S179D prolactin diminishes the effects of UV light on epidermal gamma delta T cells

Epidermal gamma delta T cells (γδT) and Langerhans cells (LC) are immune cells altered by exposure to ultraviolet radiation (UVB), a powerful stressor resulting in immune suppression. Prolactin (PRL) has been characterized as an immunomodulator, particularly during stress. In this study, we have asked whether separate administration of the 2 major forms of prolactin, unmodified and phosphorylated, to groups of 15 mice (3 experiments, each with 5 mice per treatment group) affected the number and morphology of these epidermal immune cells under control conditions, and following UV-irradiation. Under control conditions, both PRLs reduced the number of γδT, but a molecular mimic of phosphorylated PRL (S179D PRL) was more effective, resulting in a 30% reduction. In the irradiated group, however, S179D PRL was protective against a UV-induced reduction in γδT number and change in morphology (halved the reduction and normalized the morphology). In addition, S179D PRL, but not unmodified (U-PRL), maintained a normal LC:γδT ratio and sustained the dendritic morphology of LC after UV exposure. These findings suggest that S179D PRL may have an overall protective effect, countering UV-induced cellular alterations in the epidermis.     

Administration of unmodified prolactin (U-PRL) and a molecular mimic of phosphorylated prolactin (PP-PRL) during rat pregnancy provides evidence that the U-PRL:PP-PRL ratio is crucial to the normal development of pup tissues

During rat pregnancy initial high concentrations of prolactin (PRL) decline by about day 9, concomitant with an increase in the ratio of unmodified to phosphorylated PRL. The physiological significance of both the decline in total PRL and the change in ratio of the two PRLs is unknown. To test the importance of each, either unmodified PRL (U-PRL) or a molecular mimic of phosphorylated PRL (PP-PRL) were continuously administered to rats throughout pregnancy. A dose of 6 microg/24 h resulted in circulating concentrations of 50 ng/ml of each administered PRL and had little effect on the pregnancy itself. After birth, pups were killed and various tissues examined. In the pup lungs, exposure to additional PP-PRL caused a reduction in epithelial integrity and an increase in apoptosis, whereas exposure to additional U-PRL had beneficial, anti-apoptotic effects. In the heart, PP-PRL caused an apparent developmental delay, whereas U-PRL promoted tissue compaction. In the blood, U-PRL increased the number of mature red blood cells at the expense of white blood cell production. Within the white blood cell population, myelopoiesis was favored at the expense of lymphopoiesis. PP-PRL, in contrast, had a less dramatic influence on the hematopoietic compartment by promoting red blood cell maturation and granulocyte production. In the thymus, exposure to PP-PRL caused accumulation of apoptotic thymocytes in enlarged glands, whereas exposure to U-PRL resulted in smaller thymi. In the spleen, exposure to U-PRL increased cellularity, with the majority of cells belonging to the erythroid series - a finding consistent with increased red blood cells in the circulation. Exposure to PP-PRL was without discernible effect. In all of these tissues, the contrasting effects of the two PRLs indicate that the absolute concentration of PRL is not crucial, but that the ratio of U-PRL to PP-PRL has a profound effect on tissue development. In brown fat, both PRL preparations decreased the number of lipid droplets. This result is therefore probably a consequence of the increase in total PRL. The results of this study attest to the importance of the U-PRL:PP-PRL ratio normally present during pregnancy and have provided clues as to the possible pathogenesis of a variety of neonatal problems.     

