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PubMed1988

[Stimulation of prolactin secretion with thyroliberin (TRH). In vivo and in vitro studies of metastatic breast cancer].

In 120 Patients with metastatic breast cancer prolactin stimulation tests with 200 mcg TRH i.v. were done. The mechanism of the TRH-induced prolactin release was characterized in vitro. Basal prolactin levels were stimulated in all patients with an average increase of 400% (means basal level 441 mU/l, after stimulation 1753 mU/l). Hyperprolactinemic patients showed the highest absolute prolactin levels following stimulation. Patients with basal prolactin levels under 500 mU/l showed the highest relative increase of prolactin levels (delta = 550%). The prolactin stimulation tests with TRH had no diagnostic advantage compared to the basal prolactin levels in predicting the activity of the disease (basal values: sensitivity 12%, specificity 100%, stimulated values: sensitivity 10%, specificity 99%). During inhibition of plasmaprolactin with bromocriptine, an 330% increase of plasmaprolactin following TRH was observed in vivo. The same was shown in vitro: Pituitary cells cultured in vitro with 10(-6) M dopamine showed an increase of prolactin secretion after coincubation with 10(-7) M TRH. There was a linear increase of the prolactin concentration during the incubation period in dopamine-free cell cultures. The increase of prolactin concentration in vitro was constant during the whole incubation period (5 h), not influenced by the basal prolactin concentration and was seen as early as 30 min in incubation. The in vivo and in vitro results are in agreement with the hypothesis of a rapid, dopamine-independent effect of TRH on the secretion of stored prolactin in the pituitary.

Breast Neoplasms↗
PubMed1977

Prolactin release following electrical stimulation of the brain in ovarectomized and ovariectomized estrogen-treated rhesus monkeys.

Selected areas in the medial basal (MBH) and rostral (RH) hypothalamus and in the amygdala (AMYG) of long-term ovariectomized rhesus monkeys were electrically stimulated for 30 min through permanently implanted bilateral stainless steel electrodes. Stimulation of an area in the MBH extending from the dorsal part of the ventromedial nucleus through the arcurate nucleus to the upper median eminence resulted in a 200 to 400% increase within 5 min in 8 monkeys. In one monkey the elevated serum prolactin levels persisted after termination of stimulation and in 2 monkeys prolactin remained unchanged during the 30-min stimulation but increased after stimulation was discontinued. Stimulation of the paraventricular-dorsomedial nuclear area in one monkey had no effect on prolactin release. Prolactin responses to stimulation in the RH varied. In 2 monkeys the electrode tips extended into the optic chiasm but part of the uninsulated tips remained in contact with the RH; only one of these monkeys released prolactin in response to stimulation. In 4 monkeys the electrode tips were located in the suprachiasmatic-anterior hypothalamus area. Serum prolactin increased by 200 to 300% in response to stimulation in 2 of these monkeys but increased only slightly in the remaining 2 monkeys. Prolactin responses to stimulation of the AMYG varied with the location of the electrodes. Stimulation in the corticomedial region produced no change in serum prolactin but stimulation in the basal or basolateral area produced marked elevations. An increase in circulating levels of estradiol-17beta (E2) to 100 pg/ml by SC implantation of E2 capsules 72 h before stimulation had no significant effect on basal prolactin levels, but markedly enhanced the prolactin release induced by stimulation in both the MBH and RH. Sham-stimulation did not affect serum prolactin. We conclude that prolactin release in rhesus monkeys can be triggered by electrical stimulation of selected hypothalamic and amygdaloid areas and that stimulation-induced prolactin release in the RH and MBH can be enhanced by E2 pretreatment.

Amygdala↗
PubMed1975

Effect of methysergide, a blocker of serotonin receptors, on plasma prolactin levels in lactating and ovariectomized rats.

