Prolactin regulates TRPV1, TRPA1, and TRPM8 in sensory neurons in a sex-dependent manner: Contribution of prolactin receptor to inflammatory pain.

Prolactin (PRL) is a hormone produced in the anterior pituitary but also synthesized extrapituitary where it can influence diverse cellular processes, including inflammatory responses. Females experience greater pain in certain inflammatory conditions, but the contribution of the PRL system to sex-dependent inflammatory pain is unknown. We found that PRL regulates transient receptor potential (TRP) channels in a sex-dependent manner in sensory neurons. At >20 ng/ml, PRL sensitizes TRPV1 in female, but not male, neurons. This effect is mediated by PRL receptor (PRL-R). Likewise, TRPA1 and TRPM8 were sensitized by 100 ng/ml PRL only in female neurons. We showed that complete Freund adjuvant (CFA) upregulated PRL levels in the inflamed paw of both male and female rats, but levels were higher in females. In contrast, CFA did not change mRNA levels of long and short PRL-R in the dorsal root ganglion or spinal cord. Analysis of PRL and PRL-R knockout (KO) mice demonstrated that basal responses to cold stimuli were only altered in females, and with no significant effects on heat and mechanical responses in both sexes. CFA-induced heat and cold hyperalgesia were not changed in PRL and PRL-R KO compared with wild-type (WT) males, whereas significant reduction of heat and cold post-CFA hyperalgesia was detected in PRL and PRL-R KO females. Attenuation of CFA-induced mechanical allodynia was observed in both PRL and PRL-R KO females and males. Thermal hyperalgesia in PRL KO females was restored by administration of PRL into hindpaws. Overall, we demonstrate a sex-dependent regulation of peripheral inflammatory hyperalgesia by the PRL system.

Acute effects of prolactin on hypothalamic prolactin receptor expressing neurones in the mouse.

In addition to its critical role in lactation, the anterior pituitary hormone prolactin also influences a broad range of other physiological processes. In particular, widespread expression of prolactin receptor (Prlr) in the brain has highlighted pleiotropic roles for prolactin in regulating neuronal function, including maternal behaviour, reproduction and energy balance. Research into the central actions of prolactin has predominately focused on effects on gene transcription via the canonical JAK2/STAT5; however, it is evident that prolactin can exert rapid actions to stimulate activity in specific populations of neurones. We aimed to investigate how widespread these rapid actions of prolactin are in regions of the brain with large populations of prolactin-sensitive neurones, and whether physiological state alters these responses. Using transgenic mice where the Cre-dependent calcium indicator, GCaMP6f, was conditionally expressed in cells expressing the long form of the Prlr, we monitored changes in levels of intracellular calcium ([Ca2+ ]i ) in ex vivo brain slice preparations as a surrogate marker of cellular activity. Here, we surveyed hypothalamic regions implicated in the diverse physiological functions of prolactin such as the arcuate (ARC) and paraventricular nuclei of the hypothalamus (PVN), as well as the medial preoptic area (MPOA). We observed that, in the ARC of males and in both virgin and lactating females, prolactin can exert rapid actions to stimulate neuronal activity in the majority of Prlr-expressing neurones. In the PVN and MPOA, we found a smaller subset of cells that rapidly respond to prolactin. In these brain regions, the effects we detected ranged from rapid or sustained increases in [Ca2+ ]i to inhibitory effects, indicating a heterogeneous nature of these Prlr-expressing populations. These results enhance our understanding of mechanisms by which prolactin acts on hypothalamic neurones and provide insights into how prolactin might influence neuronal circuits in the mouse brain.

Prolactin stimulates the vascularisation of the retina in newborn mice under hyperoxia conditions.