Central actions of adenosine on pituitary secretion of prolactin, luteinizing hormone and thyrotropin

Pituitary hormones were measured in plasma in unanesthetized male and female rats prepared with venous and ventricular cannulae following ventricular infusions of adenosine and two adenosine receptor agonists. Adenosine (10, 100, and 200 nmol) injected intraventricularly caused dose-related rises in plasma prolactin (PRL) in males; in females, only the 100- and 200-nmol doses increased PRL. Similar doses of adenosine had little effect on plasma levels of luteinizing hormone (LH) and thyrotropin (TSH) in the same animals. The adenosine receptor agonists L-N6-phenylisopropyladenosine (L-PIA) and 5'-N-ethyl-carboxamideadenosine (NECA) potently stimulated prolactin secretion at doses of 10 and 50 nmol when administered into the lateral ventricle, and at a dose of 2.5 nmol when administered into the third ventricle. The secretion of PRL was antagonized by the adenosine receptor antagonist theophylline (25 nmol), when theophylline was coadministered with NECA and L-PIA. TSH levels were reduced slightly but significantly following the 10- and 50-nmol infusions of L-PIA into the lateral ventricle. The less potent D-isomer of PIA (D-PIA) did not significantly stimulate PRL release. Coupled with studies indicating the presence of adenosine in the basal hypothalamus, our observations indicate a potential neuroendocrine role for this purine in prolactin secretion.     

Prolactin (PRL)-releasing peptide stimulates PRL secretion from human fetal pituitary cultures and growth hormone release from cultured pituitary adenomas

The hypothalamic peptide PRL-releasing peptide (PrRP) has recently been cloned and identified as a ligand of an orphan pituitary receptor that stimulates in vitro PRL secretion. PrRP also induces PRL release in rats in vivo, especially in normal cycling females. However, no information on the effects of PrRP in the human is available. To elucidate the role of PrRP in regulating human anterior pituitary hormones, we used human PrRP-31 in primary cultures of human pituitary tissues, including fetal (20--27 weeks gestation) and normal adult pituitaries, as well as PRL- and GH-secreting adenomas. PrRP increased PRL secretion from human fetal pituitary cultures in a dose-dependent manner by up to 35% (maximal effect achieved with 10 nM), whereas TRH was slightly more potent for PRL release. Coincubation with estradiol resulted in enhanced fetal PRL response to PrRP, and GH release was only increased in the presence of estradiol. Although PRL secretion from PRL-cell adenomas was not affected by PrRP, PrRP induced PRL release from cultures of a GH-cell adenoma that cosecreted PRL. PrRP enhanced GH release in several GH-secreting adenomas studied by 25--27%, including GH stimulation in a mixed PRL-GH-cell tumor. These results show for the first time direct in vitro effects of PrRP-31 on human pituitary cells. PrRP is less potent than TRH in releasing PRL from human fetal lactotrophs and is unable to release PRL from PRL-cell adenomas in culture, but stimulated GH from several somatotroph adenomas. Thus, PrRP may participate in regulating GH, in addition to PRL, in the human pituitary.     

Effects of gonadectomy and chlorpromazine treatment on prolactin, LH, and FSH secretion in young and old rats of both sexes

Old male rats, and female rats in the states of senile persistent estrus (PE) and senile repetitive pseudopregnancy (RPD) were compared with young controls in the change of serum prolactin (PRL), LH, and FSH concentrations after gonadectomy and chlorpromazine (CPZ) treatment. Old male rats (18–21-month old) and PE (12–15-month old and 18–19-month old) and RPD (18–23-month old) female rats showed higher serum PRL levels. In both young and old male rats, orchidectomy did not cause any significant changes in serum PRL levels. The effect of ovariectomy on serum PRL levels was smaller in old female rats. CPZ treatment increased serum PRL levels in young controls of both sexes, 12-month old males and 12–15-month old PE rats. The sexual difference in PRL secretion in response to CPZ was observed at these ages. In 18–21-month old males and 18–23-month old females, CPZ did not significantly increase serum PRL levels. These results suggest that the higher serum PRL levels in intact old male and female rats may be partly due to the lowered activity of the hypothalamic dopaminergic inhibition. The changes in serum LH levels in response to ovariectomy were smaller in old female rats than in young controls, whereas male rats did not show any age-related changes in response to orchidectomy on serum LH levels. There was no appreciable age-difference in the increase in serum FSH levels after gonadectomy in either sex.     

In vitro, nitric oxide (NO) stimulates LH secretion and partially prevents the inhibitory effect of dopamine on PRL release

In recent years nitric oxide (NO) has emerged as an important intra- and intercellular transmitter involved in the control of hypothalamic-pituitary axis. In order to discriminate the potential actions of NO at hypothalamic or pituitary level in the control of PRL and LH release, we have studied PRL and LH secretion by dispersed pituitary cells obtained from males, cycling and lactating females in the presence of 1) sodium nitroprusside (SNP), a NO donor; 2) cyclic guanosine monophosphate (cGMP), the second messenger for a wide range of NO actions; 3) Nw-nitro-L-arginine methyl ester (NAME), a competitive inhibitor of NO synthase (NOS) and 4) oxadialoquinoxalione (OQD) and LY 83,583, antagonists of guanylyl cyclases. We found that SNP (at doses of 100 and 500 micromol) stimulated LH and FSH release and partially blocked the inhibitory action of dopamine (50 and 100 nmol) on prolactin secretion. These effects were not mimicked by cGMP and remained in the presence of OQD and LY 83,583. NAME alone had no significant effect on hormone secretion. These results suggest that NO plays a role in the control of gonadotropins and prolactin secretion acting directly at the pituitary level and that these effects are mediated by mechanisms other than changes in cGMP levels.     

Comparative maturation of the regulation of prolactin and thyrotropin by serotonin and thyrotropin-releasing hormone in male and female rats

The aim of this study was to compare the effects of TRH, serotonin, and haloperidol on the secretion of PRL and TSH in male and female rats from birth to puberty. Serum PRL in males and females was low from birth to 20 days; it then increased gradually until puberty. TSH did not change significantly throughout the period studied. The PRL-releasing effect of serotonin became evident at 12 days, and at all times when the response occurred, it was greater in males than in females. This was also observed in adult rats in which 2.5 mg/kg ip serotonin caused an increase in serum PRL in males but not in diestrous females. Serotonin did not modify TSH at any age. On the other hand, TRH induced the release of TSH and PRL from the first day of life and no sex difference was observed. Haloperidol caused release of PRL from birth, and the effect increased significantly with age. The PRL increase induced with haloperidol was greater than that obtained with TRH used in supramaximal doses. A clear sexual difference became evident on day 20, the PRL-releasing effect of haloperidol being more pronounced in females than in males. Haloperidol did not modify TSH values. It is concluded that the mechanisms by which TRH and dopamine regulate PRL secretion mature earlier than those influenced by serotonin. Sexual differences in the manifestation of these mechanisms were observed, the male being more sensitive to serotonin and the female to haloperidol. TRH causes release of TSH from birth; the serotoninergic and dopaminergic pathways are not of paramount importance in the regulation of TSH in the prepubertal rat.     

Modulatory effect of testosterone on the serotoninergic control of prolactin secretion in prepubertal rats

The administration of 5-hydroxytryptophan (5-HTP) induced a prolactin release in male and female prepubertal rats at 20 days of age. This response was significantly higher in male than in female rats. Neonatal androgenization of the females significantly increased the release of prolactin induced by 5-HTP treatment compared to the values observed in males; thus, the neonatal exposure to androgens seems to be responsible for the sexual differences in the prolactin response to 5-HTP. In a second series of experiments the effect of this serotoninergic precursor on prolactin release in prepubertal (16, 26, and 30 days of age), peripubertal (45-day-old) and adult male rats was studied. Castration significantly decreased the prolactin release response to 5-HTP in prepubertal rats. The administration of testosterone to castrated rats markedly increased the prolactin release response to 5-HTP. Neither castration nor testosterone administration modified the prolactin response to 5-HTP in peripubertal and adult rats. These results appear to indicate that testosterone modulates the serotoninergic control of prolactin secretion during the prepubertal stage. The control of prolactin levels could be one of the mechanisms by which testosterone participates in the sexual maturation.     

