Differential and interactive effects of ligand-bound progesterone receptor A and B isoforms on tyrosine hydroxylase promoter activity.

The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurones. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurones. The present study aimed to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK-N-SH and CV-1 cells were transfected with TH promoter constructs and PR-A or PR-B expression vectors, progesterone treatment caused three- to six-fold increases in TH-9.0 kb promoter activity in PR-B expressing cells, although only a modest increase or no change in PR-A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the -1403 to -1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated that multiple DNA elements are required for complete PR-B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR-B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation analysis indicated PR-B was recruited to the TH promoter. Two different PR-B DNA binding domain mutants had opposing effects on PR-B-mediated TH promoter activation. A GS to AA mutation located in the p-box of the first zinc finger of PR-B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR-A was able to inhibit PR-B transactivation in a dose-dependent manner, although the degree of PR-A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand-bound PR-B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene, and also that changes in the ratio of PR-A to PR-B may affect the ability of progesterone to increase TH expression.

Mu and kappa opioid receptor expression in the mediobasal hypothalamus and effectiveness of selective antagonists on prolactin release during lactation.

Endogenous opioid peptides are involved in prolactin release during lactation, in part by decreasing tuberoinfundibular dopaminergic (TIDA) neuronal activity. Both mu (mu) and kappa (kappa) opioid receptors have a role in the suckling-induced prolactin rise after 4-5 h up deprivation. The aim of this study was to investigate effects of mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA), and kappa opioid receptor antagonist, nor-binaltorphimine (nor-BNI), on prolactin secretion and TIDA neuronal activity in lactating rats after 18 h pup deprivation. After 4 h separation from pups, the suckling-induced prolactin rise was abolished by 16 microg nor-BNI and 5 microg beta-FNA, coincident with increased dihydroxyphenylacetic acid (DOPAC):dopamine ratio in the stalk-median eminence (SME). However, after 18 h pups separation, these same doses of nor-BNI and beta-FNA did not alter the prolactin surge or DOPAC:dopamine ratios in the SME. Higher doses of nor-BNI (32 microg) and beta-FNA (10 microg) were required to inhibit suckling-induced prolactin secretion. beta-FNA (10 microg) increased the DOPAC:dopamine ratio in the SME, whereas nor-BNI (32 microg) treatment had no effect. The mu and kappa opioid receptor mRNA levels in the mediobasal hypothalamus were similar to suckled control rats after 4 h pup deprivation, but increased 1.4-fold after 18 h pup deprivation. These data support involvement of endogenous opioidergic systems in the suckling-induced prolactin rise after a prolonged (18 h) period of pup deprivation, as well as the shorter (4 h) pup deprivation period previously reported. Suppression of TIDA neuronal activity likely played a part in mu opioid receptor input to the suckling-induced prolactin rise after both 4 h and 18 h separation, whereas non-dopaminergic input was implicated with kappa opioid receptors after 18 h pup deprivation. Increased mu and kappa opioid receptors gene expression in the mediobasal hypothalamus may contribute to reduced effectiveness of opioid receptor antagonists to block suckling-induced prolactin release after 18 h pup deprivation.

Gene expression profiles of intracellular and membrane progesterone receptor isoforms in the mediobasal hypothalamus during pro-oestrus.

Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRalpha, mPRbeta and mPRgamma) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRgamma. The mPRalpha mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRbeta mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRgamma and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRalpha and mPRbeta in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period.

Calmodulin and a cyclic nucleotide-dependent protein kinase facilitate the prolactin-induced increase in tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Many aspects of tuberoinfundibular dopaminergic neuronal function are increased by elevated prolactin (PRL) levels, including the activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. This study evaluated the roles of calmodulin, cyclic nucleotide-dependent protein kinase, and calcium/calmodulin-dependent protein kinase II in the PRL-induced increase in tyrosine hydroxylase activity. Ovariectomized rats were treated with haloperidol or ovine PRL (oPRL) for 20-30 h before the experiment, respectively. Treatment with haloperidol increased circulating PRL levels 8-fold and tyrosine hydroxylase activity in the stalk-median eminence 1.8-fold. Treatment with oPRL increased tyrosine hydroxylase activity 1.9-fold. W-7, a calmodulin antagonist, reversed both the haloperidol- and oPRL-induced increase in tyrosine hydroxylase activity to control levels. H-8, a cyclic nucleotide-dependent protein kinase inhibitor, also reversed the haloperidol induced increase in tyrosine hydroxylase activity. KN62, a selective calcium/calmodulin-dependent protein kinase II inhibitor, attenuated the haloperidol-induced increase in tyrosine hydroxylase activity, but KNO4, a structurally related control compound, had no effect. By contrast, the oPRL- and haloperidol-induced increases in tyrosine hydroxylase activity were not altered by KN93, a selective calcium/calmodulin-dependent protein kinase II inhibitor. These data indicate that calmodulin and a cyclic nucleotide-dependent protein kinase contribute to the PRL-induced increase in tyrosine hydroxylase activity, but the role of calcium/calmodulin-dependent protein kinase II is still unclear.

