RESUMO
Demyelination is generated in several nervous system illnesses. Developing strategies for effective clinical treatments requires the discovery of promyelinating drugs. Increased GABAergic signaling through γ-aminobutyric acid type A receptor (GABAAR) activation in oligodendrocytes has been proposed as a promyelinating condition. GABAAR expressed in oligodendroglia is strongly potentiated by n-butyl-ß-carboline-3-carboxylate (ß-CCB) compared to that in neurons. Here, mice were subjected to 0.3% cuprizone (CPZ) added in the food to induce central nervous system demyelination, a well-known model for multiple sclerosis. Then ß-CCB (1 mg/Kg) was systemically administered to analyze the remyelination status in white and gray matter areas. Myelin content was evaluated using Black-Gold II (BGII) staining, immunofluorescence (IF), and magnetic resonance imaging (MRI). Evidence indicates that ß-CCB treatment of CPZ-demyelinated animals promoted remyelination in several white matter structures, such as the fimbria, corpus callosum, internal capsule, and cerebellar peduncles. Moreover, using IF, it was observed that CPZ intake induced an increase in NG2+ and a decrease in CC1+ cell populations, alterations that were importantly retrieved by ß-CCB treatment. Thus, the promyelinating character of ß-CCB was confirmed in a generalized demyelination model, strengthening the idea that it has clinical potential as a therapeutic drug.
Assuntos
Carbolinas , Cuprizona , Doenças Desmielinizantes , Modelos Animais de Doenças , Remielinização , Animais , Cuprizona/toxicidade , Remielinização/efeitos dos fármacos , Camundongos , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/metabolismo , Carbolinas/farmacologia , Carbolinas/administração & dosagem , Bainha de Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Esclerose Múltipla/induzido quimicamente , Esclerose Múltipla/patologia , Substância Branca/efeitos dos fármacos , Substância Branca/metabolismo , Substância Branca/patologia , Imageamento por Ressonância MagnéticaRESUMO
Demyelination is typically followed by a remyelination process through mature oligodendrocytes (OLs) differentiated from precursor cells (OPCs) recruited into the lesioned areas, however, this event usually results in uncompleted myelination. Potentiation of the remyelination process is an important target for designing effective therapeutic strategies against white matter loss. Here, it was evaluated the remyelinating effect of different ß-carbolines that present differential allosteric modulation on the GABAA receptor expressed in OLs. For this, we used a focalized demyelination model in the inferior cerebellar peduncle (i.c.p.) of rats (DRICP model), in which, demyelination by ethidium bromide (0.05%) stereotaxic injection was confirmed histologically by staining with Black-Gold II (BGII) and toluidine blue. In addition, a longitudinal analysis with diffusion-weighted magnetic resonance imaging (dMRI) was made by computing fractional anisotropy (FA), apparent diffusion coefficient (ADC) and diffusivity parameters to infer i.c.p. microstructural changes. First, dMRI analysis revealed FA decreases together with ADC and radial diffusivity (RD) increases after demyelination, which correlates with histological BGII observations. Then, we evaluated the effect produced by three allosteric GABAA receptor modulators, the N-butyl-ß-carboline-3-carboxylate (ß-CCB), ethyl 9H-pyrido [3,4-b]indole-3-carboxylate (ß-CCE), and 4-ethyl-6,7-dimethoxy-9H-pyrido [3,4-b]indole-3-carboxylic acid methyl ester (DMCM). The results indicated that daily systemic ß-CCB (1 mg/Kg) or ß-CCE (1 mg/Kg) administration for 2 weeks, but not DMCM (0.35 mg/Kg), in lesioned animals increased FA and decreased ADC or RD, suggesting myelination improvement. This was supported by BGII staining analysis that showed a recovery of myelin content. Also, it was quantified by immunohistochemistry both NG2+ and CC1+ cellular population in the different experimental sceneries. Data indicated that either ß-CCB or ß-CCE, but not DMCM, produced an increase in the population of CC1+ cells in the lesioned area. Finally, it was also calculated the g-ratio of myelinated axons and observed a similar value in those lesioned animals treated with ß-CCB or ß-CCE compared to controls. Thus, using the DRICP model, it was observed that either ß-CCB or ß-CCE, positive modulators of the GABAA receptor in OLs, had a potent promyelinating effect.
