RESUMO
Sulforaphane (SFN), a bioactive phytochemical isothiocyanate, has a wide spectrum of cytoprotective effects that involve induction of antioxidant genes. Nongenomic antioxidant effects of SFN have not been investigated. Brain oxidative stress during inflammation and excitotoxicity leads to neurovascular injury. We tested the hypothesis that SNF exhibits acute antioxidant effects and prevents neurovascular injury during oxidative stress. In primary cultures of cerebral microvascular endothelial cells (CMVEC) and cortical astrocytes from the newborn pig brain, a pro-inflammatory cytokine TNF-α and an excitotoxic glutamate elevate reactive oxygen species (ROS) and cause cell death by apoptosis. Nox4 NADPH oxidase is the main Nox isoform in CMVEC and cortical astrocytes that is acutely activated by TNF-α and glutamate leading to ROS-mediated cell death by apoptosis. The Nox4 inhibitor GKT137831 blocked NADPH oxidase activity and overall ROS elevation, and prevented apoptosis of CMVEC and astrocytes exposed to TNF-α and glutamate, supporting the leading role of Nox4 in the neurovascular injury. Synthetic SFN (10-11 -10-6 mol/L) inhibited NADPH oxidase activity and reduced overall ROS production in CMVEC and astrocytes within 1-hour exposure to TNF-α and glutamate. Furthermore, in the presence of SFN, the ability of TNF-α and glutamate to produce apoptosis in CMVEC and cortical astrocytes was completely prevented. Overall, SFN at low concentrations exhibits antioxidant and antiapoptotic effects in cerebral endothelial cells and cortical astrocytes via a via a nongenomic mechanism that involves inhibition of Nox4 NADPH oxidase activity. SFN may prevent cerebrovascular injury during brain oxidative stress caused by inflammation and glutamate excitotoxicity.
Assuntos
Antioxidantes/farmacologia , Inflamação/tratamento farmacológico , Isotiocianatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Sulfóxidos/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Humanos , Inflamação/patologia , Masculino , Espécies Reativas de Oxigênio/metabolismo , SuínosRESUMO
ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.
Assuntos
Difosfato de Adenosina/metabolismo , Canais Iônicos/metabolismo , Neurônios/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Canais de Cálcio Tipo N/metabolismo , Linhagem Celular , Humanos , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Técnicas de Patch-ClampRESUMO
BACKGROUND AND PURPOSE: Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study. EXPERIMENTAL APPROACH: Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K(IR)3 and K(V)7 channels and α3ß4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na(+), Ca(2+), inward rectifier K(+), K(V)7 K(+), and TRPV1 channels, as well as GABA(A), glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons. KEY RESULTS: Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na(+) or Ca(2+) channels, inward rectifier K(+) channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K(V)7 K(+) channels to more negative potentials and the gating of GABA(A) receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K(V)7 channels and GABA(A) receptors was comparable, whereas in hippocampal neurons the effects on K(V)7 channels were more pronounced. CONCLUSIONS AND IMPLICATIONS: These results indicate that flupirtine exerts its analgesic action by acting on both GABA(A) receptors and K(V)7 channels.
Assuntos
Aminopiridinas/farmacologia , Analgésicos não Narcóticos/farmacologia , Canais de Potássio KCNQ/fisiologia , Receptores de GABA-A/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Gânglios Espinais/citologia , Hipocampo/citologia , Humanos , Células do Corno Posterior/fisiologia , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/citologiaRESUMO
BACKGROUND AND PURPOSE: P2Y(1) , P2Y(2) , P2Y(4) , P2Y(12) and P2Y(13) receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors is not available. The search for such receptors was the purpose of this study. EXPERIMENTAL APPROACH: In primary cultures of rat superior cervical ganglion neurons and in PC12 cell cultures, currents were recorded via the perforated patch clamp technique, and the release of [(3) H]-noradrenaline was determined. KEY RESULTS: ADP, 2-methylthio-ATP and ATP enhanced stimulation-evoked (3) H overflow from superior cervical ganglion neurons, treated with pertussis toxin to prevent the signalling of inhibitory G proteins. This effect was abolished by P2Y(1) antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca(2+) stores. ADP and a specific P2Y(1) agonist caused inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, (3) H overflow was also enhanced by a specific P2Y(1) agonist and by ADP, but only when the P2Y(12) receptors were blocked. ADP also enhanced K(+) -evoked (3) H overflow from PC12 cells treated with pertussis toxin, but only in a clone expressing recombinant P2Y(1) receptors. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that presynaptic P2Y(1) receptors mediate facilitation of transmitter release from sympathetic neurons most likely through inhibition of Kv7 channels.
Assuntos
Neurônios/metabolismo , Neurotransmissores/metabolismo , Norepinefrina/metabolismo , Receptores Pré-Sinápticos/fisiologia , Receptores Purinérgicos P2Y1/fisiologia , Gânglio Cervical Superior/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células , Clonagem Molecular , Estimulação Elétrica , Proteínas de Fluorescência Verde/genética , Canais de Potássio KCNQ/antagonistas & inibidores , Dose Máxima Tolerável , Neurônios/efeitos dos fármacos , Células PC12 , Técnicas de Patch-Clamp , Toxina Pertussis/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Pré-Sinápticos/genética , Receptores Pré-Sinápticos/metabolismo , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2Y1/metabolismo , Gânglio Cervical Superior/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Tionucleotídeos/farmacologiaRESUMO
Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K(V)7 K(+) and Ca(V)2 Ca(2+) channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y(1) receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca(2+) were investigated. At a membrane potential of 30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 µm. These currents had reversal potentials close to the K(+) equilibrium potential and changed direction when extracellular Na(+) was largely replaced by K(+), but remained unaltered when extracellular Cl() was changed. Currents were abolished by P2Y(1) antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca(2+), and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca(2+) stores were depleted. Blockers of K(Ca)2, but not those of K(Ca)1.1 or K(Ca)3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at 30 mV which were attenuated by P2Y(1) antagonists, depletion of Ca(2+) stores, or a blocker of K(Ca)2 channels. These results demonstrate that activation of neuronal P2Y(1) receptors may gate Ca(2+)-dependent K(+) (K(Ca)2) channels via phospholipase C-dependent increases in intracellular Ca(2+) and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y(1) receptors.
Assuntos
Neurônios/fisiologia , Canais de Potássio Cálcio-Ativados/fisiologia , Receptores Purinérgicos P2Y1/fisiologia , Difosfato de Adenosina/farmacologia , Animais , Axônios/fisiologia , Fenômenos Eletrofisiológicos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Neurônios/efeitos dos fármacos , Células PC12 , Técnicas de Patch-Clamp , Potássio/farmacologia , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/fisiologia , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Fosfolipases Tipo C/fisiologiaRESUMO
Most cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y(1), P2Y(2), P2Y(12), P2Y(13), P2X(2), A(1), A(2A) receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y(1), P2Y(12), P2Y(13), A(1), A(2A), and P2X(2) receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y(1)/P2Y(1), A(2A)/P2Y(1), A(2A)/P2Y(12)) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network.