RESUMEN
Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.
Asunto(s)
Encefalina Metionina/análogos & derivados , Encefalinas/metabolismo , Péptidos Opioides/farmacología , Útero/efectos de los fármacos , Útero/crecimiento & desarrollo , Envejecimiento/metabolismo , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Dinorfinas/farmacología , Endorfinas/farmacología , Encefalina Metionina/antagonistas & inhibidores , Encefalina Metionina/farmacología , Encefalinas/farmacología , Factor de Crecimiento Epidérmico/farmacología , Estradiol/metabolismo , Femenino , Concentración 50 Inhibidora , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Ovariectomía , Ratas , Ratas Endogámicas , Ratas Wistar , Útero/metabolismoRESUMEN
The aim of these experiments was to investigate the expression of cyclin D1 and of oestradiol receptors as well as the level of [(3)H]oestradiol binding in leiomyoma and adjacent myometrium from human uteri at different menstrual phases and at an early stage of menopause. [(3)H]oestradiol binding was determined by saturation analysis, while the oestradiol receptor (ER) alpha and beta and cyclin D1 levels were determined by Western blot analysis of 16 samples of human leiomyomas and corresponding myometria at different hormonal stages. In leiomyomas during all phases of the menstrual cycle, ERalpha expression, high affinity oestradiol binding and cyclin D1 expression were all elevated in comparison with adjacent myometrium. ERbeta expression and low affinity oestradiol binding were enhanced in leiomyomas only during the proliferative phase. During menopause, ERbeta expression and low affinity binding were enhanced in leiomyomas, while the ERalpha expression was not significantly enhanced and cyclin D1 levels were similar to that in myometrium. Only the oestradiol binding exhibited any menstrual cycle-related changes. Our data suggest the involvement of cyclin D1 in the growth of leiomyomas during the menstrual cycle. In menopause, there appears to be a switch from ERalpha to ERbeta expression in leiomyomas, and the induction of cyclin D1 is decreased. The regression of tumour may ensue from these changes at menopause.
Asunto(s)
Ciclina D1/metabolismo , Leiomioma/metabolismo , Miometrio/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias Uterinas/metabolismo , Adulto , Estradiol/metabolismo , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Femenino , Humanos , Leiomioma/patología , Menopausia , Ciclo Menstrual/fisiología , Persona de Mediana Edad , Neoplasias Uterinas/patologíaRESUMEN
Endogenous opioid peptides are negative regulators of estradiol-induced uterine cell proliferation. To investigate the possible molecular target site(s) of their anti-mitogenic action, we examined the effect of opioid peptides on epidermal growth factor-induced cell proliferation both in uterine primary cell cultures prepared from adult rats and in human myometrial smooth muscle cell lines. Epidermal growth factor (EGF) significantly increased cell density in both types of cultured monolayers. This EGF-induced stimulation of cell proliferation was blocked by [D-Met(2)-Pro(5)]enkephalinamide in a time-dependent, receptor-mediated manner. The effective concentrations were within the physiological nanomolar range. Enkephalinamide did not have any effect on the basal rate of proliferation of the uterine cells. Our results on this novel physiological cross-talk suggest that shared step(s) of the mechanism of action of estradiol and EGF might be targeted by opioid peptides and not the general machinery of cell proliferation.
Asunto(s)
Encefalina Metionina/análogos & derivados , Factor de Crecimiento Epidérmico/farmacología , Miometrio/efectos de los fármacos , Péptidos Opioides/farmacología , Receptores Opioides/efectos de los fármacos , Analgésicos/farmacología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Encefalina Metionina/farmacología , Femenino , Humanos , Miometrio/citología , Miometrio/fisiología , Ratas , Receptores Opioides/fisiología , Útero/citología , Útero/efectos de los fármacos , Útero/fisiologíaRESUMEN
The effects of a single injection or continuous infusion of opioid peptide, [D-Met(2),pro(5)]enkephalinamide (ENK) on the hormone binding and transcriptional properties of estrogen receptors were investigated in estradiol (E(2)) treated rat uterus. The level of estrogen- (ER) and progesterone receptor (PR) proteins, the hormone binding of E(2) receptors and the effects of single injection of ENK with or without naltrexone (NAL) on the E(2)-induced changes in the level of Fos and Jun proteins and the binding of AP-1 proteins to DNA were studied. The receptor proteins levels were determined by Western blots and the binding of AP-1 to DNA by electrophoretic mobility shift assay. Both the ER and PR protein concentrations and the [3H]Estradiol binding to the high affinity nuclear receptors decreased after ENK treatment during the first two days. At 72 h the PR concentration decreased further, while no significant changes were found in the level of ER, however, at this time the former competitive E(2) binding turned into positive cooperativity. The E(2)-induced increase in the level of Fos proteins and the binding of AP-1 proteins to DNA was inhibited by a single injection of ENK. We conclude that the endogenous opioid peptides may interact with E(2) in the gene regulation of rat uterus.
Asunto(s)
Encefalina Metionina/análogos & derivados , Encefalina Metionina/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Péptidos Opioides/farmacología , Útero/efectos de los fármacos , Animales , ADN/genética , ADN/metabolismo , Encefalina Metionina/administración & dosificación , Estradiol/metabolismo , Estradiol/farmacología , Moduladores de los Receptores de Estrógeno/administración & dosificación , Femenino , Péptidos Opioides/administración & dosificación , Péptidos Opioides/agonistas , Ovariectomía , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Ratas Endogámicas , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Factor de Transcripción AP-1/metabolismo , Útero/metabolismoRESUMEN
The aim of the present experiment was to investigate the effect of [D-Met2,Pro5] enkephalinamide (ENK) implantation on the development of the uterus during 8-33 days of age and the involvement of epidermal growth factor (EGF) in the effect. Administration of ENK was attained by osmotic minipumps (5 microg/h) implanted intraperitoneally. ENK resulted in a decrease in the EGF content of the uterus, which was already significant after 48 h of the implantation. The DNA content 24 and 48 h after the treatment decreased, no change at 72 h was found, however the protein/DNA ratio on the effect of ENK treatment was significantly decreased at this time in all examined age groups. High affinity and lower capacity competitive naloxone binding sites were demonstrated in the membrane fraction of the uteri. Seventy-two h after ENK treatment the binding capacity of these sites significantly dropped. The present results suggest a novel multiple interaction between estrogen and two probably paracrine hormones, EGF and opioid peptide, in the regulation of growth and development of the uterus.
Asunto(s)
Encefalina Metionina/análogos & derivados , Encefalina Metionina/farmacología , Factor de Crecimiento Epidérmico/fisiología , Útero/efectos de los fármacos , Útero/crecimiento & desarrollo , Animales , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Implantes de Medicamentos , Encefalina Metionina/administración & dosificación , Femenino , Cinética , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Ovariectomía , Ratas , Útero/metabolismoRESUMEN
The present studies demonstrate, for the first time, that the binding of activator protein-1 (AP-1)-DNA in rat uterus and the estrogen-sensitive areas of the hypothalamus, as measured by electrophoretic mobility shift assay, is increased 2 h after intraperitoneal injection of [D-Met(2),Pro(5)]enkephalinamide. The effect was prevented by the opiate antagonist naltrexone given 30 min before the administration of [D-Met(2),Pro(5)]enkephalinamide, suggesting the involvement of opioid peptide receptors in the observed effects. The present findings support the role of opioid peptides in the regulation of transcription in estrogen-sensitive cells.
Asunto(s)
ADN/metabolismo , Hipotálamo/metabolismo , Péptidos Opioides/farmacología , Factor de Transcripción AP-1/metabolismo , Útero/metabolismo , Análisis de Varianza , Animales , Unión Competitiva/efectos de los fármacos , Estrógenos/farmacología , Femenino , Hipotálamo/efectos de los fármacos , Ratas , Útero/efectos de los fármacosRESUMEN
The effect of opioid peptides on cultured, oestradiol-stimulated human myometrial cells was examined. Oestradiol increased cell densities in mixed-cell (smooth muscle cells + stromal fibroblasts) cultures by 40%. This oestradiol-induced stimulation of cell proliferation was decreased to control values by D-met2-pro5-enkephalinamide. The half-effective inhibitory concentration of enkephalinamide was 0.3 nmol/l. The opioid-induced inhibition of cell proliferation was blocked completely by the specific opiate receptor antagonist naloxone, while naloxone did not have any effect on its own. This opioid effect was mediated dominantly by the mu opiate receptor. The optimal concentration for oestradiol to stimulate uterine cell proliferation was 2.2 nM. The basal rate of cell proliferation was not affected by enkephalinamide. In saturation experiments, the parameters of specific [3H]-naloxone binding were: dissociation constant = 1.02 nM, maximal binding capacity = 2910 binding sites/cell, Hill coefficient = 1.029. In human myometrial pure smooth muscle cell cultures, oestradiol decreased the proliferation of cells. Progesterone potentiated these oestradiol effects, but had no effect on its own. Enkephalinamide was also able to block the effects of oestradiol, but naloxone did not antagonize it. In summary, here we present a novel inhibitory role of endogenous opioid peptides in the regulation of cell growth and proliferation in the human uterus.
Asunto(s)
Encefalinas/metabolismo , Estradiol/metabolismo , Miometrio/metabolismo , Adulto , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Dinorfinas/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina D-Penicilamina (2,5) , Encefalina Metionina/análogos & derivados , Encefalina Metionina/farmacología , Encefalinas/farmacología , Femenino , Humanos , Concentración 50 Inhibidora , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Miometrio/citología , Miometrio/efectos de los fármacos , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Progesterona/farmacologíaRESUMEN
The effect of opioid peptides on estradiol-induced cell proliferation in adult rat uterine primary cell cultures was studied. Estradiol increased cell density by 40%. This estradiol-induced stimulation of cell proliferation was decreased to control values by [D-Met2,Pro5]enkephalinamide. The opioid-induced inhibition of uterine cell proliferation was blocked completely by the specific opiate antagonist naloxone, while naloxone did not have any effect on its own. The inhibition of cell proliferation by enkephalinamide was apparent at each stimulatory estradiol concentration examined. This opioid effect was mediated mainly by the mu opiate receptor. The observed effects occurred within the physiological nanomolar concentration range. Enkephalinamide did not have any effect on the basal proliferation rate of adult rat uterine cells. However, enkephalinamide inhibited the basal rate of cell proliferation in cell cultures prepared from 7-day-old immature rats. In summary, here we present evidence of novel physiological direct cross-talk between the opioid and estrogenic signaling systems in the regulation of normal uterine growth.
Asunto(s)
Encefalina Metionina/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Útero/efectos de los fármacos , Factores de Edad , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Encefalina Metionina/antagonistas & inhibidores , Encefalina Metionina/farmacología , Femenino , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Ratas , Útero/metabolismoRESUMEN
The present studies demonstrate, for the first time, that the rate of DNA synthesis in rat uterus of 21-32 days of age is inhibited by opioid peptides [D-Met2, Pro5]enkephalinamide. At around the time of vaginal opening (approximately 33 days) the opioids failed to act. High-affinity nuclear [3H]naloxone binding sites with linear Scatchard plots were detected in the uteri during the opioid-sensitive periods of DNA synthesis. Characteristics of these binding sites and the opioid sensitivity of uterine DNA synthesis are dependent on the age of the animals, the level of circulatory oestradiol and/or the maturity of the nuclear oestrogen receptor system.
Asunto(s)
Encefalina Metionina/análogos & derivados , Maduración Sexual , Útero/efectos de los fármacos , Envejecimiento/fisiología , Análisis de Varianza , Animales , División Celular/efectos de los fármacos , Núcleo Celular/metabolismo , ADN/biosíntesis , Encefalina Metionina/farmacología , Estradiol/sangre , Estradiol/metabolismo , Estradiol/farmacología , Femenino , Cinética , Naloxona/metabolismo , Naloxona/farmacología , Ovariectomía , Ratas , Ratas Endogámicas , Receptores de Estradiol/metabolismo , Receptores Opioides/metabolismo , Útero/citología , Útero/crecimiento & desarrolloRESUMEN
Human myometrial smooth muscle cells contain receptors for human chorionic gonadotropin (hCG)/luteinizing hormone (LH). Exogenous hCG and LH can cause a modest hyperplasia in myometrial smooth muscle cells in culture. This response is lost after about the third subculture of the cells. The present study investigated whether the loss of hCG response could be restored by co-culturing with human follicle stimulating hormone (FSH). The results showed that co-culturing with FSH can indeed restore a modest mitogenic response of hCG. However, FSH alone was not mitogenic. The FSH restoration of hCG response can be blocked by antibodies to FSH or hCG but not by non-specific rabbit IgG. The FSH treatment resulted in an increase of steady state levels of hCG/LH receptor mRNA and protein in myometrial smooth muscle cells. Since the FSH actions could be receptor mediated, we investigated the presence of FSH receptor mRNA transcripts and protein in freshly dispersed myometrial smooth muscle cells. Northern blotting demonstrated that myometrial smooth muscle cells, just as rat ovary, a classical target of FSH action, contain multiple FSH receptor mRNA transcripts. Western immunoblotting demonstrated that myometrial smooth muscle cells also contain a 60 kDA FSH receptor protein just as rat ovary and human granulosa cells used as positive control tissues. The immunocytochemistry also demonstrated that myometrial smooth muscle cells, as rat ovary and human granulosa cells, contain FSH receptor immunostaining. In summary, it is novel that FSH could restore the mitogenic response of hCG in human myometrial smooth muscle cells and these cells contain FSH receptors. These findings may have functional implications for direct regulation of human myometrium not only by hCG/LH but also by FSH.
Asunto(s)
Gonadotropina Coriónica/farmacología , Hormona Folículo Estimulante/farmacología , Miometrio/efectos de los fármacos , Receptores de HFE/metabolismo , Animales , Anticuerpos/farmacología , Recuento de Células , División Celular , Gonadotropina Coriónica/antagonistas & inhibidores , Femenino , Hormona Folículo Estimulante/antagonistas & inhibidores , Humanos , Músculo Liso/citología , Miometrio/citología , ARN Mensajero/análisis , Ratas , Receptores de HFE/genética , Receptores de HL/metabolismo , PorcinosRESUMEN
High affinity (Kd approximately 1 nM) and low capacity [3H]naloxone binding sites were detected in the nuclear fraction of rat hypothalamus. The profile of this binding changed with age. In immature rats from 11 days of age until vaginal opening (approximately 33 day) the Scatchard plots of saturation data were linear and the Hill coefficient was 1, while just after vaginal opening (< 6 h) Scatchard analysis of [3H]naloxone binding gave a curvilinear component, with the Hill coefficient approximately 2, followed by an increase in binding sites and a decrease in Kd. In adults, after ovariectomy, the pattern of [3H]naloxone binding was similar to that observed in immature rats before vaginal opening. Following oestradiol treatment, binding sites with linear Scatchard plots disappeared and returned at least 24 h after hormone administration.
Asunto(s)
Hipotálamo/metabolismo , Naloxona/metabolismo , Antagonistas de Narcóticos/metabolismo , Receptores Opioides/metabolismo , Animales , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Estradiol/farmacología , Femenino , Hipotálamo/efectos de los fármacos , Hipotálamo/crecimiento & desarrollo , Cinética , Ovariectomía , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacosRESUMEN
The effects of a single dose of naloxone and of [D-Met2,Pro5]enkephalinamide on the DNA synthesis in the uterus of 7, 14 and 21-day-old rat were studied. After [D-Met2,Pro5]enkephalinamide treatment, an age-dependent decrease in in vitro [3H]thymidine incorporation into DNA was observed in all studied age groups. In the 21-day-old age group a reduced rate of DNA synthesis was detected for 12 h after [D-Met2,Pro5]enkephalinamide treatment followed by the return to control values at 24 h. The rate of inhibition was more marked in the younger age groups. The effect was also more pronounced in younger animals. Specific [3H]naloxone binding was detected both in membrane and nuclear fractions of uterine homogenates. While no age-related changes in binding affinities were found, the number of binding sites varied characteristically during development. Our data suggest the novel involvement of opioid peptides and their receptors in the regulation of uterine development.
Asunto(s)
ADN/biosíntesis , Péptidos Opioides/farmacología , Útero/metabolismo , Factores de Edad , Animales , Unión Competitiva , Relación Dosis-Respuesta a Droga , Femenino , Naloxona/farmacología , Ensayo de Unión Radioligante , RatasRESUMEN
The present study investigated the mechanisms involved in the mitogenic action of epidermal growth factor (EGF) in cultured human myometrial smooth muscle cells. The cells contained EGF/transforming growth factor-alpha (TGF-alpha) receptors as well as EGF and TGF-alpha mRNA transcripts and the corresponding proteins. Culturing with human EGF resulted in concentration- and time-dependent increases in cell density. The maximal increase was seen at 1 nM followed by a decrease to control levels at 100 nM EGF. The EGF increased cell density from 4 to 8 days followed by a plateau coinciding with the cells reaching confluence. EGF treatment concomitantly decreased the average size of cells. TGF-alpha mimicked EGF and there was no synergism between the two, suggesting a common mechanism of action. Although the presence of 10% fetal bovine serum enhanced overall cell growth, it was not required for EGF and TGF-alpha action. The receptor antibody, which is directed against the extracellular domain and can inhibit ligand binding to the receptors, dramatically inhibited the basal cell growth and exogenous EGF reversed the antibody effect. While TGF-alpha antibody was only marginally effective, EGF antibody had no effect on basal cell growth. Lavendustin (a tyrosine kinase inhibitor), calphostin (a protein kinase C inhibitor), but not H-89 (a protein kinase A inhibitor), inhibited EGF action. Indomethacin, a cyclo-oxygenase inhibitor, completely inhibited, whereas nordihydroguaiaretic acid, a lipoxygenase inhibitor, slightly inhibited EGF action. While estradiol-17 beta modestly inhibited basal as well as EGF-stimulated myometrial smooth muscle cell density, progesterone had no effect. In summary, mitogenic action of EGF in human myometrial smooth muscle cells does not require serum components and it involves tyrosine kinase and protein kinase C signaling and eicosanoids from the cyclooxygenase pathway of arachidonic acid metabolism.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Miometrio/citología , Miometrio/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Receptores ErbB/inmunología , Estradiol/farmacología , Femenino , Humanos , Indometacina/farmacología , Masoprocol/farmacología , Naftalenos/farmacología , Fenoles/farmacología , Progesterona/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Factor de Crecimiento Transformador alfa/inmunología , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
Human myometrium contains receptors for hCG/human LH (hLH). This suggested the possibility that hCG and hLH might regulate human myometrium, which has not previously been considered a direct target of gonadotropin regulation. To investigate such a possibility, highly pure and viable smooth muscle cells were isolated from nonpregnant human myometrium and cultured as monolayers. The cells contained hCG/LH receptor mRNA transcripts and a 50-kDa immunoreactive protein that can bind 125I-hCG in a ligand-specific manner. The presence of hCG during culture resulted in a significant increase of myometrial smooth muscle cell density. The hCG effect was time- and concentration-dependent and was mimicked by hLH but not by human FSH or human FSH or human thyroid-stimulating hormone. Human CG also greatly increased the size of a subpopulation of myometrial smooth muscle cells without affecting their chromosomal ploidy. Antibodies to hCG/LH receptors and hCG blocked hCG effects. Human prolactin and growth hormone, which do not bind to hCG/LH receptors, also increased the myometrial smooth muscle cell density. A protein kinase A inhibitor (H-89) blocked hCG response whereas calphostin (a protein kinase C inhibitor) and lavendustin A (a tyrosine kinase inhibitor) had no effect on hCG response, suggesting that a cAMP/protein kinase A signaling mechanism is involved in hCG action. Eicosanoids from cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism are probably not involved, because the inhibitors of these enzymes had no effect on hCG response. While progesterone and estradiol could not mimic or modify hCG action, epidermal growth factor did mimic hCG in increasing myometrial smooth muscle cell density.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Gonadotropina Coriónica/farmacología , Miometrio/efectos de los fármacos , Animales , Recuento de Células , División Celular/efectos de los fármacos , Células Cultivadas , Gonadotropina Coriónica/metabolismo , Eicosanoides/fisiología , Factor de Crecimiento Epidérmico/fisiología , Estradiol/farmacología , Femenino , Humanos , Cinética , Hormona Luteinizante/farmacología , Miometrio/citología , Miometrio/metabolismo , ARN Mensajero/metabolismo , Ratas , Receptores de HL/genética , Receptores de HL/metabolismo , Transducción de Señal , Tirotropina/farmacologíaRESUMEN