RESUMO
Toxin production in nematocysts by Cnidaria phylum represents an important source of bioactive compounds. Using electrophysiology and, heterologous expression of mammalian ion channels in the Xenopus oocyte membrane, we identified two main effects produced by the sea anemone Bartholomea annulata venom. Nematocysts isolation and controlled discharge of their content, revealed that venom had potent effects on both voltage-dependent Na+ (Nav) channels and GABA type A channel receptors (GABAAR), two essential proteins in central nervous system signaling. Unlike many others sea anemone toxins, which slow the inactivation rate of Nav channels, B. annulata venom potently inhibited the neuronal action potential and the Na+ currents generated by distinct Nav channels opening, including human TTX-sensitive (hNav1.6) and TTX-insensitive Nav channels (hNav1.5). A second effect of B. annulata venom was an agonistic action on GABAAR that activated distinct receptors conformed by either α1ß2γ2, α3ß2γ1 or, ρ1 homomeric receptors. Since GABA was detected in venom samples by ELISA assay at low nanomolar range, it was excluded that GABA from nematocysts directly activated the GABAARs. This revealed that substances in B. annulata nematocysts generated at least two potent and novel effects on mammalian ion channels that are crucial for nervous system signaling.
Assuntos
Venenos de Cnidários , Anêmonas-do-Mar , Animais , Venenos de Cnidários/farmacologia , Mamíferos , Receptores de GABA-A , Anêmonas-do-Mar/fisiologia , Ácido gama-AminobutíricoRESUMO
Oligodendrocytes (OLs) express functional GABAA receptors (GABAARs) that are activated by GABA released at synaptic contacts with axons or by ambient GABA in extrasynaptic domains. In both instances, the receptors' molecular identity has not been fully defined. Furthermore, data on their structural diversity in different brain regions and information on age-dependent changes in their molecular composition are scant. This lack of knowledge has delayed access to a better understanding of the role of GABAergic signaling between neurons and OLs. Here, we used functional, and pharmacological analyses, as well as gene and protein expression of GABAAR subunits, to explore the subunit combination that could explain the receptor functional profile expressed in OLs from the neonate rat. We found that GABAAR composed of α3ß2γ1 subunits mimicked the characteristics of the endogenous receptor when expressed heterologously in Xenopus laevis oocytes. Either α3 or γ1 subunit silencing by small interfering RNA transfection changed the GABA-response characteristics in oligodendrocyte precursor cells, indicating their participation in the endogenous receptor conformation. Thus, α3 subunit silencing shifted the mean EC50 for GABA from 75.1 to 46.6 µM, whereas γ1 silencing reduced the current amplitude response by 55%. We also observed that ß-carbolines differentially enhance GABA responses in oligodendroglia as compared with those in neurons. These results contribute to defining the molecular and pharmacological properties of GABAARs in OLs. Additionally, the identification of ß-carbolines as selective enhancers of GABAARs in OLs may help to study the role of GABAergic signaling during myelination. SIGNIFICANCE STATEMENT: GABAergic signaling through GABAA receptors (GABAARs) expressed in the oligodendroglial lineage contributes to the myelination control. Determining the molecular identity and the pharmacology of these receptors is essential to define their specific roles in myelination. Using GABAAR subunit expression and silencing, we identified that the GABAAR subunit combination α3ß2γ1 conforms the bulk of GABAARs in oligodendrocytes from rat neonates. Furthermore, we found that these receptors have differential pharmacological properties that allow specific positive modulation by ß-carbolines.
Assuntos
Encéfalo/citologia , Neurônios/citologia , Oligodendroglia/citologia , Receptores de GABA-A/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Carbolinas/farmacologia , Células Cultivadas , Feminino , Inativação Gênica , Camundongos , Neurônios/metabolismo , Oligodendroglia/metabolismo , Ratos , Receptores de GABA-A/genética , Xenopus laevisRESUMO
Myelin facilitates the fast transmission of nerve impulses and provides metabolic support to axons. Differentiation of oligodendrocyte progenitor cells (OPCs) and Schwann cell (SC) precursors is critical for myelination during development and myelin repair in demyelinating disorders. Myelination is tightly controlled by neuron-glia communication and requires the participation of a wide repertoire of signals, including neurotransmitters such as glutamate, ATP, adenosine, or γ-aminobutyric acid (GABA). GABA is the main inhibitory neurotransmitter in the central nervous system (CNS) and it is also present in the peripheral nervous system (PNS). The composition and function of GABA receptors (GABARs) are well studied in neurons, while their nature and role in glial cells are still incipient. Recent studies demonstrate that GABA-mediated signaling mechanisms play relevant roles in OPC and SC precursor development and function, and stand out the implication of GABARs in oligodendrocyte (OL) and SC maturation and myelination. In this review, we highlight the evidence supporting the novel role of GABA with an emphasis on the molecular identity of the receptors expressed in these glial cells and the possible signaling pathways involved in their actions. GABAergic signaling in myelinating cells may have potential implications for developing novel reparative therapies in demyelinating diseases.
RESUMO
Remyelination is common under physiological conditions and usually occurs as a response to a pathological demyelinating event. Its potentiation is an important goal for the development of therapies against pathologies such as multiple sclerosis and white matter injury. Visualization and quantification in vivo of demyelination and remyelination processes are essential for longitudinal studies that will allow the testing and development of pro-myelinating strategies. In this study, ethidium bromide (EB) was stereotaxically injected into the caudal cerebellar peduncle (c.c.p.) in rats to produce demyelination; the resulting lesion was characterized (i) transversally through histology using Black-Gold II (BGII) staining, and (ii) longitudinally through diffusion-weighted magnetic resonance imaging (dMRI), by computing fractional anisotropy (FA) and diffusivity parameters to detect microstructural changes. Using this characterization, we evaluated, in the lesioned c.c.p., the effect of N-butyl-ß-carboline-3-carboxylate (ß-CCB), a potentiator of GABAergic signaling in oligodendrocytes. The dMRI analysis revealed significant changes in the anisotropic and diffusivity properties of the c.c.p. A decreased FA and increased radial diffusivity (λâ¥) were evident following c.c.p. lesioning. These changes correlated strongly with an apparent decrease in myelin content as evidenced by BGII. Daily systemic ß-CCB administration for 2â¯weeks in lesioned animals increased FA and decreased λâ¥, suggesting an improvement in myelination, which was supported by histological analysis. This study shows that structural changes in the demyelination-remyelination of the caudal cerebellar peduncle (DRCCP) model can be monitored longitudinally by MRI, and it suggests that remyelination is enhanced by ß-CCB treatment. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
Assuntos
Doenças Desmielinizantes , Remielinização , Substância Branca , Animais , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/diagnóstico por imagem , Imageamento por Ressonância Magnética , Bainha de Mielina , Ratos , Substância Branca/diagnóstico por imagemRESUMO
Background and Objectives: Acute lymphoblastic leukemia (ALL) is the most common type of cancer in childhood. The majority of patients respond to treatment, but those with resistant phenotypes suffer relapse or death. The antifolate methotrexate (MTX) is the most commonly used drug against ALL due to its efficacy. Once inside leukemic cells, MTX is metabolized into methotrexate polyglutamates (MTX-PG) by action of the enzyme folylpolyglutamate synthetase (FPGS), leading to a longer action compared to that of MTX alone. Materials and Methods: In this work, we demonstrated that the combination treatment of methotrexate and 5 and 10 mM glutamic acid could enhance methotrexate cytotoxicity in CCRF-SB (B-ALL) cells. In addition, MTX plus 20 mM glutamic acid was able to improve the synthesis of MTX-PG5. Results: All treatments induced an increase in FPGS expression compared to that of the control group. Furthermore, we detected different cellular expression patterns of FPGS in the different treatments. Conclusion: Based on these findings, we demonstrated that levels of methotrexate polyglutamates (MTX-PGs) could be a key determinant of methotrexate-induced cytotoxicity in CCRF-SB acute lymphoblastic leukemia cells.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Metotrexato/análogos & derivados , Ácido Poliglutâmico/análogos & derivados , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Linhagem Celular , Humanos , Metotrexato/farmacologia , Ácido Poliglutâmico/farmacologiaRESUMO
Extracellular purines through specific receptors have been recognized as new regulators of ovarian function. It is known that P2Y2 receptor activity induces theca cell proliferation, we hypothesized that purinergic signaling controls the changes related to hyperthecosis in polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of UTP-sensitive P2Y receptors and their role in theca cells (TC) proliferation in experimentally-induced PCOS (EI-PCOS). In primary cultures of TC from intact rats, all the transcripts of P2Y receptors were detected by polymerase chain reaction; in these cells, UTP (10 µM) induced extracellular signal-regulated kinases (ERK) phosphorylation. Rats with EI-PCOS showed a reduced expression of P2Y2R in TC whereas P2Y4R did not change. By analyzing ERK phosphorylation, it was determined that P2Y2R is the most relevant receptor in TC. UTP promoted cell proliferation in TC from control but not from EI-PCOS rats. The in silico analysis of P2yr2 promoter indicated the presence of androgen response elements; the stimulation of TC primary cultures with testosterone promoted a significant reduction in the expression of the P2yr2 transcript. We concluded that P2Y2R participates in controlling the proliferative rate of TCs from healthy ovaries, but this regulation is lost during EI-PCOS.
Assuntos
Síndrome do Ovário Policístico/patologia , Receptores Purinérgicos P2Y2/metabolismo , Células Tecais/patologia , Células Tecais/fisiologia , Uridina Trifosfato/farmacologia , Animais , Proliferação de Células/fisiologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fosforilação , Regiões Promotoras Genéticas/genética , Ratos , Ratos Wistar , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Testosterona/farmacologiaRESUMO
Inwardly rectifying K+ (Kir) channel expression signals at an advanced stage of maturation during oligodendroglial differentiation. Knocking down their expression halts the generation of myelin and produces severe abnormalities in the central nervous system. Kir4.1 is the main subunit involved in the tetrameric structure of Kir channels in glial cells; however, the precise composition of Kir channels expressed in oligodendrocytes (OLs) remains partially unknown, as participation of other subunits has been proposed. Kir channels are sensitive to H+; thus, intracellular acidification produces Kir current inhibition. Since Kir subunits have differential sensitivity to H+, we studied the effect of intracellular acidification on Kir currents expressed in cultured OLs derived from optic nerves of 12-day-old rats. Unexpectedly, Kir currents in OLs (2-4 DIV) did not change within the pH range of 8.0-5.0, as observed when using standard whole-cell voltage-clamp recording or when preserving cytoplasmic components with the perforated patch-clamp technique. In contrast, low pH inhibited astrocyte Kir currents, which was consistent with the involvement of the Kir4.1 subunit. The H+-insensitivity expressed in OL Kir channels was not intrinsic because Kir cloning showed no difference in the sequence reported for the Kir4.1, Kir2.1, or Kir5.1 subunits. Moreover, when Kir channels were heterologously expressed in Xenopus oocytes they behaved as expected in their general properties and sensitivity to H+. It is therefore concluded that Kir channel H+-sensitivity in OLs is modulated through an extrinsic mechanism, probably by association with a modulatory component or by posttranslational modifications.
Assuntos
Oligodendroglia/fisiologia , Nervo Óptico/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Nervo Óptico/citologia , Ratos , Ratos Sprague-Dawley , Xenopus laevisRESUMO
Carybdea marsupialis is a widely distributed box jellyfish found in the Mediterranean and in the tropical waters of the Caribbean Sea. Its venom is a complex mixture of biologically active compounds that are used to catch prey. In order to evaluate the activity of the neurotoxins in the venom, bioassays were carried out using the marine crab Ocypode quadrata. The proteins with neurotoxic effect were partially purified using low-pressure liquid chromatography techniques. Gel filtration (Sephadex G-50M) was used as the first step and the active fraction in crabs was passed through a QAE Sephadex A-25 column. Finally, the active fraction was run onto a Fractogel EMD SO3- column. No further purification step could be carried out due to the loss of neurotoxic activity. The Fractogel EMD SO3- fraction was analyzed electrophysiologically using the voltage-clamp technique in Xenopus laevis oocytes expressing membrane proteins from rat brain through mRNA injection. The crude venom and a fraction were observed to affect crustaceans and showed at least two types of bioactivity in oocytes expressing brain proteins. The effects were dose-dependent and completely reversible. These results evidence the presence of neurotoxins in Carybdea marsupialis venom that act on membrane proteins of the vertebrate nervous system.
Assuntos
Braquiúros/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Venenos de Cnidários/toxicidade , Cubomedusas/metabolismo , Proteínas do Tecido Nervoso/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Neurotoxinas/toxicidade , Xenopus laevis/metabolismo , Animais , Bioensaio , Encéfalo/metabolismo , Cromatografia em Gel , Cromatografia Líquida , Venenos de Cnidários/isolamento & purificação , Relação Dose-Resposta a Droga , Técnicas de Transferência de Genes , Potenciais da Membrana , Microinjeções , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Síndromes Neurotóxicas/fisiopatologia , Neurotoxinas/isolamento & purificação , Oócitos , Técnicas de Patch-Clamp , Ratos , Xenopus laevis/genéticaRESUMO
BACKGROUND: The Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx. RESULTS: Endogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48-72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated Ca2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the T in response, a Ca2+-influx-dependent Cl- current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology. CONCLUSIONS: It was found that STIM2, but not STIM1, was essential in both responses, and T in currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the T in response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.
Assuntos
Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Oócitos/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Técnicas de Silenciamento de Genes , Oócitos/crescimento & desenvolvimento , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Xenopus laevisRESUMO
P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 µM, respectively; 10 µM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 µM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Cálcio/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/farmacologia , Adulto , Idoso , Apirase/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pessoa de Meia-Idade , Comunicação Parácrina/efeitos dos fármacosRESUMO
In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla (central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered. Distinction of the two new species is based on morphological characters and supported with phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered the two species in different clades strongly supported by posterior probabilities and bootstrap values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal fungi associated with Alnus acuminata. Information about these taxa includes the morphological variation achieved along 16 monitories 2010-2013. Descriptions are provided. They are accompanied by photos including SEM photomicrographs of basidiospores and information on differences between them and other related taxa from Europe and the United States.
Assuntos
Alnus/microbiologia , Basidiomycota/classificação , Micorrizas/classificação , Basidiomycota/genética , Basidiomycota/isolamento & purificação , Basidiomycota/ultraestrutura , DNA Fúngico/química , DNA Fúngico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Carpóforos , Proteínas Fúngicas/genética , México , Microscopia Eletrônica de Varredura , Micorrizas/genética , Micorrizas/isolamento & purificação , Micorrizas/ultraestrutura , Filogenia , RNA Polimerase II/genética , Análise de Sequência de DNA , Esporos FúngicosRESUMO
P2X receptors are trimeric, ATP-gated cation channels. In mammals seven P2X subtypes have been reported (P2X1-P2X7), as well as several variants generated by alternative splicing. Variants confer to the homomeric or heteromeric channels distinct functional and/or pharmacological properties. Molecular biology, biochemical, and functional analysis by electrophysiological methods were used to identify and study a new variant of the P2X1 receptor named P2X1b. This new variant, identified in rat optic nerve, was also expressed in other tissues. P2X1b receptors lack amino acids 182 to 208 of native P2X1, a region that includes residues that are highly conserved among distinct P2X receptors. When expressed in Xenopus oocytes, P2X1b was not functional as a homomer; however, when co-expressed with P2X1, it downregulated the electrical response generated by ATP compared with that of oocytes expressing P2X1 alone, and it seemed to form heteromeric channels with a modestly enhanced ATP potency. A decrease in responses to ATP in oocytes co-expressing different ratios of P2X1b to P2X1 was completely eliminated by overnight pretreatment with apyrase. Thus, it is suggested that P2X1b regulates, through a use-dependent mechanism, the availability, in the plasma membrane, of receptor channels that can be operated by ATP.
Assuntos
Processamento Alternativo , Ativação do Canal Iônico , Nervo Óptico/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/metabolismo , Clonagem Molecular , Humanos , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Xenopus laevisRESUMO
Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion-current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage-clamp technique. All agonists elicited Cl(-) currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca(2+) concentration ([Ca(2+) ](i)), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl(-) (F(Cl)) currents, while AII activated an oscillatory response; a robust Ca(2+) influx linked specifically to F(Cl) activation elicited an inward current (I(iw,Ca)) which was carried mainly by Cl(-) ions, through channels with a sequence of permeability of SCN(-) > I(-) > Br(-) > Cl(-). Like F(Cl), I(iw,Ca) was not dependent on oocyte [Ca(2+) ](i) ; instead both were eliminated by preventing [Ca(2+) ](i) increase in the follicular cells, and also by U73122 and 2-APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F(Cl) and I(iw,Ca) were produced by the expected, PLC-stimulated Ca(2+) -release and Ca(2+) -influx, respectively, and by the opening of I(Cl(Ca)) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca(2+) -influx appeared to be driven through store-operated, calcium-like channels. The AII response, which is also known to require PLC activation, did not activate I(iw,Ca) and was strictly dependent on oocyte [Ca(2+) ](i) increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap-junction channels.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Junções Comunicantes/metabolismo , Folículo Ovariano/metabolismo , Acetilcolina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte de Íons/fisiologia , Oócitos/metabolismo , Receptores Colinérgicos/metabolismo , Xenopus laevisRESUMO
BACKGROUND: Purinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC). METHODS: TIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA. RESULTS: RT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 microM, while that for UDP was 3.24 +/- 0.82 microM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 microM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation. CONCLUSION: Here, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets.
Assuntos
Receptores Purinérgicos P2/genética , Células Tecais/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Espaço Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células Tecais/efeitos dos fármacos , Difosfato de Uridina/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent K(+) current (I(K,cAMP)), whereas ATP activates a Cl(-) current (F(Cl)) and has a dual effect on I(K,cAMP), provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes (i(etr)/i(wf)) in the two preparations. For ATP activation of I(K,cAMP) and F(Cl), the ratios i(etr)/i(wf) were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. (I(K,cAMP)) was 14 +/- 3.8 microM [Hill coefficient (nH) = 2.7 +/- 0.61], and that in e.t.r. follicles (F(Cl)) was 1.8 +/- 0.68 microM (nH = 0.76 +/- 0.09), whereas Ado-response parameters did not change. Responses to UTP and beta,gamma-methylene-ATP, specific agonists for I(K,cAMP) inhibition and activation, respectively, indicated that in e.t.r. follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle.
Assuntos
Folículo Ovariano/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Purinérgicos/metabolismo , Xenopus laevis/metabolismo , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , AMP Cíclico/metabolismo , Feminino , Cinética , Técnicas de Patch-Clamp , Uridina Trifosfato/metabolismoRESUMO
Xenopus follicle-enclosed oocytes are endowed with purinergic receptors located in the follicular cell membrane; their stimulation by ATP elicits an electrical response that includes generation of a fast inward current (F(Cl)) carried by Cl(-). Here, it was found that mechanical stimulation of the follicle provoked a native electrical response named I(mec). This was dependent on coupling between oocyte and follicular cells, because I(mec) was eliminated by enzymatic defolliculation or application of uncoupling drugs, such as heptanol or carbenoxolone. Moreover, the characteristics of I(mec) suggested that it corresponded with opening of the Cl(-) channel involved in F(Cl). For example, I(mec) showed cross-talk with the membrane mechanism that activates the F(Cl) response and anionic selectivity similar to that displayed by F(Cl). Also like F(Cl), I(mec) was independent of extracellular or intracellular Ca(2+). Furthermore, I(mec) was inhibited by superfusion with a purinergic antagonist, suramin, or by an enzyme that rapidly hydrolyzes ATP, apyrase. The response to mechanical stimulation was reconstituted in defolliculated oocytes expressing P2X channels as an ATP sensor. Recently, it has been shown that ATP release from the Xenopus oocyte is triggered by mechanical stimulation. Together, these observations seemed to indicate that I(mec) is activated through a mechanism that involves oocyte release of ATP that diffuses and activates purinergic receptors in follicular cells, with subsequent opening of F(Cl) channels. Thus, I(mec) generation disclosed a paracrine communication system via ATP between the oocyte and its companion follicular cells that might be of physiological importance during the growth and development of the gamete.
Assuntos
Trifosfato de Adenosina/metabolismo , Ativação do Canal Iônico/fisiologia , Folículo Ovariano/fisiologia , Receptores Purinérgicos/metabolismo , Xenopus laevis/fisiologia , Animais , Apirase/farmacologia , Cálcio/farmacologia , Conexinas/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Feminino , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X , Estresse Mecânico , Suramina/farmacologiaRESUMO
Folliculogenesis modulation via distinct neurotransmitters is a well-documented phenomenon. Intraovarian purinergic signaling mechanisms have been identified previously in different species. However, the molecular elements involved and the physiological role of this purinergic signaling remain to be elucidated. Here, studies using RT-PCR amplification, immunoblotting, and immunofluorescence microscopy showed that murine and porcine ovaries express the P2X7 subtype receptor, a cationic receptor-channel operated by ATP. Using immunofluorescence it was demonstrated that P2X7 protein expression, in both mouse and pig, occurs specifically in the theca cells from antral follicles. Isolated porcine theca cells maintained in primary cultures and tested with 1 mM ATP or 250 microM Bz-ATP, a specific agonist of P2X7, responded with an increase in intracellular calcium concentration, as demonstrated in cells loaded with fluo-4 as calcium indicator. This strongly suggested that P2X7 receptors in theca cells are functional. Moreover, application for 24 hr of 1 mM ATP or 250 microM Bz-ATP induced apoptotic cell death as indicated by the DNA fragmentation pattern, positive TUNEL test, and annexin V binding. This ATP effect was antagonized by 300 microM PPADS and 200 microM oxidized ATP. Also, addition of 5 mM EGTA in the external medium to chelate free Ca++ decreased death cell to 24% of that produced by 200 microM Bz-ATP, suggesting that Ca++ influx participates in the phenomenon. The highly specific and functional expression of P2X7 receptors in theca cells suggest a role for ATP in modulating follicular physiology.
Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose/fisiologia , Receptores Purinérgicos P2/metabolismo , Suínos , Células Tecais/fisiologia , Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade/metabolismo , Animais , Anexina A5/metabolismo , Cálcio/metabolismo , Células Cultivadas , Fragmentação do DNA , Feminino , Marcação In Situ das Extremidades Cortadas , Camundongos , Receptores Purinérgicos P2X7 , Células Tecais/citologiaRESUMO
The Xenopus follicular cell membrane is endowed with ATP-sensitive K+ channels, which are operated by various transmitters. These generate the ionic response named IK,cAMP via a mechanism that involves intracellular cAMP synthesis. It is known that opening these K+ channels favors oocyte maturation. Follicle stimulation by adenosine (Ado) or ATP consistently generates a strong IK,cAMP via activation of P1 and P3 purinergic receptors; however, ATP can also inhibit IK,cAMP, apparently acting on a third receptor type. Here, we show that IK,cAMP might be elicited by ATP released within the follicle, and that current activation by ATP was entirely dependent on the presence of epithelial and/or theca layers. Morphological studies confirmed that removal of epithelium/theca in these follicles (e.t.r.) was complete, and activation of fast Cl- (Fin) currents by ATP in e.t.r. follicles confirmed that communication between oocyte and follicular cells remained unchanged. Thus, dependence on epithelium/theca was specific for ATP-elicited K+ current. Using UTP and betagamma-MeATP as specific purinergic agents for IK,cAMP inhibition and activation, respectively, it was found that inhibition of IK,cAMP elicited by ATP or UTP was robustly present in e.t.r. follicles but was absent or strongly decreased in whole follicles (w.f.). Accordingly, this indicated that in w.f., epithelium and/or theca downregulated the IK,cAMP inhibition evoked by ATP, and that this control mechanism was absent in e.t.r. follicles. We suggest that this notable action on follicular cells involves one or both of two mechanisms, a paracrine transmitter released from epithelial and/or theca layers and action of ecto-ATPases.