Calcitonin inhibition of prolactin secretion in lactating rats: mechanism of action.

The effects of intracerebroventricular (10 ng/rat) or intravenous (10 or 40 microg/15 min/rat) administration of salmon calcitonin (sCT) on the prolactin (PRL) response to suckling and the activity of tyrosine hydroxylase (TH) were examined in lactating rats. Plasma concentration of PRL increased dramatically in control rats after the onset of the suckling stimulus, while administration of sCT resulted in inhibition of PRL response to suckling. The action of sCT was much more effective with intracerebroventricular administration, which totally blocked PRL release, compared to intravenous administration. The intracerebroventricular administration of sCT increased TH activity of tuberoinfundibular dopamine neuron (TIDA) in the stalk-median eminence, as measured by DOPA accumulation, while completely suppressing the PRL response to suckling. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of TH and thus dopamine synthesis, increased PRL levels, and suckling caused a further increase in plasma concentrations of PRL. Injection of sCT (intracerebroventricularly) did not inhibit the PRL response to suckling in the presence of a depletion of dopamine. These results suggest that sCT inhibition of PRL secretion in lactating rats is mediated mainly by TIDA neurons without involvement of other neuroendocrine mechanisms.

Estradiol attenuates the forskolin-induced increase in hypothalamic tyrosine hydroxylase activity.

The purpose of this study was to evaluate interactions between estradiol and the 3',5' cyclic adenosine monophosphate (cAMP) signaling pathway to regulate tyrosine hydroxylase (TH) activity in hypothalamic dopaminergic neurons. The first experiment examined the ability of forskolin to activate TH in the tuberoinfundibular dopaminergic neurons of adult ovariectomized rats with or without estradiol treatment. Estradiol treatment reduced both basal and forskolin-stimulated TH activity in the median eminence. The second group of experiments examined the effect of estradiol on the forskolin-induced activation of TH in fetal hypothalamic cells cultures. Estradiol decreased basal TH activity in the hypothalamic cell cultures to 80% of control levels. Forskolin treatment for 1 h increased TH activity in a concentration-dependent manner in control and estradiol-treated cells, but estradiol attenuated the stimulatory response to 0.01-10 microM forskolin. The suppressive effect of estradiol on cAMP-dependent activation of TH was evident with 1-12 h of forskolin treatment. The responses to other activators of the cAMP- protein kinase A pathway, including dibutyryl cAMP and 8-bromo-cAMP, and to a depolarizing stimulus were blunted in estradiol-treated cultures. Forskolin treatment for 1 h increased radiolabeled phosphate incorporation into TH protein in control but not estradiol-treated cells, suggesting that estradiol interferes with the ability of the cAMP pathway to phosphorylate TH. Forskolin caused a time-dependent increase in TH mRNA signal levels in control cultures. The magnitude of the forskolin-induced increase in TH mRNA levels was less in the estradiol-treated cells after 6 h of forskolin treatment, indicating that estradiol hinders cAMP-regulated TH gene expression. These data indicate that estradiol attenuates the ability of hypothalamic dopaminergic neurons to respond to cAMP-dependent stimulation by interfering with phosphorylation mechanisms in the short term and control of TH mRNA levels in the long term.

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function.

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.

3,5 cyclic adenosine monophosphate mediates the salmon calcitonin-induced increase in hypothalamic tyrosine hydroxylase activity.

This study examined the effect of salmon calcitonin (sCT) on hypothalamic tyrosine hydroxylase (TH) activity and evaluated the cellular signaling mechanisms involved in the response. Fetal hypothalamic cells were cultured in a defined medium and treated with sCT and/or specific protein kinase inhibitors on day 14 in vitro. sCT (0.1-10 nM) increased both TH activity and cellular cAMP content in a concentration-dependent manner. sCT (10 nM) increased TH activity to 150-175% of control values and resulted in a 10-fold increase in cellular cAMP content. Both the C1a and C1b CT receptor isoforms were present in the cultures, as assessed by RT-PCR. Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a cAMP antagonist, and H-8, a cyclic nucleotide kinase inhibitor, blocked the sCT-induced increase in TH activity, with complete abolition of the response observed at concentrations of 1 mM and 5 microM, respectively. sCT (10 nM) increased radiolabeled phosphate incorporation into TH protein to 169% of control values and 1 mM Rp-cAMPS completely blocked this effect. In contrast, neither Calphostin C, a protein kinase C inhibitor, nor U-73122, a phospholipase C inhibitor, significantly altered the ability of sCT to increase TH activity. Likewise, the sCT-induced increase in TH activity was observed after pretreating the cells with either BAPTA/AM, an intracellular calcium chelator, or thapsigargin, an inhibitor of the endoplasmic reticulum calcium pump. These data indicate that sCT has a profound stimulatory effect on TH activity in fetal hypothalamic cells and that enhanced phosphorylation of TH coincides with the sCT-induced increase in enzyme activity. Moreover, CT receptors, which are linked to cAMP production, are expressed in the hypothalamic cells and a cAMP-dependent mechanism mediates the sCT-induced activation and phosphorylation of TH.

Endogenous opioid peptides contribute to suckling-induced prolactin release by suppressing tyrosine hydroxylase activity and messenger ribonucleic acid levels in tuberoinfundibular dopaminergic neurons.

The endogenous opioid peptides have been implicated in the control of the suckling-induced PRL rise during lactation. This study examined the role of the endogenous opioid peptides in suppressing tuberoinfundibular dopaminergic neuronal activity during lactation. In the first experiment, lactating rats were constantly exposed to pups. Naloxone (NAL; 60 mg/kg x h; i.v.), an opioid receptor antagonist, or saline was infused for 12 h. Blood was collected before and at 2-h intervals during the infusion. NAL suppressed circulating PRL levels to less than 36% of control values at 4, 6, 8, and 12 h after the onset of the infusion. Tyrosine hydroxylase (TH) activity in the stalk-median eminence and TH messenger RNA signal levels in the arcuate nucleus were determined at the end of the NAL infusion. TH activity and TH messenger RNA signal levels were increased 2.5- and 2.7-fold, respectively, after the 12-h NAL infusion. Even though the time spent with their pups was similar between the two groups, the pups in the NAL-treated group failed to gain weight during the 12-h NAL infusion period, whereas the control litters (8 pups) gained 5 g. In a second experiment, pups were removed from the dams before the 12-h NAL infusion and were returned after 11 h. Blood was collected before the infusion, at 3-h intervals during the pup separation period, and at 15-min intervals after reunion with the pups. Plasma PRL in control and NAL-treated rats was low (1-15 ng/ml) and similar during the separation period. The suckling-induced PRL surge in NAL-treated rats was markedly attenuated to 9-25% of control levels (350-650 ng/ml). After a 1-h suckling episode, TH activity in the stalk-median eminence of NAL-treated rats was 4.5-fold greater than controls. Litter weight gains were significantly less in NAL-treated rats during the 1-h suckling episode. These data indicate that the endogenous opioid peptides are an integral component for increasing PRL release in response to suckling and they act to decrease tuberoinfundibular dopaminergic neuronal activity during lactation, in part, by suppressing TH gene expression.

Semicircadian rhythms of c-Fos expression in several hypothalamic areas during pregnancy in the rat: relationship to prolactin secretion.