The effect of methysergide (MES, 2.5 mg/100 g body wt), a serotonin antagonist, on prolactin release has been studied in lactating and ovariectomized rats. MES caused significant increases in prolactin release in both animals. Studies in ovariectomized, hypophysectomized rats indicate that this effect is not due to a decrease in the peripheral metabolism of prolactin. In vitro incubations of anterior pituitary fragments with MES failed to demonstrate any increase in prolactin release, suggesting that MES does not act directly on the anterior pituitary. Parachlorophenylalanine (PCPA; 32 mg/100 g body wt) decreased brain serotonin levels in ovariectomized rats 5, 24, and 70 h after its administration, yet did not alter plasma prolactin levels. L-tryptophan (6.3 mg/100 g body wt) given 1 and 1 1/2 h prior to sacrifice increased brain serotonin levels, yet did not affect plasma prolactin levels. Neither PCPA nor L-tryptophan altered MES-induced prolactin release. In lactating rats, suckling caused marked increases in plasma prolactin levels, an effect completely abolished by the administration of MES to the mother rats 3 1/4 h prior to suckling. However, MES-induced prolactin release was not altered by prior treatment with MES, either in lactating or ovariectomized rats. Others have shown that suckling releases prolactin through an excitatory serotonergic mechanism. Therefore, the failure of suckling to release prolactin in MES-pretreated rats suggests that MES can block brain serotonin receptors. However, the ability of methysergide to release prolactin in rats with serotonin receptors presumably blocked, suggests that the serotonin receptor-blocking and the prolactin-releasing actions of MES are not related.

Animals↗
PubMed1976

[The histaminergic mechanisms in the regulation of prolactin secretion in urethan-anesthetized rats (author's transl)].

In an attempt to detect the existence of histaminergic mechanisms in the regulation of prolactin secretion in rats, the effects of histidine and histamine-receptor antagonists (chlorpheniramine and metiamide) on plasma prolactin levels in urethan-anesthetized rats were investigated in relation to other aminergic mechanisms. Chlorpheniramine was used as an H1-receptor antagonist and metiamide as an H2-receptor antagonist. Wistar male rats were used under urethan anesthesia and blood was obtained by cardiac puncture. Rat plasma prolactin was measured by radioimmunoassay. As already reported, L-DOPA and diethyldithiocarbamate markedly decreased plasma prolactin levels in rats. Dihydroxyphenylserine increased prolactin levels. Brocresine phosphate, a histidine decarboxylase inhibitor, markedly stimulated prolactin secretion in urethan-anesthetized rats. This stimulation was not blocked by pretreatment with histidine or L-DOPA. Histidine alone did not affect plasma levels of prolactin or methyldopamine-induced increase in plasma prolactin levels. On the other hand, histidine significantly stimulated prolactin secretion in chlorpheniramine-treated rats. In contrast, histidine depressed plasma prolactin levels in metiamide-treated rats. These findings indicate that a dopaminergic component is inhibitory and a noradrenergic one is stimulatory in prolactin secretion in rats and further that there exist the histaminergic mechanisms, relatively independent from other aminergic mechanisms, which might also play an important role in the regulation of prolactin secretion in rats. Furthermore, it is suggested that H1-receptor is stimulatory and H2-receptor is inhibitory in prolactin secretion in rats.

Animals↗
PubMed1986

[Studies on prolactin secreting capacity in the ovulatory infertile patients with transient hyperprolactinemia].

The changes in serum prolactin levels during the menstrual cycle have not been clarified yet. The present study was conducted to investigate whether the changes in serum prolactin levels during the menstrual cycle exist in ovulatory infertile patients having high prolactin release due to TRH administration described below (transient hyperprolactinemia) and control cases. Serum prolactin levels in both groups were less than 25 ng/ml at daytime. In the patients with transient hyperprolactinemia, serum prolactin levels showed more than 150 ng/ml at 30 min. after the administration of 500 micrograms of TRH, and those were less than 150 ng/ml in the normal control group. The daily changes of serum FSH, LH, prolactin, estradiol and progesterone levels were determined by radioimmunoassay in 6 cases of transient hyperprolactinemia and 5 controls which showed normal ovulatory cycles in the patterns of the BBT charts and other hormones. An estrogen feedback test was also carried out at the mid-luteal phase in 9 cases of transient hyperprolactinemia and the controls to determine serum levels of FSH, LH, prolactin and estradiol. In the patients with transient hyperprolactinemia, 5 mg of bromocriptine was administered every day for more than 30 days, and the effects of bromocriptine on the changes in serum gonadotropin levels by the estrogen feedback test were also analysed. Serum prolactin levels in the follicular, ovulatory and mid-luteal phases increased significantly in the patients with transient hyperprolactinemia, compared to the controls (p less than 0.005). The pattern of serum prolactin levels in the patients with transient hyperprolactinemia was almost synchronized with that of serum estradiol levels. There was also a significant correlation (r = 0.5782, p less than 0.005) between the prolactin and estradiol levels in the serum of the patients. The obvious change was not noted in serum prolactin levels during the menstrual cycle of the controls. No significant change in other hormones was observed during the cycle between these two groups. After the administration of estradiol benzoate (100 micrograms/kg), serum estradiol levels increased markedly, serum prolactin levels increased with the similar change in serum estradiol levels, and serum prolactin levels in the patients with transient hyperprolactinemia were significantly higher compared to those of the controls (p less than 0.01 approximately 0.005). The responses of serum gonadotropin levels by the administration of estradiol benzoate had good positive and negative feedback effects in the patients with transient hyperprolactinemia as well as those of the control group.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1978

