2--3--dialdehyde-atp and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

Brain ischemia leading to stroke is a major cause of disability in developed countries. Therapeutic strategies have most commonly focused on protecting neurons from ischemic damage. However, ischemic damage to white matter causes oligodendrocyte death, myelin disruption, and axon dysfunction, and it is partially mediated by glutamate excitotoxicity. We have previously demonstrated that oligodendrocytes express ionotropic purinergic receptors. The objective of this study was to investigate the role of purinergic signaling in white matter ischemia. We show that, in addition to glutamate, enhanced ATP signaling during ischemia is also deleterious to oligodendrocytes and myelin, and impairs white matter function. Thus, ischemic oligodendrocytes in culture display an inward current and cytosolic Ca(2+) overload, which is partially mediated by P2X7 receptors. Indeed, oligodendrocytes release ATP after oxygen and glucose deprivation through the opening of pannexin hemichannels. Consistently, ischemia-induced mitochondrial depolarization as well as oxidative stress culminating in cell death are partially reversed by P2X7 receptor antagonists, by the ATP degrading enzyme apyrase and by blockers of pannexin hemichannels. In turn, ischemic damage in isolated optic nerves, which share the properties of brain white matter, is greatly attenuated by all these drugs. Ultrastructural analysis and electrophysiological recordings demonstrated that P2X7 antagonists prevent ischemic damage to oligodendrocytes and myelin, and improved action potential recovery after ischemia. These data indicate that ATP released during ischemia and the subsequent activation of P2X7 receptor is critical to white matter demise during stroke and point to this receptor type as a therapeutic target to limit tissue damage in cerebrovascular diseases.

Topics: Action Potentials; Adenosine Triphosphate; Animals; Animals, Newborn; Axons; Calcium; Cell Death; Connexins; Excitatory Amino Acid Antagonists; Glucose; Hypoxia; L-Lactate Dehydrogenase; Microscopy, Electron, Transmission; Nerve Tissue Proteins; Oligodendroglia; Optic Nerve; Optic Neuropathy, Ischemic; Patch-Clamp Techniques; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Reactive Oxygen Species; Receptors, Purinergic P2; Receptors, Purinergic P2X7

Abstract A gap junction is composed of two hemichannels and possesses a relatively large pore size ( approximately 10-15 A), allowing passage of ions and molecules up to 1 kDa. Here, we report that connexin hemichannels and gap junctions in the guinea pig cochlea had significant charge selectivity among permeating molecules. In coincubation with anionic and cationic fluorescent dyes, hemichannel permeability in isolated cochlear supporting cells showed significant charge selectivity; 31% of cells had only cationic dye influx and 6% of cells had only anionic dye influx. Charge-selective influx contrary to dye size was also found, indicating charge as a dominant determinant in permeability. The cell-cell gap junctional permeability was consistent with hemichannel permeability and also showed strong charge selectivity; the permeation of anionic dyes was slower than that of cationic probes in the cochlear sensory epithelium. With a combination of immunofluorescent staining for connexin26 (Cx26) and Cx30, which are the predominant connexin isoforms in the cochlea, Cx26 was demonstrated to correlate with anionic permeability. The data indicated that cochlear gap junctions have strong charge selectivity in molecular permeability and metabolic communication. Cx26 mutation may induce specific, irreparable impairment in intercellular signalling and energy and nutrient supplies in the cochlea, causing cell degeneration and hearing loss, given that many important cell-signalling and nutrient and energy molecules (e.g. IP3, ATP, cAMP and cGMP) are anions.

Topics: Adenosine Triphosphate; Affinity Labels; Animals; Anions; Anti-Inflammatory Agents; Calcium; Cations; Cell Communication; Cell Count; Cell Membrane Permeability; Cells, Cultured; Cochlea; Connexin 26; Connexins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorescent Dyes; Gap Junctions; Glycyrrhetinic Acid; Guinea Pigs; Intracellular Membranes; Platelet Aggregation Inhibitors; Proadifen; Pyridoxal Phosphate; Spectrometry, Fluorescence; Time Factors

We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes.. Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3x as much ATP into the supernatants compared with fibroblasts from healthy subjects.. We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate; Adenosine Triphosphate; Apoptosis; Apyrase; Autocrine Communication; Cell Shape; Cytidine Triphosphate; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Interleukin-6; Membrane Potentials; Paracrine Communication; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Uridine Diphosphate; Uridine Triphosphate

Secondary injury exacerbates the extent of spinal cord insults, yet the mechanistic basis of this phenomenon has largely been unexplored. Here we report that broad regions of the peritraumatic zone are characterized by a sustained process of pathologic, high ATP release. Spinal cord neurons expressed P2X7 purine receptors (P2X7R), and exposure to ATP led to high-frequency spiking, irreversible increases in cytosolic calcium and cell death. To assess the potential effect of P2X7R blockade in ameliorating acute spinal cord injury (SCI), we delivered P2X7R antagonists OxATP or PPADS to rats after acute impact injury. We found that both OxATP and PPADS significantly improved functional recovery and diminished cell death in the peritraumatic zone. These observations demonstrate that SCI is associated with prolonged purinergic receptor activation, which results in excitotoxicity-based neuronal degeneration. P2X7R antagonists inhibit this process, reducing both the histological extent and functional sequelae of acute SCI.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Benzoxazines; Disease Models, Animal; Electrophysiology; Female; Immunohistochemistry; In Situ Nick-End Labeling; Luminescent Measurements; Microscopy, Confocal; Neurons; Oxazines; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X7; Spinal Cord Injuries

ATP activates the mouse P2X7 receptor and induces a nonselective-cation current in NG108-15 cells. We investigated the effects of five receptor antagonists on the ATP-induced nonselective-cation current through P2X7 receptor (I(NS.P2X7)) in NG108-15 cells. Nonselective P2 receptor antagonists, RB-2, PPADS and suramin inhibited the I(NS.P2X7) with IC50 values of 4.3, 53 and 40 microM, respectively. However, KN-04, which is a potent antagonist of human P2X7 receptors but is not that of rat P2X7 receptors, had only a weak blocking effect. Furthermore, oxidized-ATP (300 microM), an antagonist of the P2X7 receptor-mediated pore-formation, did not affect the I(NS.P2X7). Prolonged ATP application did not increase the membrane permeability to large molecules, N-methyl-D-glucamine or Yo-Pro-1, indicating that pore-formation was not promoted by the P2X7 receptor activation in NG108-15 cells. These results suggest that antagonist sensitivities and pore-forming properties of the P2X7 receptors in NG108-15 cells are different from those of other cells types.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Animals; Benzoxazoles; Cations; Cell Membrane Permeability; Fluorescent Dyes; Mice; Patch-Clamp Techniques; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinolinium Compounds; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Suramin; Triazines; Tumor Cells, Cultured

We have examined the interaction of P2 antagonists with the human P2X(7) receptor by studying their effect on 2' and 3'-O-benzoyl-benzoyl-ATP (DbATP) stimulated cellular accumulation of the fluorescent, DNA binding dye, YO-PRO-1 (MW=375Da). In suspensions of HEK293 cells expressing human recombinant P2X(7) receptors, DbATP produced time and concentration-dependent increases in YO-PRO-1 fluorescence. This response presumably reflects YO-PRO-1 entry through P2X(7) receptor channels and binding to nucleic acids. When studies were performed in a NaCl-free, sucrose-containing buffer, full concentration-effect curves to DbATP could be constructed. The P2 antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and periodate oxidized ATP (oATP), reduced the potency of DbATP and decreased its maximum response. 1-[N,O-bis(1, 5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62) and its analogue, KN04, reduced the potency of DbATP. Schild slopes for KN62 and KN04 were shallow and exhibited a plateau at concentrations of compound greater than 1 microM, indicating that these compounds were not competitive antagonists. Calmidazolium and a monoclonal antibody to human P2X(7) receptors attenuated DbATP-stimulated YO-PRO-1 accumulation but they were not competitive antagonists and only produced 2 - 3 fold decreases in the potency of DbATP. The effects of PPADS and KN62 were partially reversible whereas those of oATP were not. PPADS protected cells against the irreversible antagonist effects of oATP suggesting a common site of action. In contrast KN62 was not effective suggesting that it may bind at a different site to oATP and PPADS. This study has demonstrated that P2X(7) receptor function can be quantified by measuring DbATP stimulated YO-PRO-1 accumulation and has provided additional information about the interaction of P2 receptor antagonists with the human P2X(7) receptor.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Antibodies, Blocking; Benzoxazoles; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Enzyme Inhibitors; Fluorescent Dyes; Humans; Imidazoles; In Vitro Techniques; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinolinium Compounds; Receptors, Purinergic P2X7; Stimulation, Chemical

Signaling by extracellular nucleotides through P2 purinergic receptors affects diverse macrophage functions; however, its role in regulating antimicrobial radicals during bacterial infection has not been investigated. Mycobacterium tuberculosis-infected macrophages released ATP in a dose-dependent manner, which correlated with nitrite accumulation. P2 receptor inhibitors, including oxidized ATP, blocked NO synthase (NOSII) up-regulation and NO production induced by infection with M. tuberculosis or bacille Calmette-Guérin, or treatment with LPS or TNF-alpha. Oxidized ATP also inhibited oxygen radical production and activation of NF-kappaB and AP-1 in response to infection and inhibited NO-dependent killing of bacille Calmette-Guérin by macrophages. Experiments using macrophages derived from P2X7 gene-disrupted mice ruled out an essential role for P2X7 in NOSII regulation. These data demonstrate that P2 receptors regulate macrophage activation in response to bacteria and proinflammatory stimuli, and suggest that extracellular nucleotides released from infected macrophages may enhance production of oxygen radicals and NO at sites of infection.

Topics: Adenosine Triphosphate; Animals; Cell Line; Free Radicals; Macrophage Activation; Macrophages; Mice; Mycobacterium bovis; Mycobacterium tuberculosis; Nitric Oxide; Nitric Oxide Synthase; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Reactive Oxygen Species; Receptors, Purinergic P2; Receptors, Purinergic P2X7; RNA, Messenger; Signal Transduction; Suramin

1. We have utilized the human monocytic cell line, THP-1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate-6-azophenyl-2',4'-disuphonic acid (PPADS), oxidized ATP, and 1-(N, O-bis[5-isoquinolinesufonyll]-N-methyl-L-tyrosyl)-4-phenylpiper azi ne (KN-62) to pharmacologically characterize the P2 receptor involved in ATP-induced release of interleukin 1beta (IL-1beta). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)-induced IL-1beta release from both cell types. 2. ATP caused release of IL-1beta from LPS primed THP-1 cells in both a time- and concentration-dependent manner, with a minimal effective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL-1beta in cell supernatants within 30 min. 3. The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X7 receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL-1beta release. 4. KN-62 (1 micro M), PPADS (100 microM) or oxidized ATP (100 uM) significantly inhibited 5 mM ATP-induced IL-1beta release by 81, 90 and 66% respectively, but failed to significantly inhibit LPS-induced IL-1beta release in both THP-1 cells and in freshly isolated human monocytes. 5. In both THP-1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml(-1)) to cell supernatants prior to LPS activation failed to significantly inhibit the LPS-induced IL-1beta release. In addition there was no correlation between extracellular ATP concentrations and IL-1beta release in THP-1 cells when studied over a 6 h time period. 6. In conclusion our data confirm the involvement of P2X7 receptors in ATP-induced IL-1beta release in human monocytes. However no evidence was obtained which would support the involvement of either endogenous ATP release or P2X7 receptor activation as the mechanism by which LPS-induces IL-1beta release in either the THP-1 cell line or in freshly isolated human monocytes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Cell Line; Enzyme Inhibitors; Humans; Interleukin-1; L-Lactate Dehydrogenase; Lipopolysaccharides; Monocytes; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7