Prolactin (PRL) serves an important luteotrophic function in the rat during early pregnancy, expressed as a nocturnal surge in the early morning and a diurnal surge in the late afternoon. Several areas of the hypothalamus, including the preoptic area (POA), the suprachiasmatic nucleus (SCN) and the ventromedial and dorsomedial nuclei (VM-DM) have been implicated in PRL surges. We investigated the temporal relationship between neuronal activity as measured by c-Fos immunocytochemistry in these areas and PRL secretion during early and late pregnancy. Brains were collected at nine time points (24:00, 02:00, 04:00, 06:00, 10:00, 14:00, 16:00, 18:00 and 20:00 h) on days 6-7 and three time points (02:00, 14:00 and 18:00 h) on days 14-15 of pregnancy. Plasma PRL levels determined by radioimmunoassay revealed two surges with peaks at 02:00 and 18:00 h and a trough at 14:00 h on days 6-7, which were absent on days 14-15 of pregnancy. The number of neurons expressing c-Fos in the anterior medial preoptic nucleus, the medial preoptic area and the medial preoptic nucleus, but not the anteroventral preoptic nucleus of the POA, and the VM-DM, showed a semicircadian rhythm which was maximal at 02:00 h or/and 04:00 and 18:00 h and reached the lowest value at 14:00 h, in parallel with the PRL surges in early pregnancy. However, the temporal pattern of c-Fos in these areas was reversed during late pregnancy, with a peak at 14:00 h and low levels at 02:00 and 18:00 h. PRL surges were absent and levels were uniformly low during these times. Neuronal activity in the SCN did not show any correlation with PRL surges. The dorsomedial subdivision of the SCN showed high neuronal activity during the daytime in both stages of pregnancy. Neuronal activity in the ventrolateral subdivision of the SCN was high during the nighttime in early pregnancy, however it exhibited high levels during the daytime in late pregnancy. These results suggest that the two daily surges of PRL secretion during the first half of pregnancy might be related to the temporal rhythm of neuronal activity in the POA and the VM-DM, and a major change in the pattern of neuronal activity in these hypothalamic areas might result in termination of the PRL surges at midpregnancy.

Prolactin (PRL) receptors are colocalized in dopaminergic neurons in fetal hypothalamic cell cultures: effect of PRL on tyrosine hydroxylase activity.

This study examined the responsiveness of dopaminergic neurons to PRL and the expression of PRL receptors in fetal hypothalamic cells. Hypothalamic cells were cultured in medium containing 5 or 25 mM potassium (K+) with or without 5% FBS. Rat PRL (rPRL) treatment (10-1000 ng/ml) for 10 days increased tyrosine hydroxylase (TH) activity 1.6- to 1.8-fold in dopaminergic neurons cultured in serum-containing medium with 25 mM K+, but not in defined medium or any medium with 5 mM K+. The rPRL-induced increase in TH activity was observed at 10-1000 ng/ml after both 1 and 10 days of rPRL treatment, whereas 1 ng/ml was not effective. TH activity was not altered after 1-12 h of rPRL treatment (100 ng/ml), but was increased 1.4-fold after 1-3 days and 1.8-fold after 5-10 days. The colocalization of PRL receptors and TH was evaluated by double labeled immunocytochemistry. PRL receptor immunostaining was observed in most TH-immunoreactive cells cultured in either defined or serum-containing medium with or without 10 days of rPRL treatment (100 ng/ml). As assessed by reverse transcriptase-PCR, the long form, but not the short form, of the PRL receptor was expressed in the hypothalamic cells regardless of medium composition, similar to the expression pattern in adult mediobasal hypothalamus from ovariectomized rats. These data indicate that a factor present in FBS imparts PRL responsiveness to hypothalamic dopaminergic neurons in vitro. The effective PRL concentrations and the time course for PRL's action in vitro are within the physiological range in vivo. The colocalization of PRL receptor in dopaminergic neurons provides anatomical evidence for a direct effect of PRL, with the long form of the PRL receptor being the predominant form in the hypothalamic cells.

The responsiveness of tuberoinfundibular dopaminergic neurons to prolactin feedback is diminished between early lactation and midlactation in the rat.

Lactation is a state of hyperprolactinemia resulting in part from suppressed tuberoinfundibular dopaminergic (TIDA) neuronal activity. The suckling stimulus contributes to this suppression despite the fact that the TIDA neurons are a potential site for PRL feedback to increase neuronal activity. This study examined the influence of PRL feedback and the suckling stimulus on tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, during early and midlactation. On day 3 or 10 of lactation, rats were injected intracerebroventricularly with medium (control) or MMQ cells (200,000 cells), a PRL-secreting cell line. On day 6 of lactation, TH activity in the stalk-median eminence was increased 2- or 1.4-fold by MMQ cells or prior treatment with ovine PRL (oPRL; 4 mg/kg, sc), respectively. Removal of pups for 24 h increased TH activity 70% above levels in pup-exposed rats, and MMQ cells or oPRL caused an additional 60% increase. TH messenger RNA (mRNA) levels in the arcuate nucleus were increased 3-fold after removing the pups, but MMQ cells did not alter mRNA levels in either pup-exposed or pup-deprived dams. In contrast to early lactation, MMQ cells did not alter TH activity or mRNA levels in the pup-exposed dams on day 13 and only marginally increased enzyme activity in pup-derived dams. Circulating PRL levels were markedly reduced after removing pups. MMQ cells suppressed circulating PRL levels in both groups of dams on day 6 and in pup-deprived dams on day 13, but had no effect in pup-exposed dams at this time. In a second experiment, pup-exposed dams were injected with bromocriptine, a dopamine agonist, and killed after 4 or 12 h on day 5 or after 12 h on day 12. In some rats, PRL was replaced by injecting oPRL simultaneously with and 8 h after bromocriptine treatment. On day 5 of lactation, bromocriptine reduced and oPRL restored TH activity, whereas on day 12, oPRL was not able to reverse the effect of bromocriptine. These data indicate that the suckling stimulus suppresses TH activity and gene expression in the TIDA neurons in pup-exposed dams. The high endogenous PRL levels associated with the suckling stimulus may activate TH activity in TIDA neurons during early lactation. However, the responsiveness of the TIDA neurons to PRL feedback is attenuated by day 13 of lactation.