LH from anterior pituitary and hCG from placenta bind to a common receptor in gonadal and nongonadal reproductive tissues. There have been numerous examples suggesting that the brain may also contain hCG/LH receptors, yet there has been no evidence for their existence so far. We now demonstrate by reverse transcription-nested polymerase chain reaction and northern blotting that the rat brain contains hCG/LH receptor mRNA. A major receptor transcript of 2.6 kilobases and minor transcripts of 1.8 and 4.4 kilobases were found. Western immunoblotting, ligand blotting, and covalent receptor cross-linking studies have shown that rat brain also contains an 80-kilodalton receptor protein that can bind hCG and hLH, but not hFSH. Rat testis used as a positive control showed a higher abundance of multiple transcripts and an 80-kilodalton receptor protein that can bind [125I]hCG. Rat liver used as a negative control did not contain any receptor transcripts or protein. In situ hybridization, dot blotting, immunocytochemistry, and topical autoradiography have revealed that hCG/LH receptors are present in rat hippocampus; dentate gyrus; hypothalamus; cerebellum; choroid plexus; ependymal cells of third, fourth, and lateral ventricles; cortex; brainstem; bovine hypothalamus; and human area postrema. These novel findings could potentially explain numerous previous observations and suggest new possibilities concerning the regulation of brain functions by hCG and LH.
Asunto(s)
Encéfalo/metabolismo , Expresión Génica , Receptores de HL/genética , Animales , Northern Blotting , Western Blotting , Gonadotropina Coriónica/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Hormona Luteinizante/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de HL/metabolismo , Distribución TisularRESUMEN
Type II nuclear-estrogen binding sites (type II EBS) have been postulated to play a crucial regulatory role in estrogen-stimulated uterine growth. The pathogenesis of endometrial cancer is known to be connected to estrogens. However, there are no data on type II EBS in human endometrial cancer. Therefore, we investigated the presence of nuclear type II EBS and nuclear type I estrogen receptors (ER) by radioligand-binding assays in endometrial tumorous tissues (n = 135). Determination of cytoplasmic ER and progesterone receptor (PR) concentrations were also included in the study. Estradiol (E2) and progesterone (P) hormone levels in sera of patients were determined by RIAs. In addition, all data were analyzed on 5-year survival rates. Saturation analyses of 3H-E2 binding in crude nuclear pellets (n = 5) showed two types of estradiol binding: type I ER having high affinity and low capacity and type II EBS binding estradiol with lower affinity and high capacity in a positive cooperative fashion. The nuclear type II EBS and type I ER concentrations were significantly higher in cancers of increasing grade. In contrast, a significant decrease of cytoplasmic 3H-E2 binding was detected. Cytoplasmic PR binding capacities were high in G1 and G2, but low in G3 tumors. The 5-year survival data showed nuclear type I ER to have the best correlation for prognostic value (cut-off, 0.5 pmol/mg DNA), while type II EBS had no significant impact on it. Serum E2 concentrations decreased with tumors of higher grade, but were generally still higher than the control values. The serum P levels did not alter. None of the parameters investigated here differed from control values in adenoacanthoma (n = 6), suggesting a separate pathomechanism. As type II EBS are known to be stimulated by E2 under conditions that cause uterine hyperplasia, we concluded that the higher "runaway" nuclear type II EBS levels, in contrast with the lower serum estradiol concentrations, might be connected with the strong proliferative and invasive character of higher grade endometrial adenocarcinomas. This is the first demonstration of the presence of nuclear type II EBS and their possible pathological role in human endometrial cancer, and of the high prognostic significance of nuclear type I ER to identify a subgroup having a fatal form of the disease in a 5-year survival study.
Asunto(s)
Neoplasias Endometriales/metabolismo , Receptores de Estradiol/metabolismo , Receptores de Estrógenos/metabolismo , Anciano , Análisis de Varianza , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Neoplasias Endometriales/mortalidad , Neoplasias Endometriales/patología , Estradiol/sangre , Femenino , Humanos , Persona de Mediana Edad , Progesterona/sangre , Análisis de SupervivenciaRESUMEN
Two types of oestradiol binding sites were identified in human myometrium and myoma by saturation analysis. Nuclear type I receptors bind Oe with high affinity (KD = 1.4 nM) and low capacity (0.1-2.0 pmole/mg DNA) in a competitive fashion (Hill coeff. = 1.0), while nuclear type II sites bind the hormone with reduced affinity (KD: approximately 20 nM in myometrium and KD: approximately 40 nM in myoma), but high capacity (1-15 pmole/mg DNA) by positive cooperation (Hill coeff.: 4-5 in myometrium and 2-3 in myoma). Binding properties of type II sites in myoma are similar to those found in endometrium in previous studies of our laboratory, rather than those found in normal myometrium. The concentration of both nuclear Oe binding sites varied with the menstrual cycle. In myometrium, maximal concentration of nuclear type I and type II sites occurred between 10-14 days of cycle, when blood Oe level is the highest. In myoma the concentration of type I receptors was highest during the late follicular phase, but type II sites were uniformly high during the first 14 days of the cycle. In the luteal phase, receptor concentrations were low and apparently unaltered. It is possible that these changes in Oe binding capability of leiomyoma play a role in the pathomechanism.
Asunto(s)
Leiomioma/metabolismo , Miometrio/metabolismo , Receptores de Estradiol/metabolismo , Receptores de Estrógenos/metabolismo , Neoplasias Uterinas/metabolismo , Adulto , Núcleo Celular/metabolismo , Estradiol/metabolismo , Femenino , Humanos , Cinética , Ciclo MenstrualRESUMEN
The effect of an in vitro application of oestradiol-17-beta on the soluble protein synthesis in human endometrium during the menstrual cycle was investigated. Results obtained by double-label technique and SDS-polyacrylamide gel or Cellogel electrophoresis show that synthesis of soluble proteins in human endometrium is affected by oestradiol in both follicular and luteal phases of the cycle. One of the proteins obtained after oestradiol treatment seems to be similar to IP of rat uterus as judged from its molecular weight (41,000 daltons) and electrophoretic mobility (RF:1.18-1.24 relative to BSA on Cellogel). The magnitude of this protein synthesis correlates significantly with cytoplasmic oestradiol receptor concentration in endometrium throughout the cycle. The highest induction of IP like protein(s) synthesis at follicular phase and a decreased induction during the luteal phase was detected. No quantitative relationship between the changes of progesterone receptor concentration and IP like protein synthesis was found.
Asunto(s)
Endometrio/efectos de los fármacos , Estradiol/farmacología , Proteínas Musculares/biosíntesis , Proteínas , Endometrio/metabolismo , Femenino , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico , Humanos , Menstruación , Chaperonas Moleculares , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , SolubilidadRESUMEN
The effect of oestradiol treatment on protein synthesis in the cytosol fraction (10(5) g supernatant) from ovariectomized mature and developing female rat hypothalami were studied. Results obtained on adults by double-label technique and SDS polyacrylamide gel or cellogel electrophoresis show that, as in the uterus, oestradiol-induced protein synthesis (IP) occurred in the cytosol fraction of the hypothalamus. The molecular weight of IP was about 40 000 dalton. During postnatal development, IP was also observed in cytosol fractions from the hypothalami of female rats 14, 21 and 28 days old. No clear-cut effect of oestradiol was found in the 7-day-old animals.