Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: A microdialysis study

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5 min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.     

Systemic pregabalin attenuates facial hypersensitivity and noxious stimulus-evoked release of glutamate in medullary dorsal horn in a rodent model of trigeminal neuropathic pain

Pregabalin is effective in treating many neuropathic pain conditions. However, the mechanisms of its analgesic effects remain poorly understood. The aim of the present study was to determine whether pregabalin suppresses facial mechanical hypersensitivity and evoked glutamate release in the medullary dorsal horn (MDH) in a rodent model of trigeminal neuropathic pain. Nociceptive mechanical sensitivity was assessed pre-operatively, and then post-operatively 1 h following pregabalin or vehicle (saline) treatment on post-operative days 2 and 5 following infraorbital nerve transection (IONX). In addition, an in vivo microdialysis probe was inserted into the exposed medulla post-operatively and dialysate samples were collected. Glutamate release was then evoked by mustard oil (MO) application to the tooth pulp, and the effects of pregabalin or vehicle were examined on the MDH glutamate release. Glutamate concentrations in the dialysated samples were determined by HPLC, and data analyzed by ANOVA. IONX animals (but not control animals) showed facial mechanical hypersensitivity for several days post-operatively. In addition, tooth pulp stimulation with MO evoked a transient release of glutamate in the MDH of IONX animals. Compared to vehicle, administration of pregabalin significantly attenuated the facial mechanical hypersensitivity as well as the MO-evoked glutamate release in MDH. This study provides evidence in support of recent findings pointing to the usefulness of pregabalin in the treatment of orofacial neuropathic pain.     

The role of pannexin 1 in the purinergic regulation of synaptic transmission in mouse motor synapses

The role of pannexin 1 in the release to the extracellular space of ATP/adenosine modulating the acetylcholine (ACh) secretion was studied in mouse diaphragm motor synapses. Using neuromuscular preparations obtained from wild-type and pannexin-1 knockout mice, the miniature endplate potential (MEPPs) and evoked endplate potentials (EPPs) were recorded in combination with pharmacological modulation of P2-type ATP receptors and A₁-type adenosine receptors. Selective inhibition of A₁ receptors with DPCPX or P2 receptors with PPADS increased quantal content of EPPs in wild-type mice. MRS 2211, selective antagonist of P2Y13 receptors, produced the same effect. Activation of receptors A₁ or P2Y₁₃ by their agonists (2-CADO and IDP, respectively) decreased the EPP quantal content. It means that the activity of endogenous ATP and adenosine is synergistic and directed to depression of the ACh release. ARL67156, an inhibitor of synaptic ecto-ATPases, which blocks the hydrolysis of ATP to adenosine and increases the level of ATP in the synaptic cleft, prolonged EPPs without changing their quantal content. In pannexin-1 knockout mice there were no changes in the EPP quantal content and in other parameters of synaptic transmission as compared to wildtype mice. However, downregulation of purinergic effects with antagonists of A₁ or P2 receptors (DPCPX, PPADS, MRS 2211) did not change EPP quantal content and any other parameters of spontaneous or evoked ACh release in all cases. ARL67156 did not alter the temporal parameters of EPPs, either. Nevertheless, 2-CADO, the A₁-type receptor agonist, decreased the EPP quantal content, while the agonist of P2Y₁₃ receptors decreased the MEPP amplitude. Thus, in mice lacking pannexin 1, procedures revealing the presence and regulatory activity of synaptic ATP/adenosine did not change the parameters of synaptic transmission. The obtained data substantiate a mandatory role of pannexin 1 in the purinergic regulation of motor synapse activity by endogenous ATP/adenosine.     

Modulatory Role of Adenosine Receptors in Insect Motor Nerve Terminals

The effects of adenosine and ATP were studied on blowfly larvae Calliphora vicina neuromuscular preparation. Adenosine diminished (IC₅₀ = 40 ± 3 M) the amplitude of nerve-evoked postsynaptic currents (EPSCs) and slightly decreased the frequency of spontaneous currents without affecting their amplitude. EPSCs were slightly reduced by ATP, and this effect was prevented by concanavalin A. Presynaptic inhibition by adenosine was temperature-dependent and insensitive to pertussis toxin. A₁ agonists of vertebrate adenosine receptor CPA and NECA failed to reproduce the effect of adenosine, and 2-CADO enhanced the EPSCs. A₁ antagonist DPCPX competitively inhibited adenosine action. A₂ agonist DPMA potentiated EPSCs, and its effect was abolished by A₂ antagonist DMPX. Adenosine and ATP failed to affect the nonquantal release of glutamate. The results show for the first time the presence of presynaptic adenosine receptors regulating transmitter release at insect motor nerve terminals and point to differences in pharmacological properties of adenosine receptor subtypes in insects and vertebrates.     

ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line.

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.     

Modulating influence of adenosine triphosphate on the oscillating properties of hippocampal neurons during early ontogenesis

Extracellular ATP was used to test the involvement of purinergic P2 receptors in the modulation of GAB Aergic giant depolarizing potentials (GDPs) in rat neonatal hippocampus. Bath application of ATP reduced the GDP frequency in a concentration-dependent manner. The inhibitory effect of ATP persisted in the presence of adenosine P1 receptor antagonist DPCPX indicating the action of ATP on GDPs. It was suggested that, during development, ATP can modulate network-driven activity in the neonatal hippocampus through the activation of purinergic P2 receptors.     

Electrophysiological-anatomic correlates of ATP-triggered vagal reflex in the dog. V. Role of purinergic receptors

The mechanism of extracellular ATP-triggered vagal depressor reflex was further studied in a closed-chest canine model. Adenosine and ATP were administered individually in equimolar doses (0.01-1.0 mumol/kg) into the right coronary artery (RCA) and left circumflex coronary artery (LCA). When administered into the RCA, adenosine and ATP exerted an identical and relatively small negative chronotropic effect on sinus node automaticity; the time to peak negative chronotropic effect was >/=7 s. When administered into the LCA, adenosine had no effect on sinus node automaticity, whereas ATP markedly suppressed sinus node automaticity. This effect of ATP 1) reached its peak in <2 s after its administration, 2) was short lasting, and 3) was completely abolished by either intravenous administration of the muscarinic cholinergic blocker atropine (0.2 mg/kg) or intra-LCA administration of 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), a potent P2X(2/3) purinergic receptor (P2X(2/3)R) antagonist, but not by diinosine pentaphosphate (Ip(5)I), a potent inhibitor of P2X(1)R and P2X(3)R. Repetitive administrations of ATP were not associated with reduced effects, indicative of receptor desensitization, thereby excluding the involvement of the rapidly desensitized P2X(1)R in the action of ATP. It was concluded that ATP triggers a cardio-cardiac vagal depressor reflex by activating P2X(2/3)R located on vagal sensory nerve terminals localized in the left ventricle. Because these terminals mediate vasovagal syncope, these data could suggest a mechanistic role of extracellular ATP in this syndrome and, in addition, give further support to the hypothesis that endogenous ATP released from ischemic myocytes is a mediator of atropine-sensitive bradyarrhythmias associated with left ventricular myocardial infarction.     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

Water induces autocrine stimulation of tumor cell killing through ATP release and P2 receptor binding

Although exposure of cells to extreme hypotonic stress appears to be a purely experimental set up, it has found an application in clinical routine. For years, surgeons have washed the abdominal cavity with distilled water to lyse isolated cancer cells left after surgery. No data are available supporting this practice or evaluating the potential mechanisms of cell injury under these circumstances. Recent evidence indicates that increases in cell volume stimulate release of adenosine triphosphate and autocrine stimulation of purinergic (P2) receptors in the plasma membrane of certain epithelial cell types. Under physiological conditions, purigenic stimulation can contribute to cell volume recovery through activation of solute efflux. In addition, adenosine triphosphate-P2 receptor binding might trigger other mechanisms affecting cell viability after profound hypotonic stress. This study demonstrates a novel pathway of cell death by apoptosis in human colon cancer cells following a short hypotonic stress. This pathway is induced by transitory cell swelling which leads to extracellular release of adenosine triphosphate (ATP) and specific binding of ATP to P2 receptors (probably P2X7). Extracellular ATP induced activation of caspases 3 and 8, annexin V, release of cytochrome c, and eventually cell death. The effect of ATP can be blocked by addition of (i) apyrase to hydrolyse extracellular ATP and (ii) suramin, a P2 receptor antagonist. Finally, (iii) gadolinium pretreatment, a blocker of ATP release, reduces sensitivity of the cells to hypotonic stress. The adenosine triphosphate-P2 receptor cell death pathway suggests that autocrine/paracrine signaling may contribute to regulation of viability in certain cancer cells disclosed with this pathway.     

Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.     

Characterization of ATP release from cultures enriched in cholinergic amacrine-like neurons.

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine-like neurons. Depolarization of the cells with KCl, or activation of alpha-amino-3-hydroxy- 5-methyl-4-isoxazole-propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca(2+) dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and omega-Agatoxin IVA, but not by omega-Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca(2+) influx necessary to trigger the release of ATP occurs in part through the L- and the P/Q types of voltage-sensitive Ca(2+) channels (VSCC), but not through N-type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A(1) receptor antagonist, showing that the release is tonically inhibited by the adenosine A(1) receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation.     

Enhancement of purinergic signalling by excessive endogenous ATP in resiniferatoxin (RTX) neuropathy

ATP is a ligand of P2X family purinoceptors, and exogenous ATP administration evokes pain behaviors. To date, there is a lack of systematic studies to address relationships between endogenous ATP and neuropathic pain. In this report, we took advantage of a mouse model of resiniferatoxin (RTX)-induced neuropathic pain to address the role of endogenous ATP in neuropathic pain. After RTX administration, endogenous ATP markedly increased in dorsal root ganglia (DRGs) (p?<?0.01) and skin tissues (p?<?0.001). The excessive endogenous ATP was removed by apyrase, an ATP hydrolyzing enzyme, administration via either a lumbar puncture route (p?<?0.001) or an intraplantar injection (p?<?0.001), which led to the normalization of neuropathic pain. In addition, intraplantar treatment with apyrase caused mechanical analgesia. Linear analyses showed that the densities of P2X3(+) neurons (r?=??0.72, p?<?0.0001) and P2X3(+) dermal nerves (r?=??0.72, p?<?0.0001) were inversely correlated with mechanical thresholds. Moreover, the contents of endogenous ATP in skin tissues were linearly correlated with P2X3(+) dermal nerves (r?=?0.80, p?<?0.0001) and mechanical thresholds (r?=??0.80, p?<?0.0001). In summary, this study demonstrated that enhanced purinergic signalling due to an increase in endogenous ATP after RTX-induced nerve injury contributed to the development of neuropathic pain. The data in this report provide a new therapeutic strategy for pain control by targeting the endogenous ligand of purinergic signalling.     

Inhibition mechanism of the recombinant rat P2X(2) receptor in glial cells by suramin and TNP-ATP

P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.     

Characterization of ATP release from cultures enriched in cholinergic amacrine‐like neurons

Adenosine triphosphate (ATP) has been proposed to play a role as a neurotransmitter in the retina, but not much attention has been given to the regulation of ATP release from retinal neurons. In this work, we investigated the release of ATP from cultures enriched in amacrine‐like neurons. Depolarization of the cells with KCl, or activation of α‐amino‐3‐hydroxy‐ 5‐methyl‐4‐isoxazole‐propionate (AMPA) receptors, evoked the release of ATP, as determined by the luciferin/luciferase luminescent method. The ATP release was found to be largely Ca²⁺ dependent and sensitive to the botulinum neurotoxin A, which indicates that the ATP released by cultured retinal neurons originated from an exocytotic pool. Nitrendipine and ω‐Agatoxin IVA, but not by ω‐Conotoxin GVIA, partially blocked the release of ATP, indicating that in these cells, the Ca²⁺ influx necessary to trigger the release of ATP occurs in part through the L‐ and the P/Q types of voltage‐sensitive Ca²⁺ channels (VSCC), but not through N‐type VSCC. The release of ATP increased in the presence of adenosine deaminase, or in the presence of 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist, showing that the release is tonically inhibited by the adenosine A₁ receptors. To our knowledge, this is the first report showing the release of endogenous ATP from a retinal preparation. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 340–348, 1999     

Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.     

Tonic adenosine neuromodulation is preserved in motor nerve endings of aged rats

Neuromuscular transmission is decreased in aged subject. Since endogenous adenosine is a potent neuromodulator at motor nerve endings, either inhibiting via A₁ receptors or facilitating via A_2A receptors acetylcholine release, we now investigated if the tonic effect of endogenous adenosine was modified at phrenic nerve endings of aged rats. The A_2A receptor antagonist (ZM241385, 50 nM) inhibited (77 ± 9%) and the A₁ receptor antagonist (DPCPX, 50 nM) facilitated (74 ± 13%) acetylcholine release from young adult (6 weeks old) rat preparations, indicating a simultaneous tonic activation of A_2A and A₁ receptors. Tonic modulation by adenosine was unaltered in aged (24 months old) rats, since ZM241385 (50 nM) inhibited (73 ± 8%) and DPCPX (50 nM) facilitated (91 ± 20%) acetylcholine release in aged animals similarly to young rats. This indicates that, in contrast to the central nervous system where adenosine neuromodulation is modified in aged animals, the control by adenosine of phrenic nerve function is preserved in aged animals     

P2X7 receptor-mediated purinergic signaling promotes liver injury in acetaminophen hepatotoxicity in mice

Inflammation contributes to liver injury in acetaminophen (APAP) hepatotoxicity in mice and is triggered by stimulation of immune cells. The purinergic receptor P2X7 is upstream of the nod-like receptor family, pryin domain containing-3 (NLRP3) inflammasome in immune cells and is activated by ATP and NAD that serve as damage-associated molecular patterns. APAP hepatotoxicity was assessed in mice genetically deficient in P2X7, the key inflammatory receptor for nucleotides (P2X7-/-), and in wild-type mice. P2X7-/- mice had significantly decreased APAP-induced liver necrosis. In addition, APAP-poisoned mice were treated with the specific P2X7 antagonist A438079 or etheno-NAD, a competitive antagonist of NAD. Pre- or posttreatment with A438079 significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury in wild-type but not P2X7-/- mice. Pretreatment with etheno-NAD also significantly decreased APAP-induced necrosis and hemorrhage in APAP liver injury. In addition, APAP toxicity in mice lacking the plasma membrane ecto-NTPDase CD39 (CD39-/-) that metabolizes ATP was examined in parallel with the use of soluble apyrase to deplete extracellular ATP in wild-type mice. CD39-/- mice had increased APAP-induced hemorrhage and mortality, whereas apyrase also decreased APAP-induced mortality. Kupffer cells were treated with extracellular ATP to assess P2X7-dependent inflammasome activation. P2X7 was required for ATP-stimulated IL-1β release. In conclusion, P2X7 and exposure to the ligands ATP and NAD are required for manifestations of APAP-induced hepatotoxicity.     

Impairment of ATP hydrolysis decreases adenosine A1 receptor tonus favoring cholinergic nerve hyperactivity in the obstructed human urinary bladder

This study was designed to investigate whether reduced adenosine formation linked to deficits in extracellular ATP hydrolysis by NTPDases contributes to detrusor neuromodulatory changes associated with bladder outlet obstruction in men with benign prostatic hyperplasia (BPH). The kinetics of ATP catabolism and adenosine formation as well as the role of P1 receptor agonists on muscle tension and nerve-evoked [³H]ACh release were evaluated in mucosal-denuded detrusor strips from BPH patients (n = 31) and control organ donors (n = 23). The neurogenic release of ATP and [³H]ACh was higher (P < 0.05) in detrusor strips from BPH patients. The extracellular hydrolysis of ATP and, subsequent, adenosine formation was slower (t_1/2 73 vs. 36 min, P < 0.05) in BPH detrusor strips. The A₁ receptor-mediated inhibition of evoked [³H]ACh release by adenosine (100 μM), NECA (1 μM), and R-PIA (0.3 μM) was enhanced in BPH bladders. Relaxation of detrusor contractions induced by acetylcholine required 30-fold higher concentrations of adenosine. Despite VAChT-positive cholinergic nerves exhibiting higher A₁ immunoreactivity in BPH bladders, the endogenous adenosine tonus revealed by adenosine deaminase is missing. Restoration of A₁ inhibition was achieved by favoring (1) ATP hydrolysis with apyrase (2 U mL⁻¹) or (2) extracellular adenosine accumulation with dipyridamole or EHNA, as these drugs inhibit adenosine uptake and deamination, respectively. In conclusion, reduced ATP hydrolysis leads to deficient adenosine formation and A₁ receptor-mediated inhibition of cholinergic nerve activity in the obstructed human bladder. Thus, we propose that pharmacological manipulation of endogenous adenosine levels and/or A₁ receptor activation might be useful to control bladder overactivity in BPH patients.     

Pharmacological Properties and Physiological Function of a P2X-Like Current in Single Proximal Tubule Cells Isolated from Frog Kidney

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2X(f)) that was also Ca2+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2X(f) was inhibited by suramin, PPADS and TNP-ATP. Activation of P2X(f) attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2X(f) plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2X(f) inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells.