Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells

Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).     

Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells.

The decidualized endometrium during the first trimester of pregnancy synthesizes and secretes a 32-kDa insulin-like growth factor-binding protein (termed hIGFBP-1) at high levels. IGFBP-1 is the major soluble protein product of this tissue and is principally localized to the differentiated endometrial stromal cell, the decidual cell. In the present study long term culture of stromal cells from the nonpregnant endometrium have been employed to elucidate the hormonal requirements for IGFBP-1 production. Immunoreactive IGFBP-1 was undetectable in control cultures. However, inclusion of medroxyprogesterone acetate (MPA) induced rates of 0.35 +/- 0.09 microgram/0.1 mg cell DNA.day (mean +/- SEM; n = 5) after 20-30 days. In these cultures cells exhibited morphological changes consistent with decidual cell differentiation. In all cultures removal of MPA after exposure for 10-16 days, with or without subsequent inclusion of relaxin (RLX), increased production of IGFBP-1 450- to 4600-fold to rates of 150-710 micrograms/0.1 mg cell DNA.day or 26-131 micrograms/10(6) cells.day on days 24-26. The rates tended to be higher with the inclusion of RLX and were sustained in contrast to cultures without RLX, where rates fell by day 30. Individual cultures responded differently to RLX when added from the initiation of culture, with either a response similar to MPA alone or a cyclical change in production, achieving maximal rates of 190-290 micrograms/0.1 mg cell DNA.day. Cultures in which RLX alone induced high IGFBP-1 high production were obtained from endometrium during the progesterone-dominated luteal phase. In cultures exhibiting high rates of immunoreactive IGFBP-1 production, the protein represented their major secretory protein product. This was confirmed by [35S]methionine incorporation and the presence of IGFBP-1 as the predominant protein in serum-free culture medium. The immunoreactive IGFBP-1 isolated from culture medium was found to be identical, by a number of criteria, with IGFBP-1 derived from decidual tissue. These results were consistent with a primary role of progestin exposure, whether in vivo or in vitro, in converting endometrial stromal cells to cells potentially able to exhibit the high rates of IGFBP-1 production typical of the decidualized endometrium of pregnancy.     

Differential release of prolactin variants in postpartum and early follicular phase women

Several molecular forms of immunoreactive human (h) PRL with differences in biological activities have been described. To investigate these differences in PRL secretion, we have characterized the molecular forms of circulating PRL in postpartum women during breastfeeding (n = 5) and sleep (n = 5) and in eumenorrheic women with galactorrhea (n = 5) after TRH stimulation (n = 4), metaclopramide infusion (n = 5), and morphine administration (n = 2). All provocative testing in eumenorrheic women were carried out during the early follicular phase of the menstrual cycle (days 1-5). PRL variants were identified and semiquantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting and autoradiography. Mean basal levels of PRL were significantly elevated in nursing women (P less than 0.01) compared to those in nonlactating postpartum women and women with galactorrhea. The relative proportions of PRL variants [glycosylated PRL with an apparent mol wt of 25K and a nonglycosylated species (hPRL) with an apparent mol wt of 23 K] were similar for all three groups. A third immunoreactive PRL variant with an apparent mol wt of 21K was identified in nonlactating postpartum women. With breastfeeding or sleep, PRL concentrations increased more than 4-fold and were accompanied by a shift toward the lower mol wt, nonglycosylated species. In response to TRH (200 micrograms, iv), PRL levels increased by 493 +/- 86% (mean +/- SE) over basal levels. This acute release of PRL was positively correlated (r = 0.06074; P less than 0.001) with a significant increase in secretion of the hPRL species (P less than 0.05). During metaclopramide infusion, a similar increment in hPRL was observed during acute PRL release. Administration of the opiate agonist morphine was associated with a small increment in PRL release and did not alter the proportions of PRL variants in the circulation. Because glycosylation is a posttranslational step in the processing of PRL, our studies suggest that the acute release of PRL in response to physiological and pharmacological secretagogues may reflect the output of more recently synthesized PRL from the pituitary. The physiological role of the 21K PRL species remains to be determined.     

Effect of relaxin on aromatase activity in human endometrial stromal cells

Previous studies have shown that the aromatase activity in human endometrial stromal cells was stimulated by progestin and enhanced by estrogen and forskolin (Fk), an agent that stimulates the accumulation of intracellular cAMP. Present study was undertaken to investigate whether any peptide hormone would affect endometrial aromatase activity. Stromal cells were isolated from normal proliferative and secretory endometria and cultured in nutrient medium. Porcine relaxin (RLX) was added to culture medium individually or in combination with medroxyprogesterone acetate (MPA) and estradiol (E2). Cells treated with RLX alone did not affect the aromatase activity. RLX, however, exerted a synergistic effect on aromatase activity in the presence of MPA or MPA plus E2. On the other hand, human CG, epidermal growth factor, human PRL, and insulin did not increase the aromatase activity in the presence or absence of MPA and E2 studied in a limited number of specimens. The progestin-dependent effect of RLX on aromatase activity was dose dependent indicating that the biological effect of RLX is mediated through a saturable mechanism. When RLX was added to MPA-pretreated cells, additional increase of aromatase activity was seen after 24 h incubation indicating that the action of RLX on stromal cells is not an acute effect. Antiprogestin, RU486, inhibited the stimulation of aromatase activity in both MPA and MPA plus RLX treated cells. RLX has either no effect or a moderate increase (up to 2-fold over the control) on intracellular cAMP content. On the other hand, Fk increased the intracellular cAMP level and enhanced the aromatase activity in the presence of progestin. Also RLX did not replace the effect of Fk since additional increase of aromatase activity was noted when stromal cells were incubated with MPA plus RLX plus Fk in comparison to MPA plus RLX or MPA plus Fk. These results suggest that the action of RLX on stromal cells may be mediated through an intracellular messenger independent of cAMP. Present studies provide evidence that RLX exerts a synergistic effect on aromatase activity in the presence of progestin in human endometrial stromal cells. It is evident that human endometrium is a target organ of RLX.     

Pharmacokinetic studies of highly purified human prolactin in normal human subjects

Previous estimates of PRL pharmacokinetics have been made using radioiodinated human PRL (hPRL) infusions or by measuring serum PRL disappearance following prolactinoma resection. The recent purification of hPRL in significant quantities made it possible to measure the clearance and volume of distribution directly. Studies were also carried out to determine absorption and clearance after im injection. In five normal men whose endogenous PRL secretion was suppressed by dopamine, a loading dose of hPRL (70-90 micrograms) followed by a constant infusion (1.39-2.9 ng/min) produced steady state serum PRL levels of 15.2-25.4 ng/mL by 30-60 min. The calculated mean volume of distribution was 7.3 +/- 2.9 (+/- SD) L. The calculated MCR was 71 +/- 19 mL/min X m2, and the calculated production rate was 802 +/- 377 micrograms/24 h X m2. The plasma disappearance half-life following discontinuation of the infusion was 37 +/- 10 min. The PRL infusate consisted primarily (75.6%) of a 22.5 K dalton species, probably PRL monomer, a component eluting at 45K daltons (16.1% of the total radioimmunoreactivity), probably dimer, and a small amount of a larger mol wt species. Serum obtained during dopamine infusion but before hPRL infusion contained 68.1% of the 22.5K, 7.2% of the 45K, and 24.7% of the larger mol wt moieties. During hPRL infusion in two men there was a relative decrease in the proportion of PRL monomer to 55% and 69% and a relative increase in the PRL dimer to 33% and 18%, respectively. hPRL was injected im in doses of 1, 2, 4, and 8 micrograms/kg without prior dopamine infusion. No significant changes in serum PRL levels occurred after the 1 and 2 micrograms/kg doses (n = 5). After the 4 micrograms/kg dose (n = 8), mean serum PRL levels rose from 10.0 +/- 1.8 (+/- SEM) ng/mL to peak levels of 13.1 +/- 1.8 ng/mL (P less than 0.01). After the 8 micrograms/kg dose (n = 7), PRL levels rose from 9.3 +/- 1.6 to 16.5 +/- 1.8 ng/mL (P less than 0.01). The PRL rise began between 60 and 80 min after injection; peak levels occurred at 160-180 min. In two men given 8 micrograms/kg who were sampled for an additional 3 h, PRL levels peaked at 200-220 min and began to fall by 220-240 min, but had not returned to baseline by 6 h. There were no side-effects of PRL administration, although the 8 micrograms/kg dose caused transient local discomfort.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prolactin variants in ram adenohypophyses vary with season.

Secretion of PRL in sheep is affected by photoperiod being highest during the spring and summer, lowest in fall and winter. The objectives of this study were to determine if 1) the production of variant forms of PRL, and 2) immuno- and bioactivities of PRL (iPRL and bPRL) differ during times of the year selected to represent periods of low, transitional and high PRL secretion. Twelve mature rams were maintained on pasture and killed in October, December, and April (n = 4/month). Individual pituitary glands were dispersed, cells obtained, and fixed for immunocytochemical flow cytometry, extracted with 0.01 N NaHCO3 or cultured in serum-free, defined media. The Mr of PRL extracted from cells immediately following dispersion ranged from 14-140K, with significantly more bands greater than 40K being detected from rams sacrificed in December than from those killed in October and April (P less than 0.01). No bands of PRL greater than 25K were observed when samples were reduced with beta-mercaptoethanol prior to electrophoresis, indicating that the high Mr forms were disulfide-linked aggregates. Culture media from October and April contained variants of PRL that ranged from 22-40K but those greater than 25K were generally not observed from cells harvested during December. Extracts of cells after 24 h in culture contained fewer high Mr species during December than had been present in initial extracts from that month. In contrast, during April more high Mr intracellular forms were present after culture than had been detected prior to culture during that month. The percentage of lactotrophs averaged 50.0 +/- 2.5, 47.4 +/- 5.7, and 59.4 +/- 5.5 for October, December, and April, respectively. Initial lactotroph content (pg/lactotroph) of iPRL was higher (P = 0.06) in April (46.0 +/- 17.0) when compared to October and December (8.0 +/- 2.0 and 20.0 +/- 10.0, respectively). In contrast, the bPRL content of initial extracts was higher (P = 0.05) in December (267.0 +/- 68.0) than in October (101.0 +/- 35.0), but not than in April (190.0 +/- 70.0). Although iPRL and bPRL concentrations in culture media were similar for the 3 months, the intracellular iPRL (P less than 0.001) and bPRL (P less than 0.0001) content after culture was greatest during April. In summary, in addition to the well-documented seasonal changes in blood concentrations of PRL, different molecular forms of PRL were found within the pituitary at different times of the year and seasonal variations in iPRL and bPRL did not occur in parallel.     

Immunoreactive insulin-like growth factor I (IGF-I) and IGF-I-binding protein content in human thyroid tissue

We measured immunoreactive insulin-like growth factor I (IGF-I) in extracts of normal and nodular thyroid tissue obtained at surgery from patients with nontoxic goiter. The nodular tissues contained a higher concentration [mean, 279.0 +/- 69.7 (+/- SE) mU/g] than paired normal tissues (115.5 +/- 17.9 mU/g; P = 0.024; n = 12); a difference was evident in all but one patient. Sephadex G-50 gel filtration of tissue extracts revealed two immunoreactive peaks, the first in the void volume of the column, and the second in the elution volume of authentic IGF-I. The first peak was identified as IGF-I-binding protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cross-linking with iodinated IGF-I. Isolated thyroid cell membranes contained high affinity IGF-I-binding sites of similar affinity and numbers in both normal and nodular thyroid tissue. The IGF-I content of six thyroid cancer extracts was higher than that of normal thyroid tissue, but the IGF-I content of thyroid tissue from six patients with Graves' disease and five patients with Hashimoto's thyroiditis was similar to that in normal thyroid tissue. These data suggest that the stimulatory effect of TSH on thyroid cell proliferation could be mediated through IGF-I action and suggest that an increase in IGF-I production could sustain the goitrogenic process.     

Electrophoretic analyses of secreted human endometrial proteins: identification and characterization of luteal phase prolactin

Endometrial protein synthesis and secretion throughout the menstrual cycle was studied by slab gel electrophoretic analysis of [35S]methionine incorporation into protein during short term culture of human endometrial tissue. A minimum of five protein bands that fluctuate during the menstrual cycle were identified on one-dimensional gels. Those with mol wt of 28K, 35K, 51K, and 59K decreased in the luteal phase, whereas a broad 25K band was induced in the luteal phase. This broad band was identified as two species of glycosylated PRL by antihuman PRL immunostaining and [3H]glucosamine incorporation.     

Insulin-like growth factor-binding proteins (IGF-BPs) produced by human skin fibroblasts: immunological relationship to other human IGF-BPs

