Differential effects of estrogen receptors in the rostral ventrolateral medulla in Goldblatt hypertension.

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.

Produção e caracterização de protease fibrinolítica de Streptomyces parvulus DPUA 1573 / Production and characterization of fibrinolytic protease from Streptomyces parvulus DPUA 1573

As proteases fibrinolíticas são capazes de degradar coágulos de fibrina formados dentro dos vasos sanguíneos, evitando a trombose intravascular. Em animais, a tromboflebite, que acomete frequentemente os equinos, ocasiona, em seus casos graves, a obstrução jugular e também um edema de laringe, derivando a obstrução das vias aéreas, o que possibilita um edema cerebral, ocorrendo o óbito do animal. Devido ao fato de o tratamento ser de custo elevado, faz-se necessária a investigação de outras fontesde proteases fibrinolíticas com custos menores e com menos efeitos colaterais. Diante disso, este estudo tem como objetivo produzir e caracterizar proteases fibrinolíticas obtidas de Streptomyces parvulus DPUA 1573. Para produção da enzima, foi utilizado um planejamento fatorial 24 avaliando a concentração da farinha de soja (0,5, 1,0 e 1,5%) e da glicose (0, 0,5 e 1,0g/L), temperatura (28, 32 e 37ºC) e agitação (150, 200 e 250rpm) sobre a biomassa e a atividade fibrinolítica. Pode-se verificar que a protease fibrinolítica apresentou atividade máxima (835U/mL) nas condições de concentração de 1,5% de soja, 1g/L de glicose, 28°C e 150rpm com 48 horas de fermentação. A protease fibrinolítica obtida teve temperatura e pH ótimos de 55°C e pH 9,0, respectivamente. A atividade enzimática foi inibida pelo EDTA, pelo íon Fe2+ e pelo SDS, o que indicou a enzima ser uma metaloprotease. A linhagem Streptomyces parvulus DPUA 1573 foi capaz de produzir protease fibrinolítica, possuindo características bioquímicas favoráveis à aplicação na medicina veterinária e possivelmente humana.(AU)

Fibrinolytic proteases are able to degrade fibrin clot formed in the blood vessel, avoiding intravascular thrombosis. In animals, thrombophlebitis often affects horses, and in severe cases causes obstruction of the jugular and laryngeal edema leading to airway obstruction allowing cerebral edema resulting in the death of the animal. Since treatment is costly, the investigation of other sources of fibrinolytic proteases at lower cost and with fewer side effects is needed. Thus, this study aims to produce and characterize fibrinolytic proteases from Streptomyces parvulus DPUA 1573. For enzyme production, a factorial design was performed to evaluate 24 soybean flour concentration (0.5, 1.0 and 1.5%) and glucose (0, 0.5 and 1.0g/L), temperature (28, 32 and 37°C) and agitation (150, 200 and 250rpm) on biomass and fibrinolytic activity. Fibrinolytic protease showed maximum activity (835 U/mL) under these conditions: 1.5% soybean flour, 1g/L glucose, 28°C, and 150rpm 48 hours of fermentation. The optimal temperature was 55°C and optimal pH was 9.0. Fibrinolytic protease activity was inhibited by EDTA, the ion Fe2+, and by SDS, which indicated that the enzyme is a metallo-protease. The strain Streptomyces parvulus DPUA 1573 was able to produce fibrinolytic protease with biochemical characteristics favorable for application in veterinary and human medicine.(AU)

Expression and regulation of the estrogen receptors in PC-3 human prostate cancer cells.

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.

Effects of the oestrogen receptor antagonist Fulvestrant on expression of genes that affect organization of the epididymal epithelium.

The role of oestrogens in epididymal function is still unclear. Knockout of the oestrogen receptor ESR1 (Esr1(-/-) ) or treatment with the anti-oestrogen Fulvestrant affect epididymal milieu and sperm motility. We investigated the effect of in vivo treatment of rats with Fulvestrant on: (i) expression of genes that may be important for the architecture and function of the epididymal epithelium: prominins 1 and 2, metalloproteinase 7, claudin 7, beta-catenin and cadherin 13, and (ii) levels of oestradiol and testosterone, and expression of oestrogen and androgen receptors, in the initial segment (IS), caput, corpus and cauda epididymis. Fulvestrant (i) reduced gene expression of prominin 1 (variant 1) in the caput, reduced prominin 1 protein content in the caput epididymis and in the efferent ductules, and increased the localization of prominin 1 in microvilli of the caput and corpus; (ii) reduced gene expression of prominin 2 in the corpus and cauda epididymis; (iii) increased the metalloproteinase 7 content in the apical region of principal cells from IS/caput; (iv) reduced in the corpus epididymis, but increased in the efferent ductules, the cadherin 13 mRNA level; (v) reduced testosterone but increased oestradiol levels in the corpus and cauda; (vi) increased the androgen receptor protein content in all regions of the epididymis, and the oestrogen receptor GPER in the corpus and cauda epididymis. In conclusion, treatment with Fulvestrant induced regional-specific changes in hormonal and steroid receptor content, and affected expression of proteins important for epithelial organization and absorption/secretion. The mechanisms of oestrogen action may differ among epididymal regions, which may contribute to determine region-specific sperm functions.

Produção de biocompostos com atividade antimicrobiana de Streptomyces sp. ante isolados de mastite caprina / Production of biocompounds with antimicrobial activity of Streptomyces sp. Isolated in face of goat mastitis

Actinomicetos são um dos principais produtores de enzimas, vitaminas e metabólitos secundários, destacando-se o gênero Streptomyces, o qual tem uma ampla capacidade de produção de antibióticos eficazes no combate a diferentes microrganismos, entre eles o Staphylococcus sp. Em virtude dessa eficiência no combate a patógenos, o objetivo deste trabalho foi avaliar a produção de metabólitos com atividade antimicrobiana produzidos por 67 Streptomyces isolados de liquens da região amazônica, ante isolados de mastite caprina (Staphylococcus aureus) do estado de Pernambuco, Brasil. Foi utilizado um planejamento fatorial para avaliar a influência das fontes de carbono (glicose) 0%, 0,5% e 1% e de nitrogênio (farinha de soja) 1%, 2,5% e 4% na produção dos antimicrobianos, bem como das variáveis pH, biomassa e atividade antimicrobiana. Dos Streptomyces estudados, o DPUA 1566 foi o que se destacou por formação de halos de inibição entre 18 e 26mm ante os isolados de mastite caprina. Foi possível verificar que a fonte de carbono inibiu a produção de antimicrobianos quando submetidos a uma concentração de glicose de 1%; com a retirada desta, os Streptomyces apresentaram uma elevada capacidade de produção de metabólitos com atividade antimicrobiana tendo potencial para o tratamento de mastite caprina.

Actinomycetes are a leading producer of enzymes, vitamins and secondary metabolites, especially the genus Streptomyces, which have a large capacity for the production of natural antibiotics and are effective against various micro-organisms including Staphylococcus sp. Due to this efficiency in combating micro-organisms, the aim of this study was to evaluate the production of metabolites with antimicrobial activity produced by Streptomyces isolated from lichens 67 in the Amazonia, compared to isolates from goat mastitis (Staphylococcus aureus) in the state of Pernambuco, Brazil. We used a complete factorial design to evaluate the influence of concentrations of carbon sources (glucose) at 0%, 0.5% and 1% and nitrogen (soybean flour) at 1%, 2.5% and 4% in the production of antimicrobial metabolites, and the influence of the pH, microbial biomass and activity variables. Of the studied Streptomyces DPUA 1566 what stood out was the formation of inhibition halos between 18 to 26 mm compared to the isolates from goats mastitis. It was noted that the carbon source inhibited the production of antimicrobial metabolites when subjected to a glucose concentration of 1%. However, after the discontinuation, Streptomyces showed a high capacity to produce metabolites with antimicrobial activity, which has an excellent potential for the treatment of mastitis in goats.

Experimental varicocoele in rats affects mechanisms that control expression and function of the androgen receptor.

Varicocoele is an important cause of male infertility. Normal male reproductive function and fertility depends on a delicate balance between androgen receptor (AR) and the classic oestrogen receptors ESR1 (ERα) and ESR2 (ERß). Using a model of surgically induced varicocoele in rats, this study aimed to investigate the effects of varicocoele on the expression of AR, ESR1, ESR2 and G-protein coupled oestrogen receptor (GPER). Varicocoele did not affect the mRNA and protein expression of ESR1 and ESR2 in both testes. Varicocoele did not affect the mRNA and protein expression of GPER in the right testis, but slightly reduced the mRNA and increased the protein levels in the left testis. Varicocoele did not affect the mRNA for AR, but reduced the protein levels in both testes. A proteomic approach was used in an attempt to find differentially expressed targets with possible correlation with AR downregulation. Varicocoele caused the differential expression of 29 proteins. Six proteins were upregulated, including the receptor for activated C kinase 1 (RACK1), and 23 were downregulated, including dihydrolipoamide dehydrogenase, alpha-enolase and pyrophosphatase 1. Western blot analysis confirmed that varicocoele upregulated the expression of RACK1, a protein involved with tyrosine phosphorylation and regulation of AR transcriptional activity, AR metabolism and dynamics of the blood-testis barrier. In conclusion, this study suggests that varicocoele affects mechanisms that control AR expression and function. This regulation of AR may play an important role in the varicocoele-induced testicular dysfunction. Furthermore, varicocoele downregulates several other proteins in the testis that may be useful markers of spermatozoa function and male infertility.

Estrogen receptor 1 agonist PPT stimulates Slc2a4 gene expression and improves insulin-induced glucose uptake in adipocytes.

Type 2 diabetes mellitus is characterized by disruption in glycemic homeostasis, involving impaired insulin-induced glucose disposal. For that, reduced glucose transporter GLUT4, encoded by Slc2a4 gene, plays a fundamental role. Conversely, increase in Slc2a4/GLUT4 expression improves glycemic homeostasis. Recent studies have proposed that estradiol is able to modulate Slc2a4 expression, according to distinct effects upon estrogen receptors ESR1/ESR2. We hypothesize that ESR1-agonist effect could stimulate Slc2a4 expression; thus, increasing cellular glucose disposal, which could be beneficial to glycemic control. Differentiated 3T3-L1 adipocytes were treated (24 hours) with selective ESR1- agonist PPT 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, selective ESR1-antagonist MPP 1,3-Bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride, and selective ESR2 agonist DPN 2,3-bis(4-Hydroxyphenyl)-propionitrile, with/without 17ß-estradiol (E2). We analyzed Slc2a4 mRNA (real time PCR) and GLUT4 protein (Western blotting) expression, transcriptional activity of the Slc2a4 repressor Nuclear Factor- κB (NF-κB) (electrophoretic mobility shift assay), and cellular glucose disposal (2-deoxi-D-[(3)H]glucose uptake, 2-DG). ESR1-agonist PPT enhanced Slc2a4/GLUT4 expression (~30%) in the absence or presence of 0.1 and 10 nmol/L E2, and decreased the NF-κB binding activity (~50%). Conversely, ESR1-antagonist MPP, together with E2, decreased Slc2a4/GLUT4 expression (20-40%) and increased NF-κB binding activity (~30%). Furthermore, treatment with ESR2- agonist DPN decreased Slc2a4/GLUT4 expression (20-50%). 2-DG uptake was modulated in parallel to that observed in GLUT4 protein. The present results reveal that ESR1 activity enhances, whereas ESR2 activity represses, Slc2a4/GLUT4 expression. These effects are partially mediated by NF-κB, and allow parallel changes in adipocyte glucose disposal. Furthermore, the data provide evidences that ESR1-agonist PPT, as a Slc2a4/GLUT4 enhancer, can be a promising coadjuvant drug for diabetes mellitus therapy.

The anti-oestrogen fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation and cell proliferation in the rat ventral prostate.

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.

Alterations in calcium signaling and a decrease in Bcl-2 expression: possible correlation with apoptosis in aged striatum.

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.

Incidence of dementia in a community-dwelling Brazilian population.

The authors report the incidence of dementia in a community-dwelling Brazilian population. In 1997, 1656 individuals aged 65 years or more, the majority being of very low educational level, were screened at their homes in Catanduva, Brazil, and dementia was diagnosed in 118 cases. The remaining 1538 individuals were rescreened 3.25 years later applying a health questionnaire, the Mini-Mental State Examination (MMSE) and the Pfeffer Functional Activities Questionnaire (PFAQ). According to PFAQ and MMSE scores, selected subjects were submitted to clinical, neurologic, and cognitive evaluations. The subjects diagnosed with dementia underwent laboratory tests and brain computed tomography. A total of 1119 individuals were rescreened and 50 incident cases of dementia (28 with Alzheimer disease [AD]) were identified. The incidence rate of dementia was 13.8 and of AD was 7.7 per 1000 person-years for individuals aged 65 years or older. The incidence rates of dementia almost doubled with every 5 years of age. There was no difference according to gender, but women had a higher incidence of dementia, predominantly AD, in very old age. There was a trend for higher incidence of dementia in illiterates (p = 0.07), but multivariate analysis disclosed significant association only between age and higher incidence of dementia. The incidence rates of dementia in this Brazilian community are comparable to those reported in Western and Asian studies.

Prevalence of Y chromosome deletions in a Brazilian population of nonobstructive azoospermic and severely oligozoospermic men

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 percent) 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

Angiotensin receptor in the heart of Bothrops jararaca snake.

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.

Effect of estrogen on intracellular signaling pathways linked to activation of M(2)- and M(3)-muscarinic acetylcholine receptors in the rat myometrium.

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.

Receptors for androgen-binding proteins: internalization and intracellular signalling.

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.

Binding of sex-hormone-binding globulin (SHBG) to testicular membranes and solubilized receptors.

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.

Binding of an extracellular steroid-binding globulin to membranes and soluble receptors from human breast cancer cells (MCF-7 cells).

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).

Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells.

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.

Effects of castration and antiandrogens (RU 23908 and cyproterone acetate) on glandulan activity and on the secretory epithelium of the rat submandibular gland

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 secretory activity of the gland. 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-teated (RU 23908 or cyproterone acetate) male adult albino rats. 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. 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