RESUMO
Carbonic anhydrase IV (CA IV) is a glycosylphosphatidylinositol-linked isozyme previously identified on the surface of renal tubular epithelium and certain populations of vascular endothelium. This report identifies the regional, cellular, and subcellular localization of CA IV in the rat gut. Northern blot and RT-PCR analyses demonstrated little CA IV expression in stomach or proximal small intestine, but abundant expression in distal small and large intestine. In contrast, CA II mRNA was abundant in stomach, decreased in proximal small intestine, low in distal small intestine, and abundant in large intestine. CA I mRNA was detected only in large intestine. The regional distribution of CA IV activity correlated with distribution of CA IV mRNA. Immunohistochemistry localized CA IV to the apical plasma membrane of the mucosal epithelium in distal small intestine and large intestine. Signal intensity was greatest in colon. CA IV was additionally found in submucosal capillary endothelium of all gastrointestinal regions. Immunohistochemical findings in human stomach and colon paralleled those in the rat. These studies demonstrate pre-translational isozyme-specific regulation of CA expression along the cranial-caudal axis of the gastrointestinal tract. The regional, cellular, and subcellular localizations are consistent with participation of CA IV in the extensive ion and fluid transport in the distal small and large intestine.
Assuntos
Anidrases Carbônicas/biossíntese , Sistema Digestório/enzimologia , Mucosa Gástrica/enzimologia , Expressão Gênica , Mucosa Intestinal/enzimologia , Isoenzimas/biossíntese , Animais , Capilares , Anidrases Carbônicas/análise , Colo/enzimologia , Endotélio Vascular , Epitélio/enzimologia , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Mucosa Intestinal/irrigação sanguínea , Intestino Grosso/enzimologia , Intestino Delgado/enzimologia , Isoenzimas/análise , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Estômago/enzimologia , Frações Subcelulares/enzimologiaRESUMO
Tamoxifen-induced DNA adducts were analyzed with the (32)P-postlabeling method using high-performance liquid chromatography (HPLC)-radioactivity detection from endometrial tissue of breast cancer patients and controls. Liver DNA from tamoxifen-treated rats was used as a positive standard. In blind analysis, five of the seven samples from tamoxifen-treated patients showed DNA adducts; none of the five controls were positive. The identity of the tamoxifen adduct was confirmed by using different chromatographic systems, isolating the HPLC fractions and running them on TLC, with or without spiked rat liver samples. The level of adducts in the treated patients was 2.7 adducts/10(9) nucleotides in the HPLC analysis.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Carcinógenos/farmacologia , Adutos de DNA , Dano ao DNA , Endométrio/efeitos dos fármacos , Antagonistas de Estrogênios/farmacologia , Tamoxifeno/farmacologia , Idoso , Animais , Antineoplásicos Hormonais/efeitos adversos , Antineoplásicos Hormonais/uso terapêutico , Carcinógenos/efeitos adversos , Carcinógenos/uso terapêutico , Cromatografia Líquida de Alta Pressão , Endométrio/química , Antagonistas de Estrogênios/efeitos adversos , Antagonistas de Estrogênios/uso terapêutico , Feminino , Humanos , Pessoa de Meia-Idade , Ratos , Método Simples-Cego , Tamoxifeno/efeitos adversos , Tamoxifeno/uso terapêuticoRESUMO
Rat ovarian and testicular particles prelabelled with human [125I]CG (ovary) or [131I]CG (testis) were incubated in low ionic strength medium and the water-soluble components obtained from the particulate receptor-human CG complexes were characterized and compared. Sedimentation coefficients determined by sucrose density gradient centrifugation were 4.3 S for the ovarian and 4.9 S for the testicular component run in the same sample. Gel filtration analyses displayed, however, that both from the ovarian and testicular particles two components of different sizes were released, the smaller form (hydrodynamic radius of 3.9 nm) being dominant for the ovary, and the larger one (hydrodynamic radius of 4.8 nm) dominant for the testis. The molecular weights determined by SDS-polyacrylamide gel electrophoresis were 100 000 and 72 000 for the testicular, and 94 000 and 72 000 for the ovarian components. Analyses of the ovarian components by gel filtration, ion exchange chromatography and reversed-phase HPLC technique showed further that these components differ also from each other in their charge and solubility to organic solvents, and that both of them contain intact human CG as a part of their structure. These results demonstrate that two water-soluble components are released from rat testis and ovary, both of which are distinctly smaller in size than detergent-solubilized receptor-human CG complexes. Our interpretation is that they are formed by endogenous proteolysis of the receptor, at two distinct sites, respectively.
Assuntos
Gonadotropina Coriônica/metabolismo , Ovário/metabolismo , Receptores de Superfície Celular/metabolismo , Testículo/metabolismo , Animais , Membrana Celular/metabolismo , Feminino , Masculino , Fragmentos de Peptídeos/análise , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/isolamento & purificação , Receptores do LH , SolubilidadeRESUMO
Pseudopregnant rats were injected intravenously with radioactively-labelled human choriogonadotropin (CG). The animals were killed 2 h after the injection and the ovaries, liver and kidney were subjected to perifusion. Radioactivity was released from the ovaries at an increasing rate during perifusion, mainly in a complex form with a molecular size between human CG and the solubilized receptor-human CG complex. The increase in the rate of radioactivity release was inhibited by N-ethylmaleimide and CuCl2, but not by MgCl2, Trasylol, N alpha-tosyl-L phenylalanine chloromethyl ketone or N alpha-p-tosyl-L-lysine and CuCl2 chloromethyl ketone. Intact hormone dissociation from the complex at pH 3. After perifusion the ovarian tissue radioactivity only as receptor-hormone complexes. Only free radioiodine released from the control tissues, liver and kidney during perifusion. The low molecular weight hormone complex was also released from a homogenate of pseudopregnant rat ovaries prelabelled in vivo with radioactivity-labelled human CG during incubation in a hypotonic medium. The release of this complex was likewise inhibited by alkylating agents and heavy metals, and intact hormone dissociated from the complex at pH 3. A similar human CG complex was released also from purified receptor-human CG complex during incubation with ovarian homogenate, and presence of N-ethylmaleimide or use of heat inactivated ovarian homogenate inhibited this process. The present results indicate that the in vivo bound human CG sheds from the luteal tissue in perifusion and incubation as a low molecular weight complex. This may be a facet in the processing and elimination of occupied LH receptors from the ovary.
Assuntos
Gonadotropina Coriônica/metabolismo , Ovário/metabolismo , Pseudogravidez/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Cromatografia em Gel , Cobre/farmacologia , Etilmaleimida/farmacologia , Feminino , Rim/metabolismo , Cinética , Fígado/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores do LHRESUMO
In vitro degradation of LH receptor after occupancy by hCG was studied. Rat ovarian membranes labeled with [125I]iodo-hCG were incubated at 37 C; as a result, 30-40% of the radioactivity initially bound was rendered soluble in the medium. The molecular complexes in the medium and in incubated membranes solubilized with 1% Triton X-100 were then cross-linked with glutaraldehyde and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Particulate receptor-[125I]iodo-hCG complex exhibiting an apparent mol wt of 125,000 was cleaved during incubation into two distinct components (mol wt, 96,000 and 74,000) which appeared in the medium. Using tritiated hCG (beta-subunit labeled) instead of radioiodinated hCG (alpha-subunit labeled), these same two components were also observed, indicating that they both contain intact hCG (alpha and beta) as a part of their structure. In addition to the hormone (mol wt, 48,000), these two components contain receptor fragments with mol wt of 64,000 or 38,000, demonstrated directly by labeling the particulate receptor itself with periodate-tritiated borohydride before tagging with unlabeled hCG and in vitro incubation. These receptor fragments were purified from the medium by hCG-directed immunoaffinity chromatography and detached from the hormone by pH treatment. The intact receptor extracted from the membranes with detergent and purified identically in the absence of proteolysis migrated as a 90,000 mol wt polypeptide. These results demonstrate that after hormone occupancy, proteolytic cleavage of the 90,000 mol wt receptor polypeptide occurs at two specific sites. Thiol-blocking agents selectively prevented the appearance of the larger component (hCG coupled to 64,000 mol wt receptor fragment), while metal-chelating agents markedly decreased the appearance of the smaller component (hCG coupled to 38,000 mol wt receptor fragment) in the medium. Identical observations, obtained upon incubation of plasma membranes purified by sucrose density gradient centrifugation, suggest that plasma membrane enzymes are involved.
Assuntos
Membrana Celular/enzimologia , Receptores de Superfície Celular/metabolismo , Animais , Gonadotropina Coriônica/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Peso Molecular , Ovário/ultraestrutura , Inibidores de Proteases/farmacologia , Pseudogravidez , Ratos , Ratos Endogâmicos , Receptores do LHRESUMO
Antiserum to purified LH (hCG) receptor was raised in rabbits and used to localize the receptor with the peroxidase-antiperoxidase complex method in pseudopregnant rat ovary. The receptor was purified using immobilized antibodies to hCG as an immunoaffinity using immobilized antibodies to hCG as an immunoaffinity matrix for solubilized receptor-hCG complex. The unoccupied receptor was eluted from the column by acetic acid and used to immunize rabbits. The antibodies produced inhibited binding of [125I]iodo-hCG to pseudopregnant rat ovarian particles in a dose-dependent manner, mainly by reducing the number of available binding sites. The antireceptor serum gave a positive peroxidase staining at the luteal cell periphery on ovarian sections bearing unoccupied receptors, while no staining was seen on spleen, kidney, or liver sections. No reaction was seen on ovarian sections when the receptors were saturated with hCG in vivo, while the anti-hCG serum gave a distinct peripheral reaction in luteal cells on these sections. This fact and the idea that the antibodies produced did not bind to purified receptor-[125I]iodo-hCG complex suggest that the hormone-binding site and the antibody-binding site on the receptor are very near each other or identical. The immunocytochemical findings suggest that the majority of the LH (hCG) receptors in luteal cells are located at the cell surface.
Assuntos
Anticorpos , Corpo Lúteo/metabolismo , Receptores de Superfície Celular/análise , Animais , Complexo Antígeno-Anticorpo , Membrana Celular/metabolismo , Feminino , Imunoensaio , Cinética , Pseudogravidez/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/imunologia , Receptores do LHRESUMO
A polyclonal antiserum against the affinity-purified nondenatured rat 90K LH/CG receptor polypeptide was raised in rabbits, characterized, and used to study the location of the LH/CG receptor in pseudopregnant rat luteal cells and the fate of the receptor-hCG complex together with the specific anti-hCG serum during hCG-induced down-regulation by immunochemical techniques. Even at a 1:3000 dilution, the antiserum recognized a single 90K polypeptide on Western blots of both the affinity-purified receptor and the initial detergent extract of the pseudopregnant rat ovarian membranes. It recognized sodium dodecyl sulfate-denatured and reduced, sodium dodecyl sulfate-denatured, and native forms of the receptor on dot blots; the immunoreaction was the most intense with the native receptor. The antiserum also contained antibodies that recognized the hormone-binding site, or a region near to it, and the occupied receptor. The majority of the LH/CG receptors were located on the luteal cells in pseudopregnant rat ovaries before the induction of down-regulation. The receptor content seemed to vary among the luteal cells, however, and on single cells, suggesting both functional heterogeneity and a functional polarization of the luteal cells. Upon induction of down-regulation with hCG both the receptors and the bound hormone disappeared from the luteal cell surfaces at a very slow rate, without any simultaneous appearance of receptor- or hCG-specific immunostaining in the luteal cell interior. No accumulation of receptor degradation products capable of [125I]iodo-hCG or antibody binding could be detected on Western blots of the tissue. The polyclonal LH/CG receptor antiserum described here is useful for studying the structure and function of this receptor, particularly for immunohistochemical investigations into receptor location and regulation.
