RESUMO
OBJECTIVES: To evaluate the cognitive performance of a group of patients with Wilson's disease (WD) and to correlate the cognitive findings with changes in magnetic resonance imaging (MRI). METHODS: All patients with WD consecutively attended in a Movement Disorders Clinic between September 2006 and October 2007 were invited to participate in the study, together with a group of matched healthy controls. Patients and controls were submitted to comprehensive neuropsychological assessment. MRI was performed in all patients, and abnormalities (high-intensity signal, low-intensity signal and atrophy) were semi-quantitatively rated. Performance of patients and controls in each cognitive test was compared, and correlations between cognitive scores and MRI changes were investigated within the patients' group. RESULTS: Twenty patients with WD (11 men) and 20 controls (nine men) were evaluated. Mean age in the WD and control groups was 30.05 ± 7.25 and 32.15 ± 5.37 years, respectively. Mean schooling years were 11.15 ± 3.73 among WD cases and 10.08 ± 2.62 among controls. Patients with WD performed significantly worse than controls in the Mini-Mental State Examination, Dementia Rating Scale, phonemic verbal fluency (FAS), verb generation, digit span forward, Stroop test, Frontal Assessment Battery and in the Brief Cognitive Screening Battery. A significant correlation emerged between global cognitive impairment and MRI scale (r = 0.535), being higher for high-intensity signal plus atrophy (r = 0.718). CONCLUSION: Patients with WD presented cognitive impairment, especially in executive functions, with good correlation between cognitive abnormalities and MRI changes.
Assuntos
Transtornos Cognitivos/etiologia , Transtornos Cognitivos/patologia , Degeneração Hepatolenticular/patologia , Degeneração Hepatolenticular/psicologia , Adulto , Estudos de Casos e Controles , Escolaridade , Função Executiva , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Testes Neuropsicológicos , Adulto JovemRESUMO
Previous studies have shown that 17ß-estradiol plays a cardioprotective role in the central nervous system (CNS) of male rats. The aim of the present study was to determine the influence of 17ß-estradiol on sympathetic vasomotor activity and blood pressure in a renovascular hypertensive Goldblatt two-kidney one-clip (2K-1C) male rat model. We also determined the influence of angiotensin II AT1 receptor on the expression of estrogen receptors (ERα, ERß, and G protein-coupled ER (GPER)) in the rostral ventrolateral medulla (RVLM) of Goldblatt rats. Experiments were performed in Goldblatt and age-matched control rats six weeks after clipping of renal artery to induce hypertension. Microinjection of 17ß-estradiol into the RVLM led to a greater reduction in mean arterial pressure and renal sympathetic nerve activity in controls than in 2K-1C rats. Microinjection of the GPER agonist G-1 into the RVLM led to a significantly greater increase in mean arterial pressure and renal sympathetic nerve activity in 2K-1C rats. Expression levels of estrogen receptors GPER and ERα, but not ERß, were significantly higher in the RVLM of 2K-1C rats than in that of the control rats. Chronic treatment with losartan significantly reduced the expression levels of estrogen receptors in the RVLM of 2K-1C rats. Taken altogether, the data suggest that the imbalance of actions between ERα and GPER, particularly with the predominance of GPER in the RVLM, contributes to sympathetic overactivation in male rats with Goldblatt hypertension. AT1-Angiotensin II receptor in the RVLM upregulated estrogen receptor expression in male Goldblatt rats.
Assuntos
Hipertensão Renovascular , Hipertensão , Ratos , Masculino , Animais , Hipertensão Renovascular/metabolismo , Receptores de Estrogênio , Receptor alfa de Estrogênio , Pressão Sanguínea , Estradiol/farmacologiaRESUMO
This study proposed to investigate further the role of oestrogens during pubertal growth of rat ventral prostate, by analysing the effect of anti-oestrogen fulvestrant (ICI 182,780) on the expression of androgen (AR) and oestrogen receptors (ESR1 and ESR2), mitogen-activated protein kinase (ERK1/2) phosphorylation, and expression of Ki-67, a biomarker for cell proliferation. Ventral prostates were obtained from 90-day-old rats treated once a week for 2 months with vehicle (control) or ICI 182,780 (10 mg/rat, s.c.). Transcripts for AR, ESR1 and ESR2 were evaluated by quantitative real-time polymerase chain reaction. Expression of AR, ESR1, ESR2, total and phospho-ERK1/2 was analysed by Western blot or immunofluorescence. Ki-67-positive cells and myosin heavy chain were detected by immunohistochemistry. Cylindrical epithelial cells slightly taller, epithelial dysplasia and an increase in smooth muscle layer were observed in the ventral prostate from ICI 182,780-treated rats. ICI 182,780 did not change the mRNA, but decreased the protein levels for AR in the ventral prostate. The expression of ESR1 (mRNA and protein) was upregulated by ICI 182,780, but no changes were observed on ESR2 expression (mRNA and protein). ICI 182,780 decreased the phosphorylation state of ERK1/2, with no changes in total ERK1/2 levels. Ki-67-positive cells in the ventral prostate were also decreased by ICI 182,780. In conclusion, ICI 182,780 induces downregulation of AR expression and may block the translocation of ESR1 and ESR2 from the nucleus to the plasma membrane, decreasing ERK1/2 phosphorylation and prostatic epithelial cell proliferation. These findings provide a basis for physiological roles of oestrogen in the ventral prostate. Further studies with fulvestrant are necessary in benign prostate hyperplasia and prostatic cancer models.
Assuntos
Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Próstata/efeitos dos fármacos , Animais , Western Blotting , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Imunofluorescência , Fulvestranto , Imuno-Histoquímica , Masculino , Fosforilação , Próstata/enzimologia , Próstata/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Testosterona/metabolismoRESUMO
Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca(2+) homeostasis. Thus, in the present work, we have investigated Ca(2+) signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca(2+) rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca(2+). Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca(2+) content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca(2+) handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.
Assuntos
Envelhecimento/fisiologia , Apoptose/fisiologia , Cálcio/metabolismo , Corpo Estriado/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Western Blotting , Imunofluorescência , Ácido Glutâmico/metabolismo , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Mitocôndrias/fisiologia , N-Metilaspartato/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND AND PURPOSE: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. EXPERIMENTAL APPROACH: Male Wistar rats received N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and beta-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [(3)H]inositol phosphate, NO synthase (NOS) activity, [(3)H]quinuclidinyl benzilate ([(3)H]QNB) binding and bladder morphology were also performed. KEY RESULTS: Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [(3)H]inositol phosphate in bladder tissue from rats treated with L-NAME. [(3)H]QNB was bound with an apparent K(D) twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. CONCLUSIONS AND IMPLICATIONS: Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced K(D) values for muscarinic receptors and [(3)H]inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.
