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G Buell

Publications and source records attributed to G Buell.

At least 19 recordsLinked to original sources
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

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

ATP-mediated cytotoxicity in microglial cells.

Microglial cells are known to express purinergic receptors for extracellular ATP of both the P2Y and P2X subtypes. Functional studies have shown that both primary mouse microglial cells and the N9 and N13 microglial cell lines express the pore-forming P2Z/P2X7 receptor. Here we identify the presence of this receptor in N9 and N13 cells with a specific polyclonal Ab and show that microglial cells expressing the P2Z/P2X7 receptor are exquisitively sensitive to ATP-mediated cytotoxicity while clones selected for the lack of this receptor are resistant. Transfection of HEK293 cells with P2X7 (but not P2X2) receptor cDNA confers susceptibility to ATP-mediated cytotoxicity. Morphological and biochemical analysis suggests that ATP-dependent cell death in microglial cells occurs by apoptosis. Finally, microglial cells release ATP via a non-lytic mechanism when activated by bacterial endotoxin, thus suggesting the operation of a purinergic autocrine/paracrine loop.

Adenosine Triphosphate↗
PubMed1997

Tissue distribution of the P2X7 receptor.

The P2X7 receptor is a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules as large as propidium dyes (629 Da). In situ hybridization using a digoxigenin-labelled riboprobe, and immunohistochemistry using an antibody raised against a C-terminal peptide sequence, were used to determine the distribution of the P2X7 receptor mRNA and protein in rat and mouse tissues and cell lines. The brain of newborn rats showed a 6 kb RNA by Northern blotting, but this was not detectable in adult brain. By in situ hybridization and immunohistochemistry, there was heavy labelling of ependymal cells in both newborn and adult brain, but the brain parenchyma showed no labelling. However, P2X7 receptor-immunoreactive cells appeared in the penumbral region around an area of necrosis evoked by prior occlusion of the middle cerebral artery, suggesting expression of the receptor by activated microglia. NTW8 cells, a mouse microglial cell line, strongly expressed the P2X7 receptor mRNA and protein. The P2X7 receptor mRNA and protein were also observed in the majority of bone marrow cells, including those separately identified by their expression of other antigens as granulocytes, monocyte/macrophages and B lymphocytes. The expression of P2X7 receptor by brain macrophages rather than neurons would be consistent with a role in brain repair following inflammation, infarction or immune insult.

Animals↗
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↗
PubMed1996

The involvement of an ATP-gated ion channel, P(2X1), in thymocyte apoptosis.

In the immune system, apoptosis is involved in intrathymic elimination of self-reactive thymocytes and in peripheral T cell tolerance to exogenous antigens. Here, we describe the role in T cell apoptosis of P(2x1), a nonselective cation channel activated by ATP. P(2X1) molecules are up-regulated in thymocytes during dexamethasone-induced apoptosis, and antagonists to these receptors protect thymocytes from cell death. Moreover, P(2X1) mRNA and protein levels increase in thymocytes induced to die in vivo by the superantigen staphylococcal enterotoxin B. In contrast, T cells undergoing apoptosis in the periphery do not express P(2X1). The demonstration that P(2X1) ion channels play a role in the apoptosis of thymocytes but not peripheral T cells illustrates a novel mechanism contributing to thymocyte cell death and opens new possibilities for investigating clonal deletion in the thymus.

Adenosine Triphosphate↗
PubMed1996

Differences in the operational characteristics of the human recombinant somatostatin receptor types, sst1 and sst2, in mouse fibroblast (Ltk-) cells.

1. The human recombinant somatostatin (SRIF) receptors, sst1 and sst2, have been stably expressed in mouse fibroblast (Ltk-) cells. Two stable clones, LSSR 1/20 and LSSR 11/13, expressing sst1 and sst2 receptors, respectively, have been used to characterize these receptor types using radioligand binding assays as well as measurements of changes in extracellular acidification rates using microphysiometry. 2. [125I]-[Tyr11]-SRIF bound to sst1 and sst2 receptors expressed in Ltk- cells with high affinity, Kd values being 1.52 nM, and 0.23 nM respectively. 3. In Ltk- cells expressing sst1 receptors, SRIF, SRIF-28, [D-Trp8]-SRIF and CGP 23996 all displaced [125I]-[Tyr11]-SRIF binding with high potency (IC50 values of 0.43 - 1.27 nM) whilst seglitide, BIM-23027, BIM-23056 and L-362855 were either weak inhibitors of binding or were ineffective. 4. In contrast MK-678 (seglitide) and BIM-23027 were the most potent inhibitors of [125I]-[Tyr11]-SRIF binding in Ltk- cells expressing sst2 receptors with IC50 values of 0.014 and 0.035 nM, respectively. 5. SRIF and a number of SRIF agonists, including seglitide and BIM-23027, caused concentration-dependent increases in extracellular acidification rates in Ltk- cells expressing sst2 receptors but not in Ltk- cells expressing sst1 receptors. The maximum increase in acidification rate produced by SRIF was 11.3 +/- 0.7% above baseline (0.1-0.28 pH unit min-1). The relative potencies of the SRIF agonists examined in causing increases in extracellular acidification rates in Ltk- cells expressing sst2 receptors correlated well with their relative potencies in inhibiting [125I]-[Tyr11] -SRIF binding (r = 0.94). 6. The increase in extracellular acidification produced by SRIF was markedly inhibited by pretreatment of cells with pertussis toxin (100 ng ml-1) indicating the involvement of pertussis toxin-sensitive G proteins. 7. SRIF (1 microM) had no effect on basal cyclic AMP levels in Ltk- cells expressing sst1 or sst2 receptors nor did it inhibit forskolin stimulated increases in cyclic AMP levels in either cell type. 8. The results from the present study describe the operational characteristics of human sst2 receptors expressed in Ltk- cells where receptor activation causes increases in extracellular acidification rates. This receptor is coupled to a pertussis toxin-sensitive G protein. In contrast, activation of sst1 receptors, at a similar transfection density, did not cause increases in extracellular acidification rates.

Amino Acid Sequence↗
PubMed1996

D2, D3, and D4 dopamine receptors couple to G protein-regulated potassium channels in Xenopus oocytes.

Human D2, D3, D4 and dopamine receptors were individually coexpressed in Xenopus oocytes with a G protein-regulated inwardly rectifying potassium channel (GIRK1). At -100 mV in 96 mM potassium, dopamine (0.1-100 nM) evoked an inward current; the current showed inward rectification, reversed polarity at 0 mV, and was blocked by barium (50% inhibition by 10 microM). The concentrations of dopamine activating 50% of the maximal current (EC50) were not different (2-4 nM) for D2, D3, and D4 receptors, but the maximal current was 3-fold larger for D2 and D4 than for D3 receptors. Dopamine evoked reproducible inward currents at D2 and D4 receptors when applied repeatedly, but second responses could not be observed in oocytes expressing D3 receptors. 7-Hydroxy-N,N-di-n-propyl-2-aminotetralin mimicked the effect of dopamine (EC50 of approximately 2, approximately 3, and approximately 19 nM at D2, D3, and D4, respectively). (-) Sulpiride reversibly blocked the dopamine-induced current with IC50 values of 5, 300, and 2000 nM for D2, D3, and D4 receptors, respectively. Dopamine was ineffective in oocytes injected 2 hr previously with pertussis toxin. We concluded that all three D2-like dopamine receptors share the potential to activate inwardly rectifying potassium channels.

Animals↗
PubMed1995

Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons.

Cation-selective P2X receptor channels were first described in sensory neurons where they are important for primary afferent neurotransmission and nociception. Here we report the cloning of a complementary DNA (P2X3) from rat dorsal root ganglia that had properties dissimilar to those of sensory neurons. We also found RNA for (P2X1)(ref. 7), (P2X2)(ref. 8) and P2X4 (ref. 9) in sensory neurons; channels expressed from individual cDNAs did not reproduce those of sensory ganglia. Coexpression of P2X3 with P2X2, but not other combinations, yielded ATP-activated currents that closely resembled those in sensory neurons. These properties could not be accounted for by addition of the two sets of channels, indicating that a new channel had formed by subunit heteropolymerization. Although in some tissues responses to ATP can be accounted for by homomeric channels, our results indicate that ATP-gated channels of sensory neurons may form by a specific heteropolymerization of P2X receptor subunits.

Adenosine Triphosphate↗
PubMed1995

New insights on P2X purinoceptors.

Significant advances in understanding of P2X purinoceptor pharmacology have been made in the last few years. The limitations of nucleotide agonists as drug tools have now been amply demonstrated. Fortunately, inhibitors of the degrading ecto-ATPase enzymes are becoming available and it has become apparent that the complete removal of all divalent cations can be used experimentally in some systems to prevent nucleotide breakdown. Despite these issues, convincing evidence for P2X receptor heterogeneity, from data with agonists, has recently been reported. A number of new antagonists at P2X purinoceptors have also recently been described which to some degree appear to be more specific and useful than earlier antagonists like suramin. It is now apparent that suramin is a poor antagonist of ATP in many tissues because it potently inhibits ATPase activity at similar concentrations to those at which it blocks the P2X purinoceptor. Advances in the use of radiolabelled nucleotides as radioligands for binding studies has allowed the demonstration of P2X purinoceptors in a variety of tissues throughout the body including the brain. These studies have also provided evidence for receptor heterogeneity. Excitingly, two P2X purinoceptor genes have been cloned but operational studies suggest that more than two types exist. The cloning studies have also demonstrated a unique structure for the P2X purinoceptor which differentiates it from all other ligand-gated ion channel receptors. Further studies on P2X purinoceptor operation and structure are needed to help resolve controversies alluded to regarding the characterization and classification of nucleotide receptors. Hopefully such studies will also lead to a better understanding of the physiological and pathological importance of ATP and its activation of P2X purinoceptors. This will require the identification of better drug tools, in particular antagonists which may also provide the basis for novel therapeutic agents.

Adenosine Triphosphate↗
PubMed1995

P2X receptors bring new structure to ligand-gated ion channels.

P2X receptors are cation-selective ion channels that open on binding to extracellular ATP; they play a role in fast synaptic transmission between neurones, and from autonomic nerves to smooth muscles. Isolation of cDNAs that encode P2X receptors in the smooth muscle of vas deferens and in phaeochromocytoma cells indicates that the receptors are not related to other ligand-gated ion channels. Their overall structure resembles more closely that of epithelial Na+ channels and the proteins that are thought to form mechanosensitive channels in Caenorhabditis elegans. The type of P2X RNA that is found in vas deferens is expressed preferentially by apoptotic thymocytes, and the type of P2X RNA that is found in PC12 cells is abundant in the pituitary gland, suggesting hitherto unsuspected roles for ATP-gated channels in endocrine and immune function.

Adenosine Triphosphate↗