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Peter Illes

Publications and source records attributed to Peter Illes.

At least 19 recordsLinked to original sources
PubMed2006

P2X7 receptors in the nervous system.

P2X(7) receptors are ligand-gated ion channels, expressed as homo-oligomeric assemblies of individual subunits. They are widely distributed at immunocompetent cells of the central and peripheral nervous system and are believed to be primarily involved in host-defense reaction. However, a growing amount of evidence indicates that their signaling role in the brain is more widespread than previously anticipated. In this paper, we review the present knowledge on the structural and pharmacological features of the P2X(7) receptor, as well as its cell-type specific localization in the nervous system. Subsequently, the participation of P2X(7) receptors in distinct neuronal, astroglial and microglial functions are described. Finally, since they may play a prominent role in certain neurologic disorders, such as ischemia-reperfusion injury, Alzheimer's disease, spinal cord injury and sensory neuropathies, the pathological role and potential therapeutic exploitation of P2X(7) receptors are also discussed.

Animals↗
PubMed2006

P2 receptors and neuronal injury.

Extracellular adenosine 5'-triphosphate (ATP) was proposed to be an activity-dependent signaling molecule that regulates glia-glia and glia-neuron communications. ATP is a neurotransmitter of its own right and, in addition, a cotransmitter of other classical transmitters such as glutamate or GABA. The effects of ATP are mediated by two receptor families belonging either to the P2X (ligand-gated cationic channels) or P2Y (G protein-coupled receptors) types. P2X receptors are responsible for rapid synaptic responses, whereas P2Y receptors mediate slow synaptic responses and other types of purinergic signaling involved in neuronal damage/regeneration. ATP may act at pre- and postsynaptic sites and therefore, it may participate in the phenomena of long-term potentiation and long-term depression of excitatory synaptic transmission. The release of ATP into the extracellular space, e.g., by exocytosis, membrane transporters, and connexin hemichannels, is a widespread physiological process. However, ATP may also leave cells through their plasma membrane damaged by inflammation, ischemia, and mechanical injury. Functional responses to the activation of multiple P2 receptors were found in neurons and glial cells under normal and pathophysiological conditions. P2 receptor-activation could either be a cause or a consequence of neuronal cell death/glial activation and may be related to detrimental and/or beneficial effects. The present review aims at demonstrating that purinergic mechanisms correlate with the etiopathology of brain insults, especially because of the massive extracellular release of ATP, adenosine, and other neurotransmitters after brain injury. We will focus in this review on the most important P2 receptor-mediated neurodegenerative and neuroprotective processes and their beneficial modulation by possible therapeutic manipulations.

Adenosine Triphosphate↗
PubMed2006

Modulation of NMDA receptor current in layer V pyramidal neurons of the rat prefrontal cortex by P2Y receptor activation.

Current responses to N-methyl-D-aspartate (NMDA) in layer V pyramidal neurons of the rat prefrontal cortex were potentiated by the P2 receptor agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP). The failure of these nucleotides to induce inward current on fast local superfusion suggested the activation of P2Y rather than P2X receptors. The potentiation by ATP persisted in a Ca(2+)-free superfusion medium but was abolished by 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester, cyclopiazonic acid, 7-nitroindazole, fluoroacetic acid, bafilomycin, and tetanus toxin, indicating that an astrocytic signaling molecule may participate. Because the metabotropic glutamate receptor (mGluR) agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (group I/II) and (RS)-3,5-dihydroxyphenylglycine (group I) both imitated the effect of ATP and the group I mGluR antagonist 1-aminoindan-1,5-dicarboxylic acid or a combination of selective mGluR(1) (7-(hydroxyimino)-cyclopropa[b]chromen-1a-carboxylate) and mGluR(5) (2-methyl-6-(phenylethynyl)pyridine) antagonists abolished the facilitation by ATP, it was concluded that the signaling molecule may be glutamate. Pharmacological tools known to interfere with the transduction cascade of type I mGluRs (guanosine 5'-O-(3-thiodiphosphate), U-73122, xestospongin C, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin kinase II [CAMKII] inhibitor peptide) depressed the actions of both ATP and ACPD. Characterization of the P2Y receptor by agonists (ATP and UTP), antagonists (suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid), and knockout mice (P2Y(2)(-/-)) suggested that the nucleotides act at the P2Y(4) subtype. In conclusion, we propose that exogenous and probably also endogenous ATP release vesicular glutamate from astrocytes by P2Y(4) receptor activation. This glutamate then stimulates type I mGluRs of layer V pyramidal neurons and via the G(q)/phospholipase C/inositol 1,4,5-trisphosphate/Ca(2+)/CAMKII transduction pathway facilitates NMDA receptor currents.

Adenosine Triphosphate↗
PubMed2006

Expression of purinergic receptors in the hypothalamus of the rat is modified by reduced food availability.

ATP-sensitive P2 receptors are suggested to play an important role in the cerebral signal transduction. We examined the expression of the P2Y1 receptor and the possibly downstream-related neuronal nitric oxide synthase (nNOS) in the hypothalamus of rats food-restricted for 3 or 10 days and rats refed after a restriction of 10 days. The restriction caused a reduction of the body weight and plasma triacylglyceride, an increase of non-esterified fatty acid levels correlating with a decrease of leptin levels and an enhancement of plasma corticosterone. All changes returned to basal levels after refeeding. The restriction induced an enhanced intake within 30 min after food presentation and a reduction in the latency. Interestingly, the latter was not abolished by refeeding. The daily food intake induced by refeeding was enhanced at the first day only. The expression of hypothalamic P2Y1 receptor/nNOS mRNA and protein and of leptin receptor mRNA were enhanced after restricted feeding. These changes were abolished after 3 days of refeeding. Immunofluorescence studies indicated that P2Y1 receptor and nNOS immunoreactivities are present in the dorsomedial, ventromedial and lateral hypothalamus and in the nucleus arcuatus. P2Y1 receptor-positive cells were partially also nNOS-positive. The P2Y1 receptor labeling was restricted to cell bodies of obviously non-glial cells, whereas nNOS labeling could be detected also at cellular processes of these cells. In the nucleus arcuatus, astrocytes were identified, expressing P2Y1 receptors at cell bodies and cellular processes. The data suggest that restricted feeding may enhance the sensitivity of the hypothalamus to extracellular ADP/ATP by regulation of the expression of P2Y1 receptors and possibly of their signal transduction pathway via nitric oxide production.

Adenosine Triphosphate↗
PubMed2006

The suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF110) potently blocks P2X3 receptors: subtype selectivity is determined by location of sulfonic acid groups.

We have previously identified the suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of recombinant P2X(1) receptors. Here, we characterize, by two-electrode voltage-clamp electrophysiology, three isomeric suramin analogs designated para-4,4',4'',4''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetrakis-benzenesulfonic acid (NF110), meta-(3,3',3'',3''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF448), and ortho-(2,2',2'',2''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (MK3) with respect to their potency in antagonizing rat P2X receptor-mediated inward currents in Xenopus laevis oocytes. Meta, para, and ortho refer to the position of the single sulfonic acid group relative to the amide bond linking the four symmetrically oriented benzenesulfonic acid moieties to the central, invariant suramin core. NF448, NF110, and MK3 were >200-fold less potent in blocking P2X(1) receptors than NF449, from which they differ structurally only by having one instead of two sulfonic acid residues per benzene ring. Although the meta- and ortho-isomers retained P2X(1) receptor selectivity, the para-isomer NF110 exhibited a significantly increased activity at P2X(3) receptors (K(i) approximately 36 nM) and displayed the following unique selectivity profile among suramin derivatives: P2X(2+3) = P2X(3) > P2X(1) > P2X(2) >> P2X(4) > P2X(7). The usefulness of NF110 as a P2X(3) receptor antagonist in native tissues could be demonstrated by showing that NF110 blocks alphabeta-methylene-ATP-induced currents in rat dorsal root ganglia neurons with similar potency as recombinant rat P2X(3) receptors. Together, these data highlight the importance of both the number and exact location of negatively charged groups for P2X subtype potency and selectivity.

Adenosine Triphosphate↗
PubMed2006

Changes in purinergic signaling after cerebral injury -- involvement of glutamatergic mechanisms?

Extracellular purines act as neuromodulators on transmitter release and may exert toxic effects at higher concentrations. In microdialysis studies, endogenous ATP facilitated the extracellular concentration of glutamate in the nucleus accumbens (NAc) of rats. Additionally, P2 receptors are involved in astrogliosis in vivo after a stab wound injury in the same region, suggesting that these receptors, preferentially the metabotropic P2Y(1) receptor subtype, mediate also trophic responses. Two sets of experimental findings support the involvement of purinergic and glutamatergic mechanisms in the response of brain to mechanical damage. First, in the present studies, the initial time course of extracellular ATP and glutamate was analyzed after a mechanical injury. The concentration of ATP in microdialysates was elevated only in the first 15-min sample whereas glutamate returned to a basal concentration not before a 90-min period had elapsed. We suggest, that the acute injury-evoked stimulation of P2 receptors contributes to glutamate-mediated excitotoxicity. Second, the expression of P2Y(1) receptors and their possible relation to glutamatergic structures, identified by neuronal vesicular glutamate transporters (VGLUTs), were elucidated in non-treated and mechanically injured animals after 4 days. The number of P2Y(1)-positive cells was significantly increased after injury. Furthermore, P2Y(1) receptor-labeled cells do not exhibit immunoreactivity for VGLUT1 and VGLUT2 without and after injury. However, after injury, a co-expression of the P2Y(1) receptor on VGLUT3-immunopositive cells in the NAc was observed. No VGLUT1-, 2- and 3-immunoreactivity was found on P2Y(1)-positive glial fibrillary acidic protein-immunopositive astrocytes at both conditions. Our data suggest that the expression of P2Y(1) receptors at neurons and astrocytes is modulated in response to cerebral injury. It can be assumed, that the enhanced sensitivity of neurons to purinergic signaling may be related directly or indirectly to changes of the glutamatergic transmission.

Adenosine Triphosphate↗
PubMed2006

Regulation of intracellular Ca2+ by P2Y1 receptors may depend on the developmental stage of cultured rat striatal neurons.

Mixed striatal cell cultures containing neurons and glial cells were grown either in neurobasal medium (NBM) or Dulbecco's modified Eagle's medium (DMEM). Whole-cell patch-clamp recordings indicated that, if at all, only a single, low amplitude spike was evoked shortly after starting the injection of a depolarizing current pulse into NBM neurons. In contrast, DMEM neurons fired series of high amplitude action potentials, without apparent spike frequency adaptation. The possible reason for the observed action potential failure in NBM neurons was a low density of Na+ channels per unit of membrane surface area. However, both in NBM and DMEM neurons, ATP did not induce inward current responses via P2X receptor-channels, although GABAA and N-methyl-D-aspartate (NMDA) receptor-channels could be activated by muscimol and NMDA, respectively. Ca2+ imaging experiments by means of the Fura-2 method were utilized to measure intracellular Ca2+ ([Ca2+]i) in neurons and glial cells. NBM, but not DMEM neurons responded to ATP with [Ca2+]i transients; glial cells grown in either culture medium were equally sensitive to ATP. ATP caused an increase of [Ca2+]i by a mechanism only partly dependent on external Ca2+; the residual ATP effect was blocked by cyclopiazonic acid (CPA) and was therefore due to the release of Ca2+ from its intracellular pools. The receptor involved was characterized by P2 receptor antagonists (PPADS, MRS 2179, AR-C69931MX) and was found to belong to the P2Y1 subtype. CPA caused an early [Ca2+]i response due to release from intracellular storage sites, followed by a late [Ca2+]i response due to the influx of this cation from the extracellular space, probably triggered by the opening of store-operated channels (SOCs) in the plasma membrane. It is concluded that in partial analogy with the effect of CPA, ATP releases [Ca2+]i via the Gq/phospholipase C/inositoltrisphosphate (IP3) pathway, thereby opening SOCs. It is hypothesized that this effect of ATP may have an important role for the proliferation and migration of striatal neuronal progenitors.

Adenosine Diphosphate↗
PubMed2006

Affinity of corpora amylacea for oligonucleotides: sequence dependency and proteinaceous binding motif.

Corpora amylacea (CA) have an affinity to nucleic acids as shown by in situ hybridization experiments. However, little is known about the specificity of this interaction, as well as the mechanism involved. We investigated the ability of different probes of digoxigenin-labeled oligonucleotides corresponding to some specific neuronal receptors, both sense and antisense, to bind to CA from human autopsy brain tissue. The bound nucleotides were detected with antidigoxigenin antibody and the signal was further amplified using the tyramide signal amplification system. The affinity of binding varies with the sequence of nucleotides. The most intense signal is produced by the adenosine-2A receptor antisense probe and the least intense signal is produced by the N-methyl-D-aspartate receptor sense probe. The affinity of binding for the same probe does not depend on the localization of CA in the central nervous system. Complete staining loss by proteinase K pretreatment in higher concentrations shows that the binding motif is partially proteinaceous. The circumferential but not the punctate internal staining is diminished by mild amylglucosidase pretreatment, suggesting a process of progressive apposition and condensation.

Actins↗
PubMed2006

Enhanced food intake after stimulation of hypothalamic P2Y1 receptors in rats: modulation of feeding behaviour by extracellular nucleotides.

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPbetaS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1-receptor-mediated nitric oxide production.

Adenosine Diphosphate↗
PubMed2006

The monomers of the P2X1 receptor model and KcsA protein share a similar structural fold.

There is evidence that the P2X1 receptor subunit is involved in apoptosis, platelet aggregation, and smooth muscle contraction. The conformation of the membrane-embedded, ligand-gated mouse P2X1 glycoprotein, a monovalent-bivalent cation channel-forming receptor, is predicted. The first step is based on secondary structure prediction. The secondary structure is converted into a three-dimensional geometry. Then, the secondary and tertiary structures are optimized by using the quantum chemistry RHF/3-21G minimal basic set and the all-atom molecular mechanics AMBER96 force field. The fold of the membrane-embedded protein is simulated by a suitable dielectric. The structure is refined using a conjugate gradient minimizer (Fletcher-Reeves modification of the Polak-Ribiere method). Although the mouse P2X1 receptor subunit is more complex (388 amino acids) than the KcsA protein (160 amino acids), the overall folds are similar. The geometry optimized P2X1 receptor subunit is freely available for academic researchers on e-mail request (PDB format).

Animals↗
PubMed2005

Decrease of current responses at human recombinant P2X3 receptors after substitution by Asp of Ser/Thr residues in protein kinase C phosphorylation sites of their ecto-domains.

The whole-cell patch-clamp technique was used to record current responses to nucleotides in HEK 293 cells transiently transfected with the human (h) P2X(3) receptor. When GDP-beta-S was included into the pipette solution, UTP at concentrations which did not alter the holding current, facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. The substitution of Ser/Thr residues situated within protein kinase C (PKC) consensus phosphorylation sites of the P2X(3) receptor ecto-domain by the neutral amino acid Ala either abolished (T134A, S178A) or did not alter (T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. The substitution of the same Ser/Thr residues in all four PKC sites by the negatively charged Asp prevented the potentiation by UTP. The Asp mutations abolished the first, fast offset time-constant, but did not alter, or in the case of S269D even increased, the second, slow offset time-constant; at the same time such mutations invariably increased the onset time-constant and massively depressed the peak current amplitude. None of the Ala mutations (with the exception of S269A) influenced the time-course of desensitisation or the peak current amplitude. It is concluded that constitutive activation of PKC sites at the ecto-domain of the hP2X(3) receptor both abolishes the UTP-induced potentiation of the alpha,beta-meATP current and accelerates its rate of desensitisation.

Adenosine Triphosphate↗
PubMed2005

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.

Adenosine Diphosphate↗
PubMed2005

Regulation of human recombinant P2X3 receptors by ecto-protein kinase C.

The whole-cell patch-clamp technique was used to record current responses to nucleotides and nucleosides in human embryonic kidney HEK293 cells transfected with the human purinergic P2X3 receptor. When guanosine 5'-O-(3-thiodiphosphate) was included into the pipette solution, UTP at concentrations that did not alter the holding current facilitated the alpha,beta-methylene ATP (alpha,beta-meATP)-induced current. ATP and GTP, but not UDP or uridine, had an effect similar to that of UTP. Compounds known to activate protein kinase C (PKC) acted like the nucleoside triphosphates investigated, whereas various PKC inhibitors invariably reduced the effects of both PKC activators and UTP. The substitution by Ala of Ser/Thr residues situated within PKC consensus sites of the P2X3 receptor ectodomain either abolished (PKC2 and PKC3; T134A, S178A) or did not alter (PKC4 and PKC6; T196A, S269A) the UTP-induced potentiation of the alpha,beta-meATP current. Both the blockade of ecto-protein kinase C activity and the substitution of Thr-134 or Ser-178 by Ala depressed the maximum of the concentration-response curve for alpha,beta-meATP without altering the EC50 values. Molecular simulation of the P2X3 receptor structure indicated no overlap between assumed nucleotide binding domains and the relevant phosphorylation sites PKC2 and PKC3. alpha,beta-meATP-induced currents through native homomeric P2X3 receptors of rat dorsal root ganglia were also facilitated by UTP. In conclusion, it is suggested that low concentrations of endogenous nucleotides in the extracellular space may prime the sensitivity of P2X3 receptors toward the effect of subsequently applied (released) higher agonistic concentrations. The priming effect of nucleotides might be attributable to a phosphorylation of PKC sites at the ectodomain of P2X3 receptors.

Animals↗
PubMed2005

Involvement of P2 receptors in the growth and survival of neurons in the CNS.

Extracellular adenosine 5'-triphosphate (ATP) has been recognized as a ubiquitous, unstable signalling molecule, acting as a fast neurotransmitter and modulator of transmitter release and neuronal excitability. Recent findings have demonstrated that ATP is a growth factor participating in differentiation, cell proliferation, and survival, as well as a toxic agent that mediates cellular degeneration and death. Potential sources of extracellular purines in the nervous system include neurons, glia, endothelium, and blood. A complex family of ectoenzymes rapidly hydrolyzes or interconverts extracellular nucleotides, thereby either terminating their signalling action or producing an active metabolite of altered purinoceptor selectivity. Most effects are mediated through the 2 main subclasses of specific cell surface receptors, P2X and P2Y. Members of these P2X/Y receptor families are widely expressed in the central nervous system (CNS) and are involved in glia-glia and glia-neuron communications, whereby they play important physiological and pathophysiological roles in a variety of biological processes. After different kinds of "acute" CNS injury (e.g., ischemia, hypoxia, mechanical stress, axotomy), extracellular ATP can reach high concentrations, up to the millimolar range, flowing out from cells into the extracellular space, exocytotically, via transmembrane transport, or as a result of cell damage. In this review, P2 receptor activation as a cause or a consequence of neuronal cell activation or death and/or glial activation is described. The involvement of P2 receptors is also described under different "chronic" pathological conditions, such as pain, epilepsia, toxic influence of ethanol or amphetamine, retinal diseases, Alzheimer's disease (AD), and possibly, Parkinson's disease. The relationship between changes in P2 receptor expression and the specific response of different cell types to injury is extremely complex and can be related to detrimental and/or beneficial effects. The present review therefore considers ATP acting via P2 receptors as a potent regulator of normal physiological and pathological processes in the brain, with a focus on pathophysiological implications of P2 receptor functions.

Adenosine Triphosphate↗
PubMed2005

Evidence for the existence of P2Y1,2,4 receptor subtypes in HEK-293 cells: reactivation of P2Y1 receptors after repetitive agonist application.

ATP, ADPbetaS and UTP induced a comparable rise in the intracellular Ca2+ concentration ([Ca2+]i) in HEK-293 cells using fura-2 microfluorimetry. The responses persisted in Ca2+-free medium, but were abolished following depletion of intracellular Ca2+ stores by cyclopiazonic acid. Cross-desensitisation experiments demonstrated that exposure to ADPbetaS has no marked effect on UTP-induced [Ca2+]i transients and vice versa. Whereas the P2Y1 receptor-selective antagonist 2'-deoxy-N6-methyladenosine 3',5'-diphosphate (MRS 2179) abolished the responses to ADPbetaS, it decreased and did not alter the responses to ATP and UTP respectively. Although the P2Y1/P2Y4 receptor-preferential antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) abolished the responses to ADPbetaS, and decreased those to ATP, it also depressed the UTP-induced [Ca2+]i transients. Suramin, an antagonist with preference for P2Y2 receptors decreased both the ATP- and UTP-induced [Ca2+]i reactions. After numerous splittings, HEK-293 cells failed to react to ADPbetaS; however, repeated superfusion with this P2Y1 receptor agonist restored the [Ca2+]i signals. In agreement with the functional data, real-time polymerase chain reaction and immunocytochemical studies indicated the presence of P2Y1, P2Y2 and P2Y4 receptors. Our findings raise doubt with respect to the reliability of HEK-293 cells as expression systems for recombinant P2X receptors, because of a possible functional interaction with endogenous P2Y receptors.

Adenosine Triphosphate↗
PubMed2005

Characterization of rat transient receptor potential vanilloid 1 receptors lacking the N-glycosylation site N604.

Whole-cell patch-clamp recordings were performed on HEK293 cells transiently transfected with the rat (r) wild-type transient receptor potential vanilloid 1 (TRPV1) (rTRPV1) receptor or with a mutant that lacks the potential N-glycosylation site at position N604 (rTRPV1-N604T). Replacement of Asn by Thr (N604T) depressed the maximum of the concentration-response curve for capsaicin and decreased the EC50 value of this agonist. Further, such a manipulation modified the sensitivity to the TRPV1 receptor-antagonist capsazepine and altered the dependence of the capsaicin effect on extracellular pH. Hence, glycosylation may affect the basic functional characteristics of the rTRPV1 receptor channel in accordance with the knowledge that N-glycosylation may regulate ligand binding or gating properties of ionotropic neurotransmitter receptors.

Analysis of Variance↗
PubMed2005

P2X(7) receptor-mRNA and -protein in the mouse retina; changes during retinal degeneration in BALBCrds mice.

A combined real-time PCR/immunohistochemistry study was carried out to investigate whether P2X(7) receptors, known to induce apoptosis and necrosis, may be causally related to the process of retinal degeneration in BALBCrds mice. In the retinae of BALBCrds mice, P2X(7) receptor-mRNA was the highest at an age of 20-40 days, and declined afterwards. At the same time, the P2X(7) receptor-message was constantly low in the retina of control BALBC mice until postnatal day 100. The receptor-mRNA in total brain tissue of both strains of mice was comparable with that of BALBCrds retinae. Double immunofluorescence in combination with laser scanning microscopy was used to study the distribution of P2X(7) receptor-immunoreactivity (IR) on neurons and different glial cell types of the retina. An exclusively neuronal localization of P2X(7)-IR in the ganglion cell layer was found by using either anti-neuronal nuclei or microtubule associated protein-2 as neuronal markers. There was a slight age-dependent decrease in the abundance of neuronal P2X(7)-IR both in BALBCrds or BALBC mice. P2X(7)-IR failed to co-localize with any of the non-neuronal markers used to stain microglial or Müller glial cells. No P2X(7) receptor-IR was found in the retinal ganglion cell layer of P2X(7)(-/-) animals, when compared with the control littermates. Hence, we suggest that, in BALBCrds mice, an early up-regulation of neuronal P2X(7) receptors may cause injury of retinal neurons and thereby functionally contribute to the retinal damage.

Aging↗