceding and during a progesterone- (P) induced LH and prolactin (PRL) surge were investigated in ovariectomized estrogen-primed rats. THC had no effect on basal LH levels, but it inhibited basal PRL levels and blocked the surges of both LH and PRL. The turnover of NE, as estimated by measuring NE depletion after inhibition of tyrosine hydroxylase with alpha-methyl tyrosine (250 mg/kg), in both the anterior (AH) and medial basal hypothalamus (MBH) was significantly inhibited by THC. THC did not significantly affect AH or MBH DA or 5-HT content nor MBH-DA-turnover. Hypothalamic LHRH levels were significantly elevated 4 h after THC administration as compared to the vehicle-injected controls, but pituitary response to exogenous LHRH was not affected. These data suggest that THC inhibits the steroid-induced positive feedback release of LH by reducing NE metabolism and the release of hypothalamic LHRH. Although the mechanism for the inhibition of PRL release by THC is not clear from these experiments, it does not appear that alterations in DA turnover are a contributing factor.     

Characterization of prolactin-releasing factor in the rat posterior pituitary

We previously reported that the rat posterior pituitary contains a potent PRL-releasing factor (PRF) which is distinct from oxytocin (OT), TRH, and angiotensin II (AII). The objectives of this study were 1) to examine whether posterior pituitary extracts stimulate PRL release in the presence of dopamine (DA), 2) to determine the chemical nature of PRF, and 3) to estimate its mol wt. Perifused anterior pituitary cells were used to assess PRF activity. Posterior pituitaries and medial basal hypothalamus (MBH) fragments were extracted with perchloric acid and lyophilized. Subsequent to various treatments, samples were reconstituted in the perifusion medium and introduced to the cells in short pulses. Fractions were collected and analyzed for hormone content by RIA. During a constant infusion of DA (50 nM), PRL secretion was inhibited by 75%, yet the posterior pituitary extract retained its ability to rapidly stimulate PRL release. Studies using proteolytic enzymes showed that posterior pituitary PRF was resistant to inactivation by trypsin, whereas the PRF activity of AII was abolished. Both chymotrypsin and proline-specific endopeptidase significantly reduced the PRF activity in the posterior pituitary. The PRL-releasing activity of TRH was not affected by chymotrypsin. Immunoreactive vasoactive intestinal polypeptide was undetectable in posterior pituitary extracts. Oxidation of posterior pituitary extracts with performic acid caused only a modest reduction of their PRF activity, while the ability of OT to stimulate PRL release as well as immunoreactive OT was abolished. Studies using ultrafiltration membranes showed that the PRF activity in the posterior pituitary was less than 5,000 mol wt. Furthermore, posterior pituitary PRF partitioned in nearly equal amounts across 1K membranes, as did AII and OT. In contrast, about 80% of the PRF activity in the MBH and all of the synthetic TRH passed through the 1K membranes. We conclude that 1) posterior pituitary PRF can stimulate PRL secretion from perifused anterior pituitary cells in the presence of physiological concentrations of DA; 2) PRF is a small peptide(s) of less than 5,000, and perhaps closer to 1,000, mol wt; 3) PRF is resistant to inactivation by trypsin and to oxidation by performic acid, but is hydrolyzed by both chymotrypsin and proline-specific endopeptidase; and 4) these data further distinguish posterior pituitary PRF from known PRL secretagogues.     

Pineal removal or denervation: effects on hypothalamic PRF activity in the rat.

Medial basal hypothalamic (MBH) extracts from intact, blind-anosmic, blind-anosmic-pinealectomized or nervi conarii transected female rats stimulated the release of prolactin from anterior pituitary glands in vitro. Additionally, a lower tissue level of prolactin was measured in the pituitaries incubated with the extracts. These data indicate that the activity of a prolactin-releasing factor(s) (PRF) was being assayed in the MBH extracts. Additionally, it was observed that MBH extracts from blind-anosmic-pinealectomized or nervi conarii transected rats contained significantly higher levels of PRF activity than the extracts from the intact or blind-anosmic groups. The results support the concept that the pineal control of pituitary prolactin may involve hypothalamic PRF.     

Mechanism(s) by which the transient removal of dopamine regulation potentiates the prolactin-releasing action of thyrotropin-releasing hormone

The transient removal of dopamine (DA) selectively potentiated the prolactin (PRL) releasing action of thyrotropin-releasing hormone (TRH) but not vasoactive intestinal peptide (VIP). Consistent with these findings, the PRL-stimulating actions of agents which activated the Ca2+/protein kinase C second messenger pathway but not the adenylate cyclase system were also potentiated. In the current study we have extended these findings to determine the second messenger system mediating the potentiating action of the removal of DA. Dispersed anterior pituitary cells from E2-treated Sprague-Dawley rats were cultured on plastic coverslips. Cells tonically superfused with DA (500 nM were challenged with TRH (100 nM) 20 min after no additional treatment or a 10-min treatment with 8-Br-cyclic adenosine monophosphate (8-Br-cAMP), the Ca2+ ionophore A23187,12-O-tetradecanoyl-phorbol-13-acetate (TPA), TRH, or VIP. The potentiation of the TRH response was compared to the 4- to 5-fold potentiation observed following the removal of DA for 10 min 8-Br-cAMP at the concentration used (500 microM) was unable to alter the basal rate of PRL release, but, as VIP (500 nM), potentiated 2- to 3-fold the PRL-releasing action of TRH. A prior administration of TRH (100 nM) did not affect the responsiveness of the cells to a second challenge with TRH 20 min later. Both A23187 (20 microM) and TPA (5 or 50 nM) induced a sustained rise in the rate of PRL release. TPA-treated cells showed an increased responsiveness to TRH, whereas A23187-treated cells did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovariectomy permits progesterone to increase the binding of [3H]spiperone to the anterior pituitary gland in estrogen-primed rats

The binding of the dopamine (DA) D2 antagonist spiperone to DA receptors in the anterior pituitary gland was evaluated in intact and ovariectomized (OVX) estrogen-primed rats in which circulating levels of progesterone (P) were varied. Nembutal (PB; 35 mg/kg, ip) was administered at 1130 h to intact proestrous animals to prevent the LH-stimulated release of ovarian P. Two hours later some of these intact rats were QVX to remove the endogenous ovarian steroids. Both intact and OVX rats were given exogenous P. All rats were killed on the following morning, and adenohypophysial binding of [3H]spiperone was evaluated at a saturating concentration. The binding in intact PB-blocked rats that received P was only 52% of that in PB-blocked rats that were OVX and received P. The binding of [3H]spiperone in PB-treated rats that were OVX and given P did not differ statistically from that in normal estrous rats. A parallel experiment was conducted on other rats that had been OVX 7 days before the sc implantation (on day 0) of Silastic capsules containing estradiol (E2). Two days later (day 2), some of these rats were injected with Nembutal at 1130 h. Two hours later, the E2 capsules of some rats were removed to simulate ovariectomy; Silastic capsules containing P were implanted into both E2-bearing and non-E2-bearing rats. All rats were killed the following morning (day 3), and the density of adenohypophysial DA receptors was assessed. Binding densities were greatest in rats bearing both E2 and P capsules and in rats from which E2 capsules were removed and replaced with P capsules. We conclude that treatment of OVX rats with the sequential combination of E2 and P increases the density of DA receptors in the anterior pituitary gland. However, in intact rats an additional ovarian product prevents the P-induced increase in the density of adenohypophysial DA receptors.     

The pineal gland of the blind-anosmic female rat: its influence on medial basal hypothalamic lrh, pif and/or prf activity in vivo.

25-day-old female rats were left intact, or rendered either blind-anosmic or blind-anosmic-pinealectomized. Five weeks later, blind-anosmic animals were found to possess significantly depressed body, anterior pituitary, uterine and ovarian weights. The pituitary levels of LH were elevated while pituitary prolactin was depressed in the dual sensory-deprived rats. Blind-anosmic animals also had significantly depressed plasma levels of LH and elevated plasma levels of prolactin. The effects of blinding and anosmia were effectively reversed by pinealectomy. Ovariectomized, estrogen and progesterone-treated (OVX, EP-treated) rats that received an intracarotid injection of a medial basal hypothalamic (MBH) extract from intact or from blind-anosmic-pinealectomized female rats exhibited a marked rise in plasma LH to a peak at 10 min after injection. This peak was followed by a steady decline in plasma LH concentration. Animals injected with MBH extract from blind-anosmic rats also demonstrated a rise in plasma LH with the levels remaining more or less constant for the 10 and 20-min samples. Rather than declining, the plasma LH levels in 3 out of 4 rats exhibited a further increase until the 40-min sample. The injection of either cortical extract or saline had no effect on plasma LH levels. OVX, EP-treated rats receiving either MBH extracts from intact or blind-anosmic rats or cortical extract exhibited a depression in plasma prolactin titers 5 min following injection. This response was followed by a slight increase in plasma prolactin at 10 and 20 min with a subsequent decrease. Each of the OVX, EP-treated recipients injected with MBH extract from blind-anosmic-pinealectomized rats responded with an obvious increase in plasma prolactin levels within 5 min after injection, followed by a decrease. It was concluded that the MBH of the blind-anosmic rat was high in LRH activity while PIF appeared to predominate over PRF activity. On the other hand, in the MBH of the blind-anosmic-pinealectomized rat, LRH activity was lower while PRF appeared to predominate over PIF activity.     

