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1.
Pflugers Arch ; 463(4): 585-92, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22311122

RESUMO

Pannexin 1 (Panx1) is expressed in various mammalian tissues including the brain and immune cells. Here, we present evidence that Panx1 when expressed in mammalian cells, forms anion-selective channels, with a rank order of permeabilities: NO (3) (-)> I(-) > Br (-)> Cl (-) > F (-)>> aspartate (-)≈ glutamate (-)≈ gluconate(-). Single-channel Panx1-mediated currents have a unitary conductance around 68 pS. Our results show that Panx1 assembles into a membrane anion channel with a relatively low single-channel conductance.


Assuntos
Ânions/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Ânion Dependentes de Voltagem/metabolismo , Ácido Aspártico/metabolismo , Linhagem Celular , Cloretos/metabolismo , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Canais Iônicos/metabolismo
2.
J Immunol ; 185(4): 2611-9, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20639492

RESUMO

The ATP-gated P2X(7) receptor (P2X(7)R) is a promising therapeutic target in chronic inflammatory diseases with highly specific antagonists currently under clinical trials for rheumatoid arthritis. Anti-inflammatory actions of P2X(7)R antagonists are considered to result from inhibition of P2X(7)R-induced release of proinflammatory cytokines from activated macrophages. However, P2X(7)Rs are also expressed in resting macrophages, suggesting that P2X(7)R may also signal via cytokine-independent mechanisms involved in joint disease. In this study, we examined P2X(7)R function in resting human lung macrophages and mouse bone marrow-derived macrophages and found that ATP induced rapid release of the lysosomal cysteine proteases cathepsin B, K, L, and S and that was independent of the presence of the proinflammatory cytokines IL-1beta and IL-18. Cathepsins released into the medium were effective to degrade collagen extracellular matrix. ATP-induced cathepsin release was abolished by P2X(7)R antagonists, absent from P2X(7)R(-/-) mouse macrophages, and not associated with cell death. Our results suggest P2X(7)R activation may play a novel and direct role in tissue damage through release of cathepsins independently of its proinflammatory actions via IL-1 cytokines.


Assuntos
Catepsinas/metabolismo , Citocinas/metabolismo , Artropatias/metabolismo , Macrófagos/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Western Blotting , Catepsina B/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Etanercepte , Humanos , Imunoglobulina G/farmacologia , Imunossupressores/farmacologia , Interleucina-1beta/metabolismo , Artropatias/genética , Células L , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Metotrexato/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Molecular , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Receptores do Fator de Necrose Tumoral , Tiazóis/química , Tiazóis/farmacologia
3.
J Biol Chem ; 285(23): 17514-24, 2010 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-20378545

RESUMO

P2X(7) receptors (P2X(7)R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X(7)Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X(7)R resulted from a Ca(2+)-calmodulin-dependent process and a distinct calcium-independent process. However, P2X(7)Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X(7)R as well as rat/human, human/rat chimeric, and mutated P2X(7)Rs. Facilitation at the human P2X(7)R was 5-fold slower than at the rat P2X(7)R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X(7)R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X(7)R. Replacement of three critical residues of this binding domain from the rat into the human P2X(7)R (T541I, C552S, and G559V) reconstituted the Ca(2+)-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X(7)Rs. The absence of Ca(2+)-dependent facilitation at the human P2X(7)R may represent a protective adaptation of the innate immune response in which P2X(7)R plays significant roles.


Assuntos
Calmodulina/química , Receptores Purinérgicos P2/química , Motivos de Aminoácidos , Compostos de Anilina/farmacologia , Animais , Cálcio/química , Membrana Celular/metabolismo , Citosol/metabolismo , Eletrofisiologia/métodos , Humanos , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores Purinérgicos P2X7 , Xantenos/farmacologia
4.
J Psychiatr Res ; 44(6): 347-55, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19931869

RESUMO

Genetic linkage studies have previously identified many single non-synonymous nucleotide polymorphisms (SNPs) in the human P2RX7 gene in individuals with affective mood disorders. The P2RX7 gene encodes the P2X(7) receptor (P2X(7)R) that operates as an ATP-activated Ca(2+)-permeable cationic channel and induces formation of a large pore, the two functional properties that are critical for the physiological and pathological roles of the receptor. The current knowledge regarding the effects of SNPs on the P2X(7)R functional properties, which is indispensable to help elucidate the disease mechanism, is limited. In this study, we introduced by site-directed mutagenesis twelve SNP mutations in the human P2X(7) receptor that were previously identified in or associated with affective mood disorders, expressed the resultant mutants in human embryonic kidney cells, and characterized their functional properties by electrophysiology. All mutations except Q460R gave rise to profound effects on the P2X(7)R function. G150R, E186K and I568N conferred complete loss of function. V76A, R117W, L191P, T357S and E496A resulted in strong impairment of, whereas H155Y and A348T caused significant increase in, both ATP-activated ion channel function and pore formation. Q521H reduced the receptor's sensitivity to extracellular Ca(2+) inhibition. An atomic structure model of the human P2X(7)R, based on the crystal structure of the zebrafish P2X(4) receptor, suggests that the SNP mutational effects may result from changes in subunit interaction, agonist binding and/or channel gating. These results provide essential knowledge for a better understanding of the relationships between human P2RX7 SNPs and associated pathologies as well as the receptor structure-function relationships.


Assuntos
Trifosfato de Adenosina/metabolismo , Canais de Cálcio/metabolismo , Mutação , Polimorfismo de Nucleotídeo Único , Porinas/metabolismo , Receptores Purinérgicos P2/genética , Western Blotting , Linhagem Celular , Eletrofisiologia/métodos , Humanos , Rim/citologia , Rim/embriologia , Mutagênese Sítio-Dirigida/métodos , Técnicas de Patch-Clamp , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7
5.
Nat Nanotechnol ; 4(12): 876-83, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19893513

RESUMO

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.


Assuntos
Dano ao DNA , Nanopartículas/toxicidade , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Cromo/toxicidade , Cobalto/toxicidade , Conexinas/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Transferrina/metabolismo
6.
EMBO J ; 28(14): 2114-27, 2009 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-19536133

RESUMO

In acute inflammation, extracellular ATP activates P2X(7) ion channel receptors (P2X(7)R) on M1 polarized macrophages to release pro-inflammatory IL-1beta through activation of the caspase-1/nucleotide-binding domain and leucine-rich repeat receptor containing pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X(7)R on these macrophages nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL-1beta remains high and the inflammasome is intact, P2X(7)R activation selectively uncouples to the NLRP3-inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts through its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1beta release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments.


Assuntos
Trifosfato de Adenosina/metabolismo , Polaridade Celular , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Animais , Proteínas de Transporte/metabolismo , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Receptores Purinérgicos P2 , Receptores Purinérgicos P2X7
7.
Eur J Immunol ; 39(4): 986-95, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19283779

RESUMO

ATP-gated P2X(4) receptors (P2X(4)R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X(4)R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X(4)R protein expression by Western blot and biotinylation assays and functional expression by whole-cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X(4)R expression by 2- to 7-fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN-gamma and TNF-alpha or IFN-gamma and LPS reduced surface and functional P2X(4)R expression by 3-fold without altering total P2X(4)R protein levels. Alternative activation with IL-4 or IL-13 did not alter total, surface or functional expression of P2X(4)R. This is the first study of the regulation of P2X(4)R in macrophages by physiological stimuli and presents a picture whereby P2X(4)R become functional in response to initial phagocytic stimuli but return to a non-functional state during sustained activation by classical macrophage activation.


Assuntos
Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Fagocitose/imunologia , Receptores Purinérgicos P2/metabolismo , Animais , Linhagem Celular , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos , Antagonistas do Receptor Purinérgico P2 , Ratos , Receptores Purinérgicos P2/imunologia , Receptores Purinérgicos P2X4 , Fator de Necrose Tumoral alfa/farmacologia
8.
Purinergic Signal ; 5(2): 129-37, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19212823

RESUMO

The P2X(7) receptor (P2X(7)R) is uniquely associated with two distinct cellular responses: activation of a dye-permeable pathway allowing passage of molecules up to 900 Da and rapid release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta), from activated macrophage. How this dye uptake path forms and whether it is involved in IL-1beta release has not been known. Pannexin-1 is a recently identified protein found to physically associate with the P2X(7)R. Inhibition of pannexin-1 does not alter P2X(7)R ion channel activation or associated calcium flux but blocks one component of P2X(7)R-induced dye uptake and unmasks a slower, previously undetected, dye uptake pathway. Inhibition of pannexin-1 blocks P2X(7)R-mediated IL-1beta release from macrophage as well as release mediated by other stimuli which couple to activation of capase-1 and additionally inhibits the release of interleukin-1alpha, a member of the IL-1 family whose processing does not require caspase-1 activation. Thus, pannexin-1 is linked to both dye uptake and IL-1beta release but via distinct mechanisms.

9.
J Pharmacol Exp Ther ; 328(2): 409-18, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19023039

RESUMO

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone > disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) approximately disodium 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate approximately 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X(7) receptor (P2X(7)R), DIDS was found to act as a P2X(7)R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X(7)R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X(7)R activation.