Rat placental lactogen-I abolishes nocturnal prolactin surges in the pregnant rat.

The twice-daily surges of prolactin (PRL) present during the first half of pregnancy abruptly terminate at midpregnancy concurrent with the appearance of high levels of placental lactogen-I (PL-I) in the blood. This study addressed the role PL-I and other pituitary or placental hormones have in terminating PRL surges in pregnant rats. Implantation of rat PL-I (rPL-I) or ovine PRL into the arcuate-median eminence area of the hypothalamus of day 7 pregnant rats totally eliminated nocturnal PRL surges on days 8 and 9. To assess the specificity of the inhibitory effects of hormones from the PRL-growth hormone (GH) family, rat growth hormone (rGH), human growth hormone (hGH), and rat prolactin-like protein-A (PLP-A) were tested. Only the lactogenic hormone, hGH, had any effect. Since lactogenic hormones may inhibit PRL by stimulation of dopamine synthesis and release into the hypophysial portal blood vessels leading to the anterior pituitary, the effect of these hormones on tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine activity, was determined. In pregnant rats, both ovine prolactin (oPRL) and hGH significantly increased (64%) TH activity, whereas rPL-I was less effective. In ovariectomized, bromocriptine-treated rats, both rPL-I and oPRL increased TH activity 207 and 151%, respectively. This supports the concept that termination of PRL surges at midpregnancy are owing to secretion of placental lactogens (PLs) from the placenta. However, the mechanism for the inhibition cannot be entirely attributed to an increase in tuberoinfundibular dopaminergic neuronal activity.

Hypoprolactinemia decreases tyrosine hydroxylase activity in the tuberoinfundibular dopaminergic neurons acutely by protein dephosphorylation and chronically by changes in gene expression.

This study evaluated the roles of protein dephosphorylation or suppressed gene expression in reducing tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons after acute or chronic bromocriptine (BROMO) administration. Diestrous or ovariectomized rats were injected with BROMO (3 mg/kg, s.c.) at 1000 h and were sacrificed 4 h later or were injected with BROMO at 12 h intervals for 3 days.In vitro tyrosine hydroxylase activity was assessed by incubating hypothalamic explants with brocresine, an L-aromatic amino acid decarboxylase inhibitor, and measuring 3,4-dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME). The incubation medium also contained either 2 µM okadaic acid, a protein phosphatase 1 and 2A inhibitor, or its vehicle (0.25% dimethylsulfoxide). Acute (4 h) and chronic (3 days) BROMO treatment suppressed circulating PRL levels from 10-12 ng/ml to<1 ng/ml and reduced tyrosine hydroxylase activity in the SME by 60% or 40% in diestrous or ovariectomized rats, respectively. Okadaic acid increased tyrosine hydroxylase activity in the SME 2-fold in control diestrous or ovariectomized rats. The reduced tyrosine hydroxylase activity in the SME after acute BROMO treatment was increased by okadaic acid 5-or 3-fold in diestrous or ovariectomized rats respectively, to a value similar to the controls. In sharp contrast, after chronic BROMO treatment, the response to okadaic acid was blunted. As assessed byin situ hybridization, tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of diestrous rats were not altered after acute BROMO treatment, but were reduced by 70% after chronic BROMO treatment. The acute BROMO-induced decrease in tyrosine hydroxylase activity was reversed by co-administration of oPRL or rPRL, indicating that the action of BROMO is via a reduction in PRL. The data suggest that protein dephosphorylation may be a primary mechanism for the rapid BROMO-dependent suppression of tyrosine hydroxylase activity, whereas suppression of tyrosine hydroxylase gene expression may contribute to the lower tyrosine hydroxylase activity after chronic BROMO treatment.

Pituitary adenylate cyclase-activating polypeptide (PACAP) increases prolactin release and tuberoinfundibular dopaminergic neuronal activity.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a hypothalamic peptide that affects anterior pituitary cell function. This study examined the effects of PACAP on prolactin (PRL) release in vivo and on tyrosine hydroxylase (TH) activity in tuberoinfundibular dopaminergic neurons in vivo and in vitro. In ovariectomized rats, intravenous injection of PACAP increased circulating PRL levels 3-fold and TH activity in the stalk-median eminence (SME) by 30%. Incubation of the SME with 1 microM PACAP in vitro increased TH activity 2-fold. Intravenous infusion of ovine PRL (oPRL) by an osmotic mini-pump for 2 days in ovariectomized rats increased TH activity in the SME 1.7-fold and reduced circulating concentrations of endogenous rat PRL to 20% of control levels. PACAP induced a 4-fold rise in endogenous rat PRL levels in oPRL-treated rats and a 30% increase in TH activity that was additive to the elevation caused by hyperprolactinemia. In suckled lactating rats, PACAP did not alter circulating PRL levels or TH activity in the SME. When pups were removed from the dams for 4-5 h, systemic injection of PACAP stimulated PRL release without altering TH activity. However, PACAP, when administered in vitro, stimulated TH activity in the SME of lactating rats separated from their pups. These data indicate that PACAP may play a role in augmenting PRL release in female rats. The PACAP-induced rise in PRL release is modest and not due to a decrease in tuberoinfundibular dopaminergic neuronal activity. PACAP increases TH activity in the tuberoinfundibular dopaminergic neurons, possibly by a direct action on nerve terminals within the median eminence.

Progesterone suppresses tyrosine hydroxylase messenger ribonucleic acid levels in the arcuate nucleus on proestrus.

This study examined the intracellular mechanisms for the regulation of tyrosine hydroxylase in the tuberoinfundibular dopaminergic neurons of cycling female rats. It also evaluated the hormonal influences that contribute to the control of this enzyme on proestrus. Tyrosine hydroxylase messenger RNA (mRNA) levels in the arcuate nucleus of the hypothalamus were assessed by in situ hybridization. Tyrosine hydroxylase activity in the stalk-median eminence was determined from the in vitro or in vivo rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation after inhibiting DOPA decarboxylase with brocresine or m-hydroxybenzylhydrazine, respectively. Tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation were similar on diestrous day 2 and proestrous mornings, but were reduced by 50% on estrus. Although circulating PRL concentrations were similar on the morning of each day of the estrous cycle, a broad preovulatory PRL surge was observed on the afternoon of proestrus. In vitro DOPA accumulation was similar at 1000 h before the PRL surge and at 1330 h during the peak phase of the PRL surge, but was reduced during the plateau phase of the PRL surge (1700 and 2200 h) coincident with the preovulatory progesterone rise and remained low on estrus. However, in vivo DOPA accumulation was transiently decreased only at 1700 h on proestrus. Tyrosine hydroxylase mRNA levels were similar at 1000, 1330, and 1700 h on proestrus, were reduced by 50% at 2200 h on proestrus subsequent to the decrease in enzyme activity, and remained low on the morning of estrus. Okadaic acid, a protein phosphatase-1 and -2A inhibitor, induced a similar increase in tyrosine hydroxylase activity in vitro at 1330 and 2200 h on proestrus and at 1100 h on estrus, indicating that tyrosine hydroxylase was capable of being activated in spite of decreased mRNA levels. Ovariectomy between 1100-1200 h on proestrus prevented the decrease in tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation at 2200 h. The effects of ovariectomy were completely reversed by progesterone, whereas estradiol had no effect. Circulating PRL levels at 2200 h were suppressed to basal levels after ovariectomy, but were increased by progesterone treatment at 1530 h to levels similar to those in the plateau phase of the PRL surge in control rats. Administration of the progesterone antagonist RU486 at 1200 h on proestrus did not alter tyrosine hydroxylase activity, tyrosine hydroxylase mRNA levels, or circulating PRL concentrations at 2200 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone reverses the estradiol-induced decrease in tyrosine hydroxylase mRNA levels in the arcuate nucleus.

Estradiol (E2) and progesterone (P4) interact to influence tuberoinfundibular dopaminergic neuronal activity and contribute to the control of prolactin (PRL) release. This study examined tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of the hypothalamus and tyrosine hydroxylase activity in the stalk-median eminence after 1 week of steroid treatment and related these to circulating PRL levels. Ovariectomized rats were untreated (control) or were implanted with E2, P4 or both E2 and P4 pellets and were sacrificed after 7 days at either 10:00 or 18:00 h. Some E2 + P4-treated rats were injected with either RU 486 or its vehicle at 12-hour intervals for the last 3 of the 7 days of steroid treatment. Tyrosine hydroxylase mRNA signal levels in the arcuate nucleus were decreased by 70% at both 10:00 and 18:00 h in the E2-treated rats compared to control rats. P4 alone had no effect on tyrosine hydroxylase mRNA levels, but reversed the E2-induced decrease so that mRNA levels in the E2 + P4-treated rats were similar to control levels. The progesterone antagonist RU 486 blocked this effect of P4, supporting the observation of decreased mRNA levels in E2-treated rats. Steroid treatment had no effect on tyrosine hydroxylase mRNA levels in the medial zona incerta. Tyrosine hydroxylase activity in the stalk-median eminence was similar at 10:00 and 18:00 h in control rats, and was decreased by 25 and 36% at 10:00 and 18:00 h, respectively, in E2-treated rats. P4 alone had no effect on tyrosine hydroxylase activity, but reversed the E2-induced decrease in enzyme activity to control levels at both 10:00 and 18:00 h. In contrast to the effect of RU 486 on tyrosine hydroxylase mRNA, tyrosine hydroxylase activity in E2 + P4-treated rats was not significantly altered by RU 486 at either 10:00 or 18:00 h. Circulating PRL levels were elevated in the E2-treated and E2 + P4-treated rats. A diurnal PRL rise was evident at 18:00 h in E2-treated rats, but was abolished by concomitant treatment with P4. The diurnal PRL surge was re-established in E2 + P4-treated rats after administration of RU 486, whereas basal circulating PRL levels were not altered by RU 486. These data indicate that P4 antagonizes the profound inhibitory effect or E2 on tyrosine hydroxylase mRNA content in the tuberoinfundibular dopaminergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

A factor(s) from a trophoblast cell line increases tyrosine hydroxylase activity in fetal hypothalamic cell cultures.

We previously reported that a factor(s) from rat choriocarcinoma (Rcho-1) cells suppresses circulating PRL levels and increases tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons in vivo. The purposes of this study were to determine whether this factor(s) increases tyrosine hydroxylase activity in fetal hypothalamic cells in vitro and to evaluate its chemical nature. The Rcho-1 cells are of placental origin and have the capacity to differentiate into giant cells and produce members of the placental PRL family. MMQ cells, a pituitary cell line that secretes PRL, and HRP-1, a placental cell line that does not produce any known members of the PRL family, were used as control cells. Tyrosine hydroxylase activity was assessed by incubation of hypothalamic cells for 1 h with 100 microM brocresine, an inhibitor of aromatic L-amino acid decarboxylase. Tyrosine hydroxylase activity was increased in a density-dependent manner when Rcho-1, but not HRP-1 or MMQ, cells were cocultured with hypothalamic cells for 24 h. Control and Rcho-1-stimulated tyrosine hydroxylase activities were markedly reduced with 1 mM alpha-methyl-p-tyrosine, a specific inhibitor of tyrosine hydroxylase. Tyrosine hydroxylase activity was not altered when hypothalamic cells were incubated for 24 h with rat PRL or recombinant rat placental lactogen-I, whereas a 24-h stimulation with 100,000 Rcho-1 cells and a 1-h stimulation with 5 mM (Bu)2cAMP increased tyrosine hydroxylase activity 3.7- and 3-fold, respectively. The magnitudes of the increase in tyrosine hydroxylase activity were similar when hypothalamic cells were cocultured with Rcho-1 cells for 1 and 24 h. Acetic acid extracts of Rcho-1, but not HRP-1 or MMQ, cells increased tyrosine hydroxylase activity within 1 h in a concentration-dependent manner. The 3-fold increase in tyrosine hydroxylase activity observed with 500,000 Rcho-1 cell equivalents was markedly reduced with 1 mM alpha-methyl-p-tyrosine. The mol wt range of the tyrosine hydroxylase-activating factor(s) (THAF) was estimated using ultrafiltration membranes. The majority of activity was found in the eluate from a 1,000 mol wt cut-off membrane. THAF activity in Rcho-1 cell extracts was decreased by preincubation with pronase, a nonspecific proteolytic enzyme, suggesting that the factor(s) is a peptide. THAF was resistant to inactivation by trypsin or chymotrypsin pretreatment. However, both enzymes destroyed the ability of pituitary adenylate cyclase-activating peptide, either alone or with Rcho-1 cell extracts, to increase tyrosine hydroxylase activity. Oxidation of Rcho-1 cell extracts with performic acid abolished THAF activity.(ABSTRACT TRUNCATED AT 400 WORDS)

A factor(s) from a rat trophoblast cell line inhibits prolactin secretion in vitro and in vivo.

The purpose of the present study was to measure the inhibitory action of secretions from trophoblast cells on prolactin (PRL) secretion in cycling and pregnant rats, and to determine whether factor(s) from trophoblast cells act directly on anterior pituitary cells. A rat choriocarcinoma cell line (Rcho)--a line consisting of trophoblast cells, including differentiated giant cells that secrete members of the placental PRL family--was used. When Rcho cells (1 x 10(6) cells) were transplanted under the kidney capsule of cycling rats, tumors developed and the rats went into constant diestrus. Eight days after cell injection, plasma progesterone was significantly increased in treated rats compared to controls, whereas plasma and pituitary PRL and pituitary PRL mRNA levels were significantly decreased. Similar PRL results were seen on Day 9 of pregnancy after injection of Rcho cells on Day 0 or Day 1 of pregnancy. To determine whether secretions from Rcho cells had a direct effect on anterior pituitary cells to inhibit PRL release, anterior pituitary cells were enzymatically dispersed and cultured for 4 days. Conditioned medium was obtained from 9-day Rcho cell cultures and concentrated by ultrafiltration. A fraction containing substances with molecular weights greater than 10,000 suppressed PRL release from the pituitary culture after 3 and 24 h. Conditioned medium containing substances with molecular weights between 1000 and 10,000 had no effect on PRL release, nor did conditioned medium from a placental cell line designated HRP-1. HRP-1 also contains trophoblast cells but does not contain the differentiated giant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A trophoblast-specific factor(s) suppresses circulating prolactin levels and increases tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons.

Rat choriocarcinoma (Rcho) cells, which are morphologically similar to trophoblast giant cells of the normal placenta and produce placental lactogen-I in vivo, were used to investigate placental feedback on PRL secretion and tuberoinfundibular dopaminergic neuronal activity. Rcho cells were injected into female rats either intracerebroventricularly 60-65 h before use or under the kidney capsule 10-14 days before use. The following endocrine conditions were used: 1) ovariectomized rats with or without bromocriptine treatment, 2) immature (40-44 days old) rats, 3) adult cycling (diestrous) rats, and 4) pregnant rats. Serum PRL levels in ovariectomized, diestrous, and immature female rats were suppressed to less than 20% of control levels by secretions from the Rcho cells. Tyrosine hydroxylase (TH) activity in the stalk-median eminence (SME) was increased 2-fold above control activity in Rcho-treated ovariectomized and immature female rats. When TH activity was reduced to 40% of control levels by 50 h of bromocriptine treatment, secretions from Rcho cells increased TH activity 3.5-fold to levels similar to those for Rcho alone. Even though Rcho treatment suppressed PRL levels, TH activity in the SME of cycling (diestrous) rats was not altered after either central (65 h) or peripheral (12 days) administration of cells. TH mRNA levels in the arcuate nuclei were unaltered by Rcho cells in immature female and adult cycling rats. TH mRNA levels in ovariectomized rats were markedly reduced 75% by 50 h of bromocriptine treatment and modestly reduced 33% 65 h after injection of Rcho cells. However, Rcho cells partially reversed the bromocriptine-induced decline in TH mRNA to levels seen for Rcho cells alone. On day 7 of pregnancy, secretions from Rcho cells abolished the nocturnal and diurnal PRL surges characteristic of early pregnancy and suppressed circulating PRL levels throughout the day to less than 20% of intersurge PRL levels. Rcho cells eliminated the semicircadian rhythm in TH activity in the SME, which was out of phase with the twice daily PRL surges of early pregnancy. TH activity was increased by Rcho factor(s) at 0330 h (nocturnal surge) and 1800 h (diurnal surge), but not at 1000 h (intersurge). MMQ cells, pituitary-derived clonal PRL-secreting cells, similarly terminated the biphasic rhythm of PRL release and tuberoinfundibular dopaminergic neuronal activity during early pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)