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1.
Anal Chim Acta ; 1094: 106-112, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31761035

RESUMO

Endogenous hydrogen sulfide (H2S) exists in multiple physiological processes. In order to further understand the action mechanism of H2S in cells and human body, we proposed a smart surface-enhanced Raman scattering (SERS) nanoprobe, Au core-4-mercaptobenzonitrile-Ag shell nanoparticle (Au@4-MBN@Ag), for the detection of endogenous H2S in living cells based on the reaction between Ag shell and sulfide species. 4-MBN was selected as the SERS reporter to avoid interference from cellular molecules. With the sulfide concentration increasing, the Ag2S constantly formed, and consequently the SERS signal intensity of Au@4-MBN@Ag gradually decreased owing to the weaker SERS activity of Ag2S. With the nanoprobes, this method not only offers a high sensitivity for H2S detection at an nM level, but also achieves the goal of non-background analysis. It displays satisfactory anti-interference capability and a good linear relationship with sulfide concentration ranging from 50 nM to 500 µM, and an estimated detection limit is 0.14 nM. The Au@4-MBN@Ag nanoprobes were successfully applied to detect endogenous H2S in living HepG2 cells stimulated by pyridoxal 5-phosphate monohydrate. This work offers a potential analytical method in the related research of H2S physiological function.


Assuntos
Sulfeto de Hidrogênio/análise , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Linhagem Celular Tumoral , Ouro/química , Humanos , Insulina/farmacologia , Limite de Detecção , Fosfato de Piridoxal/farmacologia , Prata/química
2.
Methods Mol Biol ; 1866: 107-131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725412

RESUMO

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 µM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 µM, respectively, from the PLP baseline of 0.3 µM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.


Assuntos
Liases de Carbono-Enxofre/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Apoenzimas/metabolismo , Liases de Carbono-Enxofre/sangue , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/isolamento & purificação , Cristalização , Escherichia coli/metabolismo , Fermentação , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Fosfato de Piridoxal/administração & dosagem , Fosfato de Piridoxal/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
3.
Glia ; 67(5): 915-934, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30632636

RESUMO

Optogenetics has been widely expanded to enhance or suppress neuronal activity and it has been recently applied to glial cells. Here, we have used a new approach based on selective expression of melanopsin, a G-protein-coupled photopigment, in astrocytes to trigger Ca2+ signaling. Using the genetically encoded Ca2+ indicator GCaMP6f and two-photon imaging, we show that melanopsin is both competent to stimulate robust IP3-dependent Ca2+ signals in astrocyte fine processes, and to evoke an ATP/Adenosine-dependent transient boost of hippocampal excitatory synaptic transmission. Additionally, under low-frequency light stimulation conditions, melanopsin-transfected astrocytes can trigger long-term synaptic changes. In vivo, melanopsin-astrocyte activation enhances episodic-like memory, suggesting melanopsin as an optical tool that could recapitulate the wide range of regulatory actions of astrocytes on neuronal networks in behaving animals. These results describe a novel approach using melanopsin as a precise trigger for astrocytes that mimics their endogenous G-protein signaling pathways, and present melanopsin as a valuable optical tool for neuron-glia studies.


Assuntos
Astrócitos/metabolismo , Rede Nervosa/metabolismo , Neurônios/metabolismo , Optogenética/métodos , Opsinas de Bastonetes/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Compostos Azo/farmacologia , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Pirimidinas/farmacologia , Opsinas de Bastonetes/genética , Potenciais Sinápticos/fisiologia , Triazóis/farmacologia , Xantenos/farmacologia
4.
Reprod Biol Endocrinol ; 16(1): 106, 2018 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-30368246

RESUMO

BACKGROUND: Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs. METHODS: Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H2O2), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment. RESULTS: Mangafodipir attenuated apoptosis induced by H2O2 and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects. CONCLUSIONS: Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.


Assuntos
Antineoplásicos/farmacologia , Ácido Edético/análogos & derivados , Células da Granulosa/efeitos dos fármacos , Ovário/efeitos dos fármacos , Fosfato de Piridoxal/análogos & derivados , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cisplatino/farmacologia , Meios de Contraste/farmacologia , Ácido Edético/farmacologia , Feminino , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Paclitaxel/farmacologia , Substâncias Protetoras/farmacologia , Fosfato de Piridoxal/farmacologia
5.
Exp Physiol ; 103(12): 1679-1691, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30242927

RESUMO

NEW FINDINGS: What is the central question of this study? ATP is known to modulate the chemosensitivity of some brain areas. However, whether the ATP contributes specifically to the mechanism of chemoreception in the lateral hypothalamus/perifornical area (LH/PFA) remains to be determined. What is the main finding and its importance? ATP, acting on the LH/PFA, enhances the hypercapnic ventilatory response in rats during wakefulness, in the dark period. Our results highlight the importance of ATP as a modulator of central chemoreception and provide new insight regarding the mechanisms involved in LH/PFA chemosensitivity and the sleep-wake differences in the CO2 /H+ -dependent drive to breathe. ABSTRACT: The lateral hypothalamus/perifornical area (LH/PFA) is a central chemoreceptor site, which acts in an arousal state-dependent manner. It has been shown that purinergic signalling through ATP influences the CO2 /H+ responsiveness of other chemosensitive regions, but it is unknown whether ATP is also involved in the mechanisms that underlie LH/PFA chemoreception. Here, we studied the effects of microdialysis of a P2X-receptor agonist [α,ß-methylene ATP (α,ß-meATP), 10 mm] and a non-selective P2-receptor antagonist [pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), 1 mm] into the LH/PFA of conscious rats on ventilation in room air and in 7% CO2 . In the dark (active) phase, but not in the light, microdialysis of α,ß-meATP caused an augmented hypercapnic ventilatory response during wakefulness, but not during non-REM sleep (P < 0.001). PPADS caused no change in CO2 ventilatory responses in either the dark period or the light period. Our data suggest that ATP in LH/PFA contributes to the hypercapnic ventilatory response in conscious rats during wakefulness in the dark phase of the diurnal cycle.


Assuntos
Trifosfato de Adenosina/metabolismo , Dióxido de Carbono/metabolismo , Células Quimiorreceptoras/metabolismo , Região Hipotalâmica Lateral/metabolismo , Ventilação Pulmonar/fisiologia , Trifosfato de Adenosina/análogos & derivados , Animais , Células Quimiorreceptoras/efeitos dos fármacos , Hipercapnia/metabolismo , Região Hipotalâmica Lateral/efeitos dos fármacos , Masculino , Ventilação Pulmonar/efeitos dos fármacos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Wistar , Respiração/efeitos dos fármacos , Sono/efeitos dos fármacos , Sono/fisiologia , Vigília/efeitos dos fármacos , Vigília/fisiologia
6.
Viruses ; 10(8)2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30115859

RESUMO

Feline calicivirus (FCV) is a major cause of upper respiratory tract disease in cats, with widespread distribution in the feline population. Recently, virulent systemic diseases caused by FCV infection has been associated with mortality rates up to 50%. Currently, there are no direct-acting antivirals approved for the treatment of FCV infection. Here, we tested 15 compounds from different antiviral classes against FCV using in vitro protein and cell culture assays. After the expression of FCV protease-polymerase protein, we established two in vitro assays to assess the inhibitory activity of compounds directly against the FCV protease or polymerase. Using this recombinant enzyme, we identified quercetagetin and PPNDS as inhibitors of FCV polymerase activity (IC50 values of 2.8 µM and 2.7 µM, respectively). We also demonstrate the inhibition of FCV protease activity by GC376 (IC50 of 18 µM). Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2'-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC50 values in the low micromolar range (0.6 µM and 2.5 µM, respectively). In conclusion, we established two in vitro assays that will accelerate the research for FCV antivirals and can be used for the high-throughput screening of direct-acting antivirals.