Assuntos
Fenômenos Bioquímicos/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Conexinas/fisiologia , Potenciais da Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Splicing de RNA/genética , Trifosfato de Adenosina/farmacologia , Animais , Fenômenos Bioquímicos/efeitos dos fármacos , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/metabolismo , Conexinas/efeitos dos fármacos , Conexinas/genética , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Células Tumorais Cultivadas
10.
Annu Rev Physiol ; 71: 333-59, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-18851707

RESUMO

P2X receptors are membrane cation channels gated by extracellular ATP. Seven P2X receptor subunits (P2X(1-7)) are widely distributed in excitable and nonexcitable cells of vertebrates. They play key roles in inter alia afferent signaling (including pain), regulation of renal blood flow, vascular endothelium, and inflammatory responses. We summarize the evidence for these and other roles, emphasizing experimental work with selective receptor antagonists or with knockout mice. The receptors are trimeric membrane proteins: Studies of the biophysical properties of mutated subunits expressed in heterologous cells have indicated parts of the subunits involved in ATP binding, ion permeation (including calcium permeability), and membrane trafficking. We review our current understanding of the molecular properties of P2X receptors, including how this understanding is informed by the identification of distantly related P2X receptors in simple eukaryotes.


Assuntos
Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/fisiologia , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/fisiologia , Humanos , Canais Iônicos/fisiologia , Camundongos , Dados de Sequência Molecular , Receptores Purinérgicos P2/análise , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X
11.
J Neurosci ; 28(25): 6393-401, 2008 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-18562610

RESUMO

The ATP-gated P2X(7) receptor (P2X(7)R) is a highly unusual calcium-permeable cationic channel in that within seconds of its activation, dramatic and reversible cytoskeletal rearrangements with prominent membrane blebbing occurs. Agonist-induced membrane currents at hyperpolarized potentials show pronounced facilitation during the initial 30-100 s of receptor activation but mechanisms responsible have not been elucidated. We measured facilitation of ATP-gated currents in HEK cells expressing rat P2X(7)R and delineated distinct calcium-dependent and independent processes. The calcium-dependent facilitation was composed of an instantaneous (millisecond time domain) and slowly developing (time constant, 20 s with maximum agonist stimulation) component. Both components were prevented when recording with a highly specific calmodulin (CaM) inhibitory peptide but only the instantaneous component was reduced by expression of the dominant-negative EF-handless CaM mutant. Coimmunoprecipitation assays detected low levels of CaM binding to unstimulated P2X(7)R, and this increased by 50% during 45 s stimulation of the receptor. We identified a novel 1-5-16 Ca(2+)-dependent CaM binding motif in the intracellular C terminus of P2X(7)R; mutations in this domain resulted in the absence of calcium-dependent facilitation and binding of CaM to unstimulated or stimulated receptor. Blockade of CaM binding also delayed membrane blebbing by threefold. Our results demonstrate that CaM binds constitutively to closed P2X(7)R channels and dynamically during channel activation to significantly enhance and prolong calcium entry. This is the first example of CaM deregulating, rather than tightly controlling, calcium entry through an ion channel.


Assuntos
Calmodulina/metabolismo , Membrana Celular/metabolismo , Receptores Purinérgicos P2/metabolismo , Sequência de Aminoácidos , Animais , Calmodulina/genética , Linhagem Celular , Membrana Celular/genética , Humanos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Ligação Proteica/genética , Ligação Proteica/fisiologia , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7
12.
J Immunol ; 180(11): 7147-57, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18490713

RESUMO

The proinflammatory IL-1 cytokines IL-1alpha, IL-1beta, and IL-18 are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X(7) receptors (P2X(7)R) by extracellular ATP is a key physiological inducer of rapid IL-1beta release from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X(7)R(-/-) mice and found that release of IL-1alpha, IL-18, as well as IL-1beta, by ATP resulted exclusively from activation of P2X(7)R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1beta release. We compared IL-1-release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage, and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL-1beta independently of panx1 but do not release mature IL-1beta because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1beta that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1beta that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1beta.


Assuntos
Caspase 1/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Morte Celular , Linhagem Celular , Conexinas/imunologia , Conexinas/metabolismo , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Interleucina-1alfa/imunologia , Interleucina-1beta/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Purinérgicos P2X7
13.
Biochem Pharmacol ; 75(5): 1198-208, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18076867

RESUMO

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.


Assuntos
Receptores Purinérgicos P2/metabolismo , Ligação Competitiva , Linhagem Celular , Humanos , Cinética , Agonistas do Receptor Purinérgico P2 , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X3 , Termodinâmica
14.
Mol Pharmacol ; 73(1): 252-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17959713

RESUMO

P2X(7) receptors are distinct from other ATP-gated P2X receptors in that they are potently inhibited by submicromolar concentrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X(7) receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X(7) receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His(62) or Asp(197) strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodomain residues of the P2X(7) receptor, primarily involving combined interaction with His(62) and Asp(197) residues.


Assuntos
Cobre/farmacologia , Antagonistas do Receptor Purinérgico P2 , Zinco/farmacologia , Linhagem Celular , Humanos , Receptores Purinérgicos P2X7
15.
J Immunol ; 179(12): 8544-53, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18056402

RESUMO

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.


Assuntos
Trifosfato de Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/análogos & derivados , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Neutrófilos/imunologia , Antagonistas do Receptor Purinérgico P2 , Suramina/análogos & derivados , Suramina/farmacologia
16.
J Immunol ; 179(9): 6016-23, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17947675

RESUMO

Macrophages play a key role in inflammation by synthesis and release of proinflammatory cytokines and chemokines. Extracellular nucleotides released at sites of tissue damage may be an early danger signal for immune cells, and ATP-gated P2X(7) receptors are well known to mediate the rapid release of proinflammatory IL-18 and IL-1beta. However, there is little direct evidence for the involvement of other purine receptor subtypes in the release of other cytokines or chemokines. We initially used protein arrays to address whether extracellular ATP can release cytokines and/or chemokines from rat NR8383 alveolar macrophage, which lack the P2X(7) receptor. ATPgammaS increased the release of the proinflammatory chemokine, MCP-1 (MCP-1/CCL2). Pharmacological profiling identified the receptor responsible as the P2Y(2) receptor. Brief activation (10 min) of P2Y(2) receptors increased MCP-1 mRNA levels within 30 min and increased its release at 60 min. Similar results were obtained from rat peritoneal macrophages. We investigated likely downstream signaling cascades that may be involved, specifically the canonical G(q)-mediated phospholipase C (PLC) and subsequent MAP kinase pathways, and G(i)/G(o)-mediated signaling. We could find no evidence for these pathways being involved in the P2Y(2)R-induced increase in mRNA levels although inhibition of PLC blocked the UTP-induced increased release of MCP-1. Thus, the PLC-activated pathway can account for the increased release of MCP-1, but a novel signaling pathway may be involved in the increase in MCP-1 mRNA by activation of P2Y(2) receptors in alveolar and peritoneal macrophage.


Assuntos
Quimiocina CCL2/biossíntese , Quimiocina CCL2/metabolismo , Dentição , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneais/metabolismo , Receptores Purinérgicos P2/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Quimiocina CCL2/genética , Interleucina-1beta/metabolismo , Cinética , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2Y2 , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
17.
Mol Immunol ; 44(6): 1286-99, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16837047

RESUMO

Mammalian interleukin-1beta (IL-1beta) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1beta-converting enzyme (ICE) after the activation of P2X(7) receptor by extracellular ATP. The mechanism of IL-1beta release in non-mammalian vertebrates is largely unknown, although most of the IL-1beta gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X(7) receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X(7) receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X(7) receptors. We used this information to further investigate the mechanisms of IL-1beta release induced by mammalian and fish P2X(7) receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1beta remained unprocessed within the cell. However, activation of rat P2X(7) receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1beta. In contrast, neither seabream nor zebrafish P2X(7) receptors induced the secretion of mammalian or fish IL-1beta when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X(7) and the intracellular region of its rat counterpart did so. These findings indicate that P2X(7) receptor-mediated activation of ICE and release of IL-1beta result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1beta secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.


Assuntos
Trifosfato de Adenosina/fisiologia , Interleucina-1beta/metabolismo , Ativação do Canal Iônico/imunologia , Sinais Direcionadores de Proteínas/fisiologia , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiologia , Dourada/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Interleucina-1beta/química , Dados de Sequência Molecular , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Xenopus , Peixe-Zebra
18.
Mol Pharmacol ; 71(1): 92-100, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17032903

RESUMO

Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Asparagina , Sequência Conservada , Humanos , Cinética , Lisina , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Receptores Purinérgicos P2/efeitos dos fármacos , Receptores Purinérgicos P2X7 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transfecção
20.
J Biol Chem ; 282(4): 2386-94, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17121814

RESUMO

Pannexin-1 is a recently identified membrane protein that can act as a nonselective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X7 receptor (P2X7R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1beta (IL-1beta) in response to P2X7R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X7R to provide the conduit for dye uptake and downstream signaling to processing and release of IL-1beta. We have pursued this hypothesis by measuring dye uptake and IL-1beta processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1beta release via the same mechanism. The experiments were carried out over time periods during which no lactate dehydrogenase was released from cells to examine only noncytolytic pathways. P2X7R activation evoked dye uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1-independent phase. Maitotoxin-evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1beta processing and release in response to all three stimuli. Thus, although pannexin-1 is required for IL-1beta release in response to maitotoxin, nigericin, and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.


Assuntos
Interleucina-1beta/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Animais , Morte Celular/fisiologia , Linhagem Celular , Conexinas , Humanos , Toxinas Marinhas/farmacologia , Camundongos , Nigericina/farmacologia , Oxocinas/farmacologia , Ligação Proteica , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Transdução de Sinais/efeitos dos fármacos
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