Metabotropic P2Y1 receptors inhibit P2X3 receptor-channels in rat dorsal root ganglion neurons

Whole-cell patch-clamp recordings from cultured rat dorsal root ganglion neurons demonstrated that the P2Y1 receptor agonists adenosine 5'-O-2-thiodiphosphate (ADP-beta-S) and 2-methylthio adenosine 5'-diphosphate (2-MeSADP) inhibit the alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP)-induced P2X3 receptor-currents. This effect could be antagonized by the wide-spectrum G protein blocker GDP-beta-S and the P2Y(1) receptor antagonist MRS 2179. The P2Y12,13 receptor antagonist AR-C6993MX and pertussis toxin, a blocker of Galphai/o, did not interact with the effect of ADP-beta-S. Hence, the results indicate that ADP-sensitive P2Y1 receptors of rat dorsal root ganglion neurons inhibit ionotropic P2X3 receptors via G protein-activation.     

Identification of P2X receptors in cultured mouse and rat parasympathetic otic ganglion neurones including P2X knockout studies

We have used patch-clamp recording from cultured neurones, immunohistochemistry and gene deletion techniques to characterize the P2X receptors present in mouse otic ganglion neurones, and demonstrated the presence of similar receptors in rat neurones. All neurones from wild-type (WT) mice responded to ATP (EC(50) 109 microM), but only 38% also responded to alpha beta-meATP (EC(50) 39 microM). The response to alpha beta-meATP was blocked by TNP-ATP with an IC(50) of 38.6 nM. Lowering extracellular pH and co-application of Zn(2+) potentiated responses to ATP and alpha beta-meATP. In P2X(3)(-/-) mouse otic ganglion, all neurones tested responded to 100 microM ATP with a sustained current, but none responded to alpha beta-meATP. In P2X(2)(-/-) mice, no sustained currents were observed, but 36% of neurones responded to both ATP and alpha beta-meATP with transient currents. In P2X(2)/P2X(3)(Dbl-/-) mice, no responses to ATP or alpha beta-meATP were detected, suggesting that other P2X subunits were not involved. In rat otic ganglia, 96% of neurones responded to both ATP and alpha beta-meATP with sustained currents, suggesting a greater proportion of neurones expressing P2X(2/3) receptors. The maximum response to alpha beta-meATP was 40-60% of that evoked by ATP in the same cell. Immunohistochemistry revealed staining for P2X(2) and P2X(3) subunits in WT mouse otic ganglion neurones, which was absent in knockout animals. In conclusion, we have shown for the first time that at least two distinct P2X receptors are present in mouse and rat otic neurones, probably homomeric P2X(2) and heteromeric P2X(2/3) receptors.     

Homomeric and heteromeric P2X3 receptors in peripheral sensory neurons

ATP contributes to nociceptive sensory processing by activating a family of ligand-gated ion channels, the P2X receptors. One of these, the P2X3 receptor, is highly localized on primary afferent neurons. In sensory neurons, P2X3 receptors function as homomeric (P2X3) and heteromeric (P2X2/3) channels. Exogenous application of ATP and related agonists excites peripheral and central nerves, and increases sensitivity to noxious stimuli. Specific targeting of P2X3 receptors by gene deletion and knockdown results in a hypoalgesic phenotype. In animal models of pain, pharmacological blockade of P2X3 receptors fully blocked specific types of chronic inflammatory and neuropathic pain. Peripheral nerve injury differentially alters functional expression of P2X3 receptors on small and large diameter primary afferent neurons. These data have delineated discrete roles for homomeric P2X3 and heteromeric P2X2/3 receptor activation in acute and chronic pain. Similar findings have also been generated from extensive research of the bladder urothelial-sensory neuron system. The urinary bladder is richly innervated by P2X3 receptor-containing neurons. Heteromeric P2X2/3 channels in the bladder contribute to both mechanosensitivity and nociceptive responses. Thus, both genetic and pharmacological approaches have provided converging evidence that activation of P2X3-containing channels is an important mediator of acute and persistent nociceptive signaling in the peripheral nervous system.     

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

BACKGROUND AND PURPOSE

The P2X receptor family consists of seven subunit types – P2X1–P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits – P2X2 and P2X4, and P2X4 and P2X7.

EXPERIMENTAL APPROACH

We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging.

KEY RESULTS

Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation.

CONCLUSIONS AND IMPLICATIONS

We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.     

大鼠下颌下腺神经节分离神经元P2X受体的药理学研究

目的研究大鼠下颌下腺副交感神经节分离神经元不同P2X受体的表达。方法用全细胞膜片钳的方法对大鼠下颌下腺神经节急性分离的神经元进行了药理学鉴定。结果在所有的神经元上,ATP都可以引起一个快速反应的缓慢失活的内向电流。但只在59%的神经元上,αβmeATP也可以引起一个类似的内向电流。降低pH值及同时给予Zn2+可以增强ATP及αβmeATP诱发的电流。而Ivermectin没有作用。结论在大鼠下颌下腺神经节急性分离的神经元上,主要表达同源二聚体P2X2受体和异源二聚体P2X2/3受体。     

Facilitation of transmitter release from rat sympathetic neurons via presynaptic P2Y(1) receptors

BACKGROUND AND PURPOSE

P2Y₁, P2Y₂, P2Y₄, P2Y₁₂ and P2Y₁₃ receptors for nucleotides have been reported to mediate presynaptic inhibition, but unequivocal evidence for facilitatory presynaptic P2Y receptors is not available. The search for such receptors was the purpose of this study.

EXPERIMENTAL APPROACH

In primary cultures of rat superior cervical ganglion neurons and in PC12 cell cultures, currents were recorded via the perforated patch clamp technique, and the release of [³H]-noradrenaline was determined.

KEY RESULTS

ADP, 2-methylthio-ATP and ATP enhanced stimulation-evoked ³H overflow from superior cervical ganglion neurons, treated with pertussis toxin to prevent the signalling of inhibitory G proteins. This effect was abolished by P2Y₁ antagonists and by inhibition of phospholipase C, but not by inhibition of protein kinase C or depletion of intracellular Ca²⁺ stores. ADP and a specific P2Y₁ agonist caused inhibition of Kv7 channels, and this was prevented by a respective antagonist. In neurons not treated with pertussis toxin, ³H overflow was also enhanced by a specific P2Y₁ agonist and by ADP, but only when the P2Y₁₂ receptors were blocked. ADP also enhanced K⁺-evoked ³H overflow from PC12 cells treated with pertussis toxin, but only in a clone expressing recombinant P2Y₁ receptors.

CONCLUSIONS AND IMPLICATIONS

These results demonstrate that presynaptic P2Y₁ receptors mediate facilitation of transmitter release from sympathetic neurons most likely through inhibition of Kv7 channels.     

Heterogeneity of alpha 1-adrenergic receptors revealed by chlorethylclonidine

Chlorethylclonidine (CEC) has previously been shown to inactivate only a subpopulation of the alpha 1-adrenergic receptor binding sites in rat brain. We compared alpha 1-adrenergic receptors in different tissues to determine whether such selective inactivation might reveal the presence of distinct receptor subtypes. Pretreatment of broken cell preparations with 10 microM CEC for 10 min caused a 70-80% decrease in the density of specific 125IBE 2254 binding sites in rat liver and spleen, a 25% decrease in neocortex, but no significant loss in kidney, hippocampus, heart, vas deferens, or caudal artery. The effect of CEC in liver was not reversed by extensive washing, suggesting irreversible inactivation. The selectivity between different tissues was due to differences in the efficacy of CEC inactivating the binding sites and not due to differences in binding affinity. To determine whether the effects on 125IBE 2254 binding reflected selective inactivation of functional receptors, contractile responses of rat spleen and vas deferens were examined. Pretreatment of intact tissues with 100 microM CEC for 30 min caused a large decrease in the potency and maximal contraction to norepinephrine in spleen but had no effect in vas deferens. Inhibition of specific 125IBE 2254 binding by various agonists and antagonists was determined in CEC-sensitive (liver, spleen) and insensitive (hippocampus, vas deferens) tissues. Although many drugs had similar affinities in all tissues, others were substantially less potent in the CEC-sensitive tissues. These experiments suggest that there are at least two subtypes of alpha 1-adrenergic receptors with different pharmacological properties in mammalian tissues, only one of which is inactivated by CEC.     

Binding thermodynamic characterization of human P2X1 and P2X3 purinergic receptors

The present study was designed to perform binding and thermodynamic characterization of human P2X1 and P2X3 purinergic receptors expressed in HEK 293 cells. The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees (standard free energy, enthalpy and entropy) of the binding equilibrium of well-known purinergic agonists and antagonists at P2X1 and P2X3 receptors were determined. Saturation binding experiments, performed in the temperature range 4-30 degrees C by using the high affinity purinergic agonist [3H]alphabetameATP, revealed a single class of binding sites with an affinity value in the nanomolar range in both cell lines examined. The affinity changed with the temperature whereas receptor density was essentially independent of it. van't Hoff plots of the purinergic receptors were linear in the range 4-30 degrees C for agonists and antagonists. The thermodynamic parameters of the P2X1 or P2X3 purinergic receptors were in the ranges -31 kJ mol(-1) < or =DeltaH degrees < or =-19 kJ mol(-1) and 17 J K(-1) mol(-1)< or =DeltaS degrees < or =51 J K(-1)mol(-1) or -26 kJ mol(-1)< or =DeltaH degrees < or =36 kJ mol(-1) and 59< or =DeltaS degrees < or =249 JK(-1) mol(-1), respectively. The results of these parameters showed that P2X1 receptors are not thermodynamically discriminated and that the binding of agonists and antagonists was both enthalpy and entropy-driven. P2X3 receptors were thermodynamically discriminated and purinergic agonist binding was enthalpy and entropy-driven while antagonist binding was totally entropy-driven. The analysis of such thermodynamic data makes it possible to obtain additional information on the nature of the forces driving the purinergic binding interaction. These data could be interesting in drug discovery programs aimed at development of novel and potent P2X1 and P2X3 purinergic ligands.     

Dissecting individual current components of co-expressed human P2X1 and P2X7 receptors

Purinergic P2X(1) and P2X(7) receptors are co-expressed in several cell types such as lymphocytes or epithelial cells. Here we examined whether these two P2X subtypes interact with each other in a manner that results in a mutual alteration of their electrophysiologic behaviour. Furthermore, since specific pharmacological tools are needed to assign distinct effects to a particular receptor subtype in native cells, we assessed a series of compounds for their capacity to separate individual current components in cells that co-expressed both receptor subtypes. In Xenopus oocytes, co-expression neither changed the time courses of activation, desensitization and deactivation nor recovery from desensitization when compared to oocytes that express either hP2X(1) or hP2X(7) receptors alone. A selective activation of hP2X(7) receptors was achieved with benzoyl-benzoyl-ATP, which did not activate P2X(1) receptor currents. P2X(7) receptors could also be selectively activated by ATP when co-applied with 1 microM NF449, a suramin derivative, which is 100,000 fold more potent in blocking P2X(1) than P2X(7) receptors. alphabeta-methylene-ATP, a reportedly hP2X(1) receptor-specific agonist, as well as oxidized-ATP, brilliant blue or KN62, reported hP2X(7) receptor antagonists, were found to be ineffective in separating hP2X(1) receptor current from the P2X(7) current. The best way for a selective activation of the hP2X(1) receptor component in cells co-expressing the P2X(7) receptor is the application of low concentrations of ATP (< 1 microM) or the addition of Mg2+ when using higher concentrations of ATP.     

Differential expression of P2X receptors on neurons from different parasympathetic ganglia

Whole-cell patch clamp recording and immunohistochemistry were used to investigate the expression of P2X receptors on rat parasympathetic ganglion neurons of the otic, sphenopalatine, submandibular, intracardiac and paratracheal ganglia. Neurons from all five ganglia responded to ATP with a rapidly activating, sustained inward current. Neurons of intracardiac and paratracheal ganglia were insensitive to alphabeta-meATP, while all neurons in the otic and some neurons of sphenopalatine and submandibular ganglia responded. Lowering pH potentiated ATP responses in neurons from all five ganglia. Co-application of Zn(2+) potentiated ATP responses in intracardiac, paratracheal and submandibular ganglion neurons. Immunohistochemistry revealed strong and specific staining for the P2X(2) subunit in all five ganglia and strong P2X(3) staining in otic, sphenopalatine and submandibular ganglia. In conclusion, there is heterogeneity in P2X receptor expression in different parasympathetic ganglia of the rat, but the predominant receptor subtypes involved appear to be homomeric P2X(2) and heteromeric P2X(2/3).     

Roles of histamine receptors in pain perception: a study using receptors gene knockout mice

To study the participation of histamine H1- and H2-receptors in pain perception, H1 and H2 receptor knockout (KO) mice were examined for pain threshold by means of three kinds of nociceptive tasks. These included assays for thermal, mechanical, and chemical nociception. H1KO mice showed significantly fewer nociceptive responses to the hot-plate, tail-flick, tail-pressure, paw-withdrawal, formalin, capsaicin, and abdominal constriction tests. Sensitivity to noxious stimuli in H1KO mice was significantly decreased when compared to wild-type mice. The antinociceptive phenotypes of H2KO were relatively less prominent when compared to H1KO mice. We also examined the antinociceptive effects of intrathecally-, intracerebroventricularly-, and subcutaneously-administered morphine in H1KO and H2KO mice. In these nociceptive assays, the antinociceptive effects produced by morphine were more enhanced in both H1KO and H2KO mice. The effects of histamine H1- and H2-receptor antagonists on morphine-induced antinociception were studied in ICR mice. The intrathecal, intracerebroventricular and subcutaneous co-administrations of d-chlorpheniramine enhanced the effects of morphine in all nociceptive assays examined. In addition, intrathecal co-administrations of cimetidine enhanced the antinociception of morphine in the hot plate tests. These results suggest that existing H1 and H2 receptors play an inhibitory role in morphine-induced antinociception in the spinal and supra-spinal levels.     

Actions of a series of PPADS analogs at P2X1 and P2X3 receptors

Seven PPADS ( P yridoxal‐5′‐ P hosphate 6‐ A zophenyl 2′,4′‐ D i S ulfonate) analogs were investigated at Group 1 P2X receptors expressed in Xenopus oocytes. All seven analogs potently inhibited P2X₁ (IC₅₀ range, 5–32 nM) and P2X₃ (IC₅₀ range, 22–345 nM), the two Group I P2X receptor subtypes. Analogs showed greater inhibitory activity where the pyridoxal moiety of PPADS contained a 5′‐phosphonate group, rather than a 5′‐phosphate group. Analogs also showed greater potency where disulfonate groups were removed from, or substituted at, the azophenyl moiety. The most active analog was MRS 2257 (pyridoxal‐5′‐phosphonate 6‐azophenyl 3′,5′‐bismethylenephosphonate) at P2X₁ (IC₅₀, 5 nM) and P2X₃ (IC₅₀, 22 nM) receptors, being 14‐fold and 10‐fold more potent than PPADS itself. MRS 2257 produced a nonsurmountable inhibition when tested against a range of ATP concentrations, although blockade was reversed by about 85% after 20 minutes of washout. TNP‐ATP and Ip₅I were equipotent with MRS 2257 at P2X₁ receptors, whereas TNP‐ATP was 64‐fold more potent than MRS 2257 at P2X₃ receptors. In conclusion, the PPADS template can be altered at the pyridoxal and phenyl moieties to produce P2X₁ and P2X₃ receptor antagonists showing higher potency and greater degree of reversibility than the parent compound at these Group I P2X receptors. Drug Dev. Res. 53:281–291, 2001. © 2001 Wiley‐Liss, Inc.     

Crosstalk between presynaptic angiotensin receptors,bradykinin receptors and alpha 2-autoreceptors in sympathetic neurons: a study in alpha 2-adrenoceptor-deficient mice

1. In mouse atria, angiotensin II and bradykinin lose much or all of their noradrenaline release-enhancing effect when presynaptic alpha(2)-autoinhibition does not operate either because of stimulation with very brief pulse trains or because of treatment with alpha(2) antagonists. We now studied this operational condition in alpha(2)-adrenoceptor-deficient mice. Release of (3)H-noradrenaline was elicited by electrical stimulation. 2. In tissues from wild-type (WT) mice, angiotensin II and bradykinin increased the overflow of tritium evoked by 120 pulses at 3 Hz. This enhancement did not occur or was much reduced when tissues were stimulated by 120 pulses at 3 Hz in the presence of rauwolscine and phentolamine, or when they were stimulated by 20 pulses at 50 Hz. 3. In tissues from mice lacking the alpha(2A)-adrenoceptor (alpha(2A)KO) or the alpha(2B)-adrenoceptor (alpha(2B)KO), the concentration-response curves of angiotensin II and bradykinin (120 pulses at 3 Hz) were unchanged. In tissues from mice lacking the alpha(2C)-adrenoceptor (alpha(2C)KO) or both the alpha(2A)- and the alpha(2C)-adrenoceptor (alpha(2AC)KO), the concentration-response curves were shifted to the same extent downwards. 4. As in WT tissues, angiotensin II and bradykinin lost most or all of their effect in alpha(2A)KO and alpha(2AC)KO tissues when rauwolscine and phentolamine were present or trains consisted of 20 pulses at 50 Hz. 5. Rauwolscine and phentolamine increased tritium overflow evoked by 120 pulses at 3 Hz up to seven-fold in WT and alpha(2B)KO tissues, three-fold in alpha(2A)KO and alpha(2C)KO tissues, and two-fold in alpha(2AC)KO tissues. 6. Results confirm that angiotensin II and bradykinin require ongoing alpha(2)-autoinhibition for the full extent of their release-enhancing effect. Specifically, they require ongoing alpha(2C)-autoinhibition. The peptide effects that remain in alpha(2C)-autoreceptor-deficient mice seem to be because of alpha(2B)-autoinhibition. The results hence also suggest that in addition to alpha(2A)- and alpha(2C)- mouse postganglionic sympathetic neurons possess alpha(2B)-autoreceptors.     

Functional P2X7 receptors at cultured hippocampal astrocytes but not neurons

P2X7 receptors have been suggested to be located both on neurons and astrocytes of the central and peripheral nervous systems. In the present Ca²⁺-imaging and patch-clamp study, we reinvestigated these findings on mixed neuronal–astrocytic cell cultures prepared from embryonic or newborn rat hippocampi. We found in a Mg²⁺-free bath medium that the prototypic P2X7 receptor agonist dibenzoyl-adenosine triphosphate (Bz-ATP) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) both in the neuronal cell bodies and in their axo-dendritic processes only to a very minor extent. However, Bz-ATP produced marked [Ca²⁺]_i transients in the neuronal processes, when they grew above a glial carpet, which was uniformly sensitive to Bz-ATP. These glial signals might be misinterpreted as neuronal responses because of the poor focal discrimination by a fluorescent microscope. Most astrocytes had a polygonal shape without clearly circumscribable boundaries, but a subgroup of them had neuron-like appearance. The cellular processes of this astrocytic subgroup, just as their cell somata and their polygonal counterparts, appeared to possess a high density of functional P2X7 receptors. In contrast to astrocytes, in a low Ca²⁺/no Mg²⁺-containing bath medium, hippocampal neurons failed to respond to Bz-ATP with membrane currents. In addition, neither the amplitude nor the frequency of spontaneous excitatory postsynaptic currents, representing the quantal release of glutamate, was modified by Bz-ATP. We conclude that cultured hippocampal neurons, in contrast to astrocytes, possess P2X7 receptors, if at all, only at a low density.     

Subunit specificity of polyclonal antisera to the carboxy terminal regions of P2X receptors,P2X1 through P2X7

Polyclonal antisera generated to peptides corresponding to the carboxy termini of the seven cloned rat P2X receptors were compared using membranes from CHO‐K1 or 1321N1 cells expressing recombinant rat or human P2X receptors (rat P2X₃, P2X₄, and P2X₅ and human P2X₁, P2X₂, P2X₆, and P2X₇). Preimmune sera failed to recognize any bands in immunoblots against the membrane fractions. Antiserum to the P2X₁ receptor recognized a band of approximately 58 kDa while those to the rP2X₂ and the rP2X₃ receptors recognized doublets of approximately 60 and 64 kDa. The antiserum to the rP2X₄ receptor recognized a similarly sized doublet as well as a higher molecular weight species at the expected size for a receptor dimer. Antiserum to the rP2X₅ receptor revealed a single band at 64 kDa while antiserum to rP2X₆ and rP2X₇ gave single bands at 50 kDa and 95 kDa, respectively. In all cases, immunoreactivity was eliminated by preincubation of the antisera with the cognate peptide and there was no cross‐reactivity of antisera with heterologous receptors. These antisera are useful reagents for the analysis of the P2X receptor subtypes in native tissues as well as for measuring receptor expression levels in transfected cell lines. Drug Dev. Res. 47:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

Modulation of P2X3 receptors by Mg2+ on rat DRG neurons in culture

On nociceptive neurons the commonest response to ATP is a rapidly desensitizing current mediated by P2X(3) receptors and believed to be involved in certain forms of pain. P2X(3) receptor recovery from desensitization is a slow process. We studied whether Mg(2+) might modulate such ATP-evoked currents on rat cultured DRG neurons, and thus account for its analgesic action in vivo. Transient increases in extracellular Mg(2+) strongly and reversibly depressed ATP currents which had not recovered from desensitization. Ca(2+)-free solution had the same action as Mg(2+). High Mg(2+) or Ca(2+)-free modulation depended on exposure length to modified divalent cation solutions, whereas it was independent from membrane potential or intracellular Ca(2+) buffering. Paired-pulse protocols showed that high Mg(2+) or Ca(2+)-free medium delayed ATP receptor recovery from desensitization, while leaving desensitization onset apparently unchanged. Tests with various concentrations of Ca(2+) and Mg(2+) showed that the depressant action by Mg(2+) was primarily due to functional antagonism of a facilitatory effect of Ca(2+) on ATP receptor function. The present results suggest that, on sensory neurons, P2X(3) receptors could be inhibited by high Mg(2+) or lack of Ca(2+), representing a negative feedback process to limit ATP-mediated nociception.