P2X Receptor Subtypes Research Articles

Functional expression of three P2X(2) receptor splice variants from guinea pig cochlea.

ATP has been suggested to act as a neurotransmitter or a neuromodulator in the cochlea. The responses to ATP in different cell types of the cochlea vary in terms of the rate of desensitization and magnitude, suggesting that there may be different subtypes of P2X receptors distributed in the cochlea. Recently three ionotropic P2X(2) receptor splice variants, P2X(2-1), P2X(2-2), and P2X(2-3,) were isolated and sequenced from a guinea pig cochlear cDNA library. To test the hypothesis that these different splice variants could be expressed as functional homomeric receptors, the three P2X(2) receptor variants were individually and transiently expressed in human embryonic kidney cells (HEK293). The biophysical and pharmacological properties of these receptors were characterized using the whole cell patch-clamp technique. Extracellular application of ATP induced an inward current in HEK293 cells containing each of the three splice variants in a dose-dependent manner indicating the expression of homomeric receptors. Current-voltage (I-V) relationships for the ATP-gated current show that the three subtypes of the P2X(2) receptor had a similar reversal potential and an inward rectification index (I(50 mV)/I(-50 mV)). However, the ATP-induced currents in cells expressing P2X(2-1) and P2X(2-2) variants were large and desensitized rapidly whereas the current in those cells expressing the P2X(2-3) variant was much smaller and desensitized slower. The order of potency to ATP agonists was 2-MeSATP > ATP > alpha,beta -MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on all three variants. Results demonstrate that three P2X(2) splice variants from guinea pig cochlea, P2X(2-1), P2X(2-2), and P2X(2-3), can individually form nonselective cation receptor channels when these subunits are expressed in HEK293 cells. The distinct properties of these P2X(2) receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.

Distribution of P2X receptor subtypes in the rat female reproductive tract at late pro-oestrus/early oestrus

Distribution of P2X receptor subtypes in the rat female reproductive tract at late pro-oestrus/early oestrus

Comparative study on the distribution patterns of P2X1-P2X6 receptor immunoreactivity in the brainstem of the rat and the common marmoset (Callithrix jacchus): Association with catecholamine cell groups

The present study investigated the topographical distribution of P2X(1)-P2X(6) receptor subtypes in the rat and common marmoset hindbrain by immunohistochemistry. In addition, double-labeling immunofluorescence was used to determine the extent of colocalization between catecholamine cell groups and the various P2X receptors. The data demonstrate a widespread distribution pattern for all six P2X receptors throughout both the rat hindbrain and the marmoset hindbrain, although distinctions between species, brain nuclei, and P2X receptor subtypes exist. In rat, dense staining for the P2X receptors was found in the nucleus of the solitary tract (NTS), medial vestibular nucleus, and medial and lateral parabrachial nuclei. Moderate staining was observed in the hypoglossal nucleus, cuneate nucleus, inferior olive, prepositus hypoglossi, rostral ventrolateral medulla (RVLM), and locus coeruleus. Staining was also observed in the gracile nucleus, the mesencephalic trigeminal nucleus, and the central pontine gray. In marmoset, prominent P2X receptor-like immunoreactivity occurred in the NTS, medial cuneate nucleus, prepositus hypoglossi, and medial vestibular nucleus. Moderate staining was observed in the area postrema, dorsal motor nucleus of the vagus, lateral cuneate, lateral reticular, spinal trigeminal nucleus, RVLM, and inferior olive. Immunofluorescent double labeling of tyrosine hydroxylase (TH)-containing cells revealed that all subtypes of P2X receptors show some degree of colocalization with TH. The highest proportion of TH and P2X receptor double labeling was in the A5 region (with the P2X(2) subunit), whereas the lowest proportion of double-labeled cells occurred in the C2 region of the NTS for the P2X(5) subunit. These findings support a role for extracellular adenosine 5'-triphosphate in fast synaptic neurotransmission within the brainstem.

Molecular and functional characterization of human P2X(2) receptors.

P2X receptors are a family of ATP-gated ion channels. Four cDNAs with a high degree of homology to the rat P2X(2) receptor were isolated from human pituitary and pancreas RNA. Genomic sequence indicated that these cDNAs represent alternatively spliced messages. Northern analysis revealed high levels of human P2X(2) (hP2X(2)) message in the pancreas, and splice variants could be detected in a variety of tissues. Two cDNAs encoded functional ion channels when expressed in Xenopus oocytes, a receptor structurally homologous to the prototype rat P2X(2) receptor (called hP2X(2a)) and a variant containing a deletion within its cytoplasmic C terminus (called hP2X(2b)). Pharmacologically, these functional human P2X(2) receptors were virtually indistinguishable, with the P2X receptor agonists ATP, 2-methylthio-ATP, 2' and 3'-O-(4-benzoylbenzoyl)-ATP, and ATP5'-O-(3-thiotriphosphate) being approximately equipotent (EC(50) = 1 microM) in eliciting extracellular Ca(2+) influx. The P2 receptor agonists alpha,beta-methylene ATP, adenosine, adenosine 5'-O-(2-thiodiphosphate), and UTP were inactive at concentrations up to 100 microM. Both hP2X(2a) and hP2X(2b) receptors were sensitive to the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (IC(50) = 3 microM). In contrast to the analogous rat P2X(2) and P2X(2b) receptors, the desensitization rates of the hP2X(2a) and hP2X(2b) receptors were equivalent. Both functional forms of the human P2X(2) receptors formed heteromeric channels with the human P2X(3) receptor. These data demonstrate that the gene structure and mRNA heterogeneity of the P2X(2) receptor subtype are evolutionarily conserved between rat and human, but also suggest that alternative splicing serves a function other than regulating the desensitization rate of the human receptor.

Distribution of P2X receptor subtypes in the rat female reproductive tract at late pro-oestrus/early oestrus.

Using immunohistochemistry techniques, we have examined the female reproductive organs of the rat in late pro-oestrus/early oestrus for the presence of purine nucleotide P2X1-7 receptors. In contrast to the male genital organs and the urinary tract, P2X1 receptors were present weakly, if at all, on smooth muscle membranes, except in blood vessels, whereas P2X2 immunoreactivity in smooth muscle was present in ovary and uterus as well as in blood vessels. Neither P2X1 nor P2X2 receptors were present in fallopian tubes. P2X5 receptors were seen in the differentiating cell layers of the stratified squamous vaginal epithelium and also in the very early stages of ovarian follicular development; P2X6 receptors were present in secondary follicles. P2X7 receptors, markers for programmed cell death, were present in the keratinised vaginal epithelium and also in the exfoliating superficial endometrial cells. The possible biological significance of these signalling molecules in the female reproductive tract is discussed.

Distribution of P2X receptors in the rat adrenal gland.

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.

Lack of specificity of [35S]-ATP?S and [35S]-ADP?S as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine-5'-O-3-thio[ 35 S]triphosphate ([ 35 S]-ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X 1 , P2X 2 , P2X 3 , and P2X 4 . Similarly, adenosine-5'-O-2-thio[ 35 S]diphosphate ([ 35 S]-ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [ 35 S]-ATPγS and [ 35 S]-ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X 4 transfected 1321N1 astrocytoma cells. [ 35 S]-ATPγS (0.2 nM) and [ 35 S]-ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [ 35 S]-ATPγS and [ 35 S]-ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y 1 and P2Y 2 receptor subtypes, to 1321N1 cells stably transfected with the human P2X 4 receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X 4 receptor, as measured by cytosolic calcium flux. These data indicate that [ 35 S]-ATPγS and [ 35 S]-ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone.

P2X receptor-mediated ionic currents in dorsal root ganglion neurons.

Nociceptive neurons in the dorsal root ganglia (DRG) are activated by extracellular ATP, implicating P2X receptors as potential mediators of painful stimuli. However, the P2X receptor subtype(s) underlying this activity remain in question. Using electrophysiological techniques, the effects of P2X receptor agonists and antagonists were examined on acutely dissociated adult rat lumbar DRG neurons. Putative P2X-expressing nociceptors were identified by labeling neurons with the lectin IB4. These neurons could be grouped into three categories based on response kinetics to extracellularly applied ATP. Some DRG responses (slow DRG) were relatively slowly activating, nondesensitizing, and activated by the ATP analogue alpha,beta-meATP. These responses resembled those recorded from 1321N1 cells expressing recombinant heteromultimeric rat P2X2/3 receptors. Other responses (fast DRG) were rapidly activating and desensitized almost completely during agonist application. These responses had properties similar to those recorded from 1321N1 cells expressing recombinant rat P2X3 receptors. A third group (mixed DRG) activated and desensitized rapidly (P2X3-like), but also had a slow, nondesensitizing component that functionally prolonged the current. Like the fast component, the slow component was activated by both ATP and alpha, beta-meATP and was blocked by the P2X antagonist TNP-ATP. But unlike the fast component, the slow component could follow high-frequency activation by agonist, and its amplitude was potentiated under acidic conditions. These characteristics most closely resemble those of rat P2X2/3 receptors. These data suggest that there are at least two populations of P2X receptors present on adult DRG nociceptive neurons, P2X3 and P2X2/3. These receptors are expressed either separately or together on individual neurons and may play a role in the processing of nociceptive information from the periphery to the spinal cord.

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.

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 termi 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 P2X 3 , P2X 4 , and P2X 5 and human P2X 1 , P2X 2 , P2X 6 , and P2X 7 ). Preimmune sera failed to recognize any bands in immunoblots against the membrane fractions. Antiserum to the P2X 1 receptor recognized a band of approximately 58 kDa while those to the rP2X 2 and the rP2X 3 receptors recognized doublets of approximately 60 and 64 kDa. The antiserum to the rP2X 4 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 rP2X 5 receptor revealed a single band at 64 kDa while antiserum to rP2X 6 and rP2X 7 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.

Pharmacological characterization of recombinant human and rat P2X receptor subtypes

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X 1, hP2X 3, hP2X 4, and hP2X 7), and two rat P2X receptor subtypes (rP2X 2 and rP2X 3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X 2 and rP2X 3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca 2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. αβ-methylene ATP (αβ-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X 1, hP2X 3 and rP2X 3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2′ and 3′ mixed isomers), which has been reported to act as a P2X 7 receptor-selective agonist, was least active at the rat and human P2X 7 receptors, but was a potent (nM) agonist at hP2X 1, rP2X 3 and hP2X 3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Characterization of "mini-nucleotides" as P2X receptor agonists in rat cardiomyocyte cultures. An integrated synthetic, biochemical, and theoretical study.

The design and synthesis of "mini-nucleotides", based on a xanthine-alkyl phosphate scaffold, are described. The physiological effects of the new compounds were evaluated in rat cardiac cell culture regarding Ca(2+) elevation and contractility. The results indicate biochemical and physiological profiles similar to those of ATP, although at higher concentrations. The biological target molecules of these "mini-nucleotides" were identified by using selective P2-R and A(1)-R antagonists and P2-R subtype selective agonists. On the basis of these results and of experiments in Ca(2+) free medium, in which [Ca(2+)](i) elevation was not observed, we concluded that interaction of the analogues is likely with P2X receptor subtypes, which causes Ca(2+) influx. Theoretical calculations analyzing electronic effects within the series of xanthine-alkyl phosphates were performed on reduced models at quantum mechanical levels. Calculated dipole moment vectors, electrostatic potential maps, and volume parameters suggest an explanation for the activity or inactivity of the synthesized derivatives and predict a putative binding site environment for the active agonists. Xanthine-alkyl phosphate analogues proved to be selective agents for activation of P2X-R subtypes, whereas ATP activated all P2-R subtypes in cardiac cells. Therefore, these analogues may serve as prototypes of selective drugs aiming at cardiac disorders mediated through P2X receptors.

P2X receptors in the rat duodenal villus.

Immunohistochemical techniques were performed on freshly frozen sections of the duodenum of the rat using specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Of the antibodies to the seven known P2X receptor subtypes that mediate extracellular signalling by nucleotides, three reacted with discrete structures in the duodenal villus of the rat. Anti-P2X1 reacted with the capillary plexus in the intestinal villus, which did not extend to the crypt region, suggesting that nucleotides may be involved in the uptake and transport of metabolites. Anti-P2X5 immunostained the membranes of the narrow "stem" of villus goblet cells, where the nucleus and cell organelles reside, possibly influencing synthesis and release of mucins. P2X7 receptor immunoreactivity was only seen in the membranes of enterocytes and goblet cells at the tip of the villus, where cells are exfoliated into the lumen, consistent with earlier findings that P2X7 is involved in apoptotic events. Thus, in complex structures such as the intestinal villus, purinoceptors appear to participate in several and diverse signalling functions.

Ca2+-Permeable P2X Receptor Channels in Cultured Rat Retinal Ganglion Cells

ATP has been identified as an excitatory neurotransmitter in both the CNS and peripheral nervous system; however, little is known about the functional properties of ATP-gated channels in central neurons. Here we used a culture preparation of the postnatal rat retina to test the responsiveness of identified retinal ganglion cells (RGCs) and putative amacrines to exogenous ATP and other purinoceptor agonists. Rapidly activating ATP-induced currents (IATP) were exclusively generated in a subpopulation (approximately 65%) of RGCs. The latter were identified by Thy1.1 immunostaining, repetitive firing patterns, and activation of glutamatergic autaptic currents. None of the putative amacrine cells was ATP-sensitive. IATP could be induced with ATP, ADP, and alpha,beta-mATP but not with adenosine. It was antagonized by suramin. The current-voltage relationship of IATP showed marked inward rectification. Dose-response analysis yielded an EC50 of 14.5 microM, with a Hill coefficient of 0.9. Noise analysis of IATP suggested a mean single channel conductance of 2.3 pS. Retinal P2X purinoceptor channels exhibited a high permeability for Ca2+. PCa/PCs obtained from reversal potentials of IATP under bi-ionic conditions amounted to 2. 2 +/- 0.7. In the majority of cells, the decay of IATP was biphasic. The degree of current inactivation during the first 2 sec of agonist application was highly variable. Heterogeneity was also found with respect to the sensitivity to ADP and alpha,beta-mATP and the blocking action of suramin, suggesting expression of multiple P2X receptor subtypes. Our results indicate that activation of P2X receptor channels represents an important pathway for Ca2+ influx in postnatal RGCs.

Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X(1) to P2X(6) receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X(1) to P2X(6) receptor subtypes (about 0.4 microm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X(1) to P2X(6) receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X(1-6) small receptor clusters so that they became colocalized, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalization. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalized P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.

Chapter 1 Current status of purinergic signalling in the nervous system

The chapter discusses the current status of purinergic signaling in the nervous system. Implicit in the concept of purinergic neurotransmission is the existence of postjunctional purinergic receptors. There is still more to be learned about the distribution of P2X receptor subtypes in different tissues. The knowledge of the distribution based on northern blots and in situ hybridization methodology is valuable, but now the selective antibodies are becoming available, immunolabeling at both light and electron microscope levels is giving significant new information in this field. For example, P2X, receptors were previously not considered to be present in the brain; however, recent cytochemical studies of the cerebellum show clear evidence of synaptic transmission involving this receptor. It has also become clear that ATP receptors in vascular smooth muscle are not only of subtype P2X1, but P2X2 and P2X4 receptors have also been demonstrated. Most studies of fast signaling in the nervous system have been concerned with the role of ATP as a cotransmitter.

Characterization of cultured dorsal root ganglion neuron P2X receptors.

P2X receptors for adenosine 5'-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1-7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-meATP) and 2-methyl-thioadenosine 5'-triphosphate (2-meSATP), and using the antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2',5' disulphonic acid (PPADS). ATP (10 microM) evoked inward currents in approximately 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, approximately 4% showed a sustained response and approximately 10% showed a combination of transient and sustained components. Rapid application of ATP, alpha,beta-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-beta,gamma-methyleneadenosine 5'-triphosphate (1-beta,gamma-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.

Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia

The distribution of P2X 1, P2X 2, P2X 3, P2X 4, P2X 5 and P2X 6 receptors, a family of ATP-gated cation channels, in rat trigeminal, dorsal root, nodose, superior cervical, and coeliac ganglia was studied immunohistochemically. It was found that polyclonal antibodies for the six P2X receptor subtypes could label over 90% of neurones in these ganglia to different intensities. There was also considerable variation in intensity of immunoreactivity in individual neurones within each ganglion. In dorsal root and trigeminal ganglia expression of the P2X 3 receptor was much higher than for the other five subtypes. P2X 3 immunoreactivity was much more intense in the small-diameter neurones than in the large-diameter neurones. In nodose ganglion, both P2X 2 and P2X 3 antibodies labelled the neurones intensely. In sympathetic superior cervical and coeliac ganglia, immunoreactivity to five P2X receptor subtypes were detected (exception, P2X 5), with P2X 2, P2X 4 and P2X 6 showing higher intensity. Low level expression of P2X 3 receptor in sympathetic ganglia indicates that this receptor subtype is not limited to the sensory ganglia where it was highly expressed. The results have demonstrated that both sensory and sympathetic ganglia express a variety of P2X receptor subtypes and that different subtypes are expressed to different levels and by different subpopulations of neurones.

P2X7 receptor activation-induced contraction and lysis in human saphenous vein smooth muscle.

In cutaneous veins where purinergic neurotransmission is more prominent compared with in deep vessels, physiological and pathological roles of nerve-released ATP have been described. Neuronally released ATP has been reported to act through activation of unidentified ionotropic P2X receptor(s). This study analyzed P2X receptor subtypes expressed in human saphenous vein smooth muscle and their physiological functions. Transcripts for both hP2X1 receptors, already identified in other smooth muscles, and, surprisingly, hP2X7 receptors known to be responsible for the cytotoxic effect of ATP in macrophages were detected by Northern blot analysis in total RNA from saphenous vein smooth muscle. ATP and other P2X receptor agonists [alphabeta-methylene-ATP, 2-methylthio-ATP, and 2',3'-(4-benzoyl)benzoyl-ATP] dose-dependently contracted venous rings, but the contraction induced by 2-methylthio-ATP was more transient than that evoked by the other P2X agonists. The effect of hP2X1 agonists involved the activation of a rapidly desensitizing cation current recorded in freshly isolated myocytes. The action of hP2X7 receptor agonists was related to a maintained nondesensitizing cation current. In addition, hP2X7 receptor activation formed membrane pores that were permeable to large molecules. hP2X1 and hP2X7 receptors coexpressed in COS cells did not associate to form heteromultimers. Our data indicate that both hP2X1 and hP2X7 receptors are expressed as 2 separated populations of channels in human saphenous vein myocytes and are involved in ATP-induced tension. We suggest that cell lysis consequent to hP2X7 receptor-induced pore formation contributes to the disorganization and decrease in the amount of contractile myocytes in the media of varicose veins.

Evidence that P2X purinoceptors mediate the excitatory effects of αβmethylene-ADP in rat locus coeruleus neurones

Extracellular and whole-cell patch clamp recordings were used to study the excitatory responses elicited by purine nucleotides in pontine slices of the rat brain containing the locus coeruleus (LC). The P2 purinoceptor agonists, α β-methyleneadenosine 5′-triphosphate ( α βmeATP) and adenosine 5′- O-(2-thiodiphosphate) (ADP βS), and a novel purinoceptor agonist, α β-methyleneadenosine 5′-diphosphate ( α βmeADP), elicited concentration-dependent increases in the spontaneous firing rate over the concentration range (1–300 μM). On vagus nerve or dorsal root preparations α βmeADP (100 μM) had no agonist activity. In the presence of both α βmeATP (300 μM), ADP βS (300 μM) elicited a further and significant increase in the firing rate of the LC neurones, whilst neither α βmeATP nor α βmeADP (of 300 μM) elicited a further response. The P2 purinoceptor antagonists, suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS; 30 μM), markedly attenuated responses to all three agonists. Whole-cell recording of membrane current showed that, at −60 mV, α βmeATP and α βmeADP (both 100 μM) elicited inward currents of a similar magnitude, whilst the inward currents elicited by a lower concentration of ADP βS (30 μM) were larger and faded in the presence of this agonist. In the presence of tetrodotoxin and a combination of other neurotransmission blockers, both α βmeATP and α βmeADP still produced inward currents. Based on the known selectivity of the agonists used in this study, there appear to be two distinct P2 purinoceptor types present on neurones in the LC, which correspond to the P2X and P2Y types. The responses elicited by α βmeADP appear to be mediated through a putative P2X purinoceptor, although further work is required to determine which P2X receptor subtype(s) are involved.