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.

Endogenous opioids mediate the nocturnal prolactin surge in the pregnant rat.

Endogenous opioids stimulate PRL release in the rat during proestrus, stress, and lactation. This study investigated the potential role of opioids in regulating the daily nocturnal PRL surges that occur during the first half of pregnancy. On day 8 of pregnancy, infusion of naloxone (NAL; 0.25, 1.0, or 2.0 mg/10 min), an opioid receptor antagonist, from 0100-0400 h blocked the nocturnal surge in a dose-dependent manner. PRL remained at basal levels throughout the infusion when the highest dose of NAL was used. A PRL surge was still present in this group, but its delayed onset occurred 1 h after termination of the NAL infusion. In another experiment NAL was used to determine whether opioids are needed to maintain the nocturnal surge or only to initiate the surge. On day 8 of pregnancy, infusion of NAL (2.0 mg/10 min) was started at 0300 h, after initiation of the surge had occurred, and was continued until 0600 h. After 1 h of infusion, PRL levels were less than 20 ng/ml, compared to more than 200 ng/ml in controls (P less than 0.002) and remained low. A PRL surge occurred approximately 2-3 h after the infusion was discontinued, at a time when the surge was completed in controls (P less than 0.01). Experimental animals did not differ in their surges relative to controls on the next day when no infusion was given. Another set of experiments was performed to determine whether blockade of endogenous opioids inhibited the nocturnal PRL surge by stimulating tuberoinfundibular dopamine (TIDA) neurons. Day 8 pregnant rats infused with NAL (2.0 mg/10 min) from 0100-0400 h demonstrated no nocturnal PRL surge. Subsequently, the medial basal hypothalamus in NAL-treated animals and saline controls was incubated in vitro with the DOPA decarboxylase inhibitor brocresine (100 microM). The greater amount of resultant DOPA accumulation in the stalk median eminence of NAL-treated animals reflected increased TIDA neuronal activity compared to that in SAL-infused controls. Similar results were obtained when rats were treated in vivo with the DOPA decarboxylase inhibitor NSD 1015 (25 mg/kg BW) just before termination of the NAL infusion. Based on these results, we conclude that endogenous opioid peptides are essential in initiating and maintaining nocturnal PRL surges in pregnant rats. Moreover, opioids appear to exert their effects by decreasing TIDA neuronal activity.

Role of serotonin in nocturnal and diurnal surges of prolactin in the pregnant rat.

Two surges of PRL designated as nocturnal (N) and diurnal (D) are observed during early pregnancy. The present study was undertaken to determine the involvement of serotonin (5-HT) in regulating these PRL surges. On day 7 of pregnancy intracarotid catheters were implanted, and blood samples were obtained at 0200 and 0400 h on day 8 and between midnight and 0600 h on day 9 to monitor the N surge. Rats were killed by decapitation, brains were quickly removed, frozen on dry ice, and stored at -70 C until later determination of indole and catecholamines. Pretreatment 24 h earlier with para-chlorophenylalanine (PCPA; 250 mg/kg BW), an inhibitor of 5-HT biosynthesis, did not affect the N PRL surge, although the hypothalamic concentrations of 5-HT and its metabolite 5-hydroxyindole acetic acid were greatly reduced. On the other hand, administration at 2400 h of ketanserin (KET; 10 mg/kg BW, ip) or LY-53857 (5 mg/kg BW, ip), two selective 5-HT2 antagonists, significantly (P less than 0.025 for KET and P less than 0.01 for LY-53857) reduced the N PRL surge. Neither KET nor LY-53857 altered the hypothalamic content of biogenic amines compared to that in saline-treated controls. Intravenous treatment with LY-53857 (1 mg/kg) at 0200 h after the onset of the N surge induced no change in plasma PRL levels compared to those in controls. To test whether 5-HT plays a role in the D surge, rats were decapitated at 1800 h after drug injection. Pretreatment with the same dose of PCPA (24 h), KET (2 h), or LY-53857 (2 h) that was given to rats in the N surge study significantly (P less than 0.01) reduced plasma PRL levels. As observed in the N study, PCPA greatly diminished the hypothalamic content of 5-HT and 5-hydroxyindole acetic acid, and also reduced dopamine, but to a much lesser extent than 5-HT. It is concluded that 5-HT contributes significantly to the generation of the D surge. Its role during the N surge remains uncertain due to the contradictory effects of the synthesis inhibitor PCPA and the receptor antagonists KET and LY-53857.

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.

Termination of prolactin surges with development of placental lactogen secretion in the pregnant rat.

It has been hypothesized that it is rat placental lactogen (rPL) which causes PRL surges to terminate at mid-pregnancy. Using a hormonally induced model of delayed implantation, the temporal relationship between the secretion of rPL and the nocturnal PRL surges was followed. Implantation was prevented by removing the ovaries, the source of estrogen, on day 3 of pregnancy. Blastocysts were maintained free-floating in the uterine lumen by sc injection of 4 mg progesterone in oil daily for 0, 5, 7, or 9 days. Implantation was induced and the subsequent pregnancy was maintained with 1 microgram estrone plus 4 mg progesterone daily. Nocturnal PRL surges (0500 h) were followed for 10 days after the first estrone injection. Control animals last exhibited PRL surges on day 10. Animals with 5, 7, or 9 days of implantation delay had their last PRL surge on days 15, 17, and 18, respectively. Levels of rPL in control animals, as measured by Nb2 lymphoma cell bioassay, were low on day 6, slightly higher on day 10, and significantly elevated on day 12. Delaying implantation delayed the increase in rPL secretion in the experimental groups. This proportionately prolonged the number of days the PRL surges were present. These data suggest that the termination of the nocturnal PRL surges requires the secretion of rPL by the developing conceptus.

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)

Feedback effects of placental lactogens on prolactin levels and Fos-related antigen immunoreactivity of tuberoinfundibular dopaminergic neurons in the arcuate nucleus during pregnancy in the rat.

PRL in the rat exerts its luteotropic action during the first half of pregnancy. After midpregnancy, placental lactogens (PLs) take the place of PRL to stimulate progesterone secretion from the corpus luteum. Simultaneously, PLs trigger a negative feedback on PRL secretion. However, the brain mechanisms for the negative feedback induced by PLs are not fully understood. Here, we report changes in plasma PRL levels, tuberoinfundibular dopaminergic (TIDA) neuronal activity as measured by Fos-related antigen (FRA)/tyrosine hydroxylase (TH) immunoreactivity, and TH catalytic activity as measured by dihydroxyphenylalanine (DOPA) accumulation in the stalk-median eminence (SME) after experimental manipulation of PL levels. On day 4 of pregnancy, animals received Rcho-1 cells intracerebroventricularly (i.c.v.) to increase the level of PLs in the brain or HRP-1 cells as controls. On day 12 of pregnancy, hysterectomy alone or i.c.v. HRP-1 injection plus hysterectomy were performed to remove the source of PLs. Rcho-1 i.c.v. injection plus hysterectomy were performed to examine the effect of replacement of the PL source. Sham-hysterectomized animals were used as a control group. Animals were killed 2 days after each treatment at 0200 and 1800 h, which represent the peak times of PRL surges, and at 1400 h, which represents the intersurge time, by either transcardial perfusion for FRA/TH immunocytochemistry or decapitation 30 min after NSD 1015 injection to assess DOPA accumulation with HPLC-electrochemical detection. Rcho-1 cells completely abolished PRL surges on day 6 of pregnancy and increased the percentage of FRA/TH immunoreactivity in the dorsomedial, ventrolateral, and caudal subdivisions of the arcuate nucleus. This change in neuronal activity reflected the amount of DOPA accumulation in the SME, which was high at all time points. On day 14 of pregnancy, removal of the PL source by hysterectomy resulted in increased PRL levels and decreased neuronal activity of TIDA neurons at all three time points. Similar profiles were observed in animals that received i.c.v. HRP-1 injection plus hysterectomy. Replacement of the source of PL with Rcho-1 cells in hysterectomized rats resulted in low PRL secretion, high neuronal activity of TIDA neurons, and high TH catalytic activity. These patterns were the same as those in sham-operated animals. Our results demonstrate that PLs induce an increase in the neuronal activity of dopaminergic neurons, as measured by FRA/TH immunoreactivity and TH catalytic activity in the SME. Removal of the PL source elevates plasma PRL levels at all times during the second half of pregnancy and does not restore PRL surges.

Potentiation of suckling-induced release of prolactin by inhibition of brain catecholamine synthesis.

The effects of a catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine (alpha-MT), on the accumulation of newly synthesized brain catecholamines and plasma prolactin concentration were studied in lactating rats. [3H]tyrosine was administered to rats 10 minutes before decapitation, and the rate of its incorporation into [3H]dopamine and [3H]norepinephrine was measured in whole brain. Injection of alpha-MT (3.15--12.50 mg/kg) via a carotid cannula increased plasma prolactin levels and decreased the accumulation of newly synthesized brain catecholamines in nonsuckled, lactating rats compared to rats injected with .9% saline solution. Mother rats pretreated with saline solution were suckled for 20 minutes by pups which previously were separated from the mother rats for 5 hours. This treatment resulted in significantly increased plasma prolactin levels compared to nonsuckled controls. In rats pretreated with alpha-MT for 20 minutes, suckling resulted in a further increase in plasma prolactin levels which was significantly greater than the increase caused by suckling or alpha-MT alone. These results suggest that inhibition or catecholamine synthesis had a facilitatory effect on the brain mechanisms responsible for prolactin release in response to suckling.

