Prolactin receptor expression by splenocytes from rats in various hormonal states

Prolactin (PRL) is mitogenic for lymphocytes in vitro , but the responsiveness of lymphocytes depends on the in vivo hormonal status of the rats from which the cells were obtained. Lymphocytes from ovariectomized (OVX) rats, but not from rats in oestrus or from male rats, respond to prolactin; administration of oestradiol to OVX rats diminishes the response. In order to determine if a correlation exists between lymphocyte responsiveness to prolactin and levels of cell surface prolactin receptors (PRL-R) expression, the percentage of splenocytes and each splenocyte subpopulation expressing surface PRL-R from rats of various hormonal states (OVX, oestradiol-injected OVX, oestrus and male) was analysed by single-colour and dual-colour flow cytometric analysis. We found that approximately 20% of splenocytes expressed surface PRL-R regardless of hormonal states ( n =16). The majority (85%) of PRL-R positive splenocytes were B lymphocytes whereas 11.1% and 4.8% of splenocytes expressing the PRL-R were CD4 positive T-helper (T_H) and CD8 positive T-cytotoxic (T_C) lymphocytes, respectively. B lymphocytes also stained more brightly than T lymphocytes. This distribution of PRL-R expression did not show significant alterations on total splenocytes or T_H and T_C lymphocytes during various hormonal stages. However, the percentage of PRL-R-positive B lymphocytes increased markedly in OVX rats (twofold), compared to rats at oestrus. In summary, no correlation was found between the responsiveness to prolactin as a mitogen and levels of PRL-R expression by lymphocytes from rats at different hormonal states. This result suggests that sex steroid hormones may control prolactin responsiveness of lymphocytes by affecting the signal transduction pathway through PRL-R rather than by altering the level of the cell surface receptor expression.     

Modulation of Fcgamma and C3b receptor expression by marine bioglycans in mouse splenocytes]

Investigation of polysacharide immunomodulators of marine origin was performed--mitilane, alpha-1,4;1,6-D-glucane, isolated from midia Crenomytilus grayanus, and translam--beta-1,3;1,6-D-glucane isolated from marine algae Laminaria cichorioides were compared. Mechanisms of phagocytes cells activation were investigated. Dose-dependent ability of investigated bioglycanes to facilitate Fc gamma R [symbol: see text] C3bR expression at mice splenocytes was demonstrated in vivo and in vitro. The effect depended on immunomodulator type, incubation conditions, dose, period of incubation in vitro and by splenocytes population used for Fc gamma R and C3bR identification. It was shown that C3bR expression was more enhanced by immunomodulators than Fc gamma R expression. For Fc gamma R induction on lymphocytes membranes the presence of phagocytes cell (macrophages and neutrophils) is obligatory. Mitilane, containing alpha-1,4;1,6-D-glucane and some amount of protein is more effective in stimulation of membrane receptors expression than translam--beta-1,3;1,6-D-glucane. The results of investigation demonstrates the possibility to use marine bioglicanes as activators of Fc gamma R and C3bR activity, that is the base for control of pathological processes, related to immune system.     

Inhibition of mammary tumorigenesis in virgin rats by adoptive transfer of splenocytes from parous donors

Summary Splenocytes from parous rats have been previously found to have cytotoxic activity against mammary tumor cells in vitro. Experiments were carried out to determine if this pregnancy-induced cytotoxic nature of the splenocytes is inherent and transferable. Splenocytes from parous rats were adoptively transferred to a group of virgin rats. Another group of age-matched, virgin rats received splenocytes from virgin donors in a similar way. After a period of rest, at the age of 55 days, the rats belonging to both of the groups, received 7,12-dimethylbenz(a)anthracene (DMBA) intragastrically. A third group of untreated virgin rats were also given the chemical carcinogen the same way as above and were considered as intact controls. The rats were monitored for development and growth of mammary tumor from 60 days of DMBA administration. After 4 months of DMBA administration the rats were sacrificed and mammary glands were examined for tumors. Mammary glands with no visible tumors were taken for whole mount preparation, to be examined for microscopic lesions. The results showed that 33 of 41 intact control rats, developed tumor and 27 of the 34 rats that received spleen cells from virgin rats developed tumors. Of the rats that received spleen cells from parous rats, only 18 out of 37 rats developed tumors, indicating an inhibition of tumor induction in these rats. Growth rate of the tumors in this group was also slower than in the control groups.This research was supported by USPHS grant CA 3613906 awarded by the National Cancer Institute     

Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression

Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological role of prolactin could contribute to pituitary tumor development.     

Growth hormone releasing hormone receptors on thymocytes and splenocytes from rats.

In the present study, we determined that rat mononuclear leukocytes possess specific receptors for growth hormone releasing hormone (GHRH). The results show that the binding of 125I-labeled GHRH to spleen and thymic cells was saturable and of a high affinity, approximately 3.5 and 2.5 nM for thymus and spleen cells, respectively. The Scatchard analysis revealed a binding capacity of approximately 54 and 35 fmol per 10(6) cells on thymus and spleen, respectively. The binding of GHRH was not competed by 10(-6) M growth hormone, corticotropin releasing factor, substance P or luteinizing hormone releasing hormone and vasointestinal peptide (VIP). Partial characterization of the receptor was accomplished by crosslinking 125I-labeled GHRH to thymus cells with disuccinimidyl suberate and polyacrylamide gel electrophoresis. Autoradiography of dried gels showed two major components in leukocytes and pituitary cells at approximately 42 and 27 kDa which could be diminished by unlabeled GHRH. The treatment of leukocytes with GHRH (10 nM) rapidly increased the intracellular free calcium concentration from a basal level of 70 +/- 20 nM to a plateau value of 150 +/- 20 nM in 6 min after stimulation. The functional activity of GHRH receptors was studied further by measuring lymphocyte proliferative responses and the increase in the level of cytoplasmic GH RNA. The presence of GHRH alone resulted in a dose-dependent increase in thymidine and uridine incorporation and a dose-dependent increase in the levels of GH RNA in the cytoplasm. Taken together, the results show that lymphocytes contain specific receptors for GHRH that are coupled to important biological responses and further support the concept of bidirectional communication between the immune and neuroendocrine tissues.     

Prolactin and somatotropin binding by granulosa cells of cows at different reproductive states

Specific binding of bovine prolactin and somatotropin by granulosa cells from the antral follicles of various diameters was studied in cows at different reproductive states, prepubertal, pubertal, and early gestation. The ability of granulosa cells to bind prolactin did not depend on the reproductive state of an animal. At the same time, the dynamics of somatotropin specific binding by granulosa cells during maturation of the antral follicles differed at dissimilar reproductive states of the cows. When the diameter of follicles increased from 3-5 to 6-10 mm, specific binding of 125I-somatotropin decreased in pubertal animals, but remained unchanged in the prepubertal and pregnant animals. The results of Scatchard analysis of the binding data suggest that sexual maturation of cows did not affect the binding of prolactin and somatotropin by granulosa cells from follicles of 1-2 mm in diameter. The data obtained suggest that the decreased sensitivity of granulosa cells to somatotropin at the terminal stages of maturation of the antral follicles is essential for their development and acquisition of the ability for ovulation.     

Prolactin modulation of the maternal-like behavior displayed by juvenile rats

Reports of elevated prolactin (Prl) levels in juvenile rats of the same strain and approximate age, together with the established role of Prl in maternal behavior in adult female rats, prompted us to examine the possible involvement of Prl in the expression of maternal-like behavior in juvenile Sprague-Dawley males and females. Experiment 1 showed that at 25 days of age both sexes exhibited a rapid onset of full maternal behavior (FMB), with males (median = 2.0 days) responding significantly more quickly than females (median = 4.0 days). Moreover, blood sampled for Prl revealed that males had significantly higher levels of circulating Prl than females, (21.0 vs 10.4 ng/ml, respectively). In Experiment 2, CB-154 treatment significantly delayed the onset of FMB in males only, causing latencies to increase to 5.0 days vs 2.0 days for Controls. Female latencies were unaffected by CB-154, 7.0 and 7.5 days for CB-154 and Control groups, respectively. A second set of both male and female juveniles was treated with either CB-154 or vehicle. CB-154 reduced Prl levels in both sexes. In the Controls, the sex difference in Prl levels (males greater than females) was again evident. In Experiment 3 juvenile males were treated with either ovine Prl (0.5 mg) + CB-154, CB-154 + Vehicle, or Vehicle + Vehicle and tested for FMB. Males treated with Prl + CB-154 required 3.0 days to exhibit FMB, significantly faster than CB-154 + Vehicle males which responded in 8.0 days. The response of Vehicle + Vehicle males was intermediate, with a latency of 5.0 days. These results provide support for the idea that Prl is involved in the maternal-like responsiveness shown by 25 day old juvenile males, but that in females a maturational factor may have prevented both heightened responsiveness to pups by 25 days of age and sensitivity to the Prl releasing mechanism(s)/Prl feedback involved in the exhibition of maternal behavior.     

Prolactin enhances uteroglobin gene expression by uteri of immature rabbits

The effect of prolactin on uteroglobin production by immature rabbits was studied with neonatal (1 day old) and juvenile (14 days old) does. The animals were divided into 11 treatment groups for each age category and exposed to a 9-day injection protocol. Each day the animals received a subcutaneous injection of oestradiol-17 beta and/or ovine prolactin and/or progesterone, or were sham-injected. Juvenile animals, which received 100 micrograms oestradiol/kg 24 h-1, plus progesterone or plus prolactin and progesterone, produced detectable amounts of uteroglobin in the uterine secretions (0.034 +/- 0.010 mg uteroglobin/mg total protein and 0.098 +/- 0.03 l mg uteroglobin/mg total protein, respectively). None of the animals in the other juvenile treatment groups or any of the neonatal groups produced uteroglobin. From this survey it was apparent that uteroglobin secretion could be induced by exogenous oestradiol and progesterone in rabbits treated as early as 14 days of age, and that the added supplementation of prolactin enhanced the response to the ovarian steroids. As a result, additional juvenile animals were injected with 100 micrograms oestradiol +/- prolactin + progesterone and the effects of these two treatments were quantitated as follows: uteroglobin mRNA levels by slot-blot hybridization; endometrial surface area by computerized image analysis; and oestrogen, progesterone and prolactin receptors by immunocytochemistry. Prolactin modified the response of the juvenile rabbit uterus to oestradiol + progesterone for all parameters tested.     

Prolactin decreases expression of Runx2, osteoprotegerin, and RANKL in primary osteoblasts derived from tibiae of adult female rats

Hyperprolactinemia caused by physiological or pathological conditions, such as those occurring during lactation and prolactinoma, respectively, results in progressive osteopenia. The underlying mechanisms, however, are controversial. Prolactin (PRL) may directly attenuate the functions of osteoblasts, since these bone cells express PRL receptors. The present study therefore aimed to investigate the effects of PRL on the expression of genes related to the osteoblast functions by using quantitative real-time PCR technique. Herein, we used primary osteoblasts that were derived from the tibiae of adult rats and displayed characteristics of differentiated osteoblasts, including in vitro mineralization. Osteoblasts exposed for 48 h to 1000 ng/mL PRL, but not to 10 or 100 ng/mL PRL, showed decreases in the mRNA expression of Runx2, osteoprotegerin (OPG), and receptor activator of nuclear factor kappaBeta ligand (RANKL) by 60.49%, 72.74%, and 87.51%, respectively. Nevertheless, PRL did not change the RANKL/OPG ratio, since expression of OPG and RANKL were proportionally decreased. These concentrations of PRL had no effect on the mRNA expression of osteocalcin and osteopontin, nor on mineralization. High pathologic concentrations of PRL (1000 ng/mL) may downregulate expression of genes that are essential for osteoblast differentiation and functions. The present results explained the clinical findings of hyperprolactinemia-induced bone loss.     

Extraglandular hormonal steroidogenesis in aged rats

We have examined the metabolism in vitro of [4-¹⁴C]pregnenolone by the following organs of 2.4-year-old rats: submandibular gland, stomach, duodenum, liver, lung, heart, spleen, kidney, skin, prostate, testis and adrenal. All tissues converted pregnenolone to progesterone, the highest yields being observed with adrenal, testis and skin. Androgen formation was intense in the testis and absent in the adrenal. Moreover, 17-hydroxylation of pregnenolone occurred moderately in kidney, skin and submandibular gland and markedly in duodenum and stomach, which also produced high amounts of dehydroepiandrosterone and/or 5-androstene-3β,17β-diol. Extratesticular synthesis of androstenedione and testosterone was very low. A significant formation of 20-dihydropregnenolone was observed in all tissues but stomach, duodenum and steroidogenic endocrines. Corticosteroids were not synthesized extraadrenally, except a small amount of 11-deoxycorticosterone in the testis. These results indicate that key steroid-biosynthetic enzymes, such as 3β-hydroxysteroid dehydrogenase/Δ⁵′Δ⁴ isomerase, 17β- and 20-hydroxysteroid dehydrogenases and steroid 17-monooxygenase/17,20-lyase are also expressed extraglandularly in the rat.     

Tissue-specific expression of hormonal carcinogenesis target genes in rats treated with polycyclic aromatic hydrocarbons

We have investigated the effect of polycyclic aromatic hydrocarbons (PAHs) on expression of the estrogen-metabolizing genes CYP1A1, CYP1B1, CYP19 and also ERα, and cyclinD1 genes, regulating cell division in estrogen-depended tissues. Treatment of rats with benzo(a)pyrene (BP) or 3-methylcholantrene (MCA) significantly up-regulated CYP1A1, CYP1B1 gene expression in liver, uterus and ovary, whereas α-naphthoflavone (α-NF) did not have any effect. The high level of aromatase gene (CYP19) expression was detected in ovary only. Treatment of rats with BP or MCA significantly down-regulated expression of this gene (15- and 5,5-fold, respectively), whereas α-NF was ineffective. Administration of BP but not MCA or α-NF increased ERα and cyclinD1 gene expression in rat liver. The levels of ERα and cyclinD1 mRNA levels remained unchanged in uterus of after treatment of rats with these PAHs. BP administration increased ERα and cyclinD1 mRNA levels (3,5- and 2,5-fold, respectively) in ovary, while MCA and α-NF were ineffective. Thus, our results give evidence for tissue-specific effects of PAHs on expression of genes, which participate in hormonal carcinogenesis. On the other hand, the fact that BP and MCA treatments influenced the expression of estrogen-metabolizing genes and genes, which control cell division, supports the viewpoint that PAHs may be one of the causes of endocrine disorders and subsequent hormonal carcinogenesis.     

Increased expression in calcitonin-like receptor induced by aldosterone in cerebral arteries from spontaneously hypertensive rats does not correlate with functional role of CGRP receptor

OBJECTIVE: To analyze the effect of aldosterone on the expression of calcitonin gene-related peptide (CGRP) receptor components, calcitonin-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1), as well as the effect of this mineralocorticoid on CGRP-mediated vasodilation in middle cerebral arteries from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was nitric oxide independent and higher in SHR middle cerebral arteries. CL receptor and RAMP1 expression were similar in both strains. The relaxation to CGRP was not modified by aldosterone 1 microM in either strain, although aldosterone 1 microM increased expression of CL receptor without modifying RAMP1 in segments from SHR rats. CONCLUSIONS: CGRP elicits greater vasodilation in middle cerebral arteries from SHR than WKY rats, that is nitric oxide independent, and by mechanism independent of CGRP receptor components expression. Although aldosterone increases the expression of CL receptor in SHR, it does not alter vasodilation to CGRP, since RAMP1 expression is not increased. These results indicate that the increase in CL receptor, without an increase in RAMP1, does not correlate with changes in functional role of the CGRP receptor.     

Mapping physiological states from microarray expression measurements

MOTIVATION: The increasing use of DNA microarrays to probe cell physiology requires methods for visualizing different expression phenotypes and explicitly connecting individual genes to discriminating expression features. Such methods should be robust and maintain biological interpretability. RESULTS: We propose a method for the mapping of the physiological state of cells and tissues from multidimensional expression data such as those obtained with DNA microarrays. The method uses Fisher discriminant analysis to create a linear projection of gene expression measurements that maximizes the separation of different sample classes. Relative to other typical classification methods, this method provides insights into the discriminating characteristics of expression measurements in terms of the contribution of individual genes to the definition of distinct physiological states. This projection method also facilitates visualization of classification results in a reduced dimensional space. Examples from four different cases demonstrate the ability of the method to produce well-separated groups in the projection space and to identify important genes for defining physiological states. The method can be augmented to also include data from the proteomic and metabolic phenotypes and can be useful in disease diagnosis, drug screening and bioprocessing applications.     

The cellular localization of the unusual estrogen-binding protein in the liver of rats under different hormonal states]

The localization of an unusual estrogen-binding protein (UEBP) was studied in the rat liver using indirect immunoperoxidase reaction in intact rats and after different hormonal influences. The distribution of the UEBR in normal males displays a form of a gradient with the maximum near the central veins. The gradient is absent in normal females. Castration or hypophysectomy of males, or their injection with estradiol or triiodyronine leads to a decrease in the UEBP concentration, though the gradient character of its distribution is persisting. There is a stable increase in the UEBP concentration in the first two layers of cells adjacent to the central veins in the female rat liver after ovariectomy and especially in combination with androgen injections. The revealed changes in the intensity of immunoperoxidase reaction after different hormonal influences correlate with the UEBP concentration in liver studies performed by the radioligand assay.     

Prolactin receptor in regulation of neuronal excitability and channels

Prolactin (PRL) activates PRL receptor isoforms to exert regulation of specific neuronal circuitries, and to control numerous physiological and clinically-relevant functions including; maternal behavior, energy balance and food intake, stress and trauma responses, anxiety, neurogenesis, migraine and pain. PRL controls these critical functions by regulating receptor potential thresholds, neuronal excitability and/or neurotransmission efficiency. PRL also influences neuronal functions via activation of certain neurons, resulting in Ca²⁺ influx and/or electrical firing with subsequent release of neurotransmitters. Although PRL was identified almost a century ago, very little specific information is known about how PRL regulates neuronal functions. Nevertheless, important initial steps have recently been made including the identification of PRL-induced transient signaling pathways in neurons and the modulation of neuronal transient receptor potential (TRP) and Ca²⁺-dependent K⁺ channels by PRL. In this review, we summarize current knowledge and recent progress in understanding the regulation of neuronal excitability and channels by PRL.