The hormone prolactin (PRL) is emerging as an important regulator of ocular blood vessels. PRL is pro-angiogenic and acquires anti-angiogenic properties after undergoing proteolytic cleavage to the PRL fragment, vasoinhibin. The vascularisation of the rodent retina develops after birth when it rapidly expands until completion at the end of the first postnatal week. Exposure of newborn mice to high oxygen levels lowers the rate of blood vessel growth. In the present study, we investigated whether PRL treatment modifies the vascularisation of the retina in newborn mice exposed to high oxygen or to normoxia and whether the retinal conversion of PRL to vasoinhibin may be altered in the neonate. Newborn mice and their nursing mothers were subjected to 75% oxygen or to normoxia from postnatal day (P) 6 to P8 (group 1) or from P2 to P5 (group 2). PRL (2 µg g-1 , i.p., twice a day) or vehicle was injected from P5 to P8 in group 1 and from P1 to P5 in group 2. PRL treatment reduced the retinal inhibition of blood vessel growth and the increase in vascular regression induced by hyperoxia as revealed by immunofluorescence staining of blood vessels and the expression of angiogenesis and apoptosis markers. The pro-angiogenic effect may involve a reduced conversion of PRL to vasoinhibin. Incubation of PRL with retinal extracts showed reduced activity of the PRL-cleaving protease, cathepsin D, in the neonate vs the adult retina that was further reduced under hyperoxia. PRL and the PRL receptor mRNA were expressed at higher levels in the retina at P8 than in the adult, whereas endogenous PRL was undetectable in the circulation at P8. We conclude that PRL has a pro-angiogenic effect in the neonate retina as a result of its reduced conversion to vasoinhibin and that PRL produced by the retina may help promote physiological vascularisation after birth.

Zinc mediation of the binding of human growth hormone to the human prolactin receptor.

Human growth hormone (hGH) elicits a diverse set of biological activities including lactation that derives from binding to the prolactin (PRL) receptor. The binding affinity of hGH for the extracellular binding domain of the hPRL receptor (hPRLbp) was increased about 8000-fold by addition of 50 micromolar ZnCl2. Zinc was not required for binding of hGH to the hGH binding protein (hGHbp) or for binding of hPRL to the hPRLbp. Other divalent metal ions (Ca2+, Mg2+, Cu2+, Mn2+, and Co2+) at physiological concentrations did not support such strong binding. Scatchard analysis indicated a stoichiometry of one Zn2+ per hGH.hPRLbp complex. Mutational analysis showed that a cluster of three residues (His18, His21, and Glu174) in hGH and His188 from the hPRLbp (conserved in all PRL receptors but not GH receptors) are probable Zn2+ ligands. This polypeptide hormone.receptor "zinc sandwich" provides a molecular mechanism to explain why nonprimate GHs are not lactogenic and offers a molecular link between zinc deficiency and its association with altered functions of hGH.

Prolactin is involved in glial responses following a focal injury to the juvenile rat brain.

A cerebral growth hormone axis is activated following brain injury in the rat and treatment with growth hormone is neuroprotective. We have now investigated whether the closely related prolactin axis has similar properties following injury to the developing rat brain. From one day following a unilateral hypoxic ischemic injury, prolactin immunoreactivity was increased in the affected cortex parallel to the development of the injury (P<0.001). Initial prolactin and prolactin receptor staining on penumbral neurons progressively decreased whereas astrocytes remained strongly immunopositive. Reactive microglia also became strongly prolactin immunoreactive. Unlike growth hormone, central treatment with prolactin failed to rescue neurons in this paradigm. This was confirmed in vitro; rat prolactin failed to protect neurons under conditions for which growth hormone was neuroprotective. However, prolactin had trophic and pro-proliferative effects on glia (P<0.001). We confirmed the expression of the prolactin receptor in vitro by reverse transcriptase polymerase chain reaction, and show its strong association with astrocytes as compared with neurons by immunocytochemistry. In summary, we show for the first time that hypoxia ischemia induces a robust activation of the prolactin axis in regions of the cerebral cortex affected by injury. The lack of neuroprotective properties in vivo and in vitro indicates that, unlike growth hormone, prolactin is not directly involved in neuronal rescue in the injured brain. Its strong relation to glial reactions and its gliatrophic effects suggest that the prolactin axis is primarily involved in a gliogenic response during recovery from cerebral injury.

Steroid-induced alterations in mRNA expression of the long form of the prolactin receptor in the medial preoptic area of female rats: Effects of exposure to a pregnancy-like regimen of progesterone and estradiol.

It is firmly established that the onset of maternal behavior in the female rat is stimulated by a combination of hormones that include prolactin (PRL), estradiol (E2), and progesterone (P(4)). Specifically, nulliparous rats display short latencies to respond to foster young when primed with Silastic capsules filled with P4 and E2 and then administered PRL centrally to the medial preoptic area (MPOA), an area integrally involved in the expression of maternal behavior in this species. PRL or P4 treatments alone are ineffective in stimulating the expression of maternal care. Since the actions of PRL in the MPOA appear to be mediated by PRL receptors, it was of interest to determine whether and how treatment with P4 and E2 together or separately might alter mRNA expression of the long form of the PRL receptor (PRL-R(L)) in the MPOA. Using in situ hybridization histochemistry (ISHH), mRNA expression of the PRL-R(L) was measured in the MPOA of ovariectomized, nulliparous rats treated with various combinations of P4 and E2. Treatment of animals with P4 alone for 10 days or with P4 followed by E2 for 1 or 4 days resulted in reductions in PRL receptor mRNA expression in the MPOA when compared with the expression in animals treated with E2 alone or blank capsules. The actions of P4 on mRNA expression of the PRL-R(L) were more pronounced in the dorsal MPOA. Circulating PRL levels collected at the time of sacrifice were elevated in all groups treated with E2, but no association between PRL levels and receptor mRNA expression within the MPOA was evident. These findings indicate that the dorsal MPOA may be one site of progesterone's action in facilitating prolactin-mediated maternal behavior.

A mutant lactogenic hormone binds, but does not activate, the prolactin receptor.

We have generated mutations in mouse placental lactogen II, a hormone in the PRL/GH family that binds to the PRL receptor, to investigate the role of the conserved cysteine residues in hormone function. Disruption of the small C-terminal disulfide loop did not significantly alter hormone activity. Substitution of serine for cysteine-51, which prevents formation of the large disulfide loop, results in a protein equivalent to placental lactogen II in receptor-binding activity; however, this mutant protein is not mitogenic in an assay for lactogenic hormones. These results indicate that PRL receptor occupancy and activation are distinct events.

Multiple regulation of prolactin receptor gene expression in rat liver.

Sex steroids are major regulators of PRL receptor expression in rat liver. Using a probe encoding the rat PRL receptor we have studied receptor mRNA levels in female rat liver during ontogeny and in response to estrogen treatment. Steady state mRNA levels were determined by Northern blot and densitometric analysis. Messenger RNA levels have been compared to the number of binding sites, which was assessed by Scatchard analysis of [125I]ovine PRL binding in membrane preparations. Our results show that steady state mRNA and binding levels of PRL receptors are both regulated by development and estrogens, but that binding does not exactly parallel mRNA levels. From the developmental stages of prepuberty to adult, receptor numbers increase 8-fold, whereas mRNA levels increase 3-fold. Estrogen treatment stimulates receptor levels 6-fold, but mRNA levels are only increased 3-fold. These results suggest that PRL receptor gene expression in rat liver is regulated at the transcriptional or posttranscriptional level as well as at the translational level.

Servomechanism of prolactin and progesterone in regulating uterine gene expression.

To investigate the interaction of PRL and progesterone in regulating uterine gene expression, we have quantitated the concentration of PRL receptor and of uteroglobin (UG) mRNA in the endometrium of rabbits of different ages and after treatment with different hormones. During uterine differentiation in 2- to 4-week old rabbits, a marked increase in unoccupied uterine PRL receptor number was observed, presumably increasing uterine sensitivity to PRL. Receptor values for 4-week old rabbits were comparable to values for sexually mature, estrous females, but were lower than in 5-day pseudopregnant (PSP) animals. When total PRL receptor was determined by Scatchard analysis after in vitro desaturation with MgCl2, PSP animals again expressed the highest receptor concentration with no changes in the dissociation constant (Kd) values. To determine whether progesterone regulates uterine PRL receptor, long term ovariectomized rabbits (greater than 12 weeks) were treated with various combinations of hormones, and unoccupied and total uterine PRL receptors were determined. Progesterone treatment resulted in the highest concentration of both unoccupied and total PRL receptor after desaturation and removal of anti-ovine PRL antibodies with MgCl2. The value for total uterine PRL receptor was equivalent to the value for mammary gland, and the Kd values (2-4 x 10(-10) M) were similar. Treatment of long term ovariectomized rabbits with progesterone, with or without estradiol, produced an increase (P less than 0.05) in the UG mRNA content, which also occurred in PSP animals. PRL alone had no effect on UG mRNA but PRL plus progesterone increased (P less than 0.05) UG mRNA in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

The forgotten lactogenic activity of growth hormone: important implications for rodent studies.

Studies of the effects of GH and the mechanisms of its actions frequently use rats or mice and various recombinant human GH preparations. Authors of many of these studies appear unaware of the fact that, in rodents, human GH signals through both GH and prolactin (PRL) receptors; thus, treatment with human GH is equivalent to a combined treatment with GH and PRL. GH receptors and PRL receptors are present in multiple cell types. Importantly, PRL exerts major effects on brain neuroendocrine action, female and male reproduction, metabolism, body composition, immune responses, and a host of other functions; thus, treatment of rodents with recombinant human GH could affect these important physiological parameters.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.

Characterization of the structure and glycosylation properties of intracellular and cell surface rat hepatic prolactin receptors.

In this study we examined the structure of the PRL receptor of rat liver. We used immunoblotting with monoclonal antibodies to binding and nonbinding site epitopes of the PRL receptor to assess receptor subtypes in different hepatic subcellular fractions. Analysis of frozen-thawed cell fractions revealed 40- and 42-kilodalton (kDa) species. Digestion with neuraminidase indicated that both species were terminally sialylated. Freshly isolated membranes exhibited a single 42-kDa species in all subcellular fractions, whereas freeze-thawing generated the 40-kDa species. The carbohydrate linkages present in the PRL receptor were examined using enzymes to deglycosylate iodinated purified receptors. These studies indicated that oligosaccharides comprise about 7 kDa of receptor mass and that they are exclusively N-linked, consisting of tri- and/or tetra-antennary complex glycans. Monoclonal antibodies to the receptor recognized the deglycosylated receptor. Maximally deglycosylated receptors retained about 85% of their binding capacity. After in vivo tunicamycin treatment of rats, total PRL receptors (as determined by immunoblot analysis and binding activity) disappeared with a half-time of about 25 min. In this circumstance, no aglycosylated receptor species were recognized by monoclonal antibodies. Since deglycosylation of mature receptors did not markedly reduce binding capacity, we infer that mature receptors do not accumulate during the blockade of glycosylation by tunicamycin. Thus, glycosylation appears to be required for the acquisition of a mature receptor status, but is not necessary for the maintenance of that status.

Prolactin regulation of pim-1 expression: positive and negative promoter elements.

The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to gamma-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2-11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5'-pim-1 promoter and a series of deletion mutants were ligated upstream of the chloramphenicol acetylase transferase (CAT) gene in an expression vector that was introduced into FDC/Nb2 cells, a premyeloid line that stably expresses the intermediate form of the PRLR. Analysis of PRL-treated cultures indicated that two elements [distal (DE), -427 to -336 bp and proximal (PE), - 104 to -1] but not several GAS or GAS-like sequences were required for hormone activation of the pim-1 promoter. Moreover, treatment of Nb2-11 cells with PRL activated protein binding to these elements assessed by gel mobility shift assay. Deoxyribonuclease I (DNase I) protection experiments revealed a motif containing a nuclear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when exposed to extracts from PRL-treated cells but protected by proteins from unstimulated cells. Gel mobility shift analysis of this sequence showed decreased protein binding after PRL stimulation. It is concluded that the PRLR initiates pim-1 transcription by a mechanism that involves transcriptional activation by factors that stimulate the DE- and PE-sites and derepress a NF-1-containing element. Moreover, this mechanism appears to be independent of an interaction between Stat transcription factors and GAS-like elements present within the promoter.

Comparative study of in vitro and in vivo modulation of lactogenic and somatotropic receptors by native human growth hormone and its modified analog prepared by recombinant deoxyribonucleic acid technology.

A modified analog of human GH (hGH), prepared by recombinant DNA technology, that lacks 13 amino acids at the amino terminus (Met14hGH), was able to compete with [125I]hGH for binding to lactogenic receptors in Nb2-11C rat lymphoma cells, to somatotropic receptors in IM-9 human lymphocytes, and to both lactogenic and somatotropic receptors in the microsomal fraction of virgin female rat liver. Exposure of intact Nb2 or IM-9 cells to Met14hGH did not reduce the number of surface or intracellular receptors, as compared to the control without hormone. A parallel exposure to 500-fold lower concentrations of hGH resulted in 77-93% reduction in both surface and intracellular receptors. In contrast to [125I]hGH, [125I]Met14hGH was not taken up by the intact Nb2 lymphoma cells. Infusion of anesthetized female virgin rats for 3 h with hGH down-regulated both lactogenic and somatotropic receptors in the liver. A similar infusion with up to 200-fold higher amounts of Met14hGH did not lower the number of total receptors, indicating lack of down-regulation. Some decrease in the binding to free receptors was observed, suggesting that Met14hGH is capable of binding to liver receptors in vivo.

Differential solubilization of placental lactogen (PL)- and growth hormone-binding sites: further evidence for a unique PL receptor in fetal and maternal liver.

Previous studies from this laboratory provided evidence for the existence of a specific placental lactogen (PL) receptor in tissues of fetal lambs and pregnant sheep. The PL receptor is structurally and functionally distinct from somatotropic (GH) and lactogenic (PRL) receptors, and there are conspicuous differences in the expression of the three receptors during ontogeny. The results of the present study indicate striking differences in the solubilization of PL- and GH-binding sites in maternal and fetal sheep liver. Radiolabeled ovine PL (oPL) bound specifically and with high affinity (Kd, 0.97 nM) to soluble detergent extracts of ovine fetal liver, but there was no specific binding of radiolabeled ovine GH (oGH) or oPRL to soluble extracts or insoluble fractions of fetal liver. When liver microsomes of pregnant sheep were extracted with Triton X-100, 80% of the [125I]oPL-binding sites were recovered in the soluble fraction, but 76% of the [125I]oGH binding sites were recovered in the insoluble pellet. Soluble extracts of maternal liver had high affinity for oPL (Kd, 1.45 nM), but low affinity for oGH (Kd 33 nM) and oPRL (Kd, 1-2 microM). On the other hand, Triton-insoluble fractions of maternal liver had high affinity for oGH (Kd, 0.95 nM) as well as oPL (Kd, 0.91 nM), but low affinity for oPRL (Kd, 1-2 microM). The subunit structure of the [125I]oPL-binding site in soluble fractions of fetal and maternal liver (mol wt, 38-47K) was distinct from that of the [125I]oGH-binding site in Triton-insoluble fractions of maternal liver (mol wt, 54/118K). These findings indicate that treatment of microsomal fractions of fetal and maternal sheep liver with Triton X-100 solubilizes the oPL receptor but not the oGH receptor. The differential solubilization of PL- and GH-binding sites may facilitate purification of the two distinct receptors and clarification of their respective roles in the regulation of fetal and postnatal growth.

Prolactin (PRL) receptor induction in cultured rat hepatocytes: dual regulation by PRL and growth hormone.

Although early work implicated PRL as the pituitary factor inducing rat hepatic PRL receptors, recent studies indicated that GH, not PRL, was responsible. The roles for these two hormones were evaluated on rat hepatocytes cultured in serum-free medium supplemented with insulin (1 microgram/ml), epidermal growth factor EGF (25 ng/ml), glucagon (500 ng/ml), cholera toxin (2 ng/ml), hydrocortisone (10(-8) M), and transferrin (1 microgram/ml) and changed daily. Ovine (o) PRL, bovine (b) GH, or human (h) GH were introduced after 2-4 days of culture, and PRL receptors were measured by determining [125I]hGH binding in the presence and absence of excess oPRL in a total particulate fraction pretreated with 3 M MgCl2. The specific binding of hGH (% per 100 micrograms protein) decreased by 8- to 10-fold (female, 17.9 +/- 0.2% to 1.5%; male, 7.0 +/- 0.1% to 0.7%) after 3 days in culture. When added after 3 days, hGH induced PRL receptors in both female and male cells with the effect being more gradual in the latter. Induction occurred with 10 ng/ml hGH and was maximal [11- to 13-fold control] at 250-1000 ng/ml. bGH and oPRL also induced PRL receptors with maximal levels attained at 250-500 ng/ml oPRL (3- to 4-fold control). The combined addition of oPRL (300 ng/ml) and bGH (300 ng/ml) yielded levels of induction comparable to that seen with hGH. Although hormone treatment restored PRL receptor levels to those seen in male rats, the much higher levels of female rats were not attained. Treatment of hepatocytes with hGH, bGH, or oPRL affected neither cell number (through 10 days of culture) nor PRL receptor affinity. At supramaximal doses hGH, PRL, and bGH down-regulated PRL receptors, but this was particularly noticeable for oPRL and hGH. 17 beta-Estradiol and testosterone added to male and female hepatocytes simultaneously with hGH had little or no effect on receptor induction. We conclude that hepatic PRL receptors are induced by both PRL and GH, each acting through its own receptor. The failure to restore receptor levels to those seen in female rats attests to the importance of other modulators. This dual regulation of the PRL receptor explains the unusual potency of hGH which binds to both PRL and GH receptors.

The 20-kilodalton (kDa) human growth hormone (hGH) differs from the 22-kDa hGH in the effect on the human prolactin receptor.

Previously we have demonstrated that 20-kDa human GH (20K-hGH) is a full agonist for hGH receptor (hGHR) even though its complex formation with hGHR and hGH-binding protein differs from that of 22-kDa human GH (22K-hGH). In this study, we focused on the effect of 20K-hGH on human PRL receptor (hPRLR). To elucidate the effects of 20K-hGH on hPRLR and compare them with those of 22K-hGH, we prepared two cells stably expressing full-length hPRLR, Ba/F3-hPRLR and CHO-hPRLR. In the proliferation of Ba/F3-hPRLR cells, which can grow in a dose-response to lactogenic hormones, both 20K- and 22K-hGH exhibited bell-shaped curves in the absence of exogenous zinc ion (Zn2+); however, the curve of 20K-hGH was shifted to a 10-fold higher concentration than that of 22K-hGH in view of EC50 value (the EC50 of 20K- and 22K-hGH were 15 nM and 1.5 nM, respectively). Addition of Zn2+ up to 25 microM increased the activities of both 20K- and 22K-hGH; however, the enhancement by Zn2+ was greater in 20K-hGH than in 22K-hGH, thereby the activities of both hGH isoforms reached the same level at 25 microM Zn2+. Nevertheless, in the presence of 0.25-1 microM free Zn2+, which is equal in human serum, the activity of 20K-hGH was still lower than that of 22K-hGH. The modest effect of 20K-hGH on activating hPRLR in the absence of Zn2+ was confirmed in the rat serine protease inhibitor 2.1 (Spi2.1) gene promoter activation and JAK2/Stat5 tyrosine phosphorylation in CHO-hPRLR. In addition, in human breast cancer cell T-47D, 20K-hGH was proved to stimulate Stat5 tyrosine phosphorylation to much lower degree than 22K-hGH via not hGHR but hPRLR. Taken together, our data suggest that 20K-hGH may be a weaker agonist for hPRLR than 22K-hGH in the human body; therefore 20K-hGH may alleviate the hPRLR-mediated side-effects such as breast cancer when administered to human body.

Direct evidence that lactogenic hormones induce homodimerization of membrane-anchored prolactin receptor in intact Nb2-11C rat lymphoma cells.

The ability of full-size prolactin receptor (PRLR) from Nb2 rat lymphoma cell line to undergo lactogenic hormone-induced dimerization in intact cells or in a partially purified microsomal fraction was tested. The stoichiometry of ovine placental lactogen (oPL) binding to PRLR was documented by SDS-PAGE of the covalently cross-linked complexes between [125I]oPL and intact Nb2-11C cells. The molecular masses of the specific bands were 82 and 141 kDa, corresponding to PRLR:oPL and (PRLR)2:oPL complexes. These results provide direct evidence for the occurrence of hormone-induced receptor dimerization in intact cells. Gel-filtration studies revealed that under non-denaturing conditions, the purified receptor forms high-molecular-mass aggregates (190 and 540 kDa) composed of receptor dimers and oligomers. Since this aggregation was not dependent on the presence of lactogenic hormone, it is possible that the receptor in the intact cells may already exist as a noncovalent dimer or oligomer and that hormone-induced dimerization stabilizes the complex or changes its conformation.

Effects of morphine on prolactin receptors in the rat brain.

The effect of chronically given morphine on the binding of ovine prolactin (oPRL) to specific areas in the male rat brain was studied. The drug was delivered through subcutaneously implanted miniosmotic pumps. The results indicated that the density of prolactin binding sites in the hypothalamus and the choroid plexus was significantly decreased in the acute phase of morphine administration but restored to control levels when tolerance to morphine was developed. The decrease in prolactin binding was contrasted by elevated plasma levels of the hormone. A negative correlation was found between the hormone concentration in plasma and the density of its binding sites in the hypothalamus and choroid plexus. The hormone-binding sites in these two regions were further characterized with regard to binding constants and molecular sizes. The relevance of the present results with respect to the hypothalamic control of prolactin secretion is discussed.

Involvement of the cAMP/protein kinase A pathway and of mitogen-activated protein kinase in the anti-proliferative effects of anandamide in human breast cancer cells.

Anandamide (ANA) inhibits prolactin- and nerve growth factor (NGF)-induced proliferation of human breast cancer cells by decreasing the levels of the 100 kDa prolactin receptor (PRLr) and the high affinity trk NGF receptor, respectively, and by acting via CB(1)-like cannabinoid receptors. However, the intracellular signals that mediate these effects are not known. Here, we show that, in MCF-7 cells: (i) forskolin and the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 prevent, and the protein kinase A inhibitor RpcAMPs mimics, the inhibitory effects of ANA on cell proliferation and PRLr/trk expression and (ii) ANA inhibits forskolin-induced cAMP formation and stimulates Raf-1 translocation and MAPK activity, in a fashion sensitive to the selective CB(1) antagonist SR141716A. ANA stimulation of MAPK was enhanced by inhibitors of ANA hydrolysis. Forskolin inhibited MAPK and ANA-induced Raf-1 translocation. These findings indicate that, in MCF-7 cells, ANA inhibits adenylyl cyclase and activates MAPK, thereby exerting a down-regulation on PRLr and trk levels and a suppression of cell proliferation.