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.

Hyperprolactinemia increases and hypoprolactinemia decreases tyrosine hydroxylase messenger ribonucleic acid levels in the arcuate nuclei, but not the substantia nigra or zona incerta.

The effects of experimentally produced hypoprolactinemia and hyperprolactinemia on tyrosine hydroxylase (TH) mRNA signal levels were examined in dopaminergic neurons ovariectomized rats. TH mRNA signal levels and relative TH quantity in the arcuate nuclei, zona incerta, and substantia nigra were evaluated by in situ hybridization and immunocytochemistry, respectively. The catalytic activity of TH in the stalk-median eminence (SME) was determined from the in vitro rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation after inhibiting DOPA decarboxylase with brocresine. Chronic administration of bromocriptine (BROMO), a dopamine (DA) agonist, for 3 days reduced circulating rat PRL (rPRL) levels compared to those in the vehicle-treated controls. BROMO treatment decreased TH mRNA signal levels in the arcuate nuclei, the intensity of TH immunostaining in the arcuate-median eminence area, and the rate of DOPA accumulation in the SME. Concomitant administration of ovine PRL (oPRL) reversed the effects of BROMO on TH, resulting in markedly increased TH mRNA signal levels, intensity of TH immunostaining, and rate of DOPA accumulation. Treatment with oPRL by itself for 3 days increased TH mRNA signal levels in the arcuate nuclei and TH activity in the SME, compared to vehicle. Chronic treatment with haloperidol, a DA antagonist, increased circulating levels of endogenous rPRL and increased TH activity in the SME to values similar to those after oPRL treatment. However, in contrast to oPRL, mRNA levels in the arcuate nuclei of haloperidol-treated rats were similar to levels in vehicle-treated animals. To evaluate whether the effect of PRL on TH was species specific, oPRL or rPRL was continuously infused into the jugular vein using an osmotic minipump. TH mRNA levels in the arcuate nuclei were elevated above control levels by either oPRL or rPRL administration. TH mRNA levels in the DA perikarya located in the zona incerta and substantia nigra were not altered by treatment with a DA agonist, a DA antagonist, or PRL. These results indicate that hypoprolactinemia or hyperprolactinemia can selectively reduce or augment, respectively, TH mRNA levels in the tuberoinfundibular dopaminergic neurons. The alterations in TH mRNA content probably contribute to the decrease or increase in TH activity associated with hypoprolactinemia or hyperprolactinemia, respectively.

Mechanisms of tyrosine hydroxylase regulation during pregnancy: evidence for protein dephosphorylation during the prolactin surges.

This study examined tyrosine hydroxylase (TH) activity in the stalk-median eminence (SME) and TH messenger RNA (mRNA) signal levels in the arcuate nuclei of the hypothalamus during early, middle, and late pregnancy and related these to circulating levels of ovarian steroids. In addition, this study evaluated the intracellular mechanism(s) which contributes to the semicircadian rhythm in tuberoinfundibular dopaminergic neuronal activity during early pregnancy. The catalytic activity of TH in the SME was determined from the in vitro rate of 3,4,dihydroxyphenylalanine accumulation after inhibiting DOPA decarboxylase with brocresine. TH mRNA signal levels were evaluated by in situ hybridization. TH mRNA signal levels in the arcuate nuclei were 30% lower at 1000 h on day 20 of pregnancy as compared to days 7 and 11, whereas TH activity in the SME at 1000 h was not significantly different on days 7, 11, 16, and 20. Serum PRL levels were low (3-6 ng/ml) and unchanged at 1000 h on days 7, 11, 16, and 20. Circulating progesterone levels increased from 111 to 191 ng/ml on days 7 and 16, respectively, and then declined to 69 ng/ml by day 20. Serum estradiol levels increased from 38 to 106 pg/ml on day 7 and 16, respectively, and then remained elevated on day 20. Thus, the reduction in TH mRNA signal levels during late pregnancy is temporally related to the increased estradiol/progesterone ratio. Elevated serum PRL levels at 0330 h and 1800 h on day 7 were characteristic of the nocturnal and diurnal PRL surges of early pregnancy. Circulating PRL levels were low during the intersurge times (2330 and 1000 h) on day 7 and at all times examined on day 11. TH activity in the SME on day 7 was lower during the PRL surges as compared to the intersurge times, whereas TH activity on day 11 was similar at all times and comparable to the intersurge levels of early pregnancy. Okadaic acid, a protein phosphatase inhibitor, reversed the reduction in TH activity during the nocturnal and diurnal PRL surges, but did not significantly alter TH activity during the intersurge period on day 7. TH mRNA signal levels in the arcuate nuclei were similar throughout day 7. These data indicate that protein dephosphorylation, but not changes in the TH gene expression, may contribute to the semicircadian rhythm in TH activity during early pregnancy.

Termination of pseudopregnancy following hypothalamic implantation of prolactin.

Pseudopregnancy (PP) in the rat is characterized by maintenance of the corpora lutea and twice daily surges of prolactin at 1500-2100 h and 0100-0900 h. Four days after PP was induced by cervical stimulation with a glass rod, rats were cannulated via the right carotid artery and one day later received an implant of ovine prolactin (200-250 mg) or albumin (controls) into the median eminence of the hypothalamus. Forty-five of 54 rats implanted with prolactin showed a termination of PP within 4 days. Ova were recovered from 19 of these rats whereas all of the controls remained pseudopregnant and did not ovulate. Ovarian and uterine weights were increased in rats implanted with prolactin. Plasma prolactin levels in the rats implanted with prolactin were lower each day (45-89%) at 0100, 0300 and 1800 h compared to controls. Plasma LH levels rose gradually after prolactin implantation, and in those rats in which ovulation occurred, a preovulatory LH rise was detected at 1800 h on the previous day. It is concluded that daily surges of prolactin play an essential role in maintaining PP in the rat, and that implantation of prolactin into the median eminence results in inhibition of prolactin. The stimulation of LH secretion, which led to ovulation, may be the result of a direct neural action of prolactin on the LH release mechanism or may simply result from the termination of PP.

Ovarian and fetal control of rat placental lactogen and prolactin secretion at midpregnancy.

The role of the fetus and ovarian steroids in stimulating rat placental lactogen (rPL) secretion and in regulating nocturnal PRL surges was investigated. On day 10, pregnant rats were ovariectomized (O) and/or fetectomized (F). Those rats injected with steroids received 0.1 microgram estradiol benzoate (E) or 4 mg progesterone (P) in oil sc, or both (PE). Blood samples were taken by cardiac puncture at 0500 h, on days 10 through 14. OPE rats showed slightly depressed serum rPL levels on days 11 and 12 and normal growth of the conceptuses. Ovariectomized rats given P alone also showed significant growth of the conceptus, although less than the OPE rats, demonstrating the necessity for estrogen during pregnancy. PRL surges were no longer present in these groups on day 11, similar to controls. Ovariectomized rats had very low rPL levels, and the nocturnal PRL surges continued for 2 extra days. Fetectomized and OF animals also had very low rPL levels and one extra PRL surge. P counteracted the detrimental effects of fetectomy on rPL secretion; yet placental uterine growth was not greater than in F animals. Thus, increased placental mass was not always correlated with increased rPL secretion. The results show that P is necessary to maintain rPL secretion in the pregnant rat, although estradiol and P in combination is most effective. The fetus also is essential to stimulate both placental growth and rPL secretion. The inverse correlation between rPL and PRL levels on days 11 and 12 suggests that rPL normally terminates the PRL surges at midpregnancy.

Serotonin-induced decrease in hypothalamic tyrosine hydroxylase activity and corresponding increase in prolactin release are abolished at midpregnancy and by transplants of rat choriocarcinoma cells.

We investigated the effect of central serotonin (5-hydroxytryptamine, 5-HT) administration on hypothalamic tuberoinfundibular dopamine neurons and related changes in neuronal activity to circulating PRL levels in two physiological models: 1) pregnant rats expressing (day 8) or not expressing (days 11 and 16) PRL surges, and 2) ovariectomized rats transplanted with rat choriocarcinoma cells, which secrete functional placental lactogen-I. Over a 4-min period between 0900 and 1400 h, rats were administered either vehicle or 5-HT (20 micrograms/6 microliters) through lateral ventricular cannulae. Plasma PRL levels were determined by RIA. NSD 1015 (25 mg/kg intraarterial), a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor, was injected 20 min after initiation of ventricular infusion. Ten min later, the stalk-median eminence (SME) was dissected. The rate of DOPA accumulation, determined by measuring DOPA levels in the SME by HPLC, was used as an index of tyrosine hydroxylase catalytic activity, indicating tubero infundibular dopamine neuronal activity. In day-8 pregnant rats 5-HT reduced DOPA accumulation to 57% of vehicle-injected controls and increased circulating PRL levels 13-fold. In contrast, on days 11 and 16 of pregnancy 5-HT did not alter DOPA accumulation in the SME or plasma PRL levels. In nonpregnant rats ovariectomized for 24 h, 5-HT decreased DOPA accumulation in the SME to 43% of vehicle-infused controls and increased PRL levels approximately 26-fold. However, in nonpregnant rats with rat choriocarcinoma cells, 5-HT produced no changes in either DOPA accumulation in the SME or in circulating PRL levels. The inability of 5-HT to reduce tyrosine hydroxylase activity after mid-pregnancy may account for the lack of a PRL response. Placental lactogens secreted at midpregnancy, particularly placental lactogen-1, may induce this loss of 5-HT effect.

Late pregnancy and rat choriocarcinoma cells inhibit nocturnal prolactin surges and serotonin-induced prolactin release.

The purpose of the present study was to determine the effect of hormonal secretion by trophoblast cells on serotonin-induced release of PRL in both pregnant and nonpregnant rats. In the first experiment, three compounds that effectively lead to stimulation of serotonin receptors were injected ip or intraarterially between 0900-1200 h on day 8 or 16 of pregnancy. These included the serotonin precursor 5-hydroxytryptophan (20 mg/kg BW); a releasor of serotonin, fenfluramine (10 mg/kg BW); and a serotonin S2 receptor agonist, DOI (2,5-dimethoxy-4-iodophenyl-2-aminopropane-HCl; 500 micrograms/kg BW). When injected on day 8, these treatments significantly (P less than 0.01) increased the level of plasma PRL within 30 min after the injection. However, on day 16 the same treatments could not induce any change in the plasma PRL level. In the second experiment, rat choriocarcinoma (Rcho) cells, which secrete placental lactogen I in vivo, were injected beneath the kidney capsule on day 1 of pregnancy. Control pregnant rats injected with the cell culture medium RPMI-1640 containing 20% FBS continued to have a nocturnal PRL surges on days 7, 8, and 9, with the peak value of plasma PRL occurring at 0400 h. Rats injected with the cells had Rcho tumors at the site of injection when analyzed on day 9. These rats also had significantly (P less than 0.05) reduced nocturnal PRL surges on days 7 and 8 of pregnancy compared to the control animals, and on day 9, the PRL surge was completely blocked. In another group of day 9 pregnant rats containing Rcho tumors, DOI-induced PRL release was blocked by Rcho cells, whereas in controls, plasma PRL increased from 5 to 47 ng/ml. The final experiment tested whether the presence of Rcho cells affected serotonergic- or TRH (1 microgram/rat)-induced PRL release in cyclic rats that were ovariectomized 1 day before drug injection. Injection of Rcho cells 8 days earlier completely inhibited 5-hydroxytryptophan- or DOI-induced PRL release, but did not affect TRH-induced PRL release. These results indicate that the absence of PRL surges after midpregnancy may be due in part to the inability of serotonin to stimulate PRL at this time compared to early pregnancy. Secretion of placental lactogens or other PRL-like peptides from the placenta in the pregnant rat may be antagonistic to the normal stimuli that cause the PRL surges of early pregnancy, resulting in a loss of surges.

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)

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.

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)

Control of prolactin release induced by suckling.

In the present study, the role of dopamine and TRH in suckling-induced PRL release was investigated. Bupropion, a dopamine reuptake blocker, increased hypophysial stalk dopamine levels and inhibited suckling-induced PRL release. A short period of suckling, thought to induce a transient decrease in hypothalamic dopamine release, led to higher PRL levels following an iv injection of TRH than in rats which had not nursed their young for a short period after 4- to 6-h separation. These results, in combination with previous data, suggest that a decrease in hypothalamic dopamine release is important for suckling-induced PRL release. Increased PRL release may be in part due to an augmented hypothalamic release of TRH. Since serotonergic mechanisms seem involved in TRH release, lactating rats were treated with drugs acting on serotonergic pathways. Parachlorophenylalanine and pizotifen did not alter suckling-induced PRL release. Methysergide, a serotonin receptor blocker, prevented this PRL release when administered ip but not when injected into the lateral brain ventricle. Since methysergide is converted peripherally into metabolite(s) with dopamine agonistic activity, its effect on suckling-induced PRL release may be due to this action, rather than to its action on serotonin receptors. Thus, these data do not indicate that serotonergic mechanisms are important for suckling-induced PRL release. Passive immunization against TRH inhibited suckling-induced PRL release, indicating that TRH is a hypophysiotropic mediator of this PRL release.

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.