RESUMEN
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.