yo-pro-1 and Disease-Models--Animal

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.

Topics: Adenosine Triphosphate; Animals; Benzoxazoles; Calcium; Carbenoxolone; Cells, Cultured; Chronic Pain; Cohort Studies; Connexins; Disease Models, Animal; Enzyme Inhibitors; Female; Genetic Linkage; Genome-Wide Association Study; Genotype; Histidine; Humans; Hyperalgesia; Macrophages; Male; Mastectomy; Mice; Mice, Inbred Strains; Mutation; Nerve Tissue Proteins; Osteoarthritis; Pain Measurement; Pain Threshold; Peptides; Polymorphism, Single Nucleotide; Quinolinium Compounds; Receptors, Purinergic P2X7; Retrospective Studies; Species Specificity; Time Factors; Transfection

Astrocytes are responsible for clearance of extracellular glutamate, primarily through glial-specific glutamate transporter-1 and the Na(+)-dependent glutamate/aspartate transporter (GLAST). After traumatic injury to the CNS, such as spinal cord injury, persistent release of ATP from damaged neurons and activated glial cells occurs, inducing detrimental and/or beneficial effects via activation of ionotropic (P2XR) and metabotropic purinergic receptors. In this study, we show a decrease in GLAST mRNA in the lesion center and caudal portions at 24 h post-spinal cord injury. In an in vitro system, the ability of astrocytes to take up glutamate and astrocytic GLAST mRNA levels were significantly decreased after exposure to ATP and its P2X(7)R agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP. ATP- or 2'-3'-O-(4-benzoylbenzoyl)-ATP-induced inhibitory effect on GLAST mRNA expression was blocked by the irreversible P2X(7)R blocker, oxidized ATP, or when P2X(7)R mRNA expression was reduced by the lentivirus-short hairpin RNA knockdown approach. Furthermore, deletion of the GLAST promoter and RNA decay assays showed that P2X(7)R signaling triggered post-transcriptional regulation of GLAST expression via the phosphoinositide 3-kinase cascade. The signaling pathway participating in the P2X(7)R effect on GLAST mRNA expression was identified as a Ca(2+)-dependent phosphoinositide 3-kinase-phospholipase Cgamma involving the inositol 1,4,5-trisphosphate receptor, calcium/calmodulin-dependent kinase II, and protein kinase C. We conclude that P2X(7)R activation by sustained release of ATP in the injured CNS may decrease GLAST mRNA stability via Ca(2+)-dependent signaling, suggesting that inhibition of P2X(7)R may allow for recovery of astrocytic GLAST function and protect neurons from glutamate-induced excitotoxicity.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Benzoxazoles; Calcium; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Transporter 1; Female; Gene Expression Regulation; Glutamic Acid; Humans; Injections, Spinal; Neurons; Phosphatidylinositol 3-Kinases; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinolinium Compounds; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X7; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Spinal Cord Injuries; Transfection

Recent evidence suggests that the P2X(7) receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X(7) antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X(7)-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC(50) values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X(7) receptor, with IC(50) values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L(5) spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X(7) receptors do play a role in animal models of pain and inflammation. Further study of P2X(7) antagonists both in preclinical and clinical studies will help elucidate the role of the P2X(7) receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

Topics: Adamantane; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis; Benzamides; Benzoxazoles; Calcium; Disease Models, Animal; Humans; Inflammation; Inhibitory Concentration 50; Pain; Purinergic P2 Receptor Antagonists; Quinolinium Compounds; Rats; Receptors, Purinergic P2X7