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Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

1. Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to alphabeta-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the alphabeta and/or betagamma oxygen atoms were replaced by other moieties (including -CH2-, -CHF-, -CHCl-, -CHBr-, -CF2-, -CCl2-, -CBr2-, -CHSO3-, -CHPO3-, -CFPO3-, -CClPO3-, -CH2-CH2-, C triple bond C, -NH-, -CHCOOH-). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except betagamma-difluoromethylene-ATP, which was a weak agonist. 5. At P2X2/3 receptors, betagamma-imido-ATP, betagamma-methylene-ATP, and betagamma-acetylene-ATP were weak agonists, whereas alphabeta,betagamma- and betagamma,gammadelta-bismethylene-AP4 were potent full agonists. betagamma-Carboxymethylene-ATP and betagamma-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 microm). 6. The results indicate (a). that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b). that the heteromeric P2X2/3 receptor is much more tolerant of alphabeta and betagamma substitution; and (c). that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the betagamma-methylene.

ATP-gated ionotropic receptors (P2X receptors) are distributed widely in the nervous system. For example, a hetero-oligomeric receptor containing both P2X2 and P2X3 subunits is involved in primary afferent sensation. Each subunit has two membrane-spanning domains. We have used disulfide bond formation between engineered cysteines to demonstrate close proximity between the outer ends of the first transmembrane domain of one subunit and the second transmembrane domain of another. After expression in HEK 293 cells of such modified P2X2 or P2X4 subunits, the disulfide bond formation is evident because an ATP-evoked channel opening requires previous reduction with dithiothreitol. In the hetero-oligomeric P2X2/3 receptor the coexpression of doubly substituted subunits with wild-type partners allows us to deduce that the hetero-oligomeric channel contains adjacent P2X3 subunits but does not contain adjacent P2X2 subunits. The results suggest a "head-to-tail" subunit arrangement in the quaternary structure of P2X receptors and show that a trimeric P2X2/3 receptor would have the composition P2X2(P2X3)2.

1 ATP-gated ion channels (P2X receptors) contain two hydrophobic segments that are presumed to span the plasma membrane (TM1 and TM2). Pairs of cysteines were introduced by mutagenesis into the rat P2X(2) receptor, one in TM1 one in TM2, at positions where single substitutions have previously been shown to confer sensitivity to methanethiosulfonates. The receptors were expressed in HEK293 cells; interactions between the cysteines were sought by measuring the effects on ionic currents of dithiothreitol and methanethiosulfonates. 2 Nine pairs gave normally functioning channels: F44C/I328C, F44C/N333C, F44C/L338C, Q37C/I328C, Q37C/N333C, Q37C/T336C, Q37C/L338C, G30C/I328C, G30C/N333C. None formed functionally detectable disulfide bonds. 3 Currents at the F44C/L338C receptor had time course and ATP-sensitivity similar to those for the F44C mutation alone. Methyl-methanethiosulfonate bound to L338C but did not inhibit ionic current. Methyl-methanethiosulfonate inhibited currents at F44C, but not at F44C/L338C. 4 Ethylammonium-methylthiosulfonate inhibited currents at both F44C and L338C, but not at F44C/L338C. It reversed the rapid current deactivation at F44C/L338C. 5 The results suggest that a methanethiosulfonate binding to L338C prevents binding to F44C; this might indicate proximity of these two residues.

1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.

ATP-gated P2X(7) purinoceptors are found in most immune cells of the periphery and the brain where their activation leads to multiple downstream events such as cell permeabilization, apoptosis, and/or cytokine release. P2X(7) receptors do not form heteromeric receptors with any of the other six P2X subunits, and it is not known what type of homomeric assemblies the P2X(7) subunit makes. We constructed and purified an ectodomain protein of the rat P2X(7) receptor (amino acids 60-323) and used this to generate a monoclonal antibody (Ab) with which to probe P2X(7) receptors in central and peripheral immune cells. In HEK cells expressing rat P2X(7) receptors, the Ab increased the maximum current evoked by BzATP by 3-8-fold with a 5-fold leftward shift in EC(50) concentration. This Ab recognized only a non-denatured, multimeric form of the receptor on blue native-PAGE but did not recognize the denatured form on SDS-PAGE. A C-terminal polyclonal P2X(7) Ab recognized both monomeric subunits on SDS-PAGE and a multimeric complex on blue native-PAGE in this heterologous expression system. With Western blotting using these two Abs, native P2X(7) receptors in peritoneal macrophage and bone marrow cells are shown to exist as a strongly bound multimeric complex, whereas P2X(7) receptors in brain glia and/or astrocytes appear to form only as monomeric subunits.

The first hydrophobic segment of the rat P2X(2) receptor extends from residue Leu(29) to Val(51). In the rat P2X(2) receptor, we mutated amino acids in this segment and adjoining flanking regions (Asp(15) through Thr(60)) individually to cysteine and expressed the constructs in human embryonic kidney cells. Whole-cell recordings were used to measure membrane currents evoked by brief (2-s) applications of ATP (0.3-100 microM). Currents were normal except for Y16C, R34C, Y43C, Y55C, and Q56C (no currents but normal membrane expression by immunohistochemistry), Q37C (small currents), and F44C (normal current but increased sensitivity to ATP, as well as alphabeta-methylene-ATP). We used methanethiosulfonates of positive, negative, or no charge to test the accessibility of the substituted cysteines. D15C, P19C, V23C, V24C, G30C, Q37C, F44C, and V48C were strongly inhibited by neutral, membrane-permeant methanethiosulfonates. Only V48C was also inhibited by positively and negatively charged methanethiosulfonates, consistent with an extracellular position; however, accessibility of V48C was increased by channel opening. V48C could disulfide with I328C, as shown by the large increase in ATP-evoked current caused by reducing agents. The results suggest that Val(48) at the outer end of the first hydrophobic segment takes part in the gating movement of channel opening.

Background & Aims: In the intestine, tachykinins regulate motility by participating in neuromuscular and neuro-neuronal transmission. The aim of this study was to test the hypothesis that colonic propulsion is regulated by an interplay between tachykinergic and cholinergic transmission., Methods: Propulsion was elicited by intraluminal distention of a thin rubber balloon, which traveled from the oral to the anal end of guinea pig isolated distal colon segments. The overall contribution of endogenous tachykinins to colonic propulsion was examined by blocking NK1, NK2, and NK3 receptors simultaneously., Results: NK2-receptor blockade by MEN 11420 inhibited propulsion, whereas blockade of NK(1) by SR 140333 or of NK3 receptors by SR 142801 had minor effects on motility. Blockade of muscarinic or nicotinic receptors by hyoscine or hexamethonium decelerated peristalsis up to propulsion arrest. In the presence of partial muscarinic receptor blockade, the NK1-receptor antagonist SR 140333 and the NK2-receptor antagonist MEN 11420 markedly inhibited propulsion. Propulsion was also inhibited by the NK3-receptor antagonist SR 142801 in the presence of partial nicotinic receptor blockade. The simultaneous administration of the 3 tachykinin antagonists inhibited propulsion by 50%., Conclusions: This study demonstrates the existence of an interplay between tachykinergic and cholinergic pathways during peristalsis and the importance of endogenous tachykinins acting at multiple receptor sites in the control of colonic propulsion.

The effect of the melatonin receptor ligand, 2-phenylmelatonin, has been assessed in isolated strips of the guinea-pig proximal colon. 2-Phenylmelatonin (0.01 nM-1 microM) caused a concentration-dependent contractile response. The potency value (-log EC50) was 9.3 +/- 1.0. The maximum effect was 25 +/- 4%, of that elicited by the maximally effective concentration (0.3 microM) of 5-HT and 43 +/- 3%, of that by the maximally effective concentration (10 microM) of melatonin. When used as an antagonist, 2-phenylmelatonin (0.01 nM and 0.1 nM) concentration-dependently inhibited melatonin-induced contractions with depression of the maximum response by 25% and 54%, respectively. Higher (1 nM) 2-phenylmelatonin concentrations failed to antagonize melatonin-induced response. Prazosin (0.3 microM), a selective antagonist of melatonin MT3 sites, antagonized melatonin-induced contractions to an extent similar to that induced by 0.01 nM 2-phenylmelatonin (with 30% reduction of the maximum effect to melatonin). The combination of 0.3 microM prazosin and 0.01 nM 2-phenylmelatonin caused antagonism similar in extent to that caused by each individual antagonist. 2-Phenylmelatonin at subnanomolar concentrations behaves as an antagonist of melatonin-induced contractile responses while at nanomolar/micromolar concentrations it behaves as a weak contractile agonist.

1. Experiments were carried out in human detrusor strips to characterize muscarinic receptor subtypes involved in the prejunctional regulation of acetylcholine (ACh) release from cholinergic nerve terminals, and in the postjunctional smooth muscle contractile response. 2. In detrusor strips preincubated with [3H]-choline, electrical field stimulation (600 pulses) delivered in six trains at 10 Hz produced a tritium outflow and a contractile response. In the presence of 10 microM paraoxon (to prevent ACh degradation) the tritium outflow was characterized by HPLC analysis as [3H]-ACh (76%) and [3H]-choline (24%). 3. Electrically-evoked [3H]-ACh release was abolished by tetrodotoxin (TTX: 300 nM) and unaffected by hexamethonium (10 microM), indicating a postganglionic event. It was reduced by physostigmine (100 nM) and the muscarinic receptor agonist, muscarone (10 nM-1 microM), and enhanced by atropine (0.1-100 nM). These findings indicate the presence of a muscarinic negative feedback mechanism controlling ACh release. 4. The effects of various subtype-preferring muscarinic receptor antagonists were evaluated on [3H]-ACh release and muscle contraction. The rank potency (-log EC50) orders at pre- and postjunctional level were: atropine > or = 4-diphenyl-acetoxy-N-piperidine (4-DAMP) > mamba toxin 3 (MT-3) > tripitramine > para-fluorohexahydrosiladiphenidol (pF-HHSiD) > or = methoctramine > or = pirenzepine > tripinamide, and atropine > or = 4-DAMP > pF-HHSiD >> pirenzepine = tripitramine > tripinamide > methoctramine >> MT-3, respectively. 5. The comparison of pre- and post-junctional potencies and the relationship analysis with the affinity constants at human cloned muscarinic receptor subtypes indicates that the muscarinic autoreceptor inhibiting ACh release in human detrusor is an M4 receptor, while the receptor involved in muscular contraction belongs to the M3 subtype.

The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 microM), was partially sensitive to tetrodotoxin (1 microM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 microM) and suramin (300 microM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 microM) was ineffective. In the presence of theophylline (100 microM) and 3-isobutyl-1-methylxanthine (10 microM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01-1 microM) and suramin (300 microM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds.