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.

The preovulatory prolactin surge: an evaluation of the role of dopamine.

This study examined the contribution of dopamine (DA) to the control of PRL secretion during the preovulatory PRL surge. Immature female rats were injected with PMSG on day 28. At selected times during the periovulatory period, rats were injected with different pharmacological agents, and jugular blood was collected at frequent intervals. Blood PRL levels in vehicle-treated rats were low on the morning of day 30, rose 15- to 20-fold to peak levels from 1400-1500 h, were maintained at a plateau from 1900-2300 h, and were reduced to basal levels on the morning of day 31. Haloperidol, a DA antagonist, induced a 20-fold rise in PRL before the surge, a 2-fold rise above peak PRL levels at 1500 h, and a 50-fold rise on the morning of day 31. In contrast, haloperidol failed to alter PRL release during the plateau phase. Apomorphine, a DA agonist, reduced PRL levels when injected during either the peak or the plateau phase. Injection of 5-hydroxytryptophan, a serotonin precursor, increased PRL levels at all times examined. Anterior pituitary PRL content was reduced to 30% and 10% of the presurge level during the peak and plateau phases, respectively, but increased on the morning of day 31. Basal PRL release by hemipituitaries incubated in vitro paralleled the anterior pituitary PRL content, with markedly less PRL secreted during the peak and plateau phases compared to the presurge period. However, the percent inhibition of PRL release by hemipituitaries incubated with 50 nM DA was similar at all times tested. These data indicate that the peak PRL surge occurs in spite of DA input to the anterior pituitary, a continued responsiveness to DA inhibition, and a diminishing pituitary PRL content. We conclude that a nondopaminergic mechanism, possibly involving a PRL-releasing factor, is responsible for the peak. The plateau phase probably results from an absence of DA input to the anterior pituitary together with a reduction in the releasable pool of PRL. The termination of the PRL surge is caused by the restoration of DA input.

Tyrosine hydroxylase in the stalk-median eminence and posterior pituitary is inactivated only during the plateau phase of the preovulatory prolactin surge.

This study examined changes in the activity of tyrosine hydroxylase (TH) in the stalk-median eminence (SME) and posterior pituitary (PP) during the preovulatory PRL surge. Immature female rats were injected with PMSG on day 28. Blood PRL levels were low on the morning of day 30, rose to a peak from 1400-1600 h, remained at a lower plateau from 1800-2400 h, and declined to basal levels on the morning of day 31. SME, PP, and striatum were removed from PMSG-treated rats at selected times during the periovulatory period and from age-matched control rats. TH activity was determined in tissue homogenates by a coupled hydroxylation-decarboxylation assay. Apparent Km and maximum velocity values with respect to 6-methyl tetrahydropterine were estimated from substrate saturation curves. The kinetic parameters for TH in either the SME or the PP of control rats were similar at 1100 and 1800 h on day 30. However, the apparent Km in both tissues was significantly lower than that in the striatum. The affinity of TH in the SME and PP was unchanged before and during the peak phase of the PRL surge, reduced significantly during the late plateau, and returned to presurge levels in the morning of day 31. TH activity in the striatum was similar at all times examined. To determine the state of activation of the enzyme, tissue homogenates were preincubated with cAMP, ATP, and magnesium. TH activity in the SME during the peak phase was unchanged by cAMP, and that in the PP was modestly increased. The relatively inactive enzyme in both tissues during the plateau phase was markedly activated by a cAMP-dependent mechanism. The low affinity of striatal TH was greatly increased by cAMP at both times. These data suggest that TH in the SME and PP exists in an activated state most of the time and is transiently inactivated during the plateau phase of the PRL surge. In contrast, TH in the striatum is relatively inactive in the basal state and is not affected by hormonal changes induced by PMSG. We conclude that the peak PRL surge occurs in spite of active dopamine (DA) neurons, suggesting that it is generated by a nondopaminergic mechanism. Decreased TH activity in DA neurons in the SME and PP may prolong the PRL surge during the plateau phase, whereas increased DA activity coincides with the termination of the surge.

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.

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)

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.

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

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

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)

Norepinephrine in the rat ovary: ontogeny and de novo synthesis.

The aims of this study were to characterize the ontogeny of catecholamines (CA) in the rat ovary, to determine the ability of the immature ovary to synthetize norepinephrine (NE) in vitro, and to correlate between ovarian CA and plasma pituitary hormones. Ovaries, spleen (as a control tissue not subjected to endocrine regulation), and trunk blood were collected at 5-day intervals between days 5 and 40. Ovarian NE concentration increased markedly between days 20 and 35 of life, whereas the major rise in splenic NE concentration occurred between days 10 and 15. Ovarian and splenic tissues from neonatal females were capable of de novo synthesis of NE from tritiated tyrosine without an appreciable accumulation of L-Dopa and dopamine. The rate of NE synthesis by ovarian tissue taken from 20-day-old rats was significantly lower than that from 30- and 40-day-old rats, whereas NE production by splenic tissue from 20-, 30-, and 40-day-old rats were similar. Plasma FSH concentration was significantly elevated between days 10 and 20, whereas the major rise in plasma LH and PRL occurred between days 25 and 40. The following conclusions were reached. The delayed elevation of ovarian NE, compared to splenic NE, is attributable to a decreased production of NE by the ovary on day 20 and may involve a suppression or a delay in development of the activity of a key CA biosynthetic enzyme such as tyrosine hydroxylase. Given the temporal relationship between plasma gonadotropins, in particular FSH, and changes in ovarian NE, it is postulated that ovarian CA during neonatal development are subjected to regulation by circulating pituitary hormones.

Plasma catecholamines in the chronically cannulated sheep fetus: predominance of L-dihydroxyphenylalanine.

Dihydroxyphenylalanine (Dopa) and catecholamines (CA) concentrations were determined in plasma collected from chronically cannulated fetal and maternal sheep from days 125-140 of gestation. Dopa was measured by a radioenzymatic assay, followed by ion exchange chromatography and high performance liquid chromatography. The assay was linear to 2.5 ng Dopa, and its sensitivity was 35-45 pg. Dopamine (DA), norepinephrine, and epinephrine were determined simultaneously by the same radioenzymatic incubation procedure, followed by solvent extraction and two-dimensional thin layer chromatography. The Dopa level in the fetal circulation was 10-25 times higher than that of DA, 5-10 times higher than that of norepinephrine, and 100 times higher than that of epinephrine. Furthermore, Dopa was the only CA that was significantly (P less than 0.05) higher in fetal (3.5-4.5 ng/ml) than in maternal plasma (1-1.5 ng/ml). The data indicate that Dopa is the predominant circulating CA in the sheep fetus. While the physiological importance of this observation is unknown at the present time, fetal Dopa might serve as the source of free DA in fetal urine and/or amniotic fluid.

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.

Tyrosine hydroxylase messenger RNA in the hypothalamus, substantia nigra and adrenal medulla of old female rats.

The effects of aging in the female rat were analyzed in terms of tyrosine hydroxylase (TH) gene expression and serum prolactin levels. The number of tuberoinfundibular dopaminergic (TIDA) neurons and the concentration of TH mRNA per cell was greater in 16- to 18-month-old rats than in 25-month-old rats. The amount of TH immunostaining was more intense in the median eminence of the 18-month-old rats compared to either younger or older rats. Plasma prolactin levels were moderately elevated in 18-month-old rats compared to 4-month-old rats, and extremely elevated in 25-month-old rats due to the occurrence of pituitary prolactinomas. There were no detectable changes in TH mRNA levels in the substantia nigra with age, whereas adrenal TH mRNA increased with age. We propose that prolactin initially exerts a stimulatory effect on the TIDA neurons as the rat ages, but eventually causes a loss in neuronal number and neuronal function as the pituitary prolactinoma secretes increased amounts of prolactin.

Central administration of serotonin decreases tyrosine hydroxylase catalytic activity and messenger ribonucleic Acid signal levels in the hypothalamus of female rats.

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 prolactin (PRL) levels. Ovariectomized rats were treated with either vehicle or 5-HT through a lateral ventricular cannula in one of two dose paradigms: 1) a bolus of 20 µg, with tissues taken at 30 min, or 2) the same bolus immediately followed by 20 µg/30 min via a syringe pump for 120 min, and tissues taken at 120 min. Blood samples were taken throughout experiments and plasma PRL determined by radioimmunoassay. Under both paradigms, NSD 1015, a dihydroxyphenylalanine (DOPA) decarboxylase inhibitor (25 mg/kg intraarterially) was injected 10 min before decapitation and brain excision followed by stalk-median eminence dissection. The rate of DOPA accumulation, determined by measuring DOPA levels in the stalk-median eminence by high-performance liquid chromatography with electrochemical detection was used as a measure of tyrosine hydroxylase (TH) catalytic activity. Stalk-median eminence DOPA accumulation in control rats was 29.9 ± 4.2 and 28.8 ± 4,4 ng/mg protein (30 and 120 min experiments, respectively). DOPA accumulation in 5-HT-treated rats was significantly reduced (P<0.05) after 30 min to 17.8 ± 1.2 ng/mg protein, but it was similar (21.7 ± 3.9) to controls after 120 min of 5-HT infusion. 5-HT levels in the stalk-median eminence of rats treated with 5-HT were 13- to 17-fold greater than controls (16.9 to 18.5 ng/mg protein). Plasma PRL levels in both groups increased 10-fold after 5-HT treatment with a peak at 5 min, returning to baseline by 120 min. TH mRNA levels were determined by in situ hybridization in a second group of rats which were treated with the 20µg bolus and subsequent 120 min infusion of 5-HT. TH mRNA signal levels in the arcuate nucleus of control rats averaged 144 ± 21 grains/cell. After treatment with 5-HT, TH mRNA levels in the arcuate nucleus were significantly lower (P<0.0001) with 69±14 grains/cell. In a third group of rats, the effects of the 30 min 5-HT treatment on TH catalytic activity and circulating PRL levels was challenged with two 5-HT(2) receptor antagonists, LY53857 (5 mg/kg intraperitoneally) or ketanserin (10 mg/kg intraperitoneally). Neither the 5-HT-induced decrease in TH catalytic activity nor the increase in PRL was altered by pretreatment (120 min) with 5-HT(2) antagonists. These data suggest that central 5-HT is capable of decreasing TH activity and TH mRNA levels in the tuberoinfundibular dopamine neurons and that the decrease in dopaminergic neuronal activity may contribute to the 5-HT-induced PRL rise. The changes in TH catalytic activity and PRL after intracerebroventricular administration of 5-HT do not appear to be mediated by 5-HT(2) receptors.

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.

Ontogeny of tyrosine hydroxylase mRNA signal levels in central dopaminergic neurons: development of a gender difference in the arcuate nuclei.

Using in situ hybridization and immunocytochemistry, this study examined the tyrosine hydroxylase (TH) mRNA signal levels and immunostaining in the arcuate nuclei of the hypothalamus, zona incerta and substantia nigra of male and female rats during neonatal, peripubertal and adult life. The catalytic activity of TH in the stalk-median eminence was also investigated using the in vitro accumulation of 3,4 dihydroxyphenylalanine (DOPA) after inhibiting aromatic amino acid decarboxylase activity. In the arcuate nuclei, TH mRNA levels increased 3.5-fold between 5 and 15 days of age and remained at a steady level between 15 and 35 days of age in both male and female rats. Similar to TH mRNA levels in the arcuate nuclei, TH activity in the stalk-median eminence increased 2-3 fold between days 10 and 15 of age and remained at a steady level between 15 and 35 days of age in both sexes. A later increase in TH mRNA levels in the arcuate nuclei and catalytic activity in the stalk-median eminence was observed between 35 and 40 days in females, but not males. During adulthood, TH mRNA levels and enzyme activity were 2.7-fold higher in the arcuate nuclei and stalk-median eminence, respectively, of diestrous females vs males. These data suggest that the changes in TH mRNA in the arcuate nuclei may contribute to the developmental alterations, as well as the adult gender differences, in enzyme activity in the stalk-median eminence. Circulating progesterone levels were low (1-10 ng/ml) between days 5 and 25 of age and increased 6-fold between 25 and 35 days of age in both males and females. Progesterone levels increased 2-fold in females, but not males, between days 35 and 40 and were 4-fold higher in diestrous females as compared to adult males. Circulating prolactin levels were low (2-3 ng/ml) between days 5 and 15, increased 15-fold between days 15 and 25 and increased an additional 2- to 3-fold between days 35 and 70 in both males and females. TH mRNA signal levels increased between days 5 and 15 of age in dopaminergic perikarya in the zona incerta and the substantia nigra of both female and male rats. The TH mRNA levels remained constant between days 15 and 70 in the zona incerta, whereas TH mRNA levels declined with age in the substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)