Prolactin and progesterone receptors in pregnancy-dependent mammary tumors in GR/A mice

In this study, cellular prolactin receptors and cytosolic progesterone receptors were examined and compared in pregnancy-dependent mammary tumors (PDMT) and in normal mammary glands of pregnant GR/A mice. PDMT and normal mammary glands were examined in the same animal, thus assuring an identical hormonal environment. The PDMT cells had a larger capacity to bind prolactin or the synthetic progesterone, R5020, than did the normal mammary gland. While the dissociation constant (Kd) value for prolactin binding to normal mammary epithelial cells was similar to that of PDMT cells, PDMT cells had 2.2 times more prolactin receptors than the normal cells. Progesterone binding activity was detected only in PDMT, but not in the normal mammary cells. The receptor concentration and the Kd value for progesterone binding of PDMT were 606 fmol/mg protein and 3.53 nM, respectively. It appears, therefore, that normal regulation of these receptors may be altered within the PDMT cells. The increased growth responsiveness of PDMT to the hormones of pregnancy, especially prolactin, progesterone, and placental lactogen, may be a function of a sharp increase in the level of cellular receptors for these mammotropic hormones.     

Cycloheximide induces accumulation of vasoactive intestinal peptide (VIP) binding sites at the cell surface of a human colonic adenocarcinoma cell line (HT29-D4). Evidence for the presence of an intracellular pool of VIP receptors

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.     

Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes.

When cultured human lymphocytes of the IM-9 line were exposed to human growth hormone (hGH) at 37 degrees, washed for 2 hours, and incubated with 125I-hGH, the binding of 125I-hGH was reduced. The magnitude of the reduction in binding was dependent on the concentration of growth hormone present as well as the duration of the exposure. As little as 2 X 10(-11) M (0.5 ng/ml) growth hormone had a discernible effect. Growth hormone at 2 X 10(-10) M (5.0 ng/ml), which is a low resting concentration of hormone in vivo and occupies about 20% of the receptors at steady state at 30 degrees, produced a 50% reduction in binding while 20 mg/ml, which occupies about 50% of the receptors under steady state conditions, produced an 80% loss of receptors. Further increases in growth hormone concentration produced little further effect on receptor loss. Thus, the loss of receptors at a given concentration of growth hormone (up to 20 ng/ml) in the preincubation at 37 degrees was greater than the occupancy produced by that concentration of growth hormone receptors under steady state conditions at 30 degrees. Analysis of the data indicated that the decrease in binding of 125I-hGH was due to a loss of receptors per cell without any change in affinity of receptor for hormone or in cell number. The concentration of insulin receptors on these cells was affected by the insulin concentration in the medium, and the concentration of growth hormone receptors was affected by growth hormone, but neither hormone had any effect on the heterologous receptors. Exposure of the cells to cycloheximide (0.1 mM) produced a progressive but smaller loss of growth hormone receptors, and the effect of cycloheximide was additive to the receptor loss induced by growth hormone, suggesting that cycloheximide inhibited synthesis of receptors while growth hormone accelerated loss of receptors. When growth hormone was removed from the medium, receptor concentrations were restored rapidly; half of the loss was restored by 6 to 8 hours and the full complement of receptors was restored by 24 hours following removal of the hormone. If the growth hormone was removed and replaced with cycloheximide, the return of the receptors was delayed until the cycloheximide was removed. Thus restoration of the receptors appeared to require the synthesis of new proteins. These data indicate that in the IM-9 lymphocytes the concentration of growth hormone receptors is very sensitive to regulation by growth hormone and also add further support to the suggestion that hormones in general actively regulate the concentration of their own receptors.     

Properties and functions of human placental opioid system.

Human placental villus tissue contains opioid receptors and peptides. Kappa opioid receptors (the only type present in this tissue) were purified with retention of their binding properties. The purified kappa receptor is a glycoprotein with an apparent molecular weight of 63,000. Two opioid receptor mediated functions were identified in trophoblast tissue, namely regulation of acetylcholine and hormonal (human chorionic gonadotrophin and human placental lactogen) release. Placental content of kappa receptors increases with gestational age. Term placental content of kappa receptors correlates with route of delivery (higher in those abdominally obtained). Opioid use and/or abuse during pregnancy affects placental receptor content at delivery, as well as its mediated functions. Opioid peptides identified in placental extracts were beta-endorphin, methionine enkephalin, leucine enkephalin and dynorphins 1-8 and 1-13. Dynorphin 1-8 seem to be the predominant opioid peptide present in placental villus tissue.     

Isolation and identification of a cDNA clone of rat placental lactogen II

The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans.     

Identification of a placental lactogen in pregnant Snell and Ames dwarf mice

Sera and placentas from pregnant dwarf mice contain a placental lactogen. This placental lactogen has immunological and electrophoretic properties similar to those of placental lactogen from normal mice.     

Ovine placental lactogen specifically binds to endometrial glands of the ovine uterus

A hormonal servomechanism has been proposed to regulate differentiation and function of the endometrial glandular epithelium (GE) in the ovine uterus during pregnancy. This mechanism involves sequential actions of estrogen, progesterone, ovine interferon tau (IFNtau), placental lactogen (oPL), and placental growth hormone (oGH). The biological actions of oPL in vitro are mediated by homodimerization of the prolactin receptor (oPRLR) and heterodimerization of the oPRLR and oGH receptor. The objectives of the study were to determine the effects of intrauterine oPL, oGH, and their combination on endometrial histoarchitecture and gene expression and to localize and characterize binding sites for oPL in the ovine uterus in vivo using an in situ ligand binding assay. Intrauterine infusion of oPL and/or oGH following IFNtau into ovariectomized ewes treated with progesterone daily differentially affected endometrial gland number and expression of uterine milk proteins and osteopontin. However, neither hormone affected PRLR, insulin-like growth factor (IGF)-I, or IGF-II mRNA levels in the endometrium. A chimeric protein of placental secretory alkaline phosphatase (SEAP) and oPL was used to identify and characterize binding sites for oPL in frozen sections of interplacentomal endometrium from pregnant ewes. Specific binding of SEAP-oPL was detected in the endometrial GE on Days 30, 60, 90, and 120 of pregnancy. In Day 90 endometrium, SEAP-oPL binding to the endometrial GE was displaced completely by oPL and prolactin (oPRL) but only partially by oGH. Binding experiments using the extracellular domain of the oPRLR also showed that iodinated oPL binding sites could be competed for by oPRL and oPL but not by oGH. Collectively, results indicate that oPL binds to receptors in the endometrial glands and that oPRL is more effective than oGH in competing for these binding sites. Thus, effects of oPL on the endometrial glands may be mediated by receptors for oPRL and oGH.     

Placental lactogen not growth hormone and prolactin regulates secretion of progesterone in vitro by the 40-45 day ovine corpus luteum of pregnancy

The study was designed to compare the direct effect of three prolactin-like hormones on steroidogenesis of ovine luteal cells collected at day 40-45 of pregnancy. 100 ng/ml of ovine placental lactogen or 100 ng/ml of ovine growth hormone or 100 ng/ml of ovine prolactin were added to the media of luteal cell cultures. After 48 h incubation, all cultures were terminated and the media were frozen until further steroid analysis. To determine to what extent growth hormone (GH), prolactin (PRL) and lactogen (PL) regulate the activity of 3 beta-HSD, an enzyme involved in progesterone synthesis, the classical steroidal competitive inhibitor of 3 beta-HSD trilostane, was investigated for its effects on basal and GH-, PRL-, and PL-stimulated progesterone biosynthesis since there is a possibility that the luteotropic effect of these hormones are mediated via 3 beta-HSD. oPL resulted in an increase of progesterone secretion in a statistically significant manner, while GH or PRL had no effect on progesterone secretion. A decrease in progesterone secretion as an effect of 100 mM trilostane was observed in all culture types. An explanation for the luteotropic effect of PL and the lack of this effect for GH is that the GH receptor associates with a different molecule within the ovarian tissue and forms a heterodimeric receptor for PL, and the possibility that physiological effects of native oPL may be mediated through its binding to specific PL receptors, which have low affinities for oGH and oPRL.     

Placental Lactogen Levels in Diabetic Pregnancy

A prospective study has been carried out of placental lactogen levels in pregnancy complicated by diabetes mellitus. The levels were higher than those in normal pregnant subjects; the higher levels were related to increased placental and fetal weight but more closely to the former; and lower levels were found when there was clinical evidence of placental dysfunction. Those patients requiring the largest insulin increment for the control of their diabetes in the pregnancy have placental lactogen levels in the higher range.     

Induction of mammary prolactin receptors and lactose synthesis after ovariectomy in the pregnant mouse.

The development of prolactin receptors in the mammary gland after ovariectomy was investigated in pregnant KA mice. Mice were ovariectomized on day 13 of pregnancy and used for the determination of the amount of specific binding of 125I-labelled prolactin to the mammary tissue, and the contents of lactose and nucleic acids in the mammary gland 0, 8, 24, and 72 hr after the operation. The specific binding of 125I-labelled prolactin, lactose and RNA contents in the mammary gland remained low until 8 hr, sharply increased 24 hr and decreased 72 hr after ovariectomy. When ovariectomized mice were treated with 0.2 mg progesterone, pregnancy was maintained and an increase (1.5-fold) in the amount of specific binding was observed with an increase of lactose content. Five mg progesterone completely inhibited lactose synthesis. Cortisol administered with progesterone did not show any specific change at the dose used (0.5 to 10 mg). Although the amount of specific binding was also increased after hysterectomy, this increase (2-fold) did not fully cover the increase after ovariectomy (3-fold). These results suggest that the recepter site for prolactin is induced before the initiation of lactose synthesis caused by ovariectomy during pregnancy.     

Alterations in the synthesis of a fibroblast surface associated 35 K protein modulates the binding of somatomedin-C/insulin-like growth factor I

Human fibroblasts, a cell type that is used extensively to determine the pleiotypic effects of the insulin-like growth factors, have been shown to secrete a 35K protein that binds somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) but not insulin. This 35 K protein is associated with the fibroblast surface and following transfer to the surface of cell types that do not have this protein on their surfaces, it alters the binding of radiolabeled Sm-C/IGF-I. In this study human fibroblast monolayers that were incubated with cyclohexamide (50 micrograms/ml) for 14 h at 37 C had no detectable 35 K protein on their cell surface, but type I Sm-C/IGF-I receptors were still present. Loss of the 35 K protein was associated with 60-70% increase in binding of Sm-C/IGF-I to type I receptors. The relative affinity of the type I receptor for Sm-C/IGF-I was apparently increased because unlabeled Sm-C/IGF-I (12 ng/ml) competitively displaced 63% of radiolabeled Sm-C/IGF-I after cycloheximide exposure, whereas in cultures not exposed to cycloheximide [125I]Sm-C/IGF-I binding was increased by 11%. Coincubation of fibroblast conditioned media containing the 35 K protein with cycloheximide-treated fibroblast monolayers resulted in restoration of the paradoxical increase in Sm-C/IGF-I binding and loss of sensitivity to competition by unlabeled Sm-C/IGF-I. Exposure of suspended fibroblasts, which do not have 35 K on their cell surface, to media conditioned by fibroblast monolayers also induced both of these changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine placental lactogen stimulates DNA synthesis of bovine mammary tissue maintained in athymic nude mice

Mammary tissue from five midpregnant heifers was transplanted subcutaneously into ovariectomized athymic mice (eight pieces/mouse). After a recovery period of 19 days, mice were injected daily for 5 days with buffer (50 mM NH4HCO3, pH 7.8) as control, 17 beta-estradiol (1 micrograms) plus progesterone (1 mg). Concurrently with the buffer or steroid hormone injections, mice were injected with bovine placental lactogen (0, 5, or 25 micrograms), bovine prolactin (0, 3.4, or 17.2 micrograms), or bovine growth hormone (0, 3.4, or 17.2 micrograms). All mice were injected with 2-bromo-alpha-ergocryptine (0.1 mg/day). Transplanted bovine mammary tissue was incubated for 4 hr in minimum essential medium containing 1 mu Ci/ml [3H]TdR. Two pieces were processed for autoradiography and the others were used for DNA assay and total [3H]TdR uptake. Bovine placental lactogen, prolactin, and growth hormone each increased [3H]TdR incorporation into DNA in a linear, dose-response manner. Addition of ovarian steroids to bPL resulted in a significant increase over protein hormones alone. Autoradiographic analysis indicated that the observed differences in DNA synthesis were due to hormonal effects on epithelial, rather than stromal, DNA synthesis. These results provide the first evidence of a mammogenic role of bovine placental lactogen.     

Alteration in the receptor binding specificity of human growth hormone by genomic exon exchange

The biological activities of the GH-PRL family of hormones are mediated by selective binding to two classes of cell membrane receptors, somatogen and lactogen. Primate GH such as human GH (hGH) are unusual in that they bind to both classes of receptors. Replacement of exons 3 or 4 of the hGH gene by the corresponding exons of the rat PRL or rat GH genes results in the synthesis of chimeric proteins which retain the ability to bind to lactogen receptors but can no longer bind to somatogen receptors. This selective loss of somatogen receptor binding in the chimeric proteins suggests that certain of the structural determinants of somatogen and lactogen receptor binding activities in hGH are distinct and can be separately modified by a limited number of amino acid substitutions.     

Evidence for nuclear processing of progesterone receptors in rat placenta

The distribution of the progesterone receptor (Rp) in cytosolic and nuclear compartments of placenta has been studied in intact and ovariectomized (Ovx) rats on the 14th day of pregnancy. Removal of estradiol (E) and progesterone (P) by Ovx caused a 50% decrease in progesterone receptors from cytosolic and nuclear compartments. Estradiol replacement restored binding to intact levels. Progesterone, given 19 h after E, induced an additional 3-fold increment in the number of cytosolic and nuclear binding sites 1 h later. Four hours after progesterone the number of receptor sites in the placenta fell 60%, signifying processing. This was followed 4 h later by reversal of processing mechanisms leading to full recovery of nuclear and cytoplasmic binding sites. Actinomycin D (0.6 mg/ani) was found to have no influence on these events. On the other hand cycloheximide (0.5 mg/ani) completely prevented processing of binding sites when administered at the same time as progesterone or 2 h before, but did not influence the unmasking of nuclear sites which occurred 1 h after a progesterone challenge. The cycloheximide block to processing was partial when given 2 or 3 h after progesterone (61 and 43% complete, respectively). The full complement of receptors was processed when cycloheximide treatment was delayed 3.75 h after progesterone administration. These findings have led to the view that processing represents rapid and reversible changes in binding properties of the receptor rather than a gain or loss of receptor protein per se. The findings of this study suggest that a hypothetical substance, "processin", whose production is blocked by cycloheximide binds to the receptor and in some undefined manner inhibits ligand-receptor interaction within 4 h after an in vivo progesterone challenge. Nuclear accumulation of receptor induced by progesterone was not accompanied by cytoplasmic depletion of receptor nor was the apparent loss of processed nuclear receptor due to recycling of receptor to cytoplasm. We propose that nuclear receptors continually recycle within the nucleus in masked and unmasked states regulated by delicate interplay between progesterone and processin.     

VPAC1 and VPAC2 receptor deficiencies negatively influence pregnancy outcome through distinct and overlapping modulations of immune,trophoblast and vascular functions

Pregnancy outcome relies on the maintenance of immune and metabolic homeostasis at the maternal fetal interface. Maternal and perinatal morbidity and mortality is associated with impaired placental development. Multiple regulatory effects of the endogenous-produced vasoactive intestinal peptide (VIP) on vascular, metabolic and immune functions at the maternal-fetal interface have been reported. Here we studied the involvement of the two primary high affinity receptors for VIP (VPAC1 and VPAC2) on maternal immune response, placental homeostasis and pregnancy outcome. Targeted disruption of each receptor gene led to altered placental structure, vascular and trophoblast functional markers and shaped the functional profiles of macrophages and neutrophils towards a proinflammatory state. Several changes in pregnant mice were receptor specific: ROS production elicited by VIP on neutrophils was selectively dependent on the presence of VPAC1 whereas apoptosis rate was associated with the VPAC2 deletion. In peritoneal macrophages from pregnant mice, levels of MHC-II, TLR2, and IL-10 were selectively altered in VPAC2 receptor-deficient mice, whereas IL-6 gene expression was reduced only in mice lacking VPAC1 receptors. Additionally, MMP9 mRNA in isolated TGCs was reduced in VPAC2 receptor deleted mice, while the percentage of IL-12 cells in post-phagocytosis macrophage cultures was selectively reduced in VPAC2 receptor deficient mice. The results indicate that manipulation of VPAC1 and VPAC2 receptor affects immune, vascular and metabolic environment at the maternal fetal interface. These mouse models offer new approaches to study pregnancy complications adding new perspectives to the development of VPAC receptor-selective drugs.     

Effect of lectins on the cobalamin-protein binding reactions: implications for the tissue uptake of cobalamin

Plant lectins have been thought to impair nutrient absorption, both by specific and nonspecific interference in the absorptive process. The main objective of this investigation was to study the effect of lectins on the various binding reactions involving cobalamin (cbl)-protein complexes and their receptors, and to identify the rate-limiting step important in maintaining tissue levels of cobalamin. Among the lectins tested in vivo, only concanavalin A (ConA) was able to inhibit the transport of cobalamin to the tissues and caused a 70% to 75% inhibition of [(57)Co] cobalamin transported to the liver and kidney. The inhibition of transport to the tissues was independent of route of administration of cobalamin, whether intragastric or systemic, and was not due to decreased gastrointestinal uptake. When tested in vitro, concanavalin A inhibited the binding of transcobalamin II-cbl to its receptor, but not the binding of cobalamin to intrinsic factor or intrinsic factor-cobalamin complex to the ileal receptor. These results suggest that late events during transcellular transport of cobalamin through the enterocytes is the rate-limiting step determining tissue levels of cobalamin and that ConA inhibits these latter events.     

A receptor site for prolactin in lactating mouse mammary tissues.

Prolactin iodinated by lactoperoxidase method showed immunologically, electrophoretically and biolo9gically similar properties to native prolactin and possessed enough specific radioactivity for receptor studies. 1251-prolactin was incubated with mouse mammary tissues at 8 days of lactation. Both binding and release of 1251-prolactin depended on incubation time and temperature and were maximal at 37 degrees C. Michaelis constant was estimated to be 1.4 X 10(-9) M from Lineweaver-Burk plot and to be 1.2 X 10(-9) M from id-value of the dose-response curve for displacement with native prolactin. Total number of binding sites for prolactin was 1.38 X 10(-15) mole per mg weight of tissue. Ovine prolactin, human growth hormone and human placental lactogen complete with 1251-prolactin and dose-response curves for these three hormones were all parallel. These results suggest the existence of a specific receptor site with high affinity for prolactin in lactating mouse mammary glands.     

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen.

The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.     

The size of the corpus luteum during pseudopregnancy and pregnancy in the rat.

The corpus luteum in mature Sprague Dawley rats was weighted at the various stages of pseudopregnancy and pregancy. The average size of these corpora lutea was 1.0 +/- 0.10 mg, 1.61 +/- 0.69 mg, 1.90 +/- 0.25 mg, 3.69 +/- 0.36 mg, and 4.37 +/- 0.50 mg on day 2 of diestrus, on days 10-15 of psuedopregnancy, on days 9-10, 14, and 20 of pregnancy, respectively. The fact that the average size of the corpus luteum on days 10-15 of pseudopregnancy was larger than that on day 2 of diestrus is thought to drive from prolonged exposure of the corpus luteum to prolactin. The average size of the corpus luteum on days 9-10 of pregnancy had a tendency to be larger than that on days 10-15 of pseudopregnancy and this seems to demonstrate that the placenta secreted placental lactogen by this stage of pregnancy. The average size of the corpus luteum on day 14 of pregnancy was larger than that on days 9-10 of pregnancy. This phenomenon might be attributed to the presence of large amounts of placental lactogen secreted from the placenta between days 10 and 14 of pregnancy. Furthermore, it was noted that the size of the corpus luteum on day 20 of pregnancy was larger than that of day 14, which suggests that further secretion of placental lactogen continued after day 14 of pregnancy. As there was a remarkable decrease in the number of fetuses on day 20 of pregnancy when overiectomy was performed on day 14 of pregnancy, the ovary was considered indispensable in maintaining pregnancy in the rat.