RESUMO
Estradiol triggers key biological responses in the endometrium, which rely on the presence and levels of its cognate receptors on target cells. Employing the receptor micro-autoradiography (RMAR) technique, we aimed to provide a temporal and spatial map of the functional binding sites for estradiol in the mouse endometrial stroma during early pregnancy. Uterine samples from days 1.5 to 7.5 of pregnancy were collected 1 h after tritiated- (3H-) estradiol administration and prepared for RMAR analysis. Autoradiographic incorporation of 3H-thymidine (after 1-h pulse) was evaluated over the same gestational interval. Combined RMAR with either histochemistry with Dolichus biflorus (DBA) lectin or immunohistochemistry for detection of the desmin further characterized 3H-estradiol binding pattern in uterine Natural Killer (uNK) and decidual cells, respectively. 3H-estradiol binding levels oscillated in the pregnant endometrial stroma between the mesometrial and antimesometrial regions as well as the superficial and deep domains. Although most of the endometrial stromal cells retained the hormone, a sub-population of them, as well as endothelial and uNK cells, were unable to do so. Rises in the levels of 3H-estradiol binding preceded endometrial stromal cell proliferation. 3H-estradiol binding and 3H-thymidine incorporation progressively decreased along the development of the antimesometrial decidua. Endothelial proliferation occurred regardless of 3H-estradiol binding, whereas pericytes proliferation was associated with high levels of hormone binding. Endometrial cell populations autonomously control their levels of 3H-estradiol binding and retention, a process associated with their proliferative competence. Collectively, our results illustrate the intricate regulatory dynamic of nuclear estrogen receptors in the pregnant mouse endometrium.
Assuntos
Autorradiografia , Endométrio/citologia , Estradiol/análise , Estradiol/metabolismo , Receptores de Estrogênio/análise , Receptores de Estrogênio/metabolismo , Células Estromais/metabolismo , Animais , Sítios de Ligação , Endométrio/metabolismo , Estradiol/administração & dosagem , Feminino , Imuno-Histoquímica , Camundongos , Gravidez , Receptores de Estrogênio/química , Células Estromais/citologiaRESUMO
Information about the distribution of biopharmaceuticals is basic for understanding their actions. Tissue and cellular localization is a key to function. Autoradiography with radiolabeled compounds has provided valuable information with both low resolution whole-body macro-autoradiography and high resolution microscopic autoradiography (micro-autoradiography). Whole-body macro-autoradiography is a uniform and expedient single method approach, providing convenient dose- and time-related overviews with data similar to those obtained with conventional bioassays - and therefore widely used. However, whole-body macro-autoradiography, like common bioassays, has limitations. High specificity-low capacity sites of binding and deposition frequently remain unrecognized. Lack of cellular resolution can cause false negatives and provide misleading results (e.g., false blood-brain barrier). For micro-autoradiography, different methods are advertised in the literature. Most of them are, however, unsuited for drug localization because of inadequate resolution and frequent artifacts. Most drugs interact with their receptors non-covalently by weak electrostatic forces. Therefore, translocation and loss can occur during tissue preparation. This has complicated the use of micro-autoradiography. Receptor micro-autoradiography has overcome these complications and is a method of choice. It has been validated through several diffusible compounds with known localization, extensively applied. It has contributed numerous discoveries, followed by new concepts and therapies. Pictorial evidence in this review indicates that cellular information is essential, a 'sine qua non' for meaningful drug distribution studies. High resolution cellular microscopic information obtained from autoradiography requires tissue dissection and the necessary precautions for preserving pristine in vivo drug deposition. Receptor micro-autoradiography fulfils these requirements. It reveals crucial information at the subcellular level that cannot currently be obtained with any other type of autoradiography or spectrometric imaging.
Assuntos
Farmacocinética , Imagem Corporal Total/métodos , Animais , Autorradiografia , Humanos , Distribuição Tecidual , Vitamina D/farmacocinéticaRESUMO
For cell and tissue localization of drugs, receptor microscopic autoradiography is reviewed, including its development history, multiple testing, extensive applications and significant discoveries. This sensitive high-resolution imaging method is based on the use of radiolabeled compounds (esp. tagged with (3)H or (125)I), preservation through freezing of in vivo localization of tissue constituents, cutting thin frozen sections, and close contact with the recording nuclear emulsion. After extensive testing of the utility of this method, the distribution of radiolabeled compounds has been identified and characterized for estradiol, progestagens, adrenal steroids, thyroid hormone, ecdysteroids, vitamin D, retinoic acid, metabolic indicators glucose and 2-deoxyglucose, as well as extracellular space indicators. Target cells and associated tissues have been characterized with special stains, fluorescing compounds, or combined autoradiography-immunocytochemistry with antibodies to dopamine-beta-hydroxylase, GABA, enkephalin, specific receptor proteins, or other cellular products. Blood-brain barrier and brain entries via capillary endothelium, ependyma, or circumventricular recess organs have been visualized for (3)H-dexamethasone, (210)Pb lead, and (3)H-1,25(OH)(2) vitamin D(3). With this histopharmacologic approach, cellular details and tissue integrative overviews can be assessed in the same preparation. As a result, information has been gained that would have been difficult or impossible otherwise. Maps of brain drug distribution have been developed and relevant target circuits have been recognized. Examples include the stria terminalis that links septal-amygdaloid-thalamic-hypothalamic structures and telencephalic limbic system components which extend as the periventricular autonomic-neuroendocrine ABC (Allocortex-Brainstem-Circuitry) system into the mid- and hindbrain. Discoveries with radiolabeled substances challenged existing paradigms, engendering new concepts and providing seminal incentives for further research toward understanding drug actions. Most notable are discoveries made during the 1980s with vitamin D in the brain together with over 50 target tissues that challenged the century-old doctrine of vitamin D's main role as 'the calcitropic hormone', when the new data made it apparent that the main biological function of this multifunctional sunshine hormone rather is maintenance of life and adapting vital functions to the solar environment. In the brain, vitamin D, in close relation to sex and adrenal steroids, participates in the regulation of the secretion of neuro-endocrines, such as, serotonin, dopamine, nerve growth factor, acetyl choline, with importance in prophylaxis and therapy of neuro-psychiatric disorders. Histochemical imaging with high cellular-subcellular resolution is necessary for obtaining detailed information, as this review indicates. New spectrometric methods, like MALDI-MSI, are unlikely to furnish the same information as receptor microautoradiography does, but can provide important correlative molecular information.
Assuntos
Autorradiografia/métodos , Encéfalo/diagnóstico por imagem , Imagem Molecular/métodos , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Droga/metabolismo , Coloração e Rotulagem/métodos , Biomarcadores/metabolismo , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Técnicas de Preparação Histocitológica , Humanos , Medicamentos sob Prescrição/farmacocinética , Radioisótopos , Cintilografia , Distribuição TecidualRESUMO
Receptor binding with 3H-1,25(OH)2 vitamin D3 (vitamin D) and its oxygen analog 3H-OCT is demonstrated in rat, hamster, and mice submandibular, sublingual and parotid glands, using receptor microautoradiography high-resolution imaging. Nuclear uptake and retention of radiolabeled compound exist strongest in epithelial cells of striated ducts, granular convoluted tubules and in myoepithelial cells throughout, scattered in epithelial cells of intercalated ducts and relatively low in cells of serous and mucous acini. Deposition and retention of radiolabeled compound is also observed in interstitial spaces. The specific nuclear localization with vitamin D and its analogue OCT, which is absent with 3H-(OH) vitamin D3 and in competition with excess non-radioactive vitamin D, indicates involvement of vitamin D in the multi-hormonal regulation of salivary gland secretion, excretion, and cell proliferation. These data--together with previously recognized similar receptor binding in esophagus, gastric glands, entero-endocrine cells, pyloric muscle, and generative and absorptive epithelium of the small intestine and colon, point to the importance of vitamin D for the digestive system regulation of functions and maintenance with related therapeutic potentials.
Assuntos
Células Epiteliais/metabolismo , Glândulas Salivares/metabolismo , Vitamina D/metabolismo , Animais , Cricetinae , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-DawleyRESUMO
Nuclear receptor binding of 1,25(OH)(2)-vitamin D(3) (vitamin D) in skin keratinocytes of epidermis, hair sheaths and sebaceous glands was discovered through receptor microscopic autoradiography. Extended experiments with (3)H-1,25(OH)(2)-vitamin D(3) and its analog (3)H-oxacalcitriol (OCT) now demonstrate nuclear receptor binding in sweat gland epithelium of secretory coils and ducts as well as in myoepithelial cells, as studied in paws of nude mice after i.v. injection. The results suggest genomic regulation of cell proliferation and differentiation, as well as of secretory and excretory functions, indicating potential therapies for impaired secretion as in hypohidrosis of aged and diseased skin.
Assuntos
Células Epiteliais/metabolismo , Glândulas Sudoríparas/metabolismo , Vitamina D/metabolismo , Animais , Autorradiografia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with (3)H-estradiol-17beta in association with (3)H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-alpha. (3)H-estradiol nuclear binding is present but variable during days 1.5-7.5 of pregnancy. Sites of strong nuclear binding of (3)H-estradiol exhibit strong immunocytochemical staining with ER-alpha antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear (3)H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in (3)H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen (3)H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in (3)H-estradiol binding corresponds to an increased labeling index with (3)H-thymidine. A highly differentiated binding of (3)H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong (3)H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.
Assuntos
Receptores de Estradiol/metabolismo , Útero/metabolismo , Animais , Autorradiografia , Núcleo Celular/química , Núcleo Celular/metabolismo , Implantação do Embrião , Desenvolvimento Embrionário , Epitélio/metabolismo , Estradiol/análise , Feminino , Imuno-Histoquímica , Cinética , Camundongos , Gravidez , Receptores de Estradiol/análise , Receptores de Estradiol/imunologia , Timidina/metabolismo , Útero/anatomia & histologiaRESUMO
A combined technique of formaldehyde-induced fluorescence (FIF) and autoradiography is described for the localization of radioactively labeled substances in relation to monoamine neurons. This method permits the simultaneous visualization of 3H-labeled steroid hormone or drug uptake sites and fluorescing monoamine neural elements (cell bodies, fiber projections, terminals) in the same tissue section. Thin frozen sections cut in a cryostat are freeze-dried, exposed to formaldehyde vapor at 80°C, and carried through dry-mount autoradiography processing steps before fluorescence microscopy screening. Subsequent histological staining of sections and light microscopy are employed for conventional autoradiogram screening. With this procedure, 3H-estradiol and 3H-dihydro-testosterone are localized in various catecholamine (CA) neurons in the diencephalon and lower brain stem of the rat. Also, catecholaminergic as well as noncatechol-aminergic sex steroid target neurons are seen to be innervated by CA terminals in various rat brain regions.