We have characterized the insulin-like growth factor-binding protein (IGF-BP) produced by neonatal human skin fibroblasts in monolayer culture using antibodies specific for the acid-stable subunit of the 150K GH-dependent IGF-BP complex, BP-53, and the amniotic fluid IGF-BP, BP-28. Fibroblasts produced 65.3 +/- 10.4 ng/ml.72 h (SE; n = 6) immunoreactive BP-53 in serum-free medium; this was stimulated by increasing fetal bovine serum in the medium up to 385.3 +/- 49.0 ng/ml.72 h at 10% serum. Epidermal growth factor (EGF) also caused dose-dependent stimulation of BP-53 production, with a maximal effect (3-fold increase) at 30 ng/ml EGF. No immunoreactive BP-28 production was detectable in the presence or absence of serum or EGF. Neutral gel chromatography of serum-free medium revealed a peak of immunoreactive BP-53 at about 50K, with a smaller species at 20-30 K. Serum- and EGF-stimulated cells produced higher levels of about 50K BP-53, and an additional peak of immunoreactivity at 150K was present in serum-stimulated, but not EGF-stimulated, samples. Comparison of IGF-I and IGF-II binding by fibroblast BP-53 revealed slightly higher IGF-II than IGF-I binding, and association constants of 3-4 x 10(10) liter/mol for both IGFs, similar to BP-53 from human plasma. Affinity labeling of acid-stripped medium followed by nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specifically cross-linked IGF-binding species of 60K (identical to labeled plasma BP-53), 42K, and 37K. Only the 60K and 42K complexes were precipitable by antiserum to plasma BP-53, and none was precipitable by anti-BP-28 serum, suggesting that the 37K band might represent a third class of IGF-BP. We conclude that neonatal skin fibroblasts produce no BP-28, but do produce two IGF-BPs immunologically homologous to human plasma BP-53, one of which shows size and IGF-binding characteristics identical to the plasma protein.     

Adrenocortical function in hyperprolactinemic women.

To study the effects of prolactin (PRL) on adrenocortical function in humans, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), androstenedione (delta) and testosterone (T) were measured in serum obtained from 35 hyperprolactinemic women with galactorrhea and amenorrhea before and after treatment with bromocriptine-induced fall in mean PRL levels from 82 +/- 8 (SE) to 14 +/- 2 ng/ml (n = 39, P less than 0.0005), DHAS fell from 322 +/- 21 to 237 +/- 21 microgram/dl (n = 39); P less than 0.0005), DHA fell from 492 +/- 47 to 378 +/- 30 ng/dl (n = 39; P less than 0.01) while T (n = 16) and delta (n = 13) levels were unchanges (44 +/- 4 vs. 49 +/- 4 ng/dl and 280 +/- 55 vs. 236 +/- 40 ng/dl, respectively). In addition, 4 women were infused iv with 25 microgram synthetic ACTH over 4 h and serial blood samples drawn while hyperprolactinemic, and again 2-4 months later following normalization of PRL levels by bromocriptine. Although pre-infusion levels of DHAS were lower when PRL levels were normalized, no significant differences in responses of circulating DHAS, DHA, T, cortisol and 17-hydroxyprogesterone concentrations were detected between the two infusions. Since DHAS is virtually an exclusive product of the adrenal cortex, and since high PRL levels appear to inhibit ovarian steroid production, the findings suggest that hyperprolactinemia selectively stimulates adrenocortical androgen production.     

Effects of disodium EDTA and calcium infusion on prolactin and thyrotropin responses to thyrotropin-releasing hormone in healthy man

To determine the impact of induced hypo- and hypercalcemia on TRH (400 micrograms)-stimulated TSH and PRL release, healthy subjects (n = 11) were infused with 5% glucose in water (n = 11), disodium EDTA (n = 11), or calcium gluconate (n = 7). TRH was given as an iv bolus 60 min (5% glucose and EDTA) and 120 min (calcium) after initiation of the respective infusion. Basal plasma concentrations of TSH remained unchanged during induced hypo- and hypercalcemia, whereas those of PRL fell during the latter (P less than 0.05). The mean sum of increments (0-90 min) in PRL and TSH was considerably greater during hypocalcemia than during hypercalcemia (PRL, P less than 0.002; TSH, P less than 0.005). The increments in the plasma hormone concentration above basal after iv TRH were increased compared to those in normocalcemia (PRL, 98.4 +/- 37.9 ng/ml; TSH, 38.9 +/- 11.8 microU/ml) during hypocalcemia [PRL, 128 +/- 47.8 ng/ml (P less than 0.002); TSH, 46.7 +/- 12.8 microU/ml; (P less than 0.005)], but were impaired during hypercalcemia [PRL, 70.1 +/- 27 ng/ml (P less than 0.002); TSH, 28.9 +/- 8.5 microU/ml (P less than 0.025)]. The mean sum of increments in PRL was related to concentrations of both serum calcium (r = -0.59; P less than 0.01) and PTH (r = 0.51; P less than 0.05). A relation was also seen between the incremental responses of TSH and serum calcium (r = -0.52; P less than 0.05), PTH (r = 0.55; P less than 0.01), and phosphorus (r = -0.55; P less than 0.01). We conclude that in healthy man, TRH-mediated release of both PRL and TSH are inversely related to serum calcium concentrations in such a manner that hormone secretion is enhanced by acute hypocalcemia, but blunted by hypercalcemia.     

Demonstration of enhanced potency of a chimeric somatostatin-dopamine molecule,BIM-23A387, in suppressing growth hormone and prolactin secretion from human pituitary somatotroph adenoma cells

In acromegaly, the combination of somatostatin (SS) and dopamine (DA) agonists has been shown to enhance suppression of GH secretion. In the present study, a new chimeric molecule, BIM-23A387, which selectively binds to the SS subtype 2 receptor (sst(2); K(i) = 0.10 nM) and to the DA D2 receptor (D2DR; K(i) = 22.1 nM) was tested in cultures prepared from 11 human GH-secreting tumors for its ability to suppress GH and prolactin (PRL) secretion. The chimeric compound was compared with individual sst(2) and D2DR agonists of comparable activity at the individual receptors. All tumors expressed both sst(2) and D2DR mRNAs (0.8 +/- 0.2 and 4.7 +/- 0.7 copy/copy beta-glucuronidase mRNA, respectively). In cell cultures from seven octreotide-sensitive tumors, the maximal inhibition of GH release induced by the individual sst(2) and D2DR analogs and by BIM-23A387 was similar. However, the mean EC(50) for GH suppression by BIM-23A387 (0.2 pM) was 50 times lower than that of the individual sst(2) and D2DR analogs, either used individually or combined. Similar data were obtained in four tumors that were only partially responsive to octreotide. The inhibition of GH release by BIM-23A387 was only partially reversed by the D2R2 antagonist, sulpiride, or by the sst(2) antagonist, BIM-23454. Only when both antagonists were combined was the GH suppressive effect of BIM-23A387 totally reversed. Finally, BIM-23A387 produced a mean 73 +/- 6% inhibition of PRL in six mixed GH plus PRL tumors. These data demonstrate an enhanced potency of the chimeric molecule, BIM-23A387, in suppressing GH and PRL secretion from acromegalic tumors, which cannot be explained merely on the basis of binding affinity for SS and/or DA receptors.     

Ovarian control of pituitary hormone secretion in early human pregnancy

To determine the influence of ovarian relaxin on the secretion of pituitary GH and PRL in vivo, we evaluated circulating serum hormone levels in 17 pregnant patients with functional corpora lutea (group I) and compared them to levels in 10 patients with premature ovarian failure (POF; group II) who became pregnant with egg donation and did not have corpora lutea. Group II patients had exogenous hormonal support. Serum relaxin (RLX), GH, PRL, estradiol (E2), and progesterone levels were measured weekly by RIA from weeks 4-8 of pregnancy. Analysis of variance and covariance were used to determine hormonal relationships. Serum RLX was present in the natural pregnancy group, with a mean of 1.94 micrograms/L over the study period. Serum RLX was undetectable in the POF patients (less than 0.16 micrograms/L). No significant difference in PRL or progesterone levels between the two groups was noted. E2 levels showed an upward trend in both groups with time and were significantly higher in patients of the POF group than in group I women (P = 0.001). GH levels were significantly higher in the natural cycle patients (P = 0.02) despite lower E2 levels. These data provide additional support for the concept that RLX production in early pregnancy originates from the corpus luteum. They suggest that a luteal product, probably RLX, stimulates GH secretion in early pregnancy. This is a previously undescribed role for RLX in pituitary physiology during human pregnancy.     

Stimulation of prolactin cell differentiation in vitro by a milk-borne peptide

We have previously reported that the normal expression of PRL-secreting cells in neonatal rats requires a maternal signal specific to the first few days of lactation. These results raised the possibility that a milk-borne factor(s) ingested by the neonate and absorbed into the circulation might induce the ontogenic appearance of PRL cells. The purpose of the present study was to determine whether milk from this period could directly stimulate the differentiation of PRL secretors in culture. Monodispersed anterior pituitary cells from 1-day-old pups were cultured for 6 days with aqueous extracts of milk from early (days 2, 3, and 4) and late (days 15 and 16) lactation and then subjected to reverse hemolytic plaque assays for PRL and GH release. We found that the addition of milk extracts (10 mg/ml) from either early or late lactation stimulated the differentiation of PRL secretors (to 6.1 +/- 1.0% and 2.4 +/- 0.7% of all pituitary cells, respectively; mean +/- SE; n = 3) above that in control cultures without milk (0.2 +/- 0.2%). Thus, early milk was more than twice as effective as late milk in this regard (P less than 0.05). This effect appeared to be specific to PRL cell differentiation, since the relative abundance of GH secretors was not different between cells treated with either early or late milk (29.3 +/- 4.8% and 33.7 +/- 3.9%, respectively). On the other hand, late milk was more than twice as effective as early milk at increasing the capacity of GH secretors to release hormone (P less than 0.05). Preliminary characterization by gel filtration chromatography and proteolytic hydrolysis indicates that the bioactivity that differentiates PRL secretors is a small peptide(s) of 2000-6000 daltons. Taken together, our results demonstrate that a milk-borne peptide(s) is capable of specifically stimulating the differentiation of PRL-secreting cells in vitro, and that this bioactivity is more prevalent in milk from early lactation.     

