Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain.

The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1.

Functional regulation of P2X6 receptors by N-linked glycosylation: identification of a novel alpha beta-methylene ATP-sensitive phenotype.

Investigation of rat recombinant P2X(6) receptors has been limited because of the difficulty in obtaining functional expression in heterologous systems. In this study, we demonstrate glycosylation-dependent regulation of recombinant P2X(6) receptor function and associated conferral of a novel phenotype that is sensitive to the P2X(1) and P2X(3) receptor agonist, alphabeta-methylene ATP. In cells functionally expressing P2X(6) receptors, ATP and alphabeta-methylene ATP evoked slowly desensitizing inward currents (EC(50) values, 0.

Cyclooxygenase-2-selective nonsteroidal anti-inflammatory drugs inhibit hepatocyte growth factor/scatter factor-induced angiogenesis.

Epidemiological studies have indicated a reduced risk of malignancies with the use of nonsteroidal anti-inflammatory drugs (NSAIDs), although the exact mechanisms are debated. NSAIDs inhibit angiogenesis, which is a key step for tumor growth. Hepatocyte growth factor/scatter factor (HGF/SF), a potent and independent angiogenic factor, has been implicated in tumorigenesis, but limited knowledge exists on the potential targets for inhibiting HGF/SF-induced pathological angiogenesis.

Characterization of bradykinin-induced prostaglandin E2 release from cultured rat trigeminal ganglion neurones.

Bradykinin and prostaglandins are both local mediators strongly implicated in pain and inflammation. Here, we have investigated the effects of bradykinin on the release of prostaglandin E(2) from cultured neurones derived from adult rat trigeminal ganglia. Bradykinin was a potent inducer of prostaglandin E(2) release, an effect that was likely mediated by bradykinin B(2) receptors, as the bradykinin-induced prostaglandin E(2) release was attenuated by the bradykinin B(2) receptor-selective antagonist, arginyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl-3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-(2 alpha, 3 beta, 7a beta)-octahydro-1H-indole-2-carbonyl-L-arginine (HOE 140), but not by the bradykinin B(1) receptor-selective antagonist, des-Arg(9),[Leu(8)]-bradykinin.

Excitation of rat colonic afferent fibres by 5-HT(3) receptors.

The gastrointestinal tract contains most of the body's 5-hydroxytryptamine (5-HT) and releases large amounts after meals or exposure to toxins. Increased 5-HT release occurs in patients with irritable bowel syndrome (IBS) and their peak plasma 5-HT levels correlate with pain episodes. 5-HT(3) receptor antagonists reduce symptoms of IBS clinically, but their site of action is unclear and the potential for other therapeutic targets is unexplored.

Somatostatin release by glutamate in vivo is primarily regulated by AMPA receptors.

1. We have used in vivo microdialysis in anaesthetized rats to investigate whether levels of striatal somatostatin (SRIF) can be increased in response to application of the ionotropic glutamate receptor agonists AMPA and NMDA. 2.

Titrating the expression of a Gi protein-coupled receptor using an ecdysone-inducible system in CHO-K1 cells.

Changes in receptor density are often associated with pathological conditions. For example, high levels of the G protein-coupled somatostatin receptor, sst2, have been detected in a number of malignant cell types, a characteristic feature that is routinely utilised as a diagnostic tool. However, how the increased receptor expression affects cellular function through alterations in G protein-coupling or changes in the intensity or duration of activated signalling pathways is poorly understood.

Species- and agonist-dependent differences in the deactivation-kinetics of P2X7 receptors.

In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist.

Adenosine A(1) receptor-mediated inhibition of protein kinase A-induced calcitonin gene-related peptide release from rat trigeminal neurons.

Calcitonin gene-related peptide (CGRP), a potent vasodilator, has been implicated in the pathogenesis of migraine. Its release from adult rat trigeminal neurons in culture was shown to be markedly increased by the activation of adenylate cyclase with forskolin. Modulation of this secretion was investigated by a number of agents with known inhibitory effects on cAMP generation mediated via receptor coupling to G(i/o) proteins.

High-intensity p38 kinase activity is critical for p21(cip1) induction and the antiproliferative function of G(i) protein-coupled receptors.

G protein-coupled receptors can stimulate the p38 kinase cascade, but the effect this has on cell growth remains poorly characterized. Here we show human somatostatin sst(2) and sst(4) receptors inhibit basic fibroblast growth factor (bFGF)-induced proliferation, via a mechanism that was blocked by the p38 inhibitor PD 169316. The sst(4) receptor could also induce a proliferative activity in the absence of bFGF, which was unaffected by PD 169316.

Somatostatin activates two types of inwardly rectifying K+ channels in MIN-6 cells.

Western blotting revealed the presence of five somatostatin receptor types, sst1, sst2, sst3, sst4 and sst5, in the mouse pancreatic -cell line MIN-6. In MIN-6 cells, glucose-induced electrical activity was potently (pEC50 = 12.7) and irreversibly reduced by somatostatin (SRIF-14); this was associated with hyperpolarization of the membrane potential (pEC50 = 11.

Adenosine nucleotides acting at the human P2Y1 receptor stimulate mitogen-activated protein kinases and induce apoptosis.

For the widely distributed P2Y receptors for nucleotides, the transductional and functional responses downstream of their coupling to G proteins are poorly characterized. Here we describe apoptotic induction and the associated differential stimulation of mitogen-activated protein (MAP) kinase family members by the human P2Y(1) receptor. The potent P2Y(1) receptor agonist, 2-methylthio-ADP (2-MeSADP), stimulated the extracellular-signal regulated kinases (ERK1/2) (EC(50) approximately 5 nm) as well as several, but not all isoforms detected, of the stress-activated protein kinase (SAPK) family.

Serum constituents can affect 2'-& 3'-O-(4-benzoylbenzoyl)-ATP potency at P2X(7) receptors.

1. 2'-& 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) is the prototypic agonist for P2X(7) receptors. In this study we demonstrate that bovine serum albumin (BSA) can affect the potency of BzATP at P2X receptors.

Ligand internalization and recycling by human recombinant somatostatin type 4 (h sst(4)) receptors expressed in CHO-K1 cells.

There is controversy as to whether somatostatin sst(4) receptors internalize. In this study, CHO-K1 cells expressing human sst(4) receptor (CHOsst(4) cells) cells internalized [(125)I]-[(11)Tyr]-SRIF in a time-dependent manner, reaching a steady state at 60 min (1.4+/-0.

Complexities of measuring antagonist potency at P2X(7) receptor orthologs.

The ability of P2 antagonists to affect agonist-stimulated fluorescent dye accumulation in cells expressing human, rat, or mouse P2X(7) receptors was examined. Several compounds, including pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), which was previously thought to be a weak P2X(7) receptor antagonist, possessed high potency (nanomolar IC(50)) at human and rat P2X(7) receptors. However, there were species differences in antagonist potency with PPADS, pyridoxal 5'-phosphate (P5P), and periodate-oxidized ATP (OxATP) exhibiting 20- to 500-fold higher potency for human than for mouse P2X(7) receptors.

Somatostatin receptor-mediated arachidonic acid mobilization: evidence for partial agonism of synthetic peptides.

1. Somatostatin and the stable octapeptide analogues, octreotide and angiopeptin, were examined for their ability to stimulate the release of tritium from [(3)H]-arachidonic acid pre-loaded CHO-K1 cells expressing human recombinant sst(2), sst(3) or sst(5) receptors. 2.

Activity of adenosine diphosphates and triphosphates on a P2Y(T) -type receptor in brain capillary endothelial cells.

1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides.

Immunohistochemical localization of adenosine A1 receptors in human brain regions.

Adenosine exerts its physiological actions by binding to G-protein coupled receptors, four of which have been identified and cloned to date (A1, A2a, A2b and A3). Here we report the development of anti-human adenosine A1, receptor anti-peptide polyclonal antibodies and their use to define the distribution of A1, receptors in human brain regions, spinal cord and trigeminal ganglia by an immunohistochemical approach. Although the distribution of adenosine A1, receptor and its mRNA in the human brain has been investigated in the past by autoradiography and in situ hybridization, this is the first demonstration of localization of the A1, receptors by immunohistochemical means.

Characterisation of somatostatin sst2 receptor splice variants.

Somatostatin is a peptide with a multitude of functions in the central nervous system and the periphery. It mediates its actions by binding to high-affinity G-protein coupled receptors, genes for five of which (sst1-sst5) have recently been cloned. The somatostatin sst2 receptor exists as two splice variants, sst2(a) and sst2(b) receptors, which differ in length and composition of their intracellular carboxy-termini.

Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways.

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) >> Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38.

Antagonist effects on human P2X(7) receptor-mediated cellular accumulation of YO-PRO-1.

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.

Selective somatostatin sst(2) receptor blockade with the novel cyclic octapeptide, CYN-154806.

The cyclic octapeptide, CYN-154806, inhibited specific [(125)I]-[Tyr(11)]-SRIF binding to CHO-K1 cell membranes expressing human recombinant somatostatin (SRIF) sst(2) receptors (pIC(50) 8. 58) or rat sst(2(a)) and rat sst(2(b)) receptors (pIC(50) 8.35 and 8.

Apparent species differences in the kinetic properties of P2X(7) receptors.

1. Apparent species differences in the responses of recombinant P2X(7) receptors to repeated application of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) have been investigated. 2.