Biomedical subjects

A Surprenant

Publications and source records attributed to A Surprenant.

At least 37 records · Page 2Linked to original sources

PubMed1998

2',3'-O-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP)--a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels.

1. P2X receptor activation by alpha,beta-meATP evoked inward currents in acutely dissociated rat mesenteric artery smooth muscle cells and contractions of whole artery rings. 2. The selective P2X1 and P2X3 receptor antagonist TNP-ATP inhibited P2X receptor mediated inward currents in response to 3 microM alpha,beta-meATP (an approximately EC90 concentration) with an IC50 of approximately 2 nM. This provides further evidence that the P2X receptor underlying membrane depolarisation associated with P2X receptor activation can be accounted for by the expression of P2X1 receptors. 3. TNP-ATP inhibited alpha,beta-meATP induced contractions with an IC50 of approximately 30 microM and had non-specific effects on smooth muscle contraction. 4. The reduced potency of TNP-ATP in whole tissue experiments probably reflects the breakdown of TNP-ATP by nucleotidases. Thus, TNP-ATP is of limited use in whole tissue experiments as a P2X receptor antagonist.

Adenosine Triphosphate↗

PubMed1998

P2X3 is expressed by DRG neurons that terminate in inner lamina II.

The P2X3 receptor subunit, a member of the P2X family of ATP-gated ion channels, is almost exclusively localized in sensory neurons. In the present study, we sought to gain insight into the role of P2X3 and P2X3-containing neurons in sensory transmission, using immunohistochemical approaches. In rat dorsal root ganglia (DRG), P2X3-immunoreactivity (-ir) was observed in small- and medium-sized neurons. Approximately 40% of DRG neuronal profiles in normal rats contained P2X3-ir. In rats that had received neonatal capsaicin treatment, the number of P2X3-positive neurons was decreased by approximately 70%. Analysis of the colocalization of P2X3-ir with cytochemical markers of DRG neurons indicated that approximately 94% of the P2X3-positive neuronal profiles were labelled by isolectin B4 from Bandeiraea simplicifolia, while only 3% contained substance P-ir, and 7% contained somatostatin-ir. In dorsal horn of rat spinal cord, P2X3-ir was observed in the inner portion of lamina II and was reduced subsequent to dorsal rhizotomy, as well as subsequent to neonatal capsaicin treatment. Finally, P2X3-ir accumulated proximal to the site of sciatic nerve ligation, and was seen in nerve fibres in skin and corneal epithelium. In summary, our results suggest that P2X3 is expressed by a functionally heterogeneous population of BSI-B4-binding sensory neurons, and is transported into both central and peripheral processes of these neurons.

Animals↗

PubMed1998

Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues.

1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and N-[1-[N-methyl-p-(5 isoquinolinesulfonyl)benzyl]-2-(4 phenylpiperazine)ethyl]-5-isoquinolinesulfonamide (KN-04) potently inhibit the human lymphocyte P2Z receptor, an ATP-gated cation channel [Br J Pharmacol 120:1483-1490 (1997)]. Although the molecular identity of the lymphocyte P2Z receptor has not been established, it shares many functional characteristics with the cloned P2X7 nucleotide receptor. We have tested whether these isoquinolines inhibit P2X receptor function in human embryonic kidney 293 cells that stably express the human or rat recombinant P2X7 receptors. ATP activation of cation currents and uptake of the organic dye ethidium were potently inhibited by KN-62 and KN-04 in human embryonic kidney cells expressing the human P2X7R but not the rat P2X7R, even though these species homologues share 80% amino acid identity. Introduction of the first 335 amino acids of the human P2X7R sequence conferred KN-62 sensitivity to the rat P2X7R; this suggests that isoquinolines interact with residues in the amino-terminal half (containing the large extracellular loop) of the human P2X7R. KN-62 and KN-04 also potently inhibited ATP-gated Ca2+ influx and ethidium uptake in several leukocyte cell lines (THP-1, BAC1.2f5, and BW5147) that natively express the human or murine P2X7R mRNA. The ability of isoquinoline sulfonamides to potently inhibit human and murine P2X7R signaling will be a useful tool for identifying P2Z/P2X7 functional responses in other cell types. The substantial differences in pharmacological sensitivity between rat and human P2X7R may also indicate structural domains important in channel/pore activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

A novel and efficient method for the stable expression of heteromeric ion channels in mammalian cells.

The 5' untranslated leader sequence from the encephalomyocarditis virus was used to engineer bicistronic or tricistronic expression vectors encoding two subunits (P2X2 and P2X3) of an ATP-gated cation channel. Human embryonic kidney (293) and chinese hamster ovary (CHO-K1) cells were transfected with the vector, and stable cell lines were generated by single cell subcloning. Selection was made in a 96-well format on the basis of a sustained increase in intracellular calcium (fluorescence of Fluo3-loaded cells) evoked by the ATP analog alpha beta methylene ATP. A high proportion of transformants expressed heteromeric receptors containing both P2X2 and P2X3 subunits, as evidenced by a nondesensitizing current in response to alpha beta methylene ATP. The method is fast and simple and could be generally useful for the stable expression of heteromultimeric channel proteins.

Adenosine Triphosphate↗

PubMed1998

Trinitrophenyl-substituted nucleotides are potent antagonists selective for P2X1, P2X3, and heteromeric P2X2/3 receptors.

There are currently seven P2X receptor subunits (P2X1-7) defined by molecular cloning. The functional identification of these receptors has relied primarily on the potency of alpha,beta-methylene-ATP relative to that of ATP and on the kinetics of receptor desensitization. In the present experiments we found that the 2', 3'-O-(2,4,6-trinitrophenyl)-substituted analogs of ATP are selective and potent antagonists at some but not all P2X receptors. The trinitrophenyl analogs of ATP, ADP, AMP, and GTP produced a reversible inhibition of ATP-evoked currents in human embryonic kidney 293 cells expressing P2X1 receptors, P2X3 receptors, or both P2X2 and P2X3 (heteromeric) receptors; IC50 values were close to 1 nM. These compounds were at least 1000-fold less effective in blocking currents in cells expressing P2X2, P2X4, or P2X7 receptors (P2X5 and P2X6 not tested). GTP, 2,4,6-trinitrophenol, and the 2',3'-trinitrophenyl analog of adenosine (0.1-10 microM) had no effect. Thus, we have identified a structural motif that confers antagonist action at P2X receptors that contain P2X1 or P2X3 subunits (the alpha,beta-methylene-ATP-sensitive subclass).

Animals↗

PubMed1997

Baculovirus expression provides direct evidence for heteromeric assembly of P2X2 and P2X3 receptors.

P2X2 and P2X3 are subunits of P2X receptors, cation channels opened by binding extracellular ATP. cDNAs encoding P2X2 and P2X3 receptor subunits, each with one of two C-terminal epitope tags, were cloned into baculovirus. Virally infected insect cells (Spodoptera frugiperda) expressed moderate to high levels of the corresponding proteins, as detected by Western blotting, by the specific binding of [35S]ATP and by whole-cell recordings of membrane current evoked by ATP or alphabetamethylene-ATP. In cells infected at the same time with two viruses encoding P2X2 and P2X3 receptors, the two proteins could be cross-immunoprecipitated with antibodies specific for either of the epitope tags. Whole-cell recordings from these cells showed that ATP and alphabetamethylene-ATP evoked currents with agonist sensitivity and desensitization quite distinct from those observed when P2X2 or P2X3 receptors were expressed alone. The results offer a method to express large amounts of P2X receptor protein, and they provide direct evidence that P2X2 and P2X3 subunits assemble to form heteromeric channels having distinct properties from those formed as homomers.

Adenosine Triphosphate↗

PubMed1997

Identification of amino acid residues contributing to the pore of a P2X receptor.

P2X receptors are ion channels opened by extracellular ATP. The seven subunits currently known are encoded by different genes. It is thought that each subunit has two transmembrane domains, a large extracellular loop, and intracellular N- and C-termini, a topology which is fundamentally different from that of other ligand-gated channels such as nicotinic acetylcholine or glutamate receptors. We used the substituted cysteine accessibility method to identify parts of the molecule that form the ionic pore of the P2X2 receptor. Amino acids preceding and throughout the second hydrophobic domain (316-354) were mutated individually to cysteine, and the DNAs were expressed in HEK293 cells. For three of the 38 residues (I328C, N333C, T336C), currents evoked by ATP were inhibited by extracellular application of methanethiosulfonates of either charge (ethyltrimethylammonium, ethylsulfonate) suggesting that they lie in the outer vestibule of the pore. For two further substitutions (L338C, D349C) only the smaller ethylamine derivative inhibited the current. L338C was accessible to cysteine modification whether or not the channel was opened by ATP, but D349C was inhibited only when ATP was concurrently applied. The results indicate that part of the pore of the P2X receptor is formed by the second hydrophobic domain, and that L338 and D349 are on either side of the channel 'gate'.

Adenosine Triphosphate↗

PubMed1997

Desensitization of the P2X(2) receptor controlled by alternative splicing.

P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X(2) receptor splice variant (P2X(2-2)) carrying a 207 bp deletion in the intracellular C-terminus and the analysis of the corresponding genomic structure of the P2X(2) gene. P2X(2-2) is as highly expressed as the original P2X(2) sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X(2) or P2X(2-2) receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X(2) isoforms.

Adenosine Triphosphate↗

PubMed1997

The permeabilizing ATP receptor, P2X7. Cloning and expression of a human cDNA.

A cDNA was isolated from a human monocyte library that encodes the P2X7 receptor; the predicted protein is 80% identical to the rat receptor. Whole cell recordings were made from human embryonic kidney cells transfected with the human cDNA and from human macrophages. Brief applications (1-3 s) of ATP and 2', 3'-(4-benzoyl)-benzoyl-ATP elicited cation-selective currents. When compared with the rat P2X7 receptor, these effects required higher concentrations of agonists, were more potentiated by removal of extracellular magnesium ions, and reversed more rapidly on agonist removal. Longer applications of agonists permeabilized the cells, as evidenced by uptake of the propidium dye YO-PRO1, but this was less marked than for cells expressing the rat P2X7 receptor. Expression of chimeric molecules indicated that some of the differences between the rat and human receptor could be reversed by exchanging the intracellular C-terminal domain of the proteins.

Amino Acid Sequence↗

PubMed1997

Immunohistochemical study of the P2X2 and P2X3 receptor subunits in rat and monkey sensory neurons and their central terminals.

Of the cloned P2X receptor subunits, six are expressed in sensory neurons, suggesting that the native channels may be heteromultimers with diverse composition. It has been proposed that P2X2 and P2X3 form heteromultimers in sensory neurons. We further tested this hypothesis by examining the relationship of P2X2 and P2X3 immunocytochemically. In rat dorsal root and nodose ganglia, P2X2- and P2X3-immunoreactivity (-ir) were highly colocalized, although single-labeled cells were also present. In dorsal root ganglia (DRG), in some cases P2X2-ir appeared to be present in satellite cells. In dorsal horn of spinal cord, at low magnification the laminar localization of P2X2- and P2X3-ir overlapped, but at high magnification colocalization was rarely observed. In contrast, in the solitary tract and its nucleus (NTS), colocalization of P2X2- and P2X3-ir was seen at low and high magnification. These results suggest that the relationship of P2X2- and P2X3-ir is different in nodose and dorsal root ganglia and might reflect differences in the targeting of P2X receptors in different sensory neurons. In monkey, P2X2-ir was observed in DRG neurons and satellite cells and in dorsal horn of spinal cord. P2X3-ir was also seen in DRG neurons. However, the presence of P2X2-ir in NTS as well as the presence of P2X3-ir in spinal cord and NTS could not be established definitively. These results suggest species differences, although a more extensive study of primate sensory systems is necessary.

Animals↗

PubMed1997

Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor.

The P2X7 receptor is a uniquely bifunctional molecule through which ATP can open a small cationic channel typical of ionotropic receptors and also induce a large pore permeable to high molecular weight molecules (> 600 Da). Activation of this large pore can lead to cell lysis within 1-2 min. We asked whether pharmacological differences existed between the cationic channel and the cell permeabilizing pore by measuring whole-cell currents and uptake of a propidium dye (YO-PRO; Mw 629) in HEK293 cells stably expressing the rat P2X7 receptor, and comparing the actions of divalent cations and protons in these two assays. Currents in response to 2'-3'-(O)-(4-benzoyl benzoyl) ATP (BzATP, 30 microM) were inhibited by extracellular calcium, magnesium, zinc, copper and protons with half-maximal inhibitory concentrations (IC50) of 2.9 mM, 0.5 mM, 11 microM, 0.5 microM and 0.4 microM, respectively. The inhibition was voltage independent in each case. YO-PRO uptake induced by BzATP was also inhibited with similar IC50 values. The rank order of potency of a range of divalents was Cu2+ > Cd2+ = Zn2+ > Ni2+ >> Mg2+ = Co2+ > Mn2+ > Ca2+ = Ba2+ >> Sr2+. These results suggest that these divalent cations and protons all act primarily as allosteric modulators to alter the affinity of ATP binding to the P2X7 receptor. In contrast, extracellular (but not intracellular) calmidazolium inhibited the BzATP-evoked current by up to 90% (IC50 = 15 nM) but had no effect on YO-PRO uptake. Thus, calmidazolium can block activation of the ionic channel but this does not prevent the formation of the large permeabilizing pore.

Adenosine Triphosphate↗

PubMed1997

Different sensitivities to pH of ATP-induced currents at four cloned P2X receptors.

The effect of changing extracellular pH was studied on the currents induced by ATP or alphabeta-methylene-ATP in HEK293 cells transfected with different P2X receptor subunits. In cells expressing P2X1, P2X3, or P2X4 receptors, the effect of ATP was decreased by acidification. In cells expressing P2X2 receptors, acidification increased the ATP-induced current; this effect was also seen in cells expressing heteromeric P2X2 and P2X3 receptors. At P2X2 receptors, acidification caused a leftward shift in the ATP concentration-response curve, without change in maximum; the pKa for this effect was 7.3. At P2X4 receptors, acidification caused a rightward shift in the ATP concentration-response curve, without change in the maximum; the pKa for this effect was 6.8. The pH dependence of the action of ATP should be taken into account in studies of synaptic transmission, and it may provide a further tool to assign molecular identity to P2X receptors expressed by brain neurons.

Adenosine Triphosphate↗

PubMed1996

Ionic permeability of, and divalent cation effects on, two ATP-gated cation channels (P2X receptors) expressed in mammalian cells.

1. Complementary DNAs for the ATP-gated ion channel subunits P2X1 (from human bladder) and P2X2 (from rat phaeochromocytoma (PC12) cells) were used to express the receptors in human embryonic kidney cells by stable transfection, and in Chinese hamster ovary cells by viral infection. 2. Membrane currents evoked by ATP were recorded by the whole-cell patch clamp method. The reversal potential of the current was measured with various intracellular and extracellular solutions and used to compute the relative permeability of the P2X receptor channels. 3. There was no difference between the two receptors with respect to their permeability to monovalent organic cations. The relative permeabilities (PX/PNa) were 2.3, 1.0, 1.0, 0.95, 0.72, 0.5, 0.29, 0.16, 0.04 and 0.03 for guanidinium, potassium, sodium, methylamine, caesium, dimethylamine, 2-methylethanolamine, tris(hydroxymethyl)-aminomethane, tetraethylammonium and N-methyl-D-glucamine, respectively (values for P2X2 receptor). 4. The calcium permeability of P2X1 receptors was greater than that of P2X2 receptors. Under biionic conditions (112 mM calcium outside, 154 mM sodium inside), PCa/PNa values were 3.9 and 2.2, respectively (corrected for ionic activities). 5. ATP-evoked currents in cells expressing the P2X2 receptor were strongly inhibited when the extracellular calcium concentration was increased (0.3-30 mM); the action of ATP could be restored by increasing the ATP concentration. ATP-evoked currents in cells expressing the P2X1 receptor were not inhibited by such increases in the extracellular calcium concentration.

Adenosine Triphosphate↗

PubMed1996

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

Amino Acid Sequence↗

PubMed1996

The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).

The P2Z receptor is responsible for adenosine triphosphate (ATP)-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules. Other ATP-gated channels, the P2X receptors, are permeable only to small cations. Here, an ATP receptor, the P2X7 receptor, was cloned from rat brain and exhibited both these properties. This protein is homologous to other P2X receptors but has a unique carboxyl-terminal domain that was required for the lytic actions of ATP. Thus, the P2X7 (or P2Z) receptor is a bifunctional molecule that could function in both fast synaptic transmission and the ATP-mediated lysis of antigen-presenting cells.

Adenosine Triphosphate↗

PubMed1996

Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels.

Two new P2X receptor cDNAs (P2X5 and P2X6) were isolated and expressed. All six proteins are 36-48 percent identical and seem to have two transmembrane segments with a large extracellular loop. Functionally, P2X5 and P2X6 receptors most resemble P2X2 and P2X4; they desensitize only slowly and do not respond to alpha beta methylene-ATP. P2X6 receptors, like P2X4, receptors, are not blocked by the antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid. P2X6 and P2X5 receptors express at lower levels than P2X1-P2X4 receptors do, perhaps indicating that they do not normally form homomultimeric channels. P2X6 and P2X4 are the receptors expressed most heavily in brain, where their RNAs have a widespread and extensively overlapping distribution. The spinal cord expresses all receptors except P2X3. P2X2, P2X4, and P2X6, are the most abundant in the dorsal horn. Sensory neurons of the trigeminal, dorsal root, and nodose ganglia express all six RNAs; P2X3 is found only there. The functional properties and tissue distribution of these six P2X receptors indicate new roles for ATP-gated ion channels.

Adenosine Triphosphate↗

PubMed1996

P2X1 receptor activation in HL60 cells.

Recent cloning of the human P2X1 receptor revealed high levels of its messenger RNA in differentiated promyelocytes (HL60 cells). We found expression of P2X1 receptor protein in HL60 cells by radioligand binding, by immunohistochemistry, using a receptor specific antibody, and by electrophysiology. The currents elicited by adenosine triphosphate (ATP) had the expected properties of P2X1 receptors (rapid desensitization, mimicked by alpha,beta-methylene-ATP). However, these currents were only observed in cells that were pretreated with apyrase, which destroys extracellular ATP, or with suramin, a P2X receptor antagonist. This implies that HL60 cells release ATP, which chronically desensitizes the receptor. ATP release was detected by direct measurement, using the luciferin-luciferase assay. It is concluded that functional P2X1 receptors are present in the membrane of differentiated HL60 cells.

Adenosine Triphosphate↗

PubMed1996

An antagonist-insensitive P2X receptor expressed in epithelia and brain.

A cDNA was cloned which encodes a new ATP-gated ion channel (P2X4 receptor). ATP induces a cationic current in HEK293 cells transfected with the P2X4 receptor. However, the current is almost completely insensitive to antagonists effective at other P2X receptors. Sensitivity to two of these antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and pyridoxal 5-phosphate) is restored by replacement of Glu249 by lysine, which occurs at the equivalent position in P2X1 and P2X2 receptors. P2X4 RNA is found by in situ hybridization in the brain, peripheral ganglia and epithelia including serosal cells of salivary glands. Recordings from rat submandibular gland cells showed ATP-induced currents that are also insensitive to antagonists. These results define a further member of P2X receptor family, and they identify an amino acid residue involved in antagonist binding. They also introduce a new phenotype for ATP responses at P2X receptors--insensitivity to currently known antagonists.

Adenosine Triphosphate↗