Heparin increases prolactin and modifies the effects of fgf-2 upon prolactin accumulation in pituitary primary cultures

We have studied the effects of heparin on prolactin accumulation in the medium from primary pituitary cultures, and whether heparin interferes with the effects of fibroblast growth factor-2 (FGF-2) on PRL regulation in vitro. In the absence of exogenous FGF-2, heparin increased prolactin accumulation in the culture medium in a dose-dependent manner. FGF-2 also increased the prolactin levels of primary cells in a time-and dose-dependent manner. However, low doses of heparin reduced the effects of FGF-2, but higher doses of heparin increased the maximal FGF-2-induced prolactin secretion and ED50. In vivo estrogenization of rats resulted in the abolition of FGF-2 capability to promote prolactin release in vitro. However, heparin restored cell responsiveness to FGF-2. Our results suggest that heparin, when present in the medium, binds FGF-2, therefore reducing its ability to interact with FGF receptors in a dose-dependent manner up to a critical molar concentration, at which heparin itself starts to activate the FGF receptor, and strengthens the activation induced by its proper ligand, FGF-2. Prolactin responses to FGF-2 are blocked by estrogen pretreatment, and it is probable that this introduces lactotroph cells in the proliferative stage. In conclusion, heparin modulates PRL secretion and PRL responses to FGF-2 in vitro.     

Rat placental lactogens initiate and maintain lactation yet inhibit suckling-induced prolactin release

Mammalian reproduction is dependent on both a successful pregnancy and on the subsequent period of lactation. In the rat, ovulation occurs shortly after parturition making it possible for a dam to be simultaneously pregnant and lactating. The present studies investigate the effect of placental hormones on suckling-induced prolactin (PRL) release and the contribution of placental hormones to milk synthesis and secretion. A rat choriocarcinoma cell line, Rcho-1, which secretes placental lactogens (PLs) following transplantation in vivo, attenuated suckling-induced PRL release on both d 9 and d 14 of lactation by 43 and 58%, respectively. When PRL secretion was completely inhibited by bromocriptine, a dopamine agonist, Rcho-1-bearing dams still maintained a normal litters weight gain, demonstrating that placental lactogens can continue an established lactation. The Rcho-1 tumors also initiated milk synthesis and secretion in nulliparous rats continuously exposed to pups. Whereas none of the 11 control virgins began lactating and had an average pup weight loss of 2.07 g, the Rcho-1-bearing rats began lactating, as evidenced by a significant reduction in pup weight loss. Thirty percent of these rats became fully lactationally competent. Northern blot analysis showed that the Rcho-1 tumors expressed both PL-I and PL-II mRNA in all experimental groups. These tumors also secreted PL-I into the circulation, as shown by radioimmunoassay.     

Melatonin Effects on Prolactin Secretion in Pituitary-Grafted Male Rats

Melatonin influences prolactin (PRL) secretion through unknown mechanisms. This work was undertaken to study the effects of melatonin administration on PRL secretion in pituitary-grafted male rats. Melatonin administration 5 hours before dark resulted in a marked decrease of previously high basal plasma PRL levels in pituitary-grafted rats, whereas a marked increase was detected in sham-operated animals. Vehicle treatment did not modify basal PRL values in grafted or sham-operated animals. Luteinizing hormone-releasing hormone (LHRH) administration resulted in a marked decrease of plasma PRL levels in vehicle-treated, sham-operated or grafted rats, as well as in melatonin-treated sham-operated rats. An increase in PRL levels was shown in grafted rats treated with melatonin. Estradiol benzoate (EB) administration resulted in an increase in plasma PRL levels both in sham-operated and grafted vehicle-treated rats. No PRL response could be detected in sham-operated, melatonin-treated animals after EB administration. In pituitary-grafted animals given melatonin, PRL response to EB administration was slight and delayed. From these data, melatonin appears to modify PRL secretion through multiple complex mechanisms that may depend on the physiological status (hormonal and neurotransmitter) of the animals. A direct effect at the pituitary level altering lactotroph sensitivity seems to be one plausible explanation for the current findings, although an hypothalamic site of action cannot be excluded.     

Comparative effects of prolactin versus ACTH, estradiol, progesterone, testosterone, and dihydrotestosterone on cortisol release and proliferation of the adrenocortical carcinoma cell line H295R

In this study, using the H295R cell line as a model system, we investigated the role of prolactin (PRL) and steroid hormones in the growth regulation and cortisol release of adrenocortical cells. H295R cells were treated with increasing doses (10(-13)-10(-6) M) of PRL, adrenocorticotropic hormone (ACTH), 17beta-estradiol (E(2)), progesterone (P(4)), testosterone (T), and dihydrotestosterone (DHT). As expected, ACTH raised cortisol secretion and increased the proliferation rate of cultured cells. Incubation with T, DHT, E(2), and P(4) for 24 h did not significantly increase cortisol release. Conversely, PRL concentrations of 10(-8)-10(-6) M caused a significant increase in the release of cortisol. Long-term (5 days) stimulation of H295R cells with E(2), P(4), and PRL was a trigger to increased cell proliferation, while T and DHT did not alter H295R cell proliferation. Taken together, these results indicate that steroid hormones exert differential effects on adrenocortical function. Additionally, the present study demonstrates that PRL had biphasic actions in regulating adrenocortical function. PRL may form a novel regulatory system for steroid hormone secretion and cell proliferation in the adrenal cortex.     

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.     

Sexual differences in the serotonergic control of prolactin and luteinizing hormone secretion in the rat

The effects of serotonin on PRL and LH release were investigated in female and male rats under different experimental conditions. At a dose of 5 mg/kg ip, serotonin increased serum PRL titers in intact males and in females during diestrus and estrus; the levels attained in the male rats were much higher than in the females. At a lower dose (2.5 mg/kg) the PRL-releasing effect of serotonin was only evident in male rats. Thus, we chose this dose for the following experiments to investigate the apparent sexual difference. To evaluate the importance of the hormonal status characteristic of male and female in conditioning the serotonin effect, an experiment was performed in gonadectomized rats, untreated or treated with estradiol benzoate (EB), or testosterone propionate (TP). In the three hormonal conditions the sexual difference was maintained: serotonin released PRL in males and failed to do so in females. However, if males were castrated within 24 h of birth, and females androgenized by a single perinatal injection of TP, the sexual difference in adulthood were reversed; thus, androgenized females responded to serotonin and males castrated at birth failed to do so. These results suggest that a male differentiated brain is more sensitive to the PRL-releasing effect of serotonin, irrespective of the hormonal environment of the rat. On the other hand, serotonin increased serum LH in female rats in estrus and in adult ovariectomized rats treated with EB; but not in females in diestrus or in ovariectomized rats, treated with TP or untreated. Neither did it modify serum LH titers in male rats whether intact, orchidectomized, or orchidectomized plus steroids. However, if male rats were castrated a few hours after birth and then treated in adulthood with EB, serotonin effectively released LH. Thus, two components, estradiol and a feminine differentiated brain, may be necessary for the facilitatory action of serotonin on LH release. Since no sex differences were observed in the increase of serum serotonin after the injection of 2.5 mg/kg of the drug, it can be discounted that the differences described for the endocrine effect of the drug could be due to different levels of circulating indolamine achieved in male and female rats. Taken together, our results indicate that serotonergic control of anterior pituitary secretion is sexually differentiated and that it presents individual characteristics for PRL and LH release.     

Temperature-dependent prolactin secretion and reproductive biology of the newt Triturus carnifex Laur

The effects of temperature on pituitary prolactin (PRL) gene expression and peripheral levels were studied in both male and female newts obtained from wild conditions during reproductive (Experiment I) and nonreproductive (Experiment II) periods; moreover, changes in parameters related to reproductive function are also described. Male and female newts were taken from a pond in February (Experiment I, reproductive period) and maintained for 1 month in tanks at 4 and 18 degrees C. In male newts kept at 4 degrees C, increase of PRL mRNA in the pituitary and plasma PRL was found compared with that measured in those kept at 18 degrees C. The increase in PRL secretion was parallel to that of plasma androgens and related secondary sexual characteristics (SSC) in males and of plasma estradiol-17beta and vitellogenin in females. On the contrary, in nonreproductive newts (Experiment II), taken from the field in May, no significant changes in plasma PRL, androgens, and SSC were found in those maintained at low temperature (4 degrees C), whereas low temperature significantly increased PRL mRNA expression in the male pituitary and PRL mRNA plus plasma PRL levels in females. These findings suggest that low temperature regulates PRL secretion in this urodele species, showing a sex- and season-related control mechanism; moreover, low temperature failed to influence the reproductive biology of newts taken from the field in May, after naturally occurring reproduction during winter months.     

The paradoxical stimulatory effect of morphine on LH secretion is dose-dependent and naloxone-reversible

We previously observed that morphine stimulated luteinizing hormone (LH) secretion from ovariectomized rats when administered intravenously at a dose of 10 mg/kg body weight. The objectives of the present study were to determine: (1) if this paradoxical effect of morphine on LH secretion could be antagonized by naloxone; (2) whether beta-endorphin also stimulated LH secretion under similar conditions; (3) what influence, if any, the ovaries have on the expression of this opiate-induced LH secretion, and (4) whether this paradoxical effect of morphine extended to prolactin (PRL) secretion. An intravenous injection of morphine, 10 mg/kg body weight, to ovariectomized rats acutely increased both plasma LH and PRL concentrations. The LH and PRL responses were completely antagonized by the concurrent administration of the opiate antagonist naloxone (1 mg/kg body weight). In contrast, morphine suppressed LH concentrations and had no effect on PRL levels when injected at a dose of 1.0 mg/kg body weight. Intravenous injections of beta-endorphin, 1 mg/kg body weight, increased PRL concentrations to a level comparable to that observed following morphine, 10 mg/kg body weight, and produced a transient but insignificant inhibition of LH release. Intraventricular injections of much lower doses of beta-endorphin resulted in a dose-dependent suppression of LH release and a dose-dependent stimulation of PRL release in ovariectomized rats. Intravenous administrations of morphine (10 mg/kg), but not beta-endorphin (1 mg/kg), to normal female rats resulted in a 2-fold increase in LH concentrations similar to that observed in ovariectomized rats, whereas both treatments similarly increased PRL concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analyses for susceptibility of rat anterior pituitary cells to prolactin-releasing peptide

We validated the effect of prolactin-releasing peptide (PrRP) on prolactin (PRL) secretion from rat anterior pituitary cells in in vitro culture. We found that culture conditions considerably influenced the response of the anterior pituitary cells to PrRP. Longer culture term (4 d) was required to obtain better responses of the anterior pituitary cells to PrRP in comparison to thyrotropin-releasing hormone (TRH). Under the culture conditions employed here, PrRP was comparable to TRH in the potency promoting PRL secretion, and the action of PrRP was very specific for PRL secretion. The susceptibility of the anterior pituitary cells to PrRP varied in female rats depending on the process of reproduction: the cells prepared from lactating rats were the most sensitive to PrRP compared with those from random-cycle and pregnant rats. Because the expression levels of PrRP receptor mRNA in the pituitary varied during the reproductive process, we speculated that the susceptibility of the anterior pituitary cells would reflect cellular changes including the expression level of PrRP receptors. In addition, treatment with estrogen in vivo enhanced the susceptibility of the cultured anterior pituitary cells in male rats. Our results indicate that the susceptibility of the rat anterior pituitary cells to PrRP is regulated by physiological mechanisms.