Assuntos
Contração Muscular/efeitos dos fármacos , Óxido Nítrico/deficiência , Receptores Adrenérgicos beta 3/metabolismo , Receptores Muscarínicos/metabolismo , Bexiga Urinária Hiperativa/fisiopatologia , Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/farmacologia , Animais , Carbacol/farmacologia , Fosfatos de Inositol/metabolismo , Masculino , Agonistas Muscarínicos/farmacologia , Músculo Liso/metabolismo , NG-Nitroarginina Metil Éster , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Wistar , Receptores Muscarínicos/efeitos dos fármacos , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologiaRESUMO
The aim of this study was to identify the expression, cellular localization and regulation of classic estrogen receptors ERα and ERß, ER-α36 isoform and GPER in the androgen-independent prostate cancer cell line PC-3. In addition, we evaluated the relative contribution of these receptors to the activation of the ERK1/2 (extracellular signal-regulated protein kinases) signaling pathway. These four estrogen receptors were detected by Western blot assays and were shown by immunofluorescence assays to localize preferentially in extranuclear regions of PC-3 cells. In addition, treatment with 17ß-estradiol (E2) (1 µM) for 24 h led to down-regulation of the classic estrogen receptors, whereas E2 at physiological concentration (0.1 nM) for 24h tended to increase the levels of ERα and ERß. Furthermore, the ERα-selective agonist PPT selectively increased the expression of ERß and the ERß-selective agonist DPN increased ERα levels. None of these treatments affected expression of the ER-α36 isoform. The unusual cytoplasmic localization of the classic estrogen receptors in these cells differs from the nuclear localization in the majority of estrogen target cells and suggests that rapid signaling pathways may be preferentially activated. In fact, treatment with selective agonists of ERα, ERß and GPER induced ERK1/2 phosphorylation that was blocked by the respective antagonists. On the other hand, activation of ERK1/2 induced by E2 may involve additional mechanisms because it was not blocked by the three antagonists. Taken together, the results indicate that there is a crosstalk between ERα and ERß to regulate the expression of each other, and suggest the involvement of other receptors, such as ER-α36, in the rapid ERK1/2 activation by E2. The identification of new isoforms of ERs, regulation of the receptors and signaling pathways is important to develop new therapeutic strategies for the castration-resistant prostate cancer.
Assuntos
Estradiol/farmacologia , Receptor alfa de Estrogênio/biossíntese , Receptor beta de Estrogênio/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genéticaRESUMO
Relaxin and its receptor RXFP1 are co-expressed in Sertoli cells, and relaxin can stimulate proliferation of Sertoli cells. In this study, we investigated a role of relaxin in spermatogenesis, using a short-term culture of testicular cells of the rat that allowed differentiation of spermatogonia to spermatids. Sertoli, germ, and peritubular myoid cells were the predominant cell types in the culture. Sertoli and germ cells expressed RXFP1. Cultures were incubated without (control) or with 0.5% fetal bovine serum (FBS) or 100 ng/mL H2 relaxin (RLN) for 2 days. Cell organization, number, and differentiation were analyzed after 2 (D2), 5 (D5) or 8 (D8) days of culturing. Although the proportion of germ cells decayed from D2 to D5, the relative contribution of HC, 1C, 2C, and 4C germ cell populations remained constant in the control group during the whole culture. RLN did not affect the proportion of germ cell populations compared with control, but increased gene and/or protein expression of the undifferentiated and differentiated spermatogonia markers PLZF and c-KIT, and of the post-meiotic marker Odf2 in D5. RLN favored organization of cells in tubule-like structures, the arrangement of myoid cells around the tubules, arrangement of c-KIT-positive spermatogonia at the basal region of the tubules, and expression of the cell junction protein ß-catenin close to the plasma membrane region. Knockdown of relaxin with small interfering RNA (siRNA) reduced expression of ß-catenin at the cell junctions, and shifted its expression to the nucleus. We propose that relaxin may affect spermatogenesis by modulating spermatogonial self renewal and favoring cell contact.
Assuntos
Células-Tronco Adultas/fisiologia , Relaxina/fisiologia , Espermatogênese , Espermatozoides/fisiologia , Animais , Membrana Basal/fisiologia , Técnicas de Cocultura , DNA/metabolismo , Masculino , Ratos Wistar , Espermatozoides/citologia , beta Catenina/metabolismoRESUMO
Studies of MCF-7 breast cancer cells demonstrated that sex hormone-binding globulin (SHBG) is internalized by receptor-mediated endocytosis. The present study demonstrated specific binding of SHBG to receptor on membranes isolated from MCF-7 cells. Scatchard analysis of these binding studies suggested that SHBG binds to a single class of sites on membranes. The analysis yielded a dissociation constant (Kd) at 37 C of 3 x 10(-8) M and a binding capacity of 48 +2- 0.12 pmol/mg protein. A procedure for solubilizing the SHBG receptor from MCF-7 membranes used buffers containing protease inhibitors, 10% glycerol, and 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (246 +/- 14 pmol/mg protein) and a 10-fold decrease in binding affinity (Kd at 37 C = 2 x 10(-7) M).
Assuntos
Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Ligação Competitiva , Neoplasias da Mama , Linhagem Celular , Feminino , Humanos , Radioisótopos do Iodo , Cinética , Receptores de Superfície Celular/isolamento & purificaçãoRESUMO
This study was designed to characterize muscarinic acetylcholine receptors (mAChRs) in primary cultured Sertoli cells from 30-d-old rats. RT-PCR was performed, and five PCR products corresponding to m1-m5 mAChR mRNA subtypes were detected in these cells. Ribonuclease protection assay further confirmed the presence of protected products for m1, m2, m3, and m4 mAChR transcripts. Radioligand binding studies and the analysis of changes in intracellular signaling pathways after cell exposure to carbachol were performed to study mAChRs at the protein level. Scatchard analysis revealed one single class of [(3)H]quinuclidinyl benzilate binding sites. Carbachol produced a reduction on forskolin-induced intracellular cAMP accumulation in Sertoli cells. This effect was reversed by atropine, methoctramine, and tropicamide but not by p-fluoro-hexahydro-sila-difenidol or pirenzepine. Carbachol also induced an increase on total [(3)H]-inositol phosphates content, an effect antagonized by atropine, p-fluoro-hexahydro-sila-difenidol, or pirenzepine but not by methoctramine. Thus, mAChR activation in Sertoli cell is linked to both adenylyl cyclase inhibition and to phosphoinositide hydrolysis. Furthermore, gel shift assays indicated that carbachol also induced a time-dependent stimulation of the activator protein-1 DNA-binding activity, suggesting that activation of mAChRs may play a role in the modulation of gene expression in Sertoli cells. Taken together, these results indicate that mAChRs are present at mRNA and protein level in rat Sertoli cells.
Assuntos
Receptores Muscarínicos/metabolismo , Células de Sertoli/metabolismo , Animais , Carbacol/farmacologia , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Fosfatos de Inositol/metabolismo , Masculino , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinuclidinil Benzilato/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Muscarínicos/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Previous studies suggested that an extracellular steroid-binding protein, testosterone-estradiol-binding globulin (TeBG), can enter a variety of cells. Experiments were conducted to determine whether uptake of TeBG occurs by nonspecific fluid phase endocytosis or via a specific receptor-mediated process. In human breast carcinoma cells (MCF-7) maintained on serum-free medium, exposure to radiolabeled TeBG resulted in cellular uptake, which reached a plateau by 6 h and could be inhibited 80% by competition with unlabeled TeBG. Uptake was temperature dependent with cell-associated radioactivity at 37 C being 1.6-fold higher than at 4 C. Subsequent exposure of cells to pronase resulted in release of the cell-associated TeBG by 88% and 44% at 4 C and 37 C, respectively. After transfer to media devoid of TeBG, approximately 35% of cell-associated radioactivity was release into the medium at 37 C; it was not possible to distinguish whether this was released from the cell surface or from inside the cell. Investigation of the localization of TeBG-gold complexes by electron microscopy revealed that TeBG first binds to the plasmalemma. Within 15 min label appears in receptosomes, which fuse to form multivesicular endosomes. By 1 h all label is observed in multivesicular endosomes and lysosomes, most of which are in the Golgi zone. Localization of the internalized radioactivity using classical cell fractionation techniques showed it appears in a symmetrical band exhibiting the same buoyant density as the lysosomal marker acid phosphatase. The observations reported here show that: 1) TeBG binds to MCF-7 cells; 2) some of the bound TeBG is taken up via a mechanism with all the characteristics of receptor-mediated endocytosis; and 3) within these cells TeBG is localized in endosomes and lysosomes.
Assuntos
Neoplasias da Mama/metabolismo , Endocitose , Receptores de Superfície Celular/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Neoplasias da Mama/ultraestrutura , Endossomos/metabolismo , Ouro , Complexo de Golgi/metabolismo , Humanos , Cinética , Lisossomos/metabolismo , Microscopia Eletrônica , Células Tumorais CultivadasRESUMO
The events involved in the processing of the angiotensin II (Ang II)-receptor complex were studied in primary cultures of rat myometrial cells. Ang II bound to rat myometrial cells in a specific, time- and temperature-dependent fashion. Pretreatment with cycloheximide did not interfere with binding up to 3 hr, but inhibited increases in binding observed over longer periods. The [3H]Ang II binding to intact cells was inhibited by dithiothreitol (DTT), and the rank order of potency of Ang II and nonpeptide antagonists to inhibit the [3H]Ang II binding was Ang II > Losartan >> PD 123319 or CGP 42112B, indicating the presence of the AT1 receptor type. Whereas most of the [3H]Ang II binding at 4 degrees was susceptible to acid or pronase treatment, binding at 35 degrees was resistant to both treatments, suggesting an internalization of the Ang II-receptor complex. Phenylarsine oxide (PAO) and N-ethylmaleimide (NEM) caused a concentration-dependent inhibition when the binding assay was performed at 35 degrees, but no effect was observed at 4 degrees, indicating that these agents did not alter cell-surface binding but actually prevented the internalization process. Simultaneous treatment with 1 mM DTT or beta-mercaptoethanol prevented the inhibitory effect of NEM, but only DTT could prevent the inhibition caused by PAO, indicating that two closely located sulfhydryl groups must be involved in the internalization process. Chloroquine (100 microM) inhibited the [3H]Ang II dissociation from cells, and monensin (25 microM) induced a 30% inhibition of [3H]Ang II binding (35 degrees, 3 hr), suggesting endosomal processing of the Ang II-receptor complex with receptor recycling to the cell surface. These results indicate that Ang II binding to AT1 receptors in rat myometrial cells is followed by internalization of the Ang II-receptor complex and recycling of the receptor to the cell surface.
Assuntos
Angiotensina II/metabolismo , Miométrio/metabolismo , Receptores de Angiotensina/metabolismo , Antagonistas de Receptores de Angiotensina , Animais , Arsenicais/farmacologia , Ligação Competitiva , Células Cultivadas , Ditiotreitol/farmacologia , Endocitose , Endossomos/metabolismo , Etilmaleimida/farmacologia , Feminino , Miométrio/ultraestrutura , Ratos , Ratos WistarRESUMO
The estrogen treatment of adult female rats induces an increase in myometrium sensitivity to cholinergic agonists and in this tissue the presence of M(2)- and M(3)-muscarinic acetylcholine (mACh) receptor was shown. We now report the effect of estrogen on intracellular signaling pathways linked to activation of M(2)- and M(3)-mACh receptor subtypes. The intracellular cyclic AMP accumulation and [3H]-inositol phosphates content were measured in myometrium strips from rats in estrus (control) and estradiol-treated rats (12.5 microg/100 g body weight, sc, 24 h before experiments) (the plasma estradiol level was 30.9+/-3.5 pg/ml and 119.3+/-14.1 pg/ml from control and estrogen-treated rats, respectively). Estrogen treatment increased 2.5-fold the intracellular cyclic AMP accumulation induced by 10 microM forskolin. The effects of muscarinic agonist and antagonists on cyclic AMP accumulation were tested. Carbachol reduced the forskolin-induced intracellular cyclic AMP content, 3.0 and 10.5-fold, in myometrium from control and estradiol-treated rats, respectively. This inhibitory effect failed to occur when carbachol was incubated in the presence of methoctramine. Carbachol also induced increase on total [3H]-inositol phosphates accumulation in myometrium from estradiol-treated rats when compared with control rats. This effect was reversed by pfHHSiD. These studies suggest the modulation by estrogen of intracellular signaling pathways linked to activation of M(2)- and M(3)-mACh receptors in the rat myometrium.
Assuntos
Estradiol/análogos & derivados , Miométrio/efeitos dos fármacos , Miométrio/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Animais , Carbacol/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Estradiol/farmacologia , Feminino , Fosfatos de Inositol/metabolismo , Líquido Intracelular/metabolismo , Antagonistas Muscarínicos/farmacologia , Ratos , Ratos Wistar , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Transdução de Sinais/efeitos dos fármacosRESUMO
UNLABELLED: Sex-hormone-binding globulin (SHBG) binds to a specific protein on the surface of prostate, epididymis, and a human breast cancer cell line (MCF-7), and is internalized by these cells. The present study demonstrated specific binding of SHBG to receptor on membranes prepared from rat testes. The binding was saturable, specific, and time and temperature dependent. Scatchard analysis of these binding studies suggested that SHBG binds to a single class of sites on testicular membranes with a Kd at 37 degrees C of 5 x 10(-8) M and a binding capacity of 30 +/- 0.6 pmol/mg protein. These binding characteristics are similar to the SHBG receptor on human prostate and MCF-7 cells. Solubilization of the receptor resulted in a 5-fold increase in its binding capacity (158 +/- 0.3 pmol/mg protein) and a 10-fold decrease in binding affinity (Kd at 37 degrees C = 6.5 x 10(-7) M). The apparent molecular weight of the testicular SHBG receptor, as estimated by gel filtration, was M(r) = 174,000. CONCLUSION: a specific binding site for SHBG was identified on testicular membranes. This binding site has been tentatively identified as a SHBG receptor based on its physical properties in testicular membrane preparations and following solubilization.
Assuntos
Membrana Celular/metabolismo , Receptores de Superfície Celular/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Testículo/metabolismo , Animais , Ácidos Cólicos , Cromatografia em Gel , Detergentes , Masculino , Peso Molecular , Ligação Proteica , Coelhos , Ratos , Ratos Wistar , Receptores de Superfície Celular/isolamento & purificação , SolubilidadeRESUMO
In plasma, most steroid hormones are bound and transported by the specific binding protein, testosterone-estradiol-binding globulin (TeBG). For years, it was believed that the only function of this protein was to regulate the concentration of free steroids in plasma. However, a number of reports have provided evidence for the presence of specific TeBG receptors on plasma membranes. Furthermore, the interaction of TeBG with its receptor was shown to be inhibited when steroids are bound to TeBG, suggesting that TeBG is an allosteric protein. The purpose of this manuscript is to review the evidence that androgen-binding proteins bind to membrane receptors, and, in some cells, this binding stimulates cAMP accumulation, and transfer TeBG/ABP into tissue as a consequence of receptor mediated endocytosis. Recent studies from our laboratories have demonstrated binding and uptake of TeBG by MCF-7 breast cancer cells. The interaction of unligated rabbit TeBG with membranes from MCF-7 cells resulted in a time and concentration-dependent increase in adenylate cyclase activity. The TeBG alone also had a reproducible effect on intact cells by increasing cAMP accumulation by 30-35%. The addition of DHT to cells, after TeBG has been allowed to bind, resulted in increases in cAMP of greater than 4-fold. This effect was not blocked by antiandrogens. These data support the hypothesis that extracellular SHBG is a regulator of cellular function through a membrane receptor that is coupled to adenylate cyclase.
Assuntos
Proteína de Ligação a Androgênios/metabolismo , Receptores de Superfície Celular/metabolismo , Globulina de Ligação a Hormônio Sexual/metabolismo , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/metabolismo , Di-Hidrotestosterona/farmacologia , Endocitose , Humanos , Coelhos , Transdução de SinaisRESUMO
Angiotensin II interacts with specific cell surface angiotensin AT1 and AT2 receptors and, in some vertebrates, with an atypical angiotensin AT receptor. This study was designed to characterize the angiotensin receptor in the heart of Bothrops jararaca snake. A specific and saturable angiotensin II binding site was detected in cardiac membranes and yielded Kd=7.34+/-1.41 nM and B(max)=72.49+/-18 fmol/mg protein. Competition-binding studies showed an angiotensin receptor with low affinity to both angiotensin receptor antagonists, losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole) and PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate). Studies on the intracellular signaling pathways showed that phospholipase C/inositol phosphate breakdown and adenylylcyclase/cyclic AMP generation were not coupled with this angiotensin receptor. An adenylylcyclase enzyme sensitive to forskolin was detected. The results indicate the presence of an angiotensin receptor in the heart of B. jararaca snake pharmacologically distinct from angiotensin AT1 and AT2 receptors. It seems to belong to a new class of angiotensin receptors, like some other atypical angiotensin AT receptors that have already been described.
Assuntos
Bothrops/metabolismo , Miocárdio/metabolismo , Receptores de Angiotensina/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Angiotensina Amida/farmacologia , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Imidazóis/farmacologia , Fosfatos de Inositol/metabolismo , Losartan/farmacologia , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Piridinas/farmacologia , Receptores de Angiotensina/efeitos dos fármacos , Saralasina/farmacologia , TrítioRESUMO
In the present study, we investigated the alpha1-adrenoceptor subtypes mediating adrenaline-induced contractions of the rat seminal vesicle by using functional studies. The reverse transcription combined with polymerase chain reaction (RT-PCR) was also used to identify of alpha1-adrenoceptor mRNA subtypes. The rank order of potency of alpha1-adrenoceptor antagonists in blocking the contractile effects of adrenaline was: prazosin = WB 4101 >>> BMY 7378 > chloroethylclonidine, indicating the presence of alpha1A-adrenoceptors in the rat seminal vesicle. In the presence of nifedipine, there was a 76% reduction in the adrenaline-induced contractions. The nifedipine-insensitive component (24%) of the contractile response to adrenaline was unaffected by chloroethylclonidine. A small pool of spare alpha1-adrenoceptors for adrenaline (0.10%) was also detected. All three alpha1-adrenoceptor subtypes were amplified when RT-PCR was performed on total RNA isolated from rat seminal vesicle. In conclusion, these data indicate the presence of three alpha1-adrenoceptor mRNA subtypes, but only alpha1A-adrenoceptors are involved in the rat seminal vesicle contraction.
Assuntos
Receptores Adrenérgicos alfa 1/biossíntese , Glândulas Seminais/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Clonidina/análogos & derivados , Clonidina/farmacologia , Dioxanos/farmacologia , Epinefrina/farmacologia , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Fenoxibenzamina/farmacologia , Piperazinas/farmacologia , Prazosina/farmacologia , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Seminais/efeitos dos fármacosRESUMO
The post-natal development-related changes in the sensitivity to sympathomimetic agonists were studied in the seminal vesicle of 40-, 60- and 120-day old rats by determining the pD2 values for adrenaline, phenylephrine and methoxamine. The age-related changes in the neuronal and extraneuronal uptake of sympathomimetic agonists were also determined. The seminal vesicle sensitivity to adrenaline, phenylephrine and methoxamine increased in sexually mature rats (60- and 120-day old) (concentration-effect curves shifted to the left and pD2 values increased) when compared with immature rats (40-day old). Cocaine, a neuronal uptake inhibitor, induced supersensitivity to adrenaline at all ages, but did not affect the sensitivity to methoxamine. The ratio between the EC50 values for adrenaline in the presence and in the absence of cocaine was reduced in tissues of mature animals. This reduction suggests a loss of function of the neuronal uptake system with sexual development. In the presence of cocaine the pD2 value for adrenaline was increased during sexual development. Thus, the age-related changes in the adrenaline sensitivity were observed even when neuronal uptake was not operative, suggesting age-related changes at the post-junctional level, which was confirmed by the increase in the seminal vesicle sensitivity to methoxamine. In the presence of the extraneuronal uptake inhibitor, corticosterone, the sensitivity to adrenaline did not change and the age-related changes persisted.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glândulas Seminais/efeitos dos fármacos , Simpatomiméticos/farmacologia , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Cocaína/farmacologia , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Ratos , Ratos Wistar , Maturidade SexualRESUMO
The sensitivity of seminal vesicles to sympathomimetic drugs and the plasma concentrations of testosterone and gonadotropins (LH and FSH) were examined in adult male albino rats divided into three groups: normal, castrated and Androcur-treated. Dose-effect curves for adrenaline and noradrenaline were obtained in the presence and absence of cocaine or corticosterone and the parameter pD2 was determined. Castration and Androcur treatment induced a decrease in seminal vesicle sensitivity to drugs (shifting of dose-effect curves to the right, decreased pD2). Cocaine shifted the adrenaline and noradrenaline dose-effect curves to the left in three groups. Corticosterone did not modify the dose-effect curves of adrenaline and noradrenaline. Plasma testosterone levels were decreased by castration and Androcur-treatment although the LH and FSH concentrations were markedly altered by castration but were unaltered by the antiandrogenic treatment. The results suggest that the androgen may play an important role in the sensitivity of the seminal vesicle to sympathomimetic drugs through control of the neuronal uptake of catecholamines.
Assuntos
Antagonistas de Androgênios , Ciproterona/análogos & derivados , Orquiectomia , Parassimpatomiméticos/farmacologia , Glândulas Seminais/efeitos dos fármacos , Animais , Ciproterona/farmacologia , Acetato de Ciproterona , Epinefrina/sangue , Masculino , Norepinefrina/sangue , Ratos , Ratos EndogâmicosRESUMO
1. The influence of hormones on the submandibular gland of rodents has attracted more attention since the observation of the sexual dimorphism of these organs. Androgens enhance both the development and the secretory activity of the gland. 2. In the present investigation we have studied the differences in wet weight, protein content, glandulain activity and morphometry of granular convoluted tubules of submandibular glands excised from control, castrated and antiandrogen-treated (RU 23908 or cyproterone acetate) male adult albino rats. 3. Castration and antiandrogenic treatment did not affect the wet weight or protein content of the organs. Castration or treatment with RU 23908, but not treatment with cyproterone acetate, decreased glandulain activity and the height of the secretory epithelium of granular ducts. 4. The morphology and glandulain activity of submandibular gland granular ducts do not seem to be related solely to plasma testosterone concentration. Different hormonal treatments are needed to identify other factors implicated in the phenomenon.
Assuntos
Aminopeptidases/metabolismo , Antagonistas de Androgênios/farmacologia , Ciproterona/análogos & derivados , Imidazóis/farmacologia , Imidazolidinas , Orquiectomia , Caracteres Sexuais , Glândula Submandibular/anatomia & histologia , Animais , Ciproterona/farmacologia , Acetato de Ciproterona , Masculino , RatosRESUMO
We determined the prevalence of Y chromosome deletions in a population of 60 Brazilian nonobstructive azoospermic and severely oligozoospermic men. PCR-based screening of microdeletions was performed on lymphocyte DNA for the presence of 14 sequence-tagged sites (STS) located in the azoospermic factor (AZF) on the Yq chromosome. All STS were amplified efficiently in samples from 12 fertile men tested, but failed to be amplified in samples from fertile women, indicating the specificity of PCR conditions for Yq screening. Overall, 4 of the 60 infertile patients tested (6.7%) exhibited deletion of the Y chromosome, 2 of them being severely oligozoospermic patients (P10 and P32) and 2 azoospermic men (patients P47 and P57). Patients P47 and P57 presented larger deletions in the AZFa, AZFb and AZFc subregions, with apparent loss of Yq material evidenced by karyotype analysis. Patients P10 and P32 presented deletions confined to the AZFc region, involving the DAZ locus. Male relatives of patients P10 and P32 had no Y chromosome deletions and presented a normal karyotype, suggesting a de novo status of the deletions found. Our data add to the growing literature showing that microdeletions of the Y chromosome can be the cause of male idiopathic infertility.