RESUMO
Nuclear binding of cytoplasmic estrogen receptors was measured in an in vitro cell-free system, using various mixtures of cytosols and nuclei from uteri of mature (6-9 month old) and senescent (24-25 month old) Wistar rats. Both nuclei and cytoplasmic receptors from senescent uteri were 25-35% less efficient in supporting nuclear binding than those obtained from mature tissues as evidenced by the concentrations of occupiable nuclear acceptor sites. No age differences in association or dissociation constants were observed for the nuclear binding reaction. However, the apparent inability of some aged receptors to bind to the full complement of mature nuclear acceptor sites may indicate a qualitative deficiency in the cytosols of senescent uteri.
Assuntos
Envelhecimento , Receptores de Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Útero/metabolismo , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Estradiol/metabolismo , Feminino , Cinética , Ratos , Ratos EndogâmicosRESUMO
Estradiol injected in vivo successfully completes for binding to uterine nuclear acceptor sites measured by the assay of Kon and Spelsberg (1). Such competition is time-and dose-dependent, with maximal inhibition (75%) 1 h after injection and at a dose of 10 micrograms of 17 beta-estradiol per 300 g of BW. Thus, this assay appears to accurately measure those uterine nuclear acceptor sites mediating estrogen action in vivo.
Assuntos
Estradiol/farmacologia , Receptores de Estrogênio/metabolismo , Útero/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , Feminino , Métodos , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
We have sought to determine whether the reported age-related increase in in vivo and in vitro PRL secretion in intact and/or postpubertally ovariectomized female rats results from increased number and/or function of lactotropic cells Immunocytochemical staining with antirat-PRL serum, goat antirabbit serum, and peroxidase-antiperoxidase using H2O2 and 4-chloro-1-naphthol or 3'3'-diaminobenzidine hydrochloride as substrates was used to compare the percentages of lactotropes in primary suspensions of heterogeneous adenohypophyseal cells from groups of intact and 3-day ovariectomized mature (6 to 7 month) Wistar rats at estrus (E) or diestrus1-2 (D) with those from the corresponding old (22 to 24 month) rats in constant estrus (CE) or constant diestrus (CD). For both intact and ovariectomized old vs. mature rats, there were small (30-35%) but significant (P less than 0.005) increases in lactotrope number. Lactotrope number did not differ (P greater than 0.05) between E vs. D rats, or CE vs. CD rats, either before or after ovariectomy; moreover, ovariectomy per se did not alter (P greater than 0.05) lactotrope number in any of the groups. Electron microscopic examination of lactotropic cells derived from ovariectomized CE rats revealed ultrastructural changes compatible with estrogenic hyperstimulation; such alterations were not observed in cells from the corresponding E rats. Basal and 17 beta-estradiol (10(-10) M)-stimulated in vitro PRL secretion was measured for 4 days in primary cultures from each of the above groups of rats. After correction for lactotrope number, PRL secretion was greater (P less than 0.01) from cells of intact and ovariectomized CD vs. D rats, whereas 17 beta-estradiol-stimulated (P less than 0.001) but not basal (P greater than 0.05) PRL secretion was greater from the corresponding CE vs. E rats. There was no effect (P greater than 0.05) of reproductive status or of short term ovariectomy on in vitro PRL secretion. These data suggest that aging in the female rat is associated with alterations in both the number and function of pituitary lactotropic cells.
Assuntos
Envelhecimento , Ovariectomia , Adeno-Hipófise/metabolismo , Prolactina/metabolismo , Animais , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Estro , Feminino , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica , Adeno-Hipófise/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
We have examined the effects of aging on the capacity of rat uterine estradiol receptors to be transformed from 8S to 4S and 5S species. Cytosol receptors from mature (6-month-old) rats or senescent (24-month-old) rats have been exposed to various KCl concentrations, ammonium sulfate precipitation and 25 degrees C heating. Estradiol receptors of both the mature and senescent age groups exist in an 8S form on linear 5-20% sucrose gradients in the absence of KCl and are converted to a 4S molecule in the presence of 0.4 M KCl. At intermediate salt concentrations a greater portion of mature receptors was converted to the 4S species. At 0.15 M KCl 62.3% +/- 2.8 of the mature receptors are converted to 4S versus 41% +/- 1.9 of the senescent receptors, and at 0.2 M KCl 79.6% +/- 3.2 of the mature receptors are converted to the 4S versus 58.2% +/- 2.1 of the senescent. Ammonium sulfate treatment in the presence of 0.3 M KCl converted about 80% of the receptors from the 4S to the 5S form, while only about half of the old receptors are affected. When ammonium sulfate precipitates were heated to 25 degrees C all to mature receptors were converted to the 5S species, while only two thirds of the senescent receptors were sedimented at 5S under the same conditions. Inclusion of 20 mM molybdate during preparation blocks conversion of about 15% of the senescent receptors from the 8S to the 4S form but does not affect the mature preparations. Similarly, molybdate treatment does not affect the conversion of the mature estradiol receptors to the 5S form but increases the percentage of senescent receptors remaining in the 4S form from 30 to 45%. Such qualitative differences in receptor conversion may be related to age associated deterioration of estradiol stimulated uterine responsiveness.
Assuntos
Envelhecimento/fisiologia , Receptores de Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Útero/ultraestrutura , Sulfato de Amônio/farmacologia , Animais , Centrifugação com Gradiente de Concentração , Citosol/metabolismo , Feminino , Temperatura Alta , Peso Molecular , Molibdênio/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Endogâmicos , Receptores de Estradiol/efeitos dos fármacos , Receptores de Estradiol/isolamento & purificaçãoRESUMO
While free radical-mediated reperfusion injury is clearly important in a variety of disparate organs, the particular cellular source of these radicals is unclear. To address this question, we subjected relatively pure (92% +/- 3% by factor VIII immunoassay) cultures of rat pulmonary artery endothelial cells to 0 to 45 minutes of anoxia (95% N2, 5% CO2), followed by reoxygenation (95% air, 5% CO2), to simulate ischemia/reperfusion. Cell injury was assayed after reoxygenation by the release of previously incorporated 51chromium and/or lactate dehydrogenase, and viability was determined by means of trypan blue exclusion. These three end points correlated closely. Without anoxia, the cells remained viable, with minimal evidence of injury for the entire experimental period, while 45 minutes of hypoxia followed by 30 minutes of reoxygenation produced substantial evidence of cell injury in 71% +/- 6% of the cells. This injury was reduced to 21% +/- 2% by treatment with the highly specific free radical scavengers superoxide dismutase and catalase together, either before anoxia or after anoxia, but just before reoxygenation. Similar protection was provided by xanthine oxidase inhibition with allopurinol. The injury was mimicked (without anoxia) by the exogenous generation of superoxide radicals with xanthine and xanthine oxidase. These experiments establish the essential components of free radical generation at reperfusion to be localized within the isolated endothelial cell in the absence of neutrophils or parenchymal cells.
Assuntos
Hipóxia/metabolismo , Oxigênio/administração & dosagem , Artéria Pulmonar/metabolismo , Superóxidos/metabolismo , Alopurinol/farmacologia , Animais , Catalase/farmacologia , Células Cultivadas , Endotélio/citologia , Endotélio/metabolismo , Masculino , Perfusão , Ratos , Ratos Endogâmicos , Superóxido Dismutase/farmacologia , Xantina Desidrogenase/metabolismo , Xantina Oxidase/farmacologiaRESUMO
The maximum ability of receptor-estradiol complexes to activate RNA polymerase II and bind to nuclear acceptor sites is reduced 40-50% in senescent rat uteri. A precise, linear stoichiometric relationship exists between acceptor site occupancy and polymerase activity, and this relationship is not altered with aging. Thus, reduced ability of estrogen to stimulate RNA polymerase II in aged rat uteri appears to be due to deficits in binding of receptor-estradiol complexes to nuclear acceptor sites.
Assuntos
Envelhecimento , Núcleo Celular/metabolismo , Estradiol/metabolismo , RNA Polimerase II/análise , Receptores de Estrogênio/metabolismo , Útero/enzimologia , Animais , Ativação Enzimática , Feminino , Técnicas In Vitro , Ratos , Ratos Endogâmicos , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
We measured in vitro release of luteinizing hormone (LH) in the presence of 1.5 mM extracellular calcium, with and without LH-releasing hormone (LHRH; 10(-10) to 10(-7) M) or the ionophore A23187 (10(-7) to 10(-4) M), in primary cultures of anterior pituitary cells from intact mature (6 mo) and old (24 mo) male and intact and ovariectomized mature and old female Wistar rats. Base-line as well as LHRH- and A23187-mediated LH secretion was decreased from cells of old rats. However, exposure to A23187 led to a nearly twofold greater augmentation of LH release from cells of old rats, thus decreasing the apparent age-related LH secretory deficit by approximately one-half. We then measured LHRH-mediated (10(-8) M) vs. A23187-mediated (10(-4) M) LH release with and without extracellular calcium (0.08-1.5 mM). For cells from both mature and old rats, there was a similar calcium dependency for A23187- and LHRH-mediated LH release, with optimal LH secretion at 1.0-1.5 mM extracellular calcium concentrations. Again, both LHRH- and A23187-stimulated LH release was significantly lower and exposure to A23187 led to a greater increase in LH release from cells of old rats. Taken together with similar findings in other systems, these data suggest that the in vitro LH secretory defect of pituitary cells from old rats results in part from one or more defects in calcium mobilization and that such alterations may be a widespread manifestation of aging.
Assuntos
Envelhecimento/metabolismo , Calcimicina/farmacologia , Hormônio Luteinizante/metabolismo , Adeno-Hipófise/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Espaço Extracelular/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Adeno-Hipófise/citologia , Ratos , Ratos Endogâmicos , ReproduçãoRESUMO
An in vitro cell-free system of uterine nuclei and cytosol receptors has been used to analyze the effects of aging on estrogen stimulation of RNA polymerase II activity. By using fixed concentrations of nuclei and cytoplasmic receptor--estrogen complexes (R-E2), it was found that mature nuclei are 3 times more efficient (155% vs. 57%) than old ones for stimulation of polymerase activity by mature R-E2. Meanwhile, mature R-E2 are 5 times more efficient (155% vs. 31%) than old ones in supporting such stimulation in mature nuclei. Stimulation by old cytosol R-E2 is so poor that it is essentially unaffected by nuclear age (31% with both mature and old nuclei). Finally, equimolar mixtures of mature and old cytosol R-E2 stimulate polymerase II activity in mature nuclei by 77%, a value intermediate between mature and old cytosols used separately. These results indicate that both nuclei and cytosol from old uteri are deficient in their ability to support estrogenic stimulation of RNA polymerase II.