Biomedical subjects

Kerstin Wirkner

Publications and source records attributed to Kerstin Wirkner.

18 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

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

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

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

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

Supersensitivity of P2X receptors in cerebrocortical cell cultures after in vitro ischemia.

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

2-Amino-5-phosphonovalerate↗

PubMed2004

D1 but not D2 dopamine receptors or adrenoceptors mediate dopamine-induced potentiation of N-methyl-d-aspartate currents in the rat prefrontal cortex.

Dopamine-glutamate interactions in the prefrontal cortex (PFC) are associated with higher order cognitive functions, and are involved in the pathophysiology of schizophrenia and addiction. Recordings with intracellular sharp microelectrodes and patch-clamp pipettes were used to investigate these interactions in layer V pyramidal cells of brain slices obtained from the rat PFC. Dopamine (100 microM) potentiated N-methyl-d-aspartate (NMDA; 10mM)-evoked depolarizations, but did not change those elicited by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA; 1mM). Dopamine (100 microM) increased the amplitude of the NMDA (30 microM)-induced currents as well, and 1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF 38393; 1, 10 microM), a D(1) receptor agonist, concentration-dependently reproduced this effect. Furthermore, 7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapine hydrochloride (SCH 23390; 10 microM), a D(1) receptor antagonist, reversed both the dopamine- and the SKF 38393-evoked potentiation. The D(2) receptor agonists lisuride and quinpirole (10 microM both), as well as noradrenaline (100 microM) failed to mimic the stimulatory effect of dopamine. Isoproterenol (1, 10 microM) concentration-dependently facilitated NMDA responses. However, neither this effect at 10 microM nor that of dopamine at 100 microM could be antagonized by propranolol (10 microM), a non-selective beta adrenoceptor blocker. The isoproterenol-induced facilitation of NMDA currents was abolished by SCH 23390 (10 microM). The results indicate that dopamine potentiates NMDA responses in layer V pyramidal cells of the PFC solely by activating D(1) receptors. D(2) receptors and alpha or beta adrenoceptors are not involved in the dopamine-NMDA interaction.

Animals↗

PubMed2004

VIP enhances both pre- and postsynaptic GABAergic transmission to hippocampal interneurones leading to increased excitatory synaptic transmission to CA1 pyramidal cells.

Vasoactive intestinal peptide (VIP) is present in the hippocampus in three subtypes of GABAergic interneurones, two of which innervate preferentially other interneurones, responsible for pyramidal cell inhibition. We investigated how pre- and postsynaptic modulation of GABAergic transmission (to both pyramidal cells and interneurones) by VIP could influence excitatory synaptