RESUMO
This paper is concerned with hormonal regulation of the developmental pattern of major proteins of the mouse vas deferens (mouse vas deferens protein: MVDP, 34.5 kD) and seminal vesicle (15.5, 120 and 140 kD) whose expression is regulated by testosterone at adulthood. The ontogeny of these proteins, studied by SDS-polyacrylamide gel electrophoresis, appeared to be uncoordinated. MVDP was not accumulated until animals were 20 days old and its concentration increased sharply from 20 to 30 days of age. In seminal vesicle, the 15.5 kD protein did not accumulate before day 30 whereas 120 and 140 kD proteins appeared and accumulated between 30 and 40 days. In 30-day-old mice castrated at birth or treated with cyproterone acetate over 29 days, MVDP levels were not abolished and were similar to those measured in 20-day-old males. Testosterone administration, from 1 to 10 days of age, did not induce precocious expression of MVDP. These results suggest that the neonatal expression of MVDP is independent of androgens. In seminal vesicle, the first expression of the 3 proteins studied was dependent upon testicular androgens as shown by neonatal castration and injection experiments. The marked increase in the levels of the 4 proteins studied, during sexual maturation, was not associated with quantitative or qualitative changes in tissular androgen concentrations, suggesting that other factors may be necessary for protein expression. Whereas thyroxine may induce a precocious accumulation of MVDP, prolactin had no stimulatory effect on the accumulation of proteins from vas deferens and seminal vesicle. The results suggest that during sexual maturation gene activation by androgens was progressive.
Assuntos
Aldeído Redutase , Proteínas Secretadas pela Próstata , Proteínas/metabolismo , Glândulas Seminais/crescimento & desenvolvimento , Testosterona/farmacologia , Ducto Deferente/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Castração , Ciproterona/análogos & derivados , Ciproterona/farmacologia , Acetato de Ciproterona , Di-Hidrotestosterona/análise , Masculino , Camundongos , Prolactina/farmacologia , Proteínas de Plasma Seminal , Glândulas Seminais/efeitos dos fármacos , Glândulas Seminais/metabolismo , Testosterona/análise , Tiroxina/farmacologia , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismoRESUMO
Pure epithelial cell cultures, obtained from primary culture of vas deferens tissue collected from 20- to 30-day-old mice, were amplified by subculturing the cells over 3T3 feeder layer in a serum-free defined medium. Adhesion and proliferation of epithelial cells did not require androgens, but a minimal concentration of 5.10(-7) M hydrocortisone. In that system, epithelial cells expressed cytokeratin but failed to produce the tissue specific mouse vas deferens protein (MVDP) in response to androgens. Various culture procedures and medium compositions were assayed for induction of MVDP expression. Culture onto microporous membrane inserts, which allow polarization of cells, is absolutely required for androgenic induction of MVDP. Androgen action did not require the presence of hydrocortisone, insulin, triiodothyronine, pituitary extracts, epidermal growth factor and acetylcholine. A minimal supplemented medium was then defined in which the expression of MVDP by epithelial cells in response to androgens was dose dependent. It has also been shown that this response at each concentration of dihydrotestosterone was heterogeneous at individual cell level. Highly reproducible results were obtained from epithelial cell cultures between 8th to 16th passages, showing that subcultured cells have maintained their ability to differentiate and express specialized functions.
Assuntos
Aldeído Redutase , Androgênios/farmacologia , Proteínas/metabolismo , Ducto Deferente/citologia , Ducto Deferente/metabolismo , Acetilcolina/análise , Acetilcolina/farmacologia , Androgênios/análise , Animais , Comunicação Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/análise , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais , Epitélio/química , Epitélio/metabolismo , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrocortisona/análise , Hidrocortisona/farmacologia , Imuno-Histoquímica , Insulina/análise , Insulina/farmacologia , Queratinas/análise , Queratinas/metabolismo , Masculino , Camundongos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Proteínas/genética , Tri-Iodotironina/análise , Tri-Iodotironina/farmacologia , Ducto Deferente/químicaRESUMO
The MVDP (mouse vas deferens protein) gene, which encodes an aldose reductase-like enzyme, is mainly expressed in vas deferens epithelium and adrenal cortex. Vas deferens MVDP gene transcription was known to be under androgenic control, we now have evidence for androgen and probable ACTH responsiveness of the MVDP gene in the adrenal. To analyze the role of potential regulatory regions in hormonal, developmental, and tissue-specific aspects of MVDP regulation, we generated transgenic mice harboring MVDP-CAT fusion genes. The constructs carried either -1.8 or -0.5 kb 5'-flanking sequence attached to the chloramphenicol acetyltransferase gene in presence or absence of a 3.5-kb intragenic fragment in a downstream position. We show that at least two regions ensure proper gene regulation in vivo. The first, located within the 1.8-kb promoter fragment, directs tissue specificity; positive elements necessary for vas deferens and adrenal expression lay within positions -1804 to -510 and -510 to +41, respectively. The second, located within the 3.5-kb intragenic fragment spanning intron 1 to intron 2, increases percentage of expressing lines and behaves as a vas deferens-specific enhancer. Hormonal and developmental control of transgenes closely parallel endogenous gene regulation. Androgen and ACTH responsiveness in adrenals is conferred by 0.5-kb promoter, whereas in vas deferens, full androgenic response of the 1.8-kb promoter required the 3.5-kb intragenic fragment. Thus, vas deferens and adrenals use distinct cis-acting elements to direct and regulate the expression of the MVDP gene.
Assuntos
Glândulas Suprarrenais/metabolismo , Aldeído Redutase , Androgênios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Análise de Sequência de DNA , Desidrogenase do Álcool de Açúcar/genética , Ducto Deferente/metabolismo , Aldo-Ceto Redutases , Animais , Fusão Gênica Artificial , Cloranfenicol O-Acetiltransferase/genética , Mapeamento Cromossômico , Masculino , Camundongos , Camundongos Transgênicos , Especificidade de ÓrgãosRESUMO
The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.
Assuntos
Aldeído Redutase/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Células Intersticiais do Testículo/enzimologia , Testículo/enzimologia , Animais , Sequência de Bases , Northern Blotting , Gonadotropina Coriônica/farmacologia , Colforsina/farmacologia , Primers do DNA , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Tumor de Células de Leydig , Masculino , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Testiculares , Testículo/embriologia , Células Tumorais CultivadasRESUMO
The understanding of androgen-regulated gene expression requires a cell cultures system that mimics the functions of cells in vivo. In the present paper we have examined a vas deferens epithelial cell subculture system. Cultured vas deferens epithelial cells have been shown to exhibit polarized properties characteristic of functioning epithelia and to display a high level of androgen receptors. Incubation of cells with androgen caused a decrease in cellular androgen receptor mRNA that was time-dependent. Total suppression was observed after 24h of exposure to androgen. By contrast, incubation of vas deferens epithelial cells with androgen resulted in a threefold increase in the cellular content of androgen receptor protein, as assayed by ligand binding. In response to androgens, vas deferens epithelial cells expressed mouse vas deferens protein mRNA (MVDP mRNA). Maximum expression of the MVDP gene, at both mRNA and protein levels, was observed after 24h of androgen induction. DEAE-dextran transfection conditions were defined using the MMTV-CAT gene in vas deferens epithelial cells in a dose- and time-dependent manner. No induction was seen when fragments of the MVDP promoter region were cloned directly in front of the CAT gene and transiently transfected into vas deferens epithelial cells. It was found that cotransfection of cells with MVDP-CAT gene and transfection of cells with MVDP-CAT constructs and with an androgen receptor expression vector resulted in a small but consistent androgen-dependent increase in reporter gene activity. Transiently transfected vas deferens epithelial cells are a suitable model with which to study the effect of androgen on gene regulatory elements.
Assuntos
Di-Hidrotestosterona/farmacologia , Regulação da Expressão Gênica , Receptores Androgênicos/biossíntese , Ducto Deferente/metabolismo , Células 3T3 , Animais , Diferenciação Celular , Células Cultivadas , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/biossíntese , Cicloeximida/farmacologia , Células Epiteliais , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Vírus do Tumor Mamário do Camundongo , Metribolona/metabolismo , Camundongos , Camundongos Endogâmicos , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , Transfecção , Ducto Deferente/citologiaRESUMO
Cortisol metabolism was studied in conscious adult male guinea-pigs subjected to a neurotrophic stress (immobilization and stimulation by light for 3 h). The disappearance curves of tracer quantities of [3H]cortisol were represented by a two-pool model. In stressed animals, there was marked increase in the mean plasma level of cortisol (184% of control value; P less than 0.001) and in the metabolic clearance rate (MCR; 17% of control value; 0.001 less than P less than 0.001). This rise in the MRC of plasma cortisol resulted from an increase in the mean total apparent volume of distribution (49%, P less than 0.001). The lack of significant differences in the slopes of the second exponential phase of the disappearance curves indicated that the stress did not significantly increase the half-lie of cortisol. The mean binding capacity of transcortin for cortisol (ST) was significantly higher in the animals which had been subjected to the neurotrophic stress than in the control guinea-pigs (0.02 less than P less than 0.05). However, ST values remained very low and accounted for the very high levels of free cortisol found after the stress. The results suggest that the raised concentrations of unbound cortisol found in the plasma of conscious adult male guinea-pigs in response to neurotrophic stress reflect a hypersecretion of corticosteroid.
Assuntos
Hidrocortisona/sangue , Estresse Fisiológico/sangue , Animais , Cobaias , Imobilização , Masculino , Taxa de Depuração Metabólica , Estimulação LuminosaRESUMO
To evaluate the role of the liver in cortisol catabolism the extraction ratio of both cortisol and cortisone by the organs of the splanchnic area was estimated in guinea-pigs anaesthetized with pentobarbitone. The [3H]cortisol and [3H]cortisone concentrations were measured in portal and sus hepatic venous plasma during a constant infusion of [3H]cortisol or [3H]cortisone. The extraction ratio of cortisol was estimated to be 10-14% in the splanchnic area and the viscera, while in the liver it had a small negative value suggesting that the liver had produced as much or more cortisol than it had taken up. All the cortisone (95%) formed from cortisol in the viscera was eliminated from the plasma compartment by the liver. Some 75-80% of the infused cortisone was converted to cortisol; rather less of the infused cortisol was converted to cortisone (32%). Using estimates of plasma flow derived from sham-operated animals, the uptake of cortisol by the various organs was calculated. The splanchnic area extracted 41% of the infused cortisol from the plasma: 25-27% as cortisol and 13-16% as cortisone. The liver appeared to take up cortisone preferentially. The conversion of cortisone into cortisol within the liver seems to be important in limiting the amount of cortisol removed from the plasma by the splanchnic area. The liver is also important in inactivating the steroids although other sites are probably also involved.
Assuntos
Cortisona/metabolismo , Hidrocortisona/metabolismo , Fígado/metabolismo , Animais , Cortisona/sangue , Cortisona/farmacologia , Feminino , Cobaias , Hidrocortisona/sangue , Hidrocortisona/farmacologia , Masculino , Taxa de Depuração Metabólica , Circulação EsplâncnicaRESUMO
Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Metribolona/farmacologia , Próstata/metabolismo , Neoplasias da Próstata/genética , Retinoides/farmacologia , Congêneres da Testosterona/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Calcitriol/farmacologia , Divisão Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Próstata/efeitos dos fármacos , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Neoplasias da Próstata/metabolismo , Tretinoína/farmacologia , Tri-Iodotironina/farmacologia , Células Tumorais CultivadasRESUMO
We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.
Assuntos
Linhagem Celular , Epididimo/citologia , Animais , Diferenciação Celular/fisiologia , Polaridade Celular/efeitos dos fármacos , Proliferação de Células , DNA/análise , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Expressão Gênica , Marcadores Genéticos/genética , Hidrocortisona/farmacologia , Junções Intercelulares/ultraestrutura , Inulina/metabolismo , Masculino , Camundongos , Permeabilidade , Poliploidia , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Transcription of the mouse vas deferens protein (MVDP) gene is stimulated by androgens and we have previously shown that in a 162 bp fragment, located at position -121 to +41, a TGAAGTtccTGTTCT sequence functions as an androgen-dependent enhancer. To determine which factors are involved in the hormonally regulated MVDP gene transcription, we have used DNase I footprinting and band-shift assays to examine in vitro binding of proteins to the enhancer and promoter sequences and have determined the functional significance of the recognized DNA sequences in transient transfection assays. Studies using recombinant proteins such as the DNA binding domain of the androgen receptor (AR-DBD) and Sp1, and crude cellular extracts from T47D and vas deferens epithelial cells (VDEC) showed that in addition to AR-DBD, the transcriptional activators NF1 and Sp1 interact with the -121/+41 fragment in a specific manner. Transient transfection assays revealed that site-directed mutations in the NF1 and Sp1 binding elements strongly reduced (NF1) or abolished (Sp1) androgen induced expression. The results demonstrate that the -121/+41 sequence is a composite site for the androgen receptor mediated transactivation of the MVDP gene.
Assuntos
Aldeído Redutase , Androgênios/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas/genética , Fator de Transcrição Sp1/genética , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Fatores de Transcrição NFI , Regiões Promotoras Genéticas/genética , Transcrição Gênica , TransfecçãoRESUMO
Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the vas deferens. To better understand androgen-regulated MVDP gene expression we have used RNA hybridization to study the effects of androgens on the steady-state levels of MVDP mRNA in vas deferens epithelial cell subcultures. Northern blot analysis revealed that these cells only express MVDP mRNA in the presence of androgens. There was a close relationship between MVDP mRNA levels and dihydrotestosterone concentrations. MVDP mRNA is induced over a period of 24h and maximal induction is about 25-fold. Treatment of cells with cycloheximide completely abolished the observed androgen effect suggesting that the induction of the MVDP gene by androgens depends on continuous protein synthesis. Transient transfection of vas deferens epithelial cells with MMTV-CAT vector showed that these cells contained functional androgen receptors and that they are a suitable system to study androgen effect on MVDP gene regulatory elements.
Assuntos
Oxirredutases do Álcool/genética , Aldeído Redutase , Androgênios/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas/genética , Células 3T3 , Aldo-Ceto Redutases , Animais , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Cicloeximida/farmacologia , Di-Hidrotestosterona/farmacologia , Células Epiteliais , Epitélio/metabolismo , Cinética , Masculino , Vírus do Tumor Mamário do Camundongo , Camundongos , Família Multigênica , Proteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ducto Deferente/citologia , Ducto Deferente/metabolismoRESUMO
Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i. e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.
Assuntos
Aldeído Redutase , Androgênios/farmacologia , Fator de Transcrição AP-1/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular , Polaridade Celular , DNA/genética , DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Genes jun/efeitos dos fármacos , Masculino , Camundongos , Proteínas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ducto Deferente/citologiaRESUMO
Insulin-related material was measured in acid ethanol extracts of brain, testis, liver, and kidney from adult rats acutely injected with insulin or saline. Insulin injection resulted in a twofold to threefold increase in plasma insulin during a two-hour period after injection. Plasma glucose was greatly depressed. Insulin injection had no effect on the insulin-related material in most areas of brain (cerebral cortex, olfactory bulbs, and medial hypothalamus) and the cerebrospinal fluid; lateral hypothalamus was an exception and paradoxically exhibited a decrease of this material. The testis insulin-related material was unaffected; purification of the testis extracts using the C18 Sep pak method revealed no further difference between the animals. In liver, the insulin-related material was not significantly different in the control and the insulin-injected group; however, we found a significant correlation between this material and plasma insulin within the insulin-injected group. In contrast, insulin injection resulted in an important increase in kidney insulin-related material that paralleled the change in plasma insulin. Thus, like chronic experiments, acute hyperinsulinemia revealed that the insulin-related material was largely independent from blood insulin in tissues that exhibit very different insulin uptake from the blood; kidney appeared to be an exception.
Assuntos
Encéfalo/metabolismo , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Rim/metabolismo , Fígado/metabolismo , Testículo/metabolismo , Animais , Masculino , Especificidade de Órgãos , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
Insulin binding and insulin receptor kinase activity were measured in solubilized and partially purified receptor preparations from liver and skeletal muscles of rats that were either fed a standard diet or subjected to a 72-hour fasting period. Insulin binding capacity was increased in both tissues from fasted rats as determined by Scatchard analysis. The affinity of the receptors was not modified by fasting. Affinity labeling of the alpha-subunit of insulin receptors also suggested an increase in the number of insulin receptors in both tissues. The ability of insulin to stimulate the autophosphorylation of the beta-subunit as well as the phosphorylation of the artificial substrate Glu80-Tyr20 was significantly impaired in liver from fasted rats and by contrast unchanged in skeletal muscles. These findings indicate that in rats, fasting produces changes in insulin receptor kinase activity in liver but not in muscle. The physiological significance of this tissue-specific regulation of receptor kinase activity in relation to insulin action during fasting remains to be established.
Assuntos
Fígado/metabolismo , Músculos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor de Insulina/metabolismo , Inanição , Marcadores de Afinidade/metabolismo , Animais , Peso Corporal , Insulina/metabolismo , Cinética , Masculino , Especificidade de Órgãos , Ratos , Ratos Endogâmicos , Receptor de Insulina/isolamento & purificação , Valores de Referência , Succinimidas/metabolismoRESUMO
Infusion of 125I-(Tyr A14)-insulin at tracer doses into the cerebrospinal fluid (CSF) resulted in a slow rate of increase in the CSF-labeled insulin during the first 2 hours with a plateau thereafter. Labeled insulin was cleared from the CSF at a higher rate than 3H-inulin, a marker of CSF bulk flow. The labeled insulin was mainly distributed in all the ventricular and periventricular brain regions. Small amounts of degraded insulin appeared in the CSF. Coinfusion with an excess of unlabeled insulin impaired the clearance and degradation of labeled insulin. It also inhibited the labeling in medial hypothalamus, olfactory bulbs and brain stem. In contrast, coinfusion of ribonuclease B (used to test the specificity of uptake) was without any effect. It was concluded that there is an active insulin intake from CSF into brain specific compartments that is presumably essential for the effects of insulin on brain function.
Assuntos
Insulina/líquido cefalorraquidiano , Anestesia , Animais , Ventrículos Cerebrais , Infusões Parenterais , Insulina/administração & dosagem , Insulina/farmacocinética , Masculino , Taxa de Depuração Metabólica , Ratos , Ratos Endogâmicos , Receptor de Insulina/metabolismo , Contagem de Cintilação , Ácido TricloroacéticoRESUMO
We used cultured vas deferens epithelial cells (VDECs) as a model system to determine the conditions that allow mouse vas deferens protein (MVDP) gene expression and acquisition of androgen responsiveness. On the basis of Northern blot analysis, the mvdp gene is constitutively expressed at very low levels in prepubertal VDECs grown on collagen-coated plastic or on microporous membrane inserts. In the presence of dihydrotestosterone (DHT), mvdp messenger RNA levels dramatically increased in cells cultured on microporous membrane inserts and stayed unchanged in cells grown on matrix-coated plastic. Epithelial cells derived from fetal vas deferens were able to synthesize MVDP in response to DHT, and the presence of fetal mesenchymal cells did not influence MVDP production. Providing the cells with a culture procedure that permits access to the basolateral membranes and caters to the polarity requirements of the cell is a prerequisite for androgen induction of MVDP gene expression. The results also point to a role for epidermal growth factor, insulin, and tyrosine kinase activity in mediating the action of androgen on mvdp gene expression. In vivo studies show that the first expression of the mvdp gene between 5 and 7 days postpartum is not associated with major structural changes in the epithelium. The acquisition of a mature phenotype by epithelial and peritubular contractile cells, between 10 and 20 days, correlates with androgen dependency of the mvdp gene. We propose that cell differentiation and polarization on a matrix-coated microporous membrane reproduces some of the events that are necessary for acquisition of androgenic responsiveness of the mvdp gene during postnatal development.
Assuntos
Envelhecimento/fisiologia , Aldeído Redutase , Animais Recém-Nascidos/fisiologia , Di-Hidrotestosterona/farmacologia , Proteínas/genética , Ducto Deferente/fisiologia , Células 3T3 , Animais , Biomarcadores , Polaridade Celular/fisiologia , Células Cultivadas , Meios de Cultura , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais/fisiologia , Insulina/metabolismo , Masculino , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ducto Deferente/efeitos dos fármacosRESUMO
Polyclonal antibodies have been generated to investigate the localization, tissue and species distribution, androgen regulation, and ontogeny of a protein secreted by mouse seminal vesicle, designated as MSVSP99 (ie, mouse seminal vesicle secretory protein of 99 amino acids). MSVSP99 is a polymorphic compound with a molecular weight of around 14 kilodaltons and a positive immunoreactivity range of 5.23 to 5.70. Positive immunoreactivity was restricted to the epithelial cells of the seminal vesicle. Western blot analysis showed organ specificity for MSVSP99, which could not be detected in several organs in the mouse. Time course decrease of MSVSP99 after castration closely followed that of its mRNA. In contrast, the length of time required to restore control levels after testosterone treatment was higher for the protein than it was for its mRNA. Whereas the MSVSP99 gene is already active in 10-day-old males, MSVSP99 is first detected at 27 days. Then, we conclude that factors other than the accumulation of the mRNA regulate MSVSP99 expression.
Assuntos
Proteínas Secretadas pela Vesícula Seminal/genética , Glândulas Seminais/crescimento & desenvolvimento , Glândulas Seminais/fisiologia , Sequência de Aminoácidos , Animais , Western Blotting , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônios Esteroides Gonadais/farmacologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Orquiectomia , Coelhos , Proteínas Secretadas pela Vesícula Seminal/análise , Proteínas Secretadas pela Vesícula Seminal/imunologia , Especificidade da Espécie , Testosterona/farmacologiaRESUMO
The effect of estradiol on adrenal secretion rate of cortisol in response to a stress induced by anesthesia, was examined by comparing the metabolic clearance rate and the production rate of cortisol between males and females and after estradiol administration in castrated animals. Metabolic clearance rates of cortisol (MCR) were significantly higher (+30%) in males than in females. Castration lowered the MCR of cortisol in males and had no significant effect in females. After estradiol administration, a fall in the MCR of cortisol concomitant with a rise in blood cortisol level was observed especially in males in which the effect of treatment was more marked than in females and highly significant. The production rate of cortisol was identical in males and females and was slightly increased in estradiol-treated males and females. The data indicate that estradiol had an inhibitory effect on metabolic clearance of cortisol, which caused an important rise of blood cortisol levels in response to stress and which prevented an increase in the adrenal response to the stress. Since the pituitary adrenal cortex can respond in a normal way to stress, the low value of MCR of cortisol could be the limiting factor in the adrenal secretion rate of cortisol in estrogen-treated guinea-pigs.
Assuntos
Anestesia/efeitos adversos , Estradiol/farmacologia , Hidrocortisona/biossíntese , Estresse Fisiológico/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Castração , Feminino , Cobaias , Cinética , Masculino , Tamanho do Órgão/efeitos dos fármacos , Fatores Sexuais , Estresse Fisiológico/etiologiaRESUMO
Metabolism of the cortisol (F) and the cortisone (E) was studied by continuous infusion of [14C]-F and [3H]-E in adult conscious male guinea-pigs. Parameters, calculated from the specific activities of F and E, are expressed in mumoles/24 h : production rate of F (PRF) = 8 +/- 1 and E (PRE) = 4.9 +/- 0.7 ; secretion rate of F (QF) = 7.1 +/- 0.9 and E (QE) = 0.9 +/- 0.4 ; rate of irreversible metabolism of F (rF) = 6.2 +/- 0.9 and E (rE) = 1.8 +/- 0.3 ; percentage of transfer of F into E = 59 +/- 4% and E into F = 80 +/- 3%. These results demonstrate that adrenal gland in the adult male guinea-pig secretes essentially cortisol and little or no cortisone. Practically all the pool of E is derived from transformation of F into E ; the major part of E production is reconverted into F.
Assuntos
Cortisona/sangue , Cobaias/sangue , Hidrocortisona/sangue , Animais , Masculino , Taxa de Depuração MetabólicaRESUMO
In Croatian archives a rich collection of registers is preserved. Among the oldest and best-conserved collections of such valuable sources in Europe, are those from the territory of Istria. Investigating these sources we focused our attention on three recipes for treatment of calculi and cuts found on pages of Kastel baptismal's record (1749-1815) in Istria. Similar to other recipes found in various other recipe collections they mirror interlace of folk experience and theurgical views of healing which was detected unexpectedly sometimes on unconventional places, have survived on Croatian territory throughout centuries.