Frequency of macroprolactinemia due to autoantibodies against prolactin in pregnant women

The frequency of macroprolactinemia related to the presence of anti-PRL autoantibodies in the serum of 209 healthy women at different stages of pregnancy was studied. Measurements were taken of serum PRL concentrations before and after chromatographic separation (gel filtration and affinity with proteins A and G) and extraction of free PRL with polyethylene glycol (PEG). Sera from 8 of the 209 women (3.8%) were found to have a significantly high proportion of precipitated PRL by PEG (macroprolactinemia); in these patients, gel filtration showed that a substantial amount of big big PRL (molecular mass >100 kDa) was present (19.0--78.2% vs. 3.8-4.9%, P = 0.009 in normal pregnant women with a normal proportion of precipitated PRL by PEG). The presence of macroprolactinemia was attributable to anti-PRL autoantibodies in 5 of the 8 women. Comparison of serum levels of direct and free PRL between women with macroprolactinemia related to anti-PRL autoantibodies and women without macroprolactinemia showed significant differences (direct PRL: 270.2 +/- 86.9 vs. 203.4 +/- 69.0 microg/L, P = 0.04; and free PRL: 107.0 +/- 75.9 vs. 173.3 +/- 67.6 microg/L, P = 0.002). On the other hand, there was no difference between women with macroprolactinemia not related to anti-PRL autoantibodies and women with macroprolactinemia caused by anti-PRL autoantibodies, nor was there a difference between women with macroprolactinemia not related to anti-PRL autoantibodies and women without macroprolactinemia. There was a positive correlation between titers of the anti-PRL autoantibody and serum PRL levels (r = 0.82, P = 0.09). The presence of the anti-PRL autoantibody had no relation to the patient's age, stage of gestation, or number of previous pregnancies. We concluded that the frequency of macroprolactinemia was 3.8% among healthy, pregnant women, which was caused by a anti-PRL autoantibodies in 62.5% of the cases. The autoantibodies were found in the bloodstream, forming a PRL-IgG complex, in accordance with the following observations: 1) immunoreactive PRL on gel filtration was eluted in the fractions corresponding to the molecular mass of IgG (150 kDa); 2) a significantly high proportion of immunoreactive PRL was retained on an affinity gel for IgG (proteins A and G); and 3) a significantly high proportion of serum PRL bound to IgG was precipitated by protein A. There was a positive correlation between titers of anti-PRL autoantibodies and serum PRL levels. Serum levels of total PRL were higher, and serum levels of free PRL were lower, in pregnant women with anti-PRL autoantibodies than in pregnant women without macroprolactinemia.     

Induction (or stimulation) of prolactin and growth hormone production in a rat pituitary tumor cell line by bromodeoxyuridine

Under basal conditions, a rat pituitary tumor cell line (C8 11RAP) does not secrete any detectable PrL, FSH, and LH, and secretes only minute amounts of GH (27.1 +/- 0.5 ng/10(6) cells.24 h), as evaluated by RIA. Bromodeoxyuridine (BrdUrd) added to the culture medium induced the accumulation of PRL into cells and medium, increased that of GH, but did not induce that of LH or FSH. The amount of radioimmunoassayable PRL and GH accumulated in the medium increased after a lag period of 15 days and was drug concentration dependent. Maximal accumulation was 232.9 +/- 36.8 and 493.6 +/- 41.5 ng/10(6) cells.24 h for PRL and GH, respectively, at 50 micrograms/ml BrdUrd. In the presence of BrdUrd (greater than or equal to 20 micrograms/ml), the cells grew more slowly and were more strongly attached to the flasks. All of the effects induced by BrdUrd were reversible. PRL and GH were characterized by three methods; 1) radiocompetition with increasing dilution of samples; 2) Sephadex chromatography, followed by RIAs; and 3) sodium dodecyl sulfate-polyacrylamide gel electrophoresis done on the immunoprecipitate of the proteins secreted by cells incubated with [3H]leucine. Chronic treatment with TRH (3 X 10(-6) M) of cells grown without BrdUrd was unable to increase the production of GH or to induce that of PRL. On the other hand, after the same treatment of cells cultured in the presence of BrdUrd, the amounts of PRL accumulated in the culture medium or cells were increased 2- to 7-fold over unstimulated levels; under the same conditions, GH accumulation in the medium was also increased, but this augmentation was less than that of PRL. These results indicate that BrdUrd simultaneously induces or stimulates the production of PRL and GH in C8 11RAP cells, and that TRH increases the production of both hormones only in BrdUrd-treated cells.     

Pooled prolactin measurements in the evaluation of short children

PRL secretion was determined in 63 children undergoing evaluation of GH status. Children were assigned to 1 of 3 groups based on GH studies: group 1, those with abnormal GH responses to provocative testing (n = 23); group 2, children with normal GH responses to provocative testing and mean 24-h GH concentrations below 2.5 micrograms/L (n = 14); or group 3, those with normal stimulated GH secretion and mean 24-h GH concentrations of 2.5 micrograms/L or more (n = 26). Serum PRL concentrations were measured in daytime (0800-1600 h), nighttime (2200-0600 h), and 24-h pools of serum specimens obtained every 20 min over a 24-h period. Mean (+/- SD) daytime (17.5 +/- 14.3 micrograms/L) and 24-h (19.2 +/- 13.0 micrograms/L) pool PRL concentrations were significantly higher in group 1 than in group 3 (daytime, 6.7 +/- 2.3; 24 h, 10.2 +/- 2.5 micrograms/L; P less than 0.01). Mean nighttime pool PRL concentrations did not differ among groups. Mean nighttime pool PRL values were significantly higher (P less than 0.01) than daytime pool values in group 3 (nighttime pool, 13.6 +/- 3.6 micrograms/L; night to day ratio, 2.2 +/- 1.0) and group 2 (16.8 +/- 9.0 micrograms/L; night to day ratio, 2.5 +/- 1.5), but not within group 1 (21.4 +/- 13.5 micrograms/L; night to day ratio, 1.4 +/- 0.5). The mean peak and increment in PRL concentrations after an iv bolus of insulin-TSH-LHRH were not different among groups. The mean decrement in serum PRL level after L-dopa ingestion was greater in group 1 than in group 3 (P less than 0.05). Two children in group 2 and 10 in group 1 had significantly elevated daytime pool PRL concentrations (greater than 11.3 micrograms/L; 2 SD above the mean value for group 3). Two additional children in group 2 and 2 in group 1 had elevated 24-h (greater than 15.2 micrograms/L) pool PRL concentrations. One child in group 2 and 3 in group 1 had low 24-h PRL concentrations (less than 5.2 micrograms/L; less than 2 SD below the mean of group 3). Fourteen of 20 children with elevated daytime and/or 24-h pool PRL levels or low 24-h pool PRL values had structural or radiation-associated insults to the hypothalamic-pituitary axis evident in the history or with brain-imaging techniques; 1 had microphallus with panhypopituitarism and 5 children had no structural abnormalities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histaminergic regulation of prolactin secretion: dose-response relationship and possible involvement of the dopaminergic system

Histamine (HA) may participate in the neuroendocrine regulation of pituitary hormone secretion. HA diphosphate infused iv for 120 min in a dose of 9, 18, 30, or 50 micrograms/kg BW.h to six normal men stimulated PRL secretion in a dose-dependent manner [absolute change in PRL (delta PRL) area = 52 X (HA dose) - 618; r = 0.9926; P less than 0.001]. The stimulatory effect of HA was modest and occurred during the second hour of infusion. This increase might be due to the opposing effects of HA on PRL secretion, specifically stimulation via H1 receptors and inhibition via H2 receptors. The PRL-releasing effect of 11 micrograms HA dihydrochloride was not significantly different from that of an equimolar dose of HA diphosphate (18 micrograms). Selective activation of H2 receptors by combined infusion of HA and the H1 receptor antagonist mepyramine inhibited PRL secretion compared to the effect of NaCl [delta PRL, -55 +/- 23 (+/- SEM) vs. -20 +/- 17 microIU/ml; P less than 0.01; n = 6). Mepyramine infused alone had no effect (delta PRL, -43 +/- 22 vs. -33 +/- 30 microIU/ml; n = 6). Selective activation of H1 receptors by combined infusion of HA and the H2 receptor antagonist cimetidine stimulated PRL secretion (delta PRL, 193 +/- 40 vs. -20 +/- 17 microIU/ml; P less than 0.0005; n = 6). When infused alone, cimetidine had only a modest and late stimulatory effect (delta PRL, 35 +/- 22 vs. -27 +/- 15; P less than 0.025; n = 6). Dopamine receptor blockade with metoclopramide (MET; 10 mg, three times daily, orally) did not prevent the PRL-inhibiting action of H2 receptor activation (delta PRL, -374 +/- 70 vs. -184 +/- 107 microIU/ml; P less than 0.01; n = 6), whereas the PRL-stimulating effect of H1 receptor activation was abolished by the drug (delta PRL, -249 +/- 64 vs. -174 +/- 54 microIU/ml; n = 6). The latter effect of MET was not due to exhaustion of the lactotrophs, since 200 micrograms TRH stimulated PRL secretion during MET treatment. These findings suggest that the H1 receptor-mediated PRL-stimulating effect of HA occurs through an inhibition of the dopaminergic system, whereas the H2 receptor-mediated PRL-inhibiting effect of HA does not involve dopaminergic neurons.     

The circadian variation of prolactin in fetal sheep is affected by the seasons

Plasma PRL concentration shows a circadian variation in fetal and adult sheep. In the adult sheep the presence of this variation depends on the season. In this paper we investigated whether season affects the presence of the circadian variation of PRL in the fetal sheep. To that effect we measured plasma PRL concentration every 2 h for 24 h during summer, fall, and winter in three groups of fetal sheep whose gestational ages ranged from 125-133 days. Mean (+/- SEM) fetal plasma PRL concentrations were 352.8 +/- 65.0 ng/ml during summer (n = 6), 98.7 +/- 12.9 during fall (n = 8), and 10.5 +/- 2.6 during winter (n = 4). A 24-h variation of plasma PRL was detected during summer [PRL (ng/ml) = 352.8 + 85.2 cos 15 (t - 18.5); P = 0.007] and fall [PRL (ng/ml) = 98.7 + 26.6 cos 15 (t - 23.6); P = 0.041] but not during winter. The mesor and amplitude of the variation are higher in summer than in fall, and the acrophases differ by 5 h, taking place at dusk in summer and close to midnight in fall. These findings show that in fetal sheep, PRL responds to seasons in utero. The signal triggering this response is most likely photoperiod.     

Evidence for the presence of gastrin-releasing peptide immunoreactivity in rat anterior pituitary corticotrophs and lactotrophs, AtT20 cells, and GH3 cells: failure to demonstrate participation in local control of hormone release.

Using antisera raised against the N-terminal (1-16) or the C-terminal part of the bombesin (BBN)-like peptide gastrin-releasing peptide (GRP) and against the C-terminus of pro-GRP, GRP- and pro-GRP-like immunoreactivity (IR) was detected by immunostaining in freshly dispersed rat anterior pituitary (AP) cells and in reaggregate AP cells cultured in serum-free medium. Depending on the antiserum used, 6.4 +/- 0.4% to 12.4 +/- 2.7% of freshly dispersed cells were immunoreactive. Solid phase preabsorption of the antisera with the respective antigens abolished the staining. GRP-IR was detectable by double immunostaining in a subpopulation of PRL cells and ACTH-containing cells. In contrast, only very few somatotrophs, gonadotrophs, and thyrotrophs were immunoreactive, and they were much smaller than the typical mature forms of these cell types. GRP- and pro-GRP-IR were also detected in the ACTH-secreting AtT20 cell line and in the PRL- and GH-secreting GH3 cell line. GRP- and pro-GRP-IR were present in AP cell aggregates maintained in culture for 4 weeks. The finding of both GRP- and pro-GRP-IR in a subpopulation of lactotrophs and corticotrophs and their persistence in culture under serum-free conditions strongly suggest that GRP-like peptides are produced in rat AP. Although the potent GRP receptor antagonist L 686,095-001C002 effectively blocks stimulation of GH and PRL release by exogenous BBN-like peptides, it failed to affect basal GH and PRL release from perifused reaggregate AP cell cultures as well as GH and/or PRL release stimulated by epinephrine, vasoactive intestinal peptide, TRH, GH-releasing factor, and angiotensin-II or PRL release inhibited by dopamine. Thus, the latter data do not support the hypothesis often suggested that peptides endogenously present in AP cells are involved in the paracrine regulation of AP hormone secretion.