Dissociation of plasma and CSF prolactin heterogeneity.

Plasma and cerebrospinal fluid (CSF) specimens from 3 patients with suprasellar extension of a pituitary tumour were fractionated by column chromatography. "Big" and "little" prolactin were found in plasma but only "little" prolactin was found in CSF. Prolactin containing CSF from 2 of the patients was incubated with prolactin-free plasma and chromatographed. The elution patterns demonstrated a striking shift toward the [125I]albumin marker resembling the "big" and "little" prolactin pattern seen in native plasma. In addition, [125I]h prolactin was incubated with prolactin-free plasma and rechromatographed. A substantial early eluting peak of radioactivity occurred in the zone of albumin elution. The presence of only "little" prolactin in CSF and the conversion of CSF "little" prolactin to "big" prolactin by exposure to plasma suggest that "big" prolactin is not a prohormone but results from binding of "little" prolactin to plasma proteins or aggregation of "little" prolactin in the presence of plasma.

Adult↗
PubMed1987

Size heterogeneity of immunoreactive prolactin in patients with prolactinoma.

We investigated the chromatographic pattern of serum prolactin in 41 patients with prolactinoma and correlated the distribution of immunoreactive prolactin with the clinical variables sex, tumour size, age, and response to bromocriptine therapy. In addition, the effect of long-term storage and repeated freezing and thawing on the different molecular weight forms of prolactin was evaluated. Gel chromatography (column 100 cm X 1.5 cm) was performed in 0.1 mol/l phosphate buffer, pH 7.5, using Ultrogel ACA 54 (LKB). No correlation of age or the response to drug therapy to the elution pattern of prolactin was found. Females showed a higher percentage of big prolactin than males (10.4 +/- 1.2% vs 6.8 +/- 0.7%, mean +/- SEM, P less than 0.05) and patients with microprolactinomas too had a higher percentage of big prolactin than those with macroprolactinomas (11.3 +/- 1.8% vs 7.7 +/- 0.7%, P less than 0.05). Serum samples kept frozen for more than 2 years showed a higher percentage of bigbig prolactin (P less than 0.01) than samples stored for less than 12 months suggesting formation in vitro. However, examination of fresh samples prior to freezing also demonstrated bigbig prolactin, indicating that bigbig prolactin circulates in vivo. Repeated freezing and thawing of bigbig prolactin led to almost complete interconversion to little prolactin without any increase in immunoreactivity. This finding supports the concept that bigbig prolactin represents little prolactin loosely associated to a carrier molecule.

Adult↗
PubMed1990

Molecular cloning and nucleotide sequence analysis of complementary DNA for bullfrog prolactin.

A prolactin cDNA was cloned from a cDNA expression library constructed from total RNA of bullfrog (Rana catesbeiana) adenohypophyses by immunoscreening with antiserum against bullfrog prolactin. The cDNA clone thus obtained contained a 249 bp insert. Using this clone as a probe, plaque hybridizations were performed and two additional clones obtained. These clones had a polyadenylation site different from that of the first obtained clone, suggesting that the 3'-untranslated sequence was heterogeneous in length. The longest clone contained 830 bp, which encoded part of the signal peptide and the entire sequence of mature prolactin. The deduced amino acid sequence was in good accord with that determined by direct protein sequencing of purified bullfrog prolactin. The length of the bullfrog prolactin mRNA was estimated by Northern blot analysis to be about 1.0 kb. Homologies of prolactin nucleotide and amino acid sequences between bullfrog and other vertebrates were 64 and 65% for man, 66 and 68% for pig, 61 and 52% for rat, 69 and 74% for chicken, and 50 and 35% for salmon respectively. Highly conserved regions reported for mammalian prolactins also existed in bullfrog prolactin. Homologies of nucleotide and amino acid sequences between prolactin and GH of bullfrog origin were 49 and 25% respectively. Using the cDNA, the content of prolactin mRNA in the pituitary glands of metamorphosing tadpoles was measured. Prolactin mRNA levels rose at the mid-climax stage, suggesting that the increase in plasma and pituitary prolactin levels known to occur at the climax stage accompanies the increase in prolactin synthesis.

Amino Acid Sequence↗
PubMed1995

The relationship between decidualization and prolactin mRNA and production at different stages of human pregnancy.

Within the human utero-placental unit only decidualized stromal cells express mRNA for prolactin. However, it is not clear if the level of prolactin production is related to the number of decidualized cells or the capacity of individual decidual cells to synthesize prolactin, either or both of which parameters may change during pregnancy. In the present study, prolactin production at different stages of human pregnancy was examined using quantitative in situ hybridization to assess decidual prolactin mRNA abundance, immunocytochemistry to examine the prolactin content inside decidual cells and RIA to measure decidual prolactin output into amniotic fluid. Throughout pregnancy the proportion of stromal cells showing positive immunostaining and mRNA for prolactin increased. There was a parallel increase in decidual cell size which was correlated with an increase in prolactin gene expression and intensity of immunostaining for prolactin in individual decidual cells. These changes in decidual cells were consistent with the changes in the concentration of prolactin in amniotic fluid. These results suggest that there is a close link between the level of prolactin gene expression and production of prolactin by individual decidual cells, which in turn is directly related to the process of decidualization that continues throughout human pregnancy.

Amniotic Fluid↗
PubMed1988

Relationships between prolactin, LH and broody behaviour in bantam hens.

The interactions between broody behaviour and changes in concentrations of plasma prolactin and LH were investigated in bantam hens. Adoption of newly hatched chicks caused incubating hens to leave their nests and prevented plasma prolactin decreasing as rapidly as in hens deprived of their nests and not given chicks. Further, the hens allowed to rear chicks came back into lay later (P less than 0.001) than the hens not allowed chicks. Plasma prolactin decreased and plasma LH increased in hens deprived of their nests: these changes were reversed when the hens re-nested. The changes in plasma LH and prolactin in nest-deprived and re-nesting birds were not always synchronous; this was particularly clear immediately after nest deprivation when the increase in plasma LH preceded the decrease in the plasma prolactin. Readiness to incubate disappeared between 48 and 72 h after nest deprivation and corresponded with the time when plasma prolactin decreased to baseline values. Administration of ovine prolactin depressed (P less than 0.01) the initial increase in plasma LH after nest deprivation, but repeated administration of prolactin for up to 72 h failed to suppress plasma LH to the values found in incubating hens. Repeated administration of ovine prolactin at 5- to 8-h intervals for 72 h maintained readiness to incubate in nest-deprived hens. It is concluded that the secretion of prolactin in broody hens is facilitated by the presence of chicks and that increased concentrations of plasma prolactin maintain incubation behaviour. In incubating hens the secretion of LH and prolactin may be partly regulated independently. In addition, LH secretion may also be inhibited by increased plasma prolactin.

Animals↗
PubMed1987

Secretion and mammary gland uptake of prolactin in dairy cows during lactogenesis.

Mammary arteriovenous differences of prolactin concentration and net mammary uptake of prolactin from blood were quantified near parturition in 9 dairy cows. Six cows were milked once daily for at least 6 d before parturition, and prepartum lactogenesis occurred in 3 of 6 cows. Prepartum milking 2 or more d before parturition abruptly increased secretion of prolactin into blood, but milkings within 1 d before or after parturition did not increase prolactin secretion. Concentrations of prolactin in whole milk sampled over 8 d before parturition (64.5 ng/ml) were substantially greater than those occurring several days after parturition (19 ng/ml). Milk prolactin concentrations were unaffected by the successful induction of prepartum lactogenesis, which greatly increased prepartum yields of milk (2 to 8 kg/milking). Therefore, the alveolar lumenal content of prolactin was greatest in pregnant cows with prepartum lactogenesis. This enhanced content of intraalveolar prolactin before parturition was associated with an absence of mammary uptake of prolactin immediately prior to ejection of the prolactin-containing milk from the alveoli. However, prolactin uptake was quickly restored to about 2 micrograms/min per half udder shortly after milk ejection. During the prepartum period, an enhanced intraalveolar reservoir of 200 to 400 micrograms prolactin, due to induction of prepartum lactogenesis, appears to saturate temporarily all putative sites for uptake of prolactin from blood.

Animals↗
PubMed1991

Bioactivity of plasma prolactin in ovariectomized, diethylstilbestrol-treated Long-Evans and Holtzman rats after thyrotropin-releasing hormone or bromocriptine administration.

The objective of this study was to determine the effects of thyrotropin-releasing hormone (TRH) and bromocriptine on plasma levels of biologically active prolactin in ovariectomized, diethylstilbestrol (DES)-treated rats. Female Long-Evans and Holtzman rats were ovariectomized and each was given a subcutaneous implant of diethylstilbestrol (DES). One week later, groups of DES-treated rats were fitted with indwelling intra-atrial catheters, and 2 days later blood samples were withdrawn before and at 1, 2, 5, 10, and 20 min after intravenous administration of TRH (250, 500, or 1000 ng/rat). Blood samples were obtained from other groups at 4 weeks of DES treatment by orbital sinus puncture under ether anesthesia before and at 30, 60, and 120 min after bromocriptine administration (2.5 mg/rat sc). Plasma was assayed for prolactin by conventional radioimmunoassay (RIA) and by Nb2 lymphoma bioassay (BA). Holtzman rats released significantly more prolactin following TRH than did Long-Evans rats when the RIA was used to measure prolactin. However, when the BA was used to assay prolactin in the same samples, the Long-Evans rats released more prolactin than did the Holtzman rats. In addition, the ratio of the BA to RIA values was significantly increased in both strains following TRH, but the greatest increase was observed in the Long-Evans rats, in which the ratio was 4.5 at the peak of the TRH-induced rise in plasma prolactin. Gel filtration chromatography of plasma obtained at 5 min after TRH treatment in Long-Evans rats revealed large molecular forms of prolactin with BA to RIA ratios of 4-5. In addition, monomeric prolactin had a BA to RIA ratio of 2. Bromocriptine treatment reduced prolactin levels in both strains, but the effect was more rapid in Holtzman than in Long-Evans rats. In addition, bromocriptine treatment of Holtzman, but not Long-Evans, rats significantly reduced the BA to RIA ratio of plasma prolactin. The results indicate that TRH and bromocriptine affect the release of biologically active prolactin to a greater extent than prolactin detected by antibody in the RIA, and that Long-Evans and Holtzman rats respond to these secretagogues differently with regard to BA to RIA comparisons.

Animals↗
PubMed1988

Prolactin synthesis in cultured pituitary cells is chloride-dependent.

Release of prolactin from both normal pituitary cells and rat pituitary tumor (GH) cells is an osmotic process that is dependent upon chloride. The long term growth rate of GH-cells in medium in which chloride was exchanged with isethionate was completely normal, but, by 48 h, isethionate substitution resulted in a 70% decrease in the concentration of internal and secreted prolactin. Isethionate caused a much smaller reduction in growth hormone production (less than 20%). These results suggest that exchange of chloride with isethionate is inhibiting the synthesis of prolactin. Reduction of intracellular levels of prolactin in cells grown in isethionate-containing medium was evident by 30 h, and the level of prolactin was reduced 92% at 96 h. This reduction in the internal concentrations of prolactin was reversed when the cells were returned to normal medium containing chloride with a t1/2 of 48 h. Addition of epidermal growth factor and the calcium channel agonist BAY K 8644 to cells in medium containing chloride increased internal prolactin by 400%, and isethionate exchange reduced the response by 85%. To confirm that isethionate exchange was inhibiting the synthesis of prolactin, mRNA concentrations for prolactin and actin were determined. Both basal and hormone-stimulated levels of prolactin mRNA were reduced 70 to 90% by isethionate exchange, while actin mRNA levels did not change. To determine whether the effect of isethionate was at the level of gene transcription, GH-cells were transfected with a prolactin-chloramphenicol acetyltransferase fusion gene and chloramphenicol acetyltransferase expression was assessed using cells in chloride and isethionate-containing media. Both basal and hormone-stimulated synthesis of chloramphenicol acetyltransferase driven by the prolactin promoter was inhibited by isethionate exchange. These studies demonstrate that exchange of medium chloride with isethionate inhibits the synthesis of prolactin at the level of transcription.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1981

[Synthesis of prolactin by human decidua in vitro (author's transl)].

Human decidua, chorion, amnion and placenta from 1st., 2nd. and 3rd. trimesters of gestation were investigated for synthesis and secretion of prolactin by in vitro incubation of these tissue fragments in medium 199. Prolactin content in decidua was found to be significantly higher than those in chorion, amnion or placenta at any stages of gestation. During 6 hours of incubation, decidua secreted significantly more prolactin into medium than did chorion, amnion or placenta. Amount of prolactin secreted by decidua was significantly higher than prolactin content in tissue before incubation. Decidua were also incubated in medium 199 with or without actinomycin-D (20-200 microgram/ml), puromycin (200 microgram/ml) or cycloheximide (100-200 microgram/ml) for 12 hours. Both total prolactin secretion into medium and prolactin content in tissue after incubation were significantly lower as compared to control without inhibitor. Decidua of 2nd. trimester of gestation was noted to secrete more prolactin into medium than decidua of 1st. or 3rd. trimester of gestation. In further studies, radioleucine (200 uCi) was incubated for 12 hours with decidua (10 grams) from 2nd. trimester of gestation, the incorporated radioprotein in medium or tissue was extracted, fractionated by Sephadex G-100 chromatography and by 10% SDS-polyacrylamide gel electrophoresis. Peaks of radioactivity and immunoreactive prolactin in gel slices were coincident with the peak of human pituitary prolactin (NIAMDD, hPRL, Friesen No. 1) at Kf = 0.62 (gel slice No. 31). In addition, mRNA was extracted from decidual tissue by oligo-d (T)-cellulose chromatography, which was then used for cell-free system translation study. The translation product of decidual mRNA and rabbit reticulocyte lysate was shown to be identical to human pituitary prolactin by analysis of gel electrophoresis. These results demonstrate that prolactin synthesized and secreted by human decidua is identical to human pituitary prolactin. The synthetic activity is most prominent in the decidua of 2nd. trimester of gestation.

Amnion↗
PubMed2005

Prolactin inhibits apoptosis of ovarian carcinoma cells induced by serum starvation or cisplatin treatment.

Prolactin, a peptide hormone essential for the development and function of reproductive organs, is involved in development of breast, prostate and colorectal cancers. However, the role of prolactin on the carcinogenesis of ovarian carcinomas is unclear. In this study, we show that mRNA of prolactin receptor is expressed in 5 out of 9 ovarian carcinoma cell lines, 15 out of 17 cases of surgical samples and all of normal ovarian surface epithelium, while prolactin transcript is detected only in 1 ovarian carcinoma cell line and in 1 of the surgical samples. For the prolactin receptor-positive ovarian carcinoma cells, exogenous prolactin did not affect the proliferation but markedly inhibited apoptosis. Therefore, actual cell growth was enhanced by prolactin in a dose-dependent manner. The blocking of prolactin receptor by antibody severely impaired the antiapoptotic and growth-promoting effects of prolactin. Interestingly, the cisplatin-induced cell death of the prolactin receptor-positive cells was significantly inhibited by pretreatment with prolactin. These findings indicate a responsiveness of ovarian carcinomas to prolactin and suggest that the prolactin/prolactin receptor system may be a new therapeutic target of ovarian carcinomas.

Apoptosis↗
PubMed1987

Biological, immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland.

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (micrograms) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with 125I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50-60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.

Amino Acid Sequence↗
PubMed1983

Effects of transglutaminase or phospholipase A2 inhibitors on down-regulation of prolactin receptors and stimulation of casein and DNA synthesis in mammary gland explants.

A number of compounds have been found to inhibit the internalization of alpha 2-macroglobulin and/or epidermal growth factor into fibroblasts. Using the same inhibitor we tried to block the down-regulation of prolactin receptors, supposed to mirror internalization of PRL receptors, in order to investigate the possible role of internalization in the mechanism of prolactin action. In rabbit mammary cells it appeared that bacitracin and ethylamine completely block prolactin receptor down-regulation, but dansylcadaverine, the most potent inhibitor of transglutaminase, was without effect. The blockage of phospholipase A2 by chlorpromazine or bromophenacyl bromide (BPB) was without effect on the down-regulation of prolactin receptor. Subcellular distribution of [125I]oPRL has been studied after incubation with or without these various inhibitors after fractionation of mammary gland homogenate on sucrose gradient. Any of these compounds was able to increase the labelling of prolactin receptors located in plasma membrane fractions, suggesting that the rate of internalization of prolactin was not modified. In addition, none of these compounds inhibited the stimulation of beta-casein and DNA synthesis by prolactin. These results suggest that both transglutaminase and phospholipase A2 are not involved in the mechanism of prolactin-induced down-regulation of prolactin receptors, although bacitracin and ethylamine are able to block this phenomenon probably by different mechanism. In all cases, inductions of mitogenesis and beta-casein synthesis by prolactin in the rabbit mammary cells were not modified by the various compounds utilized. We conclude that neither transglutaminase nor phospholipase A2 are involved in the internalization of prolactin-receptor complexes, although bacitracin, ethylamine and quinacrine are able to block the down regulation of prolactin receptors by other means.

Acyltransferases↗
PubMed1984

Structures of the somatotropin receptor and prolactin receptor on rat hepatocytes characterized by affinity labelling.

Human somatotropin competed for 125I-human somatotropin binding to hepatocytes from female or male rats. Bovine somatotropin and prolactin each inhibited part, but not all, of the uptake of 125I-human somatotropin. The binding of 125I-prolactin was inhibited by human somatotropin and prolactin, but not by bovine somatotropin. Bovine somatotropin and human somatotropin, but not prolactin, competed for 125I-bovine somatotropin binding sites. 125I-labelled hormones were covalently coupled to membrane receptors with higher efficiency on hepatocytes from female than from male rats, allowing structural descriptions of lactogenic and somatogenic binding sites that had not been possible previously. Disuccinimidyl suberate covalently coupled 125I-human somatotropin into saturable complexes of Mr 300 000, 220 000, 130 000, 65 000 and 50 000. Bovine somatotropin inhibited the incorporation of 125I-human somatotropin into complexes of Mr 300 000, 220 000 and 130 000, whereas low concentrations of prolactin competed for incorporation into the 65 000- and 50 000-Mr species. 125I-bovine somatotropin was incorporated into complexes of Mr 300 000, 220 000 and 130 000. Human somatotropin and bovine somatotropin, but not prolactin, inhibited the production of these complexes. 125I-prolactin binding produced complexes of Mr 65 000 and 50 000. Native prolactin and human somatotropin, but not bovine somatotropin, inhibited uptake of 125I-prolactin into these species. Thus direct affinity labelling, as well as competition for covalent coupling, suggests that the 300 000-, 220 000- and 130 000-Mr species are components of the somatotropin receptor and that the 65 000- and 50 000-Mr complexes result from hormone binding to the prolactin receptor. By subtracting the Mr of prolactin, it was calculated that the hormone was bound to species of Mr 43 000 and 28 000. These Mr values were not affected by reduction of solubilized membranes, suggesting that the structure of the prolactin receptor is not stabilized by interchain disulphide bonds between subunits. Subtracting the Mr of somatotropin from somatogenic complexes indicated that the hormone had bound to species of Mr 280 000, 200 000 and 100 000. The 300 000- and 220 000-Mr complexes were not isolated from reduced membranes, whereas the amount of the 130 000-Mr species was augmented. These observations could suggest that a major component of the somatotropin receptor is a trimeric aggregate in which some subunits are retained in a larger complex by interchain disulphide bonds.

Affinity Labels↗