Assuntos
Ovário/metabolismo , Receptores do LH/análise , Animais , Complexo Antígeno-Anticorpo , Gonadotropina Coriônica/metabolismo , Corpo Lúteo/citologia , Corpo Lúteo/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Feminino , Histocitoquímica , Soros Imunes , Cinética , Peso Molecular , Pseudogravidez , Coelhos/imunologia , Ratos , Ratos Endogâmicos , Receptores do LH/isolamento & purificação , Receptores do LH/metabolismoRESUMO
The role of the glycan moiety of the rat ovarian LH/CG receptor and human CG (hCG) in high-affinity receptor-hormone interaction was investigated by cross-linking and quantitative binding experiments. hCG and its derivatives, desialylated hCG and deglycosylated hCG were labeled either to the alpha-subunit (125I) or the beta-subunit (3H). The ligands were attached to ovarian membrane particles, which were treated with neuraminidase or peptide-N-glycosidase F to remove terminal sialic acids or N-linked oligosaccharides of the receptor, respectively, and the complexes formed were solubilized, cross-linked with glutaraldehyde, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All of the ligands produced similar autoradiographic patterns with the native or glycosidase-treated receptor, and only the receptor-(alpha)hCG and receptor-(alpha, beta)hCG complexes were detected. Moreover, quantitative binding studies indicated that all of the hormone derivatives had similar affinities for the native or glycosidase-treated receptor. In addition, the orientation of the carbohydrate side chains on the receptor-hormone complex was studied by digesting the complex with the glycosidases. The molecular weight of the receptor, evidenced by ligand blotting, was reduced to the same extent, whether the membrane-bound free receptor or receptor-hormone complex was treated with the glycosidases, suggesting that the oligosaccharide side chains of the receptor are apart from the hormone binding region. As peptide-N-glycosidase F treatment reduced the size of the Mr 90,000 receptor first to about Mr 67,000 and finally to about Mr 62,000, there may possibly be 2 N-linked carbohydrate chains per receptor polypeptide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the glycosidase-treated receptor-[125I]hCG complex also revealed that neuraminidase was able to remove the sialic acids from both subunits of the receptor-bound hormone. In conclusion, the results suggest that hCG interacts with the polypeptide backbone of its ovarian receptor mainly through the peptide core of its alpha-subunit. Moreover, the carbohydrate side chains of both subunits of hCG are positioned on the outward face of the receptor-hormone complex.
Assuntos
Gonadotropina Coriônica/fisiologia , Ovário/metabolismo , Polissacarídeos/fisiologia , Receptores da Gonadotropina/fisiologia , Receptores do LH/fisiologia , Animais , Interações Medicamentosas , Feminino , Glicosilação , Ratos , Ratos Endogâmicos , Receptores da Gonadotropina/química , Receptores do LH/químicaRESUMO
17 beta-Hydroxysteroid dehydrogenase (17HSD) catalyzes the reversible conversion of estrone into estradiol. The complementary DNA (cDNA) coding for rat 17HSD type 1 was cloned from a commercial rat ovarian cDNA library, using human 17HSD type 1 cDNA as a probe. The nucleotide sequence extends for 1160 basepairs (bp), including 1035 bp of open reading frame, a stop codon, and 125 bp of 3'-untranslated sequence. The cDNA encodes a protein of 344 amino acids, with a calculated molecular mass of 36,967 daltons. The overall amino acid identity and similarity between rat and human 17HSD type 1 enzymes are 68% and 80%, respectively. Immunohistochemistry and in situ and Northern hybridizations were used to study regulation of the enzyme in rat ovary in vivo. Enzyme expression was detected in granulosa cells only, whereas no expression was observed in stromal or thecal cells. The enzyme was almost undetectable in ovaries from immature hypophysectomized rats. After 2-day treatment with recombinant FSH (recFSH), an induction of 17HSD type 1 expression was observed in granulosa cells of growing antral follicles. During 5 days of diethylstilbestrol (DES) treatment, a time-dependent increase in developing follicles was observed, showing strong expression of 17HSD type 1 in granulosa cells. Treatment with recFSH for 2 days in DES-primed animals resulted in down-regulation of ovarian enzyme expression. This reduction of enzyme expression was associated with luteinization of the follicles. hCG treatment of recFSH- or DES- plus recFSH-primed animals further induced luteinization, resulting in strong down-regulation of 17HSD type 1 expression. The enzyme was not detected in corpora lutea. The data show that 17HSD type 1 expression in rat ovary is regulated by gonadotropins and estrogens. The results suggest that expression of 17HSD type 1 and that of cytochrome P450 aromatase are regulated by distinct mechanisms, and 17HSD type 1 may be down-regulated earlier than P450 aromatase during luteinization, limiting estradiol biosynthesis in luteinizing granulosa cells in rat ovary.
Assuntos
17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/genética , Dietilestilbestrol/farmacologia , Gonadotropinas/farmacologia , Ovário/enzimologia , 17-Hidroxiesteroide Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Aromatase/genética , Aromatase/metabolismo , Aromatase/fisiologia , Sequência de Bases , Northern Blotting , Gonadotropina Coriônica/farmacologia , DNA/análise , DNA/genética , Ativação Enzimática , Feminino , Hormônio Foliculoestimulante/farmacologia , Regulação Enzimológica da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Dados de Sequência Molecular , Ovário/efeitos dos fármacos , Ovário/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fatores de TempoRESUMO
LH receptors were solubilized from human corpora lutea in phosphate-buffered saline containing 1% Triton X-100, 20% glycerol, and protease inhibitors. The presence of 20% (vol/vol) glycerol was necessary for quantitative preservation of [125I]hCG-binding activity in detergent solution. The solubilized receptors were stable for several weeks at -20 C and at -80 C and for at least 18 h at 4 C. Binding of [125I]hCG to the soluble LH receptors was time and temperature dependent and varied linearly with the amount of soluble protein. Equilibrium binding studies revealed a single class of high affinity [125I]hCG-binding sites with an equilibrium dissociation constant (Kd) of 4.3 x 10(-10) mol/L (at 20 C). The molecular size of the human LH receptors was analyzed by ligand blotting. Solubilized receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose. Incubation of the protein blot with [125I]hCG to the 85/90K mol wt species was inhibited by unlabeled hCG. These results indicate that LH receptors can be solubilized in nonionic detergent while maintaining their hormone-binding activity and demonstrate that the receptors contain 85/90K hormone-binding species.
Assuntos
Corpo Lúteo/análise , Receptores do LH/isolamento & purificação , Sítios de Ligação/efeitos dos fármacos , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Feminino , Glicerol/farmacologia , Humanos , Ligantes , SolubilidadeRESUMO
Wedge resection was performed in 12 patients with polycystic ovarian disease, and cell samples from the cystic follicles were assayed for LH(hCG) receptor using [125I]iodo-hCG as a ligand hormone. Simultaneously to wedge resection, blood samples were taken for serum FSH, LH, 17 beta-estradiol, progesterone, and testosterone RIA measurements. Serum LH was regularly elevated (16.0-57.1 U/liter), whereas FSH (5.2-11.5 U/liter) was within the normal reference range. The LH to FSH ratio was between 2.1-7.8. The 17 beta-estradiol concentrations (0.12-0.23 nmol/liter) were within the normal reference range found during the early follicular phase. Only 3 patients had progesterone levels exceeding the assay sensitivity limit of 0.1 nmol/liter. Ony 3 of the 11 patients assayed for serum testosterone had values exceeding the upper limit of the reference range. Seventy-seven percent of the ovarian follicular samples showed specific binding of [125I]iodo-hCG. The number of receptors in positive samples averaged 0.67 +/- 0.11 fmol/mg homogenate protein, which is clearly lower than that in normal preovulatory follicles. Scatchard analyses revealed a single class of binding sites, with a mean equilibrium association constant of 5.4 X 10(9) M-1 at 37 C. These results suggest that the derangement of follicular development in patients with polycystic ovarian disease probably is not due to the lack of appearance of the LH(hCG) receptor. It is possible that the tonic elevation of serum LH results in a decrease in the number of available receptor sites; this would be one step in the process leading to ovarian changes characteristic of this disease.
Assuntos
Folículo Ovariano/metabolismo , Síndrome do Ovário Policístico/metabolismo , Receptores de Superfície Celular/metabolismo , Adulto , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Hormônio Luteinizante/sangue , Receptores do LHRESUMO
To elucidate the molecular mechanisms involved in the homologous regulation of LH/chorionic gonadotrophin (CG) receptors, the receptor and its mRNA levels were analysed in the same pseudopregnant rat ovarian samples after human (h)CG-induced down-regulation using a binding assay, ligand blotting, immunoblotting and Northern blotting together with the polymerase chain reaction (PCR). Treatment of the animals with 500 IU hCG resulted in a loss of 125I-labelled hCG binding and the 90 kDa receptor on the ligand and immunoblots within 12 and 24 h respectively, followed by a transient partial recovery on days 4 and 5, while a distinct decline occurred only on day 7 in the controls. Northern blots of total ovarian RNA, as probed with a 293 bp AvaI/HindIII fragment from the extracellular domain of PCR-generated full-length rat LH/CG receptor cDNA, revealed six major mRNAs of 7.0, 4.2, 2.8, 2.0, 1.4 and 1.1 kb. The 4.2 kb mRNA, which was the most abundant, possibly encodes the 90 kDa receptor, while the smaller species probably represent alternatively spliced forms of the LH/CG receptor pre-mRNA, as also supported by the finding that PCR produced three cDNA bands of 2.1, 2.0 and 1.8 kb when oligomers derived from the N and C termini of rat LH/CG receptor cDNA were used as primers and rat ovarian total RNA as a template. Treatment with hCG led to the down-regulation of all six mRNAs in a fashion parallel to the changes in receptor protein. No smaller receptor components capable of binding radiolabelled hCG or receptor antibody appeared on the ligand or immunoblots prior to or during down-regulation or the subsequent transient period of up-regulation, suggesting that the smaller mRNA species are translated in minute amounts in vivo or are not translated at all. Laser densitometric analysis of the Northern blots revealed that the amounts of the four smallest mRNA species increased during the period of down-regulation in relation to the 4.2 kb mRNA, and correspondingly decreased during the subsequent period of up-regulation, indicating changes in the alternative splicing of the primary transcript. The data suggest that hCG-induced transient down-regulation of the LH/CG receptor results in part from down-regulation of its mRNA levels, and that changes in alternative processing of the receptor pre-mRNA may play a regulatory role in the expression of functional LH/CG receptor during down- and up-regulation.
Assuntos
Gonadotropina Coriônica/fisiologia , Processamento Pós-Transcricional do RNA , Receptores do LH/metabolismo , Animais , Sequência de Bases , Northern Blotting , DNA de Cadeia Simples , Densitometria , Regulação para Baixo , Feminino , Humanos , Immunoblotting , Dados de Sequência Molecular , Ovário/metabolismo , Reação em Cadeia da Polimerase , Pseudogravidez/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Transcrição GênicaRESUMO
A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.
Assuntos
Fases de Leitura Aberta , Reação em Cadeia da Polimerase/métodos , Animais , Vírus da Mieloblastose Aviária/enzimologia , Sequência de Bases , Biotecnologia , Primers do DNA/genética , DNA Complementar/genética , Feminino , Dados de Sequência Molecular , Inibidores da Síntese de Ácido Nucleico , Ovário/metabolismo , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA/genética , DNA Polimerase Dirigida por RNA/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores do LH/genética , Sensibilidade e Especificidade , Taq PolimeraseRESUMO
We used confocal scanning microscopy to study the semi-quantitative distribution of luteinizing hormone/chorionic gonadotropin (LH/CG) receptors on rat luteal cells at both the two- and the three-dimensional level. The receptors were visualized in 6-microns sections of pseudopregnant rat ovaries using polyclonal rabbit antiserum to hCG-affinity-purified LH/CG receptor in conjunction with rhodamine-conjugated anti-rabbit immunoglobulins. Twenty to 30 optical sections were taken at different focal planes from representative luteal cells with a confocal laser scanning microscope and then processed digitally to two- and three-dimensional pseudocolored images. Distinct differences in fluorescence intensity could be demonstrated at both the two- and the three-dimensional level on the luteal cell surfaces, suggesting an uneven distribution of the LH/CG receptors on the cell membranes. This probably results in the compartmentalization and polarization of luteal cell function.
Assuntos
Imunofluorescência , Células Lúteas/ultraestrutura , Microscopia Eletrônica/métodos , Receptores da Gonadotropina/metabolismo , Receptores do LH/metabolismo , Animais , Feminino , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica/métodos , Lasers , Células Lúteas/metabolismo , Pseudogravidez/metabolismo , Ratos , Ratos EndogâmicosRESUMO
Localization of receptor-bound human chorionic gonadotropin (hCG) in pseudopregnant rat ovarian cells was studied by the peroxidase-antiperoxidase (PAP) complex method. The animals were injected, 2,6,12, and 24 hr prior to killing with a single intravenous dose of hCG. The hormone localized to the periphery of the luteal and interstitial glandular cells. The luteal cells seemed to bind different amounts of the hormone, which suggests that the number of receptor varies from one cell to another or that the receptors are not similarly accessible to the circulating hormone in all cells. The hormone seemed to disappear unequally from the luteal cells, possibly due to the variable amounts of primarily bound hormone or to different removal mechanisms. The hormone was distributed unevenly at the luteal cell periphery. This observation supports previous findings that hCG preferentially binds to the cell surface regions directed towards the capillary areas. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. Our results support the previous autoradiographic evidence of the plasma membrane localization of luteinizing hormone receptor in the luteal cells, and are compatible with the concept that only a small fraction of the receptor-hormone complexes may be internalized.
Assuntos
Gonadotropina Coriônica/metabolismo , Ovário/análise , Pseudogravidez/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Membrana Celular/metabolismo , Gonadotropina Coriônica/imunologia , Feminino , Técnicas Imunoenzimáticas , Ovário/patologia , Ratos , Receptores do LHRESUMO
We developed a double immunofluorescence technique for detection of the rat luteinizing hormone/choriogonadotropin (LH/CG) receptor and bound hCG in the same rat ovarian section and used it in conjunction with confocal laser scanning microscopy (CLSM) to study the fate of the receptor-hormone complex in luteal cells during the hCG-induced down-regulation. Pseudopregnant immature females rats were perfusion-fixed before (0 hr) and 2, 6, 12, 24, or 36 hr after a down-regulating dose of hCG (500 IU IV). The cryosections were stained for the LH/CG receptor and bound hormone by sequential incubations with a polyclonal rabbit antiserum to purified rat LH/CG receptor and a mouse monoclonal antibody (MAb) to hCG, followed by sequential incubation with TRITC- and FITC-conjugated secondary antibodies to rabbit and mouse immunoglobulins, respectively. The results were semiquantitatively analyzed by a pseudo-three-dimensional (3D) plotting of the intensities of the receptor and hormone-specific fluorescence in luteal cells by CLSM. The analysis suggested that the majority of the LH/CG receptors are located on the luteal cells before induction of the down-regulation and that their content seem to vary not only among cells but also on the surface of single cells, thus supporting the previous concept of the functional heterogeneity among the cells and their functional compartmentation. At 2 hr after injection of the down-regulating dose of hCG, the LH/CG receptor-specific and hCG-specific fluorescences clearly co-localized on the luteal cells. Both the LH/CG receptor- and hCG-specific fluorescences disappeared from the luteal cell surfaces in a parallel fashion within 36 hr without a detectable accumulation of either fluorescence deep in the cell interior. These results suggest that the LH/CG receptor and bound hCG do not differ in their manner of in vivo processing in luteal cells. Therefore, the disappearance of the receptor and bound hormone occurs in a parallel fashion and without detectable internalization.
Assuntos
Gonadotropina Coriônica/farmacologia , Corpo Lúteo/metabolismo , Receptores do LH/biossíntese , Animais , Anticorpos Monoclonais , Gonadotropina Coriônica/metabolismo , Corpo Lúteo/citologia , Corpo Lúteo/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Feminino , Imunofluorescência , Gonadotropinas Equinas/farmacologia , Imuno-Histoquímica , Camundongos/imunologia , Microscopia/métodos , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Pseudogravidez , Coelhos/imunologia , Ratos , Ratos Sprague-Dawley , Receptores do LH/metabolismoRESUMO
We studied the location of carbonic anhydrase (CA) isoenzymes I, II, and VI in human pituitary gland using specific antisera in conjunction with immunoblotting, immunoperoxidase, and double immunofluorescence staining techniques. Stainings with anti-CA II serum showed intense cytoplasmic reaction in the anterior lobe of the pituitary gland. Double immunofluorescence staining was used to identify the cells that expressed CA II. Confocal laser scanning microscopy revealed that, of the anterior pituitary hormones studied, ACTH coincides mainly with CA II in these cells. Stainings with anti-CA I and VI sera were negative in the endocrine cells of the pituitary gland. Western blotting of the pituitary gland with anti-CA II revealed a distinct 29-KD polypeptide band corresponding in molecular weight to CA II, suggesting that the antiserum does not detect any nonspecific protein. Anti-CA I serum similarly showed a major 29-KD band, possibly recognizing the enzyme, which is abundantly present in erythrocytes. The results indicate that CA II is expressed in corticotrophs of human pituitary gland, in which its physiological role may be linked to the regulation of optimal pH in the secretory vesicles for the cleavage of ACTH from its precursor.
Assuntos
Anidrases Carbônicas/análise , Isoenzimas/análise , Hipófise/enzimologia , Adulto , Idoso , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis. (J Histochem Cytochem 49:657-662, 2001)
Assuntos
Anidrases Carbônicas/metabolismo , Glândulas Salivares Menores/metabolismo , Língua/metabolismo , Animais , Western Blotting , Humanos , Imuno-Histoquímica , Isoenzimas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Glândulas Salivares Menores/enzimologia , Papilas Gustativas/enzimologia , Língua/enzimologiaRESUMO
Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.
Assuntos
Anidrases Carbônicas/metabolismo , Glândula Parótida/enzimologia , Glândula Submandibular/enzimologia , Anticorpos/imunologia , Anidrases Carbônicas/imunologia , Anidrases Carbônicas/fisiologia , Humanos , Imuno-Histoquímica/métodos , Glândula Parótida/citologia , Glândula Submandibular/citologiaRESUMO
Localization of receptor-bound human chorionic gonadotropin (hCG) in rat testis was studied by the peroxidase-antiperoxidase (PAP) complex method. The rats were injected with a single intravenous dose (1000 IU) of hCG. Three, 6, 12, and 24 hr after injection the testes were removed for localization of the hormone. The hormone localized to the periphery of the Leydig cells at all observation points. The intensity of the staining varied between the cells, suggesting that the number of receptors or the accessibility of the receptors to the circulating hormone varies from one cell to another. The staining surrounded the Leydig cells unevenly, but no progressive patching or capping was found. This observation suggests that hCG binds preferentially to the cell surface areas directed toward the capillaries. Compatible results were obtained with anti-hCG serum and with antisera against the hCG subunits. These results are consistent with previous observations that the luteinizing hormone (hCG) receptors accessible to the circulating hormone are located at the surface of the Leydig cells.