RESUMO
The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68. Herein, we have investigated the expression and function of Sam68 in human prostate cancer cells. Analysis of specimens obtained from 20 patients revealed that Sam68 is upregulated at the protein level in 35% of the samples. Real-time polymerase chain reaction confirmed the results at the mRNA level in most patients. Downregulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression and reduced the proliferation rate. Moreover, depletion of Sam68 sensitized cells to apoptosis induced by DNA-damaging agents. Similarly, stable cell lines expressing a truncated GFP-Sam68(GSG) protein displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Our results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ligação a DNA/fisiologia , Neoplasias da Próstata/patologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genéticaRESUMO
Whole-cell currents activated by bath applications of acetylcholine (ACh) (10-30 microM) were recorded from patch-clamped myotubes of the mouse C2 cell line. Increasing concentrations of forskolin caused a dose-dependent fast decay of ACh-activated currents as compared to the long-lasting ACh-currents in control cells. The forskolin-induced modulation of nicotinic ACh receptor (nAChR) desensitization was proportional to the drug-induced elevation in the cyclic AMP (cAMP) cellular content. Furthermore, an increase in the rate of decay of the ACh-current response, which paralleled an elevation in cAMP cellular content, was caused by treatment with a calcitonin gene-related peptide (1 microM), 8-Br-cAMP (0.5 mM), or by loading the myotubes with cAMP. These results therefore indicate that the desensitization of nAChR is a cAMP-related process in C2-myotubes.
Assuntos
AMP Cíclico/metabolismo , Músculos/metabolismo , Receptores Colinérgicos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/farmacologia , Citosol/metabolismo , Eletrofisiologia , Camundongos , Músculos/efeitos dos fármacos , Músculos/fisiologiaRESUMO
As the name implies, Stimulated by Retinoic Acid 8 is an early retinoic acid (RA) responsive gene pivotal for the beginning of meiosis in female and male germ cells. Its expression is strictly time-dependent and cell-specific (pre-meiotic germ cells) and likely requires a complex mechanism of regulation. In this study, we demonstrate a direct negative control of SOHLH1 and SOHLH2, 2 germ cell specific bHLH transcription factors, on Stra8 expression. We observed a negative correlation between STRA8 and SOHLH1 expression in prepuberal differentiating mouse KIT(+) spermatogonia and found that SOHLH1 and SOHLH2 were able to directly and cooperatively repress STRA8 expression in cell lines in vitro through binding to its promoter. We also identified 2 canonical E-Box motives in the Stra8 promoter that mediated the negative regulation of SOHLH1 and SOHLH2 on these gene both in the cell lines and KIT(+) spermatogonia. We hypothesize that this novel negative activity of SOHLH1 and SOHLH2 in male cooperates with that of other transcription factors to coordinate spermatogonia differentiation and the RA-induced meiosis and in female ensures STRA8 down-regulation at mid-end stages of meiotic prophase I.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Bases , Diferenciação Celular , Regulação para Baixo , Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Masculino , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Espermatogônias/fisiologiaRESUMO
The platelet-derived growth factor (PDGF) is a major mitogen for cells of mesenchymal origin. Because Leydig cells arise from mesenchymal precursors, we tested the hypothesis that these cells might be a target for PDGF. We also investigated a possible production of a PDGF-like substance by Leydig cells in culture and the distribution of PDGF-like material in the rat testis using immunohistochemistry. PDGF was found to bind specifically to high affinity receptors on the surface of purified adult rat Leydig cells. Conditioned medium from cultured Leydig cells competed with 125I-labeled PDGF for binding to the Leydig cells. The secretion of PDGF receptor-competing activity was stimulated in a dose-dependent manner by the trophic hormone hCG. Immunohistochemical studies revealed specific staining for PDGF in the Leydig cells of adult rat testis. Taken together these observations suggest that PDGF may play a role in the local control mechanisms of testicular function.
Assuntos
Células Intersticiais do Testículo/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , Masculino , Fator de Crescimento Derivado de Plaquetas/biossíntese , Ratos , Ratos Endogâmicos , Receptores do Fator de Crescimento Derivado de PlaquetasRESUMO
Pro-opiomelanocortin-derived peptides, alpha-MSH and beta-endorphin, are synthesized and secreted by Leydig cells, and are believed to have paracrine effects on Sertoli cells in the testis. Peptides with MSH activity stimulate adenylate cyclase and cAMP accumulation in Sertoli cell-enriched cultures. The purpose of the present study was to determine whether such peptides would affect Sertoli cell parameters, such as aromatase and plasminogen activator activities, that are known to be regulated by cAMP. alpha-MSH stimulated aromatase activity in Sertoli cell-enriched cultures prepared from 10-day-old rats and this effect was potentiated by methyl isobutylxanthine (MIX). The combination of alpha-MSH plus MIX was not as potent as FSH. alpha-MSH, des-acetyl-alpha-MSH, beta-MSH, ACTH(1-13), and ACTH(1-24) stimulated aromatase activity to a similar extent, suggesting that Sertoli cells do not distinguish between the activities of these peptides. alpha-MSH potentiated the action of dbcAMP and forskolin on Sertoli cell aromatase, but unexpectedly had no effect on the action of either half-maximal or maximal doses of FSH. The regulation of plasminogen activator was examined next; urokinase was markedly suppressed by FSH in 10-day-old Sertoli cells. Although neither alpha-MSH nor MIX alone had an effect on urokinase secretion, in combination they were as effective as FSH. In 10-day-old Sertoli cells each of these peptides had little or no effect on tissue plasminogen activator.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Estradiol/metabolismo , Ativadores de Plasminogênio/metabolismo , Células de Sertoli/metabolismo , alfa-MSH/farmacologia , beta-Endorfina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Aromatase/metabolismo , Bucladesina/farmacologia , Células Cultivadas , Colforsina/farmacologia , Cosintropina/farmacologia , Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante/farmacologia , Masculino , Hormônios Estimuladores de Melanócitos/farmacologia , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Endogâmicos , alfa-MSH/análogos & derivadosAssuntos
Receptores de Fator de Crescimento Neural/metabolismo , Testículo/embriologia , Testículo/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Masculino , Microscopia de Fluorescência , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor de Fator de Crescimento Neural , Receptores de Fator de Crescimento Neural/genéticaRESUMO
We report a short-term culture system that allows to define novel characteristic of programmed cell death (PCD) in fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic prophase I progression underwent apoptotic degeneration waves as revealed by TUNEL staining. TEM observations revealed recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features were also observed. Further characterization of oocyte death evidenced DNA ladder, Annexin V binding, PARP cleavage, and usually caspase activation (namely caspase-2). In the aim to modulate the oocyte death process, we found that the addition to the culture medium of the pan-caspase inhibitors Z-VAD or caspase-2-specific inhibitor Z-VDVAD resulted in a partial and transient prevention of this process. Oocyte death was significantly reduced by the antioxidant agent NAC and partly prevented by KL and IGF-I growth factors. Finally, oocyte apoptosis was reduced by calpain inhibitor I and increased by rapamycin after prolonged culture. These results support the notion that fetal oocytes undergo degeneration mostly by apoptosis. This process is, however, often morphologically atypical and encompasses other forms of cell death including caspase-independent apoptosis and autophagia. The observation that oocyte death occurs mainly at certain stages of meiosis and can only be attenuated by typical anti-apoptotic treatments favors the notion that it is controlled at least in part by stage-specific oocyte-autonomous meiotic checkpoints and when activated is little amenable to inhibition being the oocyte able to switch back and forth among different death pathways.
Assuntos
Apoptose/fisiologia , Feto/fisiologia , Prófase Meiótica I/fisiologia , Oócitos/citologia , Ovário/citologia , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Autofagia , Inibidores de Caspase , Técnicas de Cultura de Células , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Fragmentação do DNA , Feminino , Glicoproteínas/farmacologia , Imunossupressores/farmacologia , Marcação In Situ das Extremidades Cortadas , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica de Transmissão , Necrose , Oligopeptídeos/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Sirolimo/farmacologia , Fator de Células-Tronco/farmacologiaRESUMO
The effect of the antiviral agent interferon (IFN) on the function of the nicotinic acetylcholine receptor (AChR) channel, has been investigated in both mammalian cultured myotubes and adult fibres, using the single channel recording patch-clamp technique. Shortened AChR-channel lifetime, and occasionally reduced channel conductance and slowed opening frequency were seen with fibroblast IFN (IFN-beta) in the mouse myotubes, and with IFN-beta and leucocytes IFN (IFN-alpha), in the rat muscle fibres. These effects paralleled an increase in the cytosolic level of cAMP. This suggests that IFN exerts a regulatory action on AChR function. A similar regulatory action on other receptor may be responsible for some of the neurological side effects observed in patients treated with IFN.
Assuntos
Interferon Tipo I/farmacologia , Canais Iônicos/fisiologia , Músculos/fisiologia , Receptores Nicotínicos/fisiologia , Animais , Células Cultivadas , AMP Cíclico/biossíntese , Condutividade Elétrica , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos , Proteínas RecombinantesRESUMO
1. Whole-cell currents were recorded from cultures of dissociated neocortical neurones of the rat. Rat alpha-calcitonin gene-related peptide (CGRP; 1 nM-1 microM) caused significant dose-dependent decreases in the voltage-activated transient (A-current) and delayed rectifier K+ currents. Forskolin (10 nM-20 microM) mimicked this effect. Peak K+ currents were gradually decreased after loading neurones with cyclic AMP (100 microM) through patch pipettes. CGRP was ineffective in neurones loaded with cyclic AMP. 2. CGRP (0.5-2 microM) increased cytosolic cyclic AMP concentration and this effect was mimicked by forskolin (5-40 microM). 3. CGRP (0.1-1 microM) reduced high-threshold Ca2+ currents; as did forskolin (5-20 microM) and cyclic AMP loaded into the neurones. In contrast, low-threshold Ca2+ currents were not affected by any of these agents. 4. Voltage-activated Na+ currents were significantly reduced by both CGRP (0.1-1 microM) and forskolin (5-20 microM). A similar effect was observed when cells were loaded with cyclic AMP. 5. We conclude that, in neocortical neurones, CGRP attenuates voltage-activated currents by stimulating the intracellular cyclic AMP signalling system.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Canais Iônicos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Colforsina/farmacologia , AMP Cíclico/metabolismo , Citosol/metabolismo , Eletrofisiologia , Canais Iônicos/metabolismo , Neurônios/metabolismo , Potássio/metabolismo , Ratos , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia , Sódio/metabolismoRESUMO
We have studied the presence and distribution of beta 1 integrins in the seminiferous epithelium of prepubertal and adult rats. Our immunofluorescence data show that in the adult the antibody recognizes specific areas localized around the heads of elongating and maturing spermatids and above spermatogonia at stages I-VII. The following were found to be negative: a) areas adjacent to spermatogonia at stages IX-XIV and adjacent to spermatocytes and to round spermatids; b) spermiated spermatozoa. In the prepubertal rat, positive tubules are first apparent around Day 17 of age. Immunofluorescence and immunoprecipitation studies show that Sertoli cell monolayers from 3-wk-old rats express beta integrins in vitro.
Assuntos
Integrinas/biossíntese , Túbulos Seminíferos/metabolismo , Animais , Células Cultivadas , Epitélio/metabolismo , Imuno-Histoquímica , Integrina beta1 , Masculino , Ratos , Ratos Wistar , Células de Sertoli/metabolismoRESUMO
Cultured myotubes and freshly dissociated muscle fibres from adult rats were exposed to calcitonin gene-related peptide (CGRP) and studied by patch-clamp recording during the peptide-induced maximal accumulation of cellular cyclic AMP (cAMP). Acetylcholine receptor- (AChR-) channel properties in myotubes were not modified by the presence of CGRP (10(-7) M). The peptide, applied to the non-patched membrane, significantly increased the variance of the AChR-channel amplitude distribution at the synaptic region of muscle fibres, and three classes of AChR-channels were resolved immediately after peptide application. AChR-channels at extrasynaptic regions of fibres from denervated muscles were unaffected by CGRP. It is suggested that CGRP may regulate the synaptic AChR-channel conductance through second messenger systems.
Assuntos
Calcitonina/farmacologia , AMP Cíclico/metabolismo , Músculos/fisiologia , Neuropeptídeos/farmacologia , Receptores Colinérgicos/fisiologia , Sinapses/fisiologia , Animais , Peptídeo Relacionado com Gene de Calcitonina , Células Cultivadas , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Denervação Muscular , Músculos/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Receptores Colinérgicos/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
Three cDNA clones encoding isoforms of casein kinase I (CKI) were isolated from Arabidopsis thaliana. One full-length clone, designated CKI1, contained an open reading frame of 1371 bp encoding a protein of 51,949 D with an isoelectric point of 9.7. In addition to the highly conserved catalytic domain (of about 300 amino acids), the Arabidopsis CKI isoforms contain 150 to 180 amino acid carboxyl-terminal extensions, which show among themselves a lower level of sequence conservation. These extensions do not show any sequence similarity to nonplant CKI isoforms, such as rat testis CKI delta, which is their closest isolated homolog, or to yeast CKI isoforms. Three additional isoforms of Arabidopsis CKI were found in the data bases of expressed sequence tags and/or were isolated serendipitously in nonspecific screening procedures by others. One of them also shows a carboxyl-terminal extension, but of only 80 amino acids. Casein kinase activity was detected in the soluble fraction of Escherichia coli strains expressing the CKI1 protein. This activity showed the crucial properties of CKI, including the ability to phosphorylate the D4 peptide, a specific substrate of CKI, and inhibition by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific CKI inhibitor. Like several recombinant CKI isoforms from yeast, CKI1 was able to phosphorylate tyrosine-containing acidic polymers.
Assuntos
Arabidopsis/enzimologia , Isoenzimas/química , Isoenzimas/genética , Proteínas Quinases/química , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Sequência de Bases , Sítios de Ligação , Caseína Quinases , Clonagem Molecular , Sequência Conservada , Escherichia coli , Humanos , Isoenzimas/biossíntese , Masculino , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta , Filogenia , Proteínas Quinases/biossíntese , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Testículo/enzimologiaRESUMO
The cytodifferentiation of peritubular myoid cells was studied in developing rats from fetal day 18 through approachment of puberty. The parameters taken into consideration were 1) the presence of desmin, a component of intermediate filaments in contractile cells; 2) the expression of alkaline phosphatase, a cell surface enzyme present in no other cell type of the seminiferous tubule; 3) the expression of the smooth muscle specific isoform of alpha-actin, a marker of terminal differentiation in smooth muscle cells; 4) cell proliferation rate, evaluated in radioautography as labeling index after incorporation of 3H-thymidine in short-term organ culture; and 5) cytoarchitectural changes detected with scanning electron microscopy. By means of immunofluorescence and cytochemistry it was observed that the three markers are expressed early during life, long before the onset of the first spermatogenic wave; in particular desmin is already present in fetal samples and alkaline phosphatase activity appears a few days after birth, whereas alpha-smooth muscle isoactin is first detected around birth. As for myoid cell replication, the high prenatal labeling index was found to drop soon after birth and to further slow down during the first month of postnatal life, suggesting that myoid cell proliferation is not a major factor in peritubular expansion. SEM examination of developing peritubulum has shown that, when approaching puberty, the myoid cell undergoes a dramatic change in cytoarchitecture, consisting in extreme flattening and cytoplasmic expansion resulting in an apparent increase in peritubular surface.
Assuntos
Testículo/citologia , Actinas/análise , Actinas/metabolismo , Fosfatase Alcalina/análise , Fosfatase Alcalina/metabolismo , Animais , Autorradiografia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Desmina/análise , Desmina/metabolismo , Imunofluorescência , Imuno-Histoquímica , Filamentos Intermediários/química , Filamentos Intermediários/ultraestrutura , Masculino , Microscopia Eletrônica de Varredura , Ratos , Ratos Endogâmicos , Epitélio Seminífero/fisiologia , Testículo/metabolismo , Testículo/fisiologia , Timidina/metabolismo , TrítioRESUMO
Nerve growth factor (NGF) and the other members of the family of neurotrophic factors (the neurotrophin) are essential for neuronal development and differentiation. Neurotrophins interact with two types of cell surface receptors: a low-affinity receptor (p75 NGF-R) and a high-affinity tyrosine kinase receptor belonging to the trk proto-oncogene family, both expressed in the nervous system and in certain non-neuronal tissues. Recently, NGF immunoreactivity and mRNA have been detected in the testis of the adult mouse, rat and human. In the present report we demonstrate the expression of p75 NGF-R during early gonadal development, by mesenchymal cells of the embryonic mouse and rat testis. In the embryonic testis p75 NGF-R-positive cells are spread through the interstitial compartment; during postnatal development they become organized in a cellular layer that surrounds differentiating myoid cells of the seminiferous tubule. Our results also show the expression in the peripuberal and adult mouse and rat testis, of an abundant and shorter transcript of 3.2 kb that cross-hybridizes to the receptor mRNA (3.7 kb). This new mRNA species, which appears at the beginning of spermatogenesis, is expressed by pachytene spermatocytes and round spermatids.
Assuntos
Receptores de Fator de Crescimento Neural/metabolismo , Testículo/embriologia , Testículo/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Feto , Masculino , Camundongos , Fator de Crescimento Neural/metabolismo , Proto-Oncogene Mas , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/metabolismo , Receptor trkC/metabolismo , Receptores de Fator de Crescimento Neural/genética , Epitélio Seminífero/citologia , Epitélio Seminífero/metabolismo , Espermatogênese/fisiologia , Testículo/citologiaRESUMO
New information regarding primordial germ cell (PGC's) segregation and proliferation over the last decade is reviewed. Advances have been obtained in the mouse but current knowledge of human PGC's remains scant. Questions still fully or partially unresolved about the emergence of the germline in mammals are addressed. (i) When and where is the germ line set aside in the embryo? (ii) How is the germ line segregated from the somatic lineages? (iii) Which factors guide PGC's to the gonadal ridges? (iv) Which factors regulate PGC's proliferation? The main purpose of this review is to outline the information obtained using mainly in vitro culture systems about two aspects of these processes namely the segregation of PGC's and their proliferation.
Assuntos
Células Germinativas/citologia , Gônadas/citologia , Gônadas/embriologia , Animais , Divisão Celular/fisiologia , Linhagem da Célula/fisiologia , HumanosRESUMO
In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII), plant CKII is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit alpha (CKA1) and the regulatory subunit beta (CKB1) of CKII were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKB1 on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities. The combination of purified CKA1 and CKB1 proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKA1 activity alone, showing that CKB1 has biochemical properties similar to those of the beta subunit from animals. CKA1 and CKB1 spontaneously assembled into a tetrameric complex, CKA1(2)CKB12, which had properties very similar to those of the oligomeric CKII form isolated from broccoli. However, the properties of the catalytic subunit CKA1 alone differed from those of the broccoli monomeric form of CKII-like activity. Phosphorylation of transcription factor GBF1 with the reconstituted CKA1(2)CKB1(2) enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKII from broccoli.
Assuntos
Arabidopsis/enzimologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Arabidopsis/genética , Caseína Quinase II , Escherichia coli/genética , Fatores de Ligação G-Box , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Corpos de Inclusão/enzimologia , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Several lines of evidence indicate that cannabinoids, among other functions, are involved in motor control. Although cannabinoid receptors (CB(1)) mRNA has been observed in medium-sized spiny neurons of the striatum, a description of the precise localization of CB(1) at a protein level among striatal cells is still lacking. Therefore, we performed immunohistochemical studies with light and confocal microscopy to identify neuronal subpopulations that express CB(1) and to assess the distribution of the receptor within these neurons. In our single label light microscopy study, CB(1) was observed in most medium-sized neurons of the caudate-putamen. However, CB(1) was also present in large-sized neurons scattered throughout the striatum. Our dual-label study showed that 89.3% of projection neurons in matrix contain CB(1), and that 56.4% of projection neurons in patch are labeled for CB(1). To investigate the presence of CB(1) among the different subclasses of striatal interneurons we performed a double-labeling study matching CB(1) and each of the striatal interneuron markers, namely, choline acetyl-transferase, parvalbumin, calretinin, and nitric oxide synthase. Our double-label study showed that most parvalbumin immunoreactive interneurons (86.5%), more than one-third (39.2%) of cholinergic interneurons, and about one-third (30.4%) of the NOS-positive neurons are labeled for CB(1). Calretinin-immunolabeled neurons were devoid of CB(1).
Assuntos
Moduladores de Receptores de Canabinoides/metabolismo , Neostriado/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Calbindina 2 , Colina O-Acetiltransferase/metabolismo , Imuno-Histoquímica , Interneurônios/citologia , Interneurônios/metabolismo , Masculino , Microscopia Confocal , Neostriado/citologia , Vias Neurais/citologia , Vias Neurais/metabolismo , Óxido Nítrico Sintase/metabolismo , Parvalbuminas/metabolismo , Ratos , Ratos Wistar , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
Several reports have established that the action of neurotrophins is not restricted to the nervous system but can affect a broad range of non-neuronal cells. Nerve growth factor (NGF) is present in adult testis and has been suggested as a potential regulator of meiosis in rat seminiferous epithelium. Here we present an extensive immunohistochemical study on neurotrophins and their receptors (p75 and trk) in the developing mouse testis and epididymis, and in fetal human testis. During the early steps of testicular and epididymal organization in the mouse, strong p75 immunoreactivity is detectable in the gonadal ridge in the mesenchyme that is excluded from the evolving testicular cords, and in the mesenchymal cells of the mesonephros. Later in organogenesis, most of the p75-positive interstitial cells of the testis coexpress neurotrophin-3 (NT-3) and the truncated trk B receptor in a developmentally regulated pattern. Our Western blot data confirm the expression of these molecules. These findings suggest that neurotrophin receptors play a role in early inductive events during critical periods of testicular and epididymal development. During fetal and postnatal histogenesis, an increasing number of NT-3- and p75-positive mesenchymal cells start to express alpha-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.