Dopaminergic stimulation of pituitary but not hypothalamic estrogen receptors in ovariectomized rats

This study was designed to examine the role of catecholamines and serotonin in the regulation of estrogen receptors (ER) in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) of adult ovariectomized rats. Plasma PRL and LH were also determined. Injection of alpha-methyl tyrosine (alpha-MT) resulted in the significant reduction of norepinephrine and dopamine (DA) content in the MBH, and of plasma LH levels as well as in the significant elevation of plasma PRL levels. This treatment also resulted in the significant reduction of ER concentration in the AP. The elevation in plasma PRL by alpha-MT was reversed by the simultaneous injection of bromocriptine (BC), a DA agonist, which also partially reversed the alpha-MT reduction of ER concentrations in the AP. BC had no effect on plasma LH levels. The ER concentration in the MBH was not significantly changed by any of these treatments. The reduction of serotonin content in the MBH by the injection of p-chlorophenylalanine had no effect on the ER concentration in either the MBH or AP, nor did it have any effect on plasma PRL levels. However, p-chlorophenylalanine treatment did decrease plasma LH levels. Neonatal treatment of female rats with monosodium glutamate which has been reported to destroy part of the MBH, resulted in a significant reduction in body and pituitary weight and in a significant elevation of plasma PRL levels in adults (2 months old). This treatment also resulted in a significant reduction in the AP and MBH ER concentration. Injection of BC to adults reversed the effects of neonatal monosodium glutamate treatment on plasma PRL levels and on the pituitary ER concentration, but had no effect on the ER concentration in the MBH. BC had no effect on the AP or MBH ER concentration in control rats, although it did as expected reduce the plasma PRL levels in these animals. Plasma LH levels were not significantly changed by any of these treatments. Injection of the DA antagonist, haloperidol, to adult rats resulted in a significant elevation of plasma PRL and in a significant reduction of ER concentration in the AP. Haloperidol treatment did not affect the binding affinity of these receptors. Overall, these data suggest that DA is involved with the regulation of ER in the AP.     

Decreases in mediobasal hypothalamic and preoptic area opioid ([3H]naloxone) binding are associated with the progesterone-induced luteinizing hormone surge

In view of evidence implicating hypothalamic opioid systems in the control of LH release, we have examined the binding of [3H]naloxone (NAL) to slices of mediobasal hypothalamus (MBH) and preoptic area (POA) during the induction of an afternoon LH surge (1630-1700 h) in estradiol benzoate (EB)-primed ovariectomized (OVX; day 0) rats by treatment with progesterone (P; day 2). Such a surge was invariably accompanied by a decrease from early morning (1000 h) values in the number of NAL-binding sites detectable in the MBH, while the affinity of the binding site was not affected over the course of the day. Time-course studies indicated that P injection at 1000 h on day 2 was followed by a transient midday elevation in the amount of NAL bound to slices of MBH; the binding decreased significantly before the onset of and during the LH surge. A similar diurnal change was not observed in MBH slices of either oil-treated OVX rats (controls) or EB-treated OVX rats, which displayed only a 2-fold increase in LH release in the afternoon. Further studies indicated a similar change in NAL binding to slices of the POA of EBP-treated rats. Since hypothalamic opioid systems inhibit LH release, the decrease in opioid binding to MBH as well as POA slices suggests that P may curtail the existing opioid inhibitory influence in these areas before and during the course of the afternoon LH surge.     

Effect of pretreatment of long-term ovariectomized (OVX) rats with a competitive LRH antagonist on FSH release in vitro

The effect of a single sc injection of an LRH antagonist ((Ac-D-p-Cl-Phe1,2,D-Trp3,D-Phe6,D-Ala10)-LRH, Org 30093) into OVX rats on FSH release 24 h later was studied. Plasma FSH was decreased but pituitary FSH content was not changed. Incubation of the pituitary glands during 4 h resulted in a decreased basal release. FSH release induced by a low concentration of LRH (1 ng/ml) was depressed but that of a high concentration (10 000 ng/ml) was augmented in comparison to FSH release induced in control glands. However, pretreatment with the antagonist had no specific effect on FSH release in vitro induced by high K+ or high K+ plus mbcAMP and theophylline, indicating that the changes of pituitary responsiveness to LRH are not caused by those parts of the secretory mechanism which are stimulated by these secretagogues. Moreover, it is concluded that the changes of pituitary LH release induced by administration of an LRH antagonist also concern FSH.