Assuntos
Antivirais/farmacologia , Calicivirus Felino/efeitos dos fármacos , Citidina/análogos & derivados , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Peptídeo Hidrolases/metabolismo , Poliproteínas/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/veterinária , Infecções por Caliciviridae/virologia , Calicivirus Felino/genética , Calicivirus Felino/metabolismo , Doenças do Gato/tratamento farmacológico , Doenças do Gato/virologia , Gatos , Linhagem Celular , Citidina/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Flavonas/farmacologia , Expressão Gênica , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Peptídeo Hidrolases/genética , Poliproteínas/genética , Poliproteínas/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/veterinária , Infecções Respiratórias/virologia , Ácidos Sulfônicos/farmacologia
7.
J Pharmacol Exp Ther ; 366(2): 238-243, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29858389

RESUMO

The current study originates from the assumption that, in tumors, levels of naturally occurring pyridoxal 5'-phosphate (PLP) are too small to allow conversion of tetra hydro pteroylglutamate (H4PteGlu) into methylene tetra hydro pteroylglutamate (CH2-H4PteGlu) in amounts required to improve inhibition of thymidylate synthase by 5-fluorouracil (FUra) through ternary complex stabilization. The hypothesis relates to the low affinity for cofactor of the PLP-dependent serine hydroxymethyl transferase (SHMT), the enzyme that catalyzes formation of CH2-H4PteGlu by transfer of the Cß of serine to H4PteGlu. Intracellular concentrations of PLP are smaller than the dissociation constant of SHMT for cofactor, which suggests that enzyme activity should be sensitive to PLP level changes. Three cancer cell lines were supplemented with PLP to investigate the influence of this cofactor on FUra cytotoxicity. Cells were exposed to FUra, FUra and folinic acid (FA), FUra and PLP, and FUra combined with both FA and PLP. The median-effect principle for concentration-effect analysis and combination indices were used to determine interactions on cytotoxicity. FUra cytotoxicity in vitro was enhanced by FA and PLP in tandem. Synergistic cytotoxic interaction of FUra with FA and PLP was demonstrated in HT29 and L1210 cells. Summation was found in HCT116 cells. Parenteral pyridoxamine was administered in mice to explore erythrocyte production of PLP in vivo. Cofactor attained levels in the range of the KD for binding to SHMT, and it was rapidly cleared from cells. Pharmacokinetics of pyridoxamine suggests that modulation of FUra by vitamin B6 could be achieved in vivo.


Assuntos
Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Ácido Fólico/farmacologia , Leucovorina/farmacologia , Fosfato de Piridoxal/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Células HCT116 , Células HT29 , Humanos , Concentração Inibidora 50 , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos
8.
PLoS One ; 13(2): e0190893, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29466379

RESUMO

Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.


Assuntos
Trifosfato de Adenosina/metabolismo , Células Ependimogliais/metabolismo , Retina/citologia , Retina/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Ambystoma , Animais , Células Ependimogliais/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ictaluridae , Técnicas In Vitro , Transporte de Íons/efeitos dos fármacos , Lampreias , Macaca fascicularis , Macaca mulatta , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Receptores Purinérgicos P2Y/efeitos dos fármacos , Transdução de Sinais , Suramina/farmacologia
9.
Am J Respir Cell Mol Biol ; 59(1): 87-95, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29393654

RESUMO

We previously described several ionic conductances in human pulmonary fibroblasts, including one activated by two structurally distinct TRPV4 (transient receptor potential, vanilloid-type, subtype 4)-channel agonists: 4αPDD (4α-phorbol-12,13-didecanoate) and GSK1016790A. However, the TRPV4-activated current exhibited peculiar properties: it developed slowly over many minutes, exhibited reversal potentials that could vary by tens of millivolts even within a given cell, and was not easily reversed by subsequent addition of two distinct TRPV4-selective blockers (RN-1734 and HC-067047). In this study, we characterized that conductance more carefully. We found that 4αPDD stimulated a delayed release of ATP into the extracellular space, which was reduced by genetic silencing of pannexin expression, and that the 4αPDD-evoked current could be blocked by apyrase (which rapidly degrades ATP) or by the P2Y purinergic receptor/channel blocker pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), and could be mimicked by exogenous addition of ATP. In addition, we found that the 4αPDD-evoked current was blocked by pretreatment with RN-1734 or HC-067047, by Gd3+ or La3+, or by two distinct blockers of pannexin channels (carbenoxolone and probenecid), but not by a blocker of connexin hemichannels (flufenamic acid). We also found expression of TRPV4- and pannexin-channel proteins. 4αPDD markedly increased calcium flashing in our cells. The latter was abrogated by the P2Y channel blocker PPADS, and the 4αPDD-evoked current was eliminated by loading the cytosol with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or by inhibiting Ca2+/calmodulin-sensitive kinase II using KN93. Altogether, we interpret these findings as suggesting that 4αPDD triggers the release of ATP via pannexin channels, which in turn acts in an autocrine and/or paracrine fashion to stimulate PPADS-sensitive purinergic receptors on human pulmonary fibroblasts.


Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Fibroblastos/metabolismo , Pulmão/citologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Cátion TRPV/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Ésteres de Forbol/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Fatores de Tempo
10.
J Bacteriol ; 200(2)2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29061666

RESUMO

ZipA is essential for cell division in Escherichia coli, acting early in the process to anchor polymers of FtsZ to the cytoplasmic membrane. Along with FtsA, FtsZ and ZipA form a proto-ring at midcell that recruits additional proteins to eventually build the division septum. Cells carrying the thermosensitive zipA1 allele divide fairly normally at 30°C in rich medium but cease dividing at temperatures above 34°C, forming long filaments. In a search for suppressors of the zipA1 allele, we found that deletions of specific genes involved in amino acid biosynthesis could partially rescue cell growth and division at 34°C or 37°C but not at 42°C. Notably, although a diverse group of amino acid biosynthesis gene deletions could partially rescue the growth of zipA1 cells at 34°C, only deletions of genes related to the biosynthesis of threonine, glycine, serine, and methionine could rescue growth at 37°C. Adding exogenous pyridoxal 5-phosphate (PLP), a cofactor for many of the enzymes affected by this study, partially suppressed zipA1 mutant thermosensitivity. For many of the deletions, PLP had an additive rescuing effect on the zipA1 mutant. Moreover, added PLP partially suppressed the thermosensitivity of ftsQ and ftsK mutants and weakly suppressed an ftsI mutant, but it failed to suppress ftsA or ftsZ thermosensitive mutants. Along with the ability of a deletion of metC to partially suppress the ftsK mutant, our results suggest that perturbations of amino acid metabolic pathways, particularly those that redirect the flow of carbon away from the synthesis of threonine, glycine, or methionine, are able to partially rescue some cell division defects.IMPORTANCE Cell division of bacteria, such as Escherichia coli, is essential for their successful colonization. It is becoming increasingly clear that nutritional status and central metabolism can affect bacterial size and shape; for example, a metabolic enzyme (OpgH) can moonlight as a regulator of FtsZ, an essential cell division protein. Here, we demonstrate a link between amino acid metabolism and ZipA, another essential cell division protein that binds directly to FtsZ and tethers it to the cytoplasmic membrane. Our evidence suggests that altering flux through the methionine-threonine-glycine-serine pathways and supplementing with the enzyme cofactor pyridoxal-5-phosphate can partially compensate for an otherwise lethal defect in ZipA, as well as several other cell division proteins.


Assuntos
Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Mutação , Aminoácidos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Deleção de Genes , Redes e Vias Metabólicas , Fosfato de Piridoxal/farmacologia
11.
Neuropharmacology ; 128: 366-378, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29079292

RESUMO

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores Purinérgicos P2X/metabolismo , Trifosfato de Adenosina/farmacologia , Peptídeos beta-Amiloides/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Embrião de Mamíferos , Feminino , Proteínas Associadas aos Microtúbulos/metabolismo , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Inibidores da Agregação de Plaquetas/farmacologia , Gravidez , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X/genética
12.
Cell Death Dis ; 8(12): 3214, 2017 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-29238081

RESUMO

Pyridoxine 5'-phosphate oxidase (PNPO) is an enzyme that converts pyridoxine 5'-phosphate into pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 implicated in several types of cancer. However, the role of PNPO and its regulatory mechanism in epithelial ovarian cancer (EOC) are unknown. In the present study, PNPO expression in human ovarian tumour tissue and its association with the clinicopathological features of patients with EOC were examined. Further, the biological function of PNPO in EOC cells and in xenograft was evaluated. We demonstrated for the first time that PNPO was overexpressed in human EOC. Knockdown of PNPO induced EOC cell apoptosis, arrested cell cycle at G2/M phase, decreased cell proliferation, migration and invasion. Xenografts of PNPO-shRNA-expressing cells into the nude mouse attenuated tumour growth. PNPO at mRNA and protein levels in EOC cells was decreased after transforming growth factor-ß1 (TGF-ß1) treatment. The inhibitory effect of TGF-ß1 on PNPO expression was abolished in the presence of SB-431542, a TGF-ß type I receptor kinase inhibitor. Moreover, we found that TGF-ß1-mediated PNPO expression was at least in part through the upregulation of miR-143-3p. These data indicate a mechanism underlying PNPO regulation by the TGF-ß signalling pathway. Furthermore, PLP administration reduced PNPO expression and decreased EOC cell proliferation, suggesting a feedback loop between PLP and PNPO. Thus, our findings reveal that PNPO can serve as a novel tissue biomarker of EOC and may be a potential target for therapeutic intervention.


Assuntos
Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , Proteínas Serina-Treonina Quinases/genética , Piridoxaminafosfato Oxidase/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta1/genética , Animais , Antagomirs/genética , Antagomirs/metabolismo , Sequência de Bases , Benzamidas/farmacologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dioxóis/farmacologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Piridoxaminafosfato Oxidase/antagonistas & inibidores , Piridoxaminafosfato Oxidase/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Toxins (Basel) ; 9(10)2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-29048353

RESUMO

The pore forming hemolysin A, Hla, is a major virulence factor of Staphylococcus aureus. Apparently, 1-2 pore(s) per cell suffice(s) to cause cell death. Accumulated experimental evidence points towards a major role of ATP-gated purinergic receptors (P2XR) for hemolysis caused by Hla, complement and other pore forming proteins, presumably by increasing membrane permeability. Indeed, in experiments employing rabbit erythrocytes, inhibitory concentrations of frequently employed P2XR-antagonists were in a similar range as previously reported for erythrocytes of other species and other toxins. However, Hla-dependent hemolysis was not enhanced by extracellular ATP, and oxidized adenosinetriphosphate (oxATP) had only a minor inhibitory effect. Unexpectedly, P2XR-inhibitors also prevented Hla-induced lysis of pure lipid membranes, demonstrating that the inhibition did not even depend on the presence of P2XR. Fluorescence microscopy and gel-electrophoresis clearly revealed that P2XR-inhibitors interfere with binding and subsequent oligomerisation of Hla with membranes. Similar results were obtained employing HaCaT-cells. Furthermore, calorimetric data and hemolysis experiments with Hla pre-treated with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) showed that this compound directly binds to Hla. Our results call for a critical re-assessment of the appealing concept, which suggests that P2XR are general amplifiers of damage by pore-forming proteins.


Assuntos
Toxinas Bacterianas/toxicidade , Proteínas Hemolisinas/toxicidade , Hemólise/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Staphylococcus aureus/patogenicidade , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Fluoresceínas/metabolismo , Humanos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Coelhos , Receptores Purinérgicos P2X/fisiologia
14.
J Neurophysiol ; 118(4): 1952-1961, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28701543

RESUMO

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X2 purinoceptors acquire permeability to large cations, such as N-methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,ß-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.


Assuntos
Células Amácrinas/metabolismo , Colina/metabolismo , Neurônios Colinérgicos/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Neurônios Retinianos/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Células Amácrinas/fisiologia , Animais , Células Cultivadas , Neurônios Colinérgicos/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Neurônios Retinianos/fisiologia
15.
Brain Res ; 1669: 69-78, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28554806

RESUMO

Preventing damage caused by nerve degeneration is a great challenge. There is a growing body of evidence implicating extracellular nucleotides and their P2 receptors in many pathophysiological mechanisms. In this work we aimed to investigate the effects of the administration of Brilliant Blue G (BBG) and Pyridoxalphosphate-6-azophenyl-2', 4'- disulphonic acid (PPADS), P2X7 and P2 non-selective receptor antagonists, respectively, on sciatic nerve regeneration. Four groups of mice that underwent nerve crush lesion were used: two control groups treated with vehicle (saline), a group treated with BBG and a group treated with PPADS during 28days. Gastrocnemius muscle weight was evaluated. For functional evaluation we used the Sciatic Functional Index (SFI) and the horizontal ladder walking test. Nerves, dorsal root ganglia and spinal cords were processed for light and electron microscopy. Antinoceptive effects of BBG and PPADS were evaluated through von Frey E, and the levels of IL-1ß and TNF-α were analyzed by ELISA. BBG promoted an increase in the number of myelinated fibers and on axon, fiber and myelin areas. BBG and PPADS led to an increase of TNF-α and IL-1ß in the nerve on day 1 and PPADS caused a decrease of IL-1ß on day 7. Mechanical allodynia was reversed on day 7 in the groups treated with BBG and PPADS. We concluded that BBG promoted a better morphological regeneration after ischiatic crush injury, but this was not followed by anticipation of functional improvement. In addition, both PPADS and BBG presented anti-inflammatory as well as antinociceptive effects.


Assuntos
Lesões por Esmagamento/tratamento farmacológico , Regeneração Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/farmacologia , Analgésicos/farmacologia , Animais , Lesões por Esmagamento/metabolismo , Lesões por Esmagamento/patologia , Modelos Animais de Doenças , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Interleucina-1alfa/metabolismo , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Distribuição Aleatória , Receptores Purinérgicos P2X7/metabolismo , Corantes de Rosanilina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Pharmacol Biochem Behav ; 158: 1-6, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28522214

RESUMO

The involvement of purinergic signaling in several brain functions has been recognized, but the modulation on maternal behavior by the purinergic system is not established, even though there are functional interactions between the purinergic and oxytocinergic systems. Therefore, the aim of our study was to investigate whether central administration of P2 receptor antagonists affected the maternal behavior of lactating rats and c-Fos immunoreactivity in the forebrain. On day 7 of lactation, female rats were treated with vehicle (5µL; i.c.v.), suramin (9.4-75.0µg/5µL; i.c.v.) or PPADS (9.4-75.0µg/5µL; i.c.v.) 30min before the experiment began. The maternal behavior was evaluated during the 30min following suramin or PPADS administration. In addition, c-Fos-positive nuclei were counted in the medial preoptic area (MPOA) and in the bed nucleus of the stria terminalis (BNST), and neurons that were double-labeled for c-Fos/OT were counted in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus of lactating rats. The results show that P2 receptor antagonists decreased maternal care and decreased neuronal activation in the MPOA and BNST and activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the purinergic system modulates maternal behavior and neuronal activation induced by suckling during lactation.


Assuntos
Comportamento Animal , Lactação , Antagonistas do Receptor Purinérgico P2/farmacologia , Animais , Feminino , Prosencéfalo/efeitos dos fármacos , Prosencéfalo/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Wistar , Suramina/farmacologia
17.
Bull Exp Biol Med ; 162(5): 606-610, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28361417

RESUMO

We studied the role of purinergic P2X receptors in the body response to cooling. In experiments on rats, P2X receptor antagonist PPADS was administered in different modes, which resulted in changes of different characteristics of the thermoregulatory response to cold. Iontophoresis of P2X antagonist into the skin decreased the thermal thresholds of all thermoregulatory responses to cooling, which can attest to a modulating effect of P2X receptors on peripheral thermosensitive afferents. Intraperitoneal administration of P2X antagonist suppressed thermoregulatory activity of skeletal muscles (shivering) developing during cooling without changing the thresholds of thermoregulatory responses. The findings suggest that ATP and P2X receptors play an important role in the formation of the response to cooling.


Assuntos
Regulação da Temperatura Corporal , Receptores Purinérgicos P2X/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Temperatura Baixa , Resposta ao Choque Frio , Masculino , Consumo de Oxigênio , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos Wistar , Tremor por Sensação de Frio/efeitos dos fármacos , Pele/irrigação sanguínea , Temperatura Cutânea , Vasoconstrição/efeitos dos fármacos
18.
Acta Biol Hung ; 68(1): 22-34, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28322086

RESUMO

To investigate the role of metabotrophic purinergic P2Y receptors in neuroblastoma cell survival, expression of P2 receptors by normal mouse (C57BL/6) brain and human neuroblastoma SH-SY5Y cells was investigated by Western blot and real time PCR studies. Viability of SH-SY5Y cells treated with purinergic receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) was evaluated by MTT assay and flow cytometry. In the brain samples of C57BL/6 mice, expressions of P2Y4 and P2X7 were significantly reduced, whereas that of P2Y1 was significantly elevated in an age-dependent manner. SH-SY5Y cell viability was significantly reduced and necrotic cell rates were mildly increased by 400 µM suramin and 100 µM PPADS treatment. Antagonist treatment downregulated P2Y1, P2Y2 and P2Y4 and upregulated P2Y6, P2Y12 and P2X7 mRNA levels in SH-SY5Y cells on the 24th hour. These alterations were abolished for all P2 receptors except P2Y1 in the 48th hour. P2Y receptors are expressed by both normal mouse brain and human neuroblastoma cells. Purinergic receptor antagonism interferes with neuroblastoma viability through elevation of necrotic cell death and modulation of P2 receptor expression. P2Y receptors might thus be useful targets for future anti-tumor treatment trials.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Receptores Purinérgicos P2/genética , Fatores Etários , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Antagonistas do Receptor Purinérgico P2/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Receptores Purinérgicos P2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suramina/farmacologia
19.
Auris Nasus Larynx ; 44(4): 422-427, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27692399

RESUMO

OBJECTIVE: The present study aimed at investigating ATP release in response to acetylcholine (Ach) and pharmacologically elucidating the intracellular signal transduction pathway of this reaction in an ex vivo experiment. METHODS: The inferior turbinate mucosa was collected from 21 patients with chronic hypertrophic rhinitis who underwent endoscopic turbinectomy. The mucosa was shaped into a filmy round piece, and incubated with chemical(s) in Hank's balanced salt solution for 10min. After incubation, the ATP concentration was measured by a luciferin-luciferase assay. RESULTS: The baseline release of ATP without stimulus was 57.2±10.3fM. The ATP release was significantly increased by stimulation with 100µM Ach. The Ach-induced ATP release was completely inhibited by removing extracellular Ca2+. Significant inhibition of the Ach-induced ATP release was also observed by the addition of 1µM atropine, 40µM 2-APB, 10µM CBX, and 100µM PPADS, whereas 30nM bafilomycin A1 did not affect the ATP release. CONCLUSION: These results indicate that the Ach-induced ATP release from the human nasal mucosa is dependent on the pannexin-1 channel and purinergic P2X7 receptor, suggesting that these two molecules constitute a local autocrine/paracrine signaling system in the human nasal epithelium.


Assuntos
Acetilcolina/farmacologia , Trifosfato de Adenosina/metabolismo , Atropina/farmacologia , Agonistas Colinérgicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Mucosa Nasal/efeitos dos fármacos , Adolescente , Adulto , Idoso , Antiulcerosos/farmacologia , Cálcio/metabolismo , Carbenoxolona/farmacologia , Conexinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Macrolídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Antagonistas do Receptor Purinérgico P2/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Rinite/cirurgia , Transdução de Sinais , Adulto Jovem
20.
Neurosci Res ; 115: 5-12, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27720754

RESUMO

ATP activates P2X receptors and acts as a neurotransmitter in the nervous system. We have previously reported that P2X receptors modulate the firing rate of retinal ganglion cells. Since many subtypes of P2X receptors are distributed in the mouse retina, it is likely that the modulatory effects of P2X receptor-mediated signaling can occur at multiple synaptic levels in the retina. In this study, we investigated whether P2X receptors expressed between the photoreceptor layer and the inner nuclear layer in the mouse retina were physiologically functional, by electroretinography (ERG). In the combined rod-cone ERG and the scotopic ERG, intravitreal injection of PPADS, an antagonist of P2X receptors, had no effects on the amplitude of the a-wave, but decreased the amplitude of the b-wave. In the photopic ERG, intravitreal injection of PPADS significantly decreased the amplitude of both the a-wave and the b-wave. In ex vivo recordings, a decrease in the b-wave amplitude was observed at 20µM PPADS, confirming that the inhibition of the b-wave by intravitreal injection of PPADS is due to the inhibition of P2X receptors. Our findings suggest that P2X receptor-mediated signaling has a physiological effect in both the rod and the cone pathways in postreceptoral processing.


Assuntos
Receptores Purinérgicos P2X/fisiologia , Retina/fisiologia , Animais , Eletrorretinografia , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/fisiologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Retina/efeitos dos fármacos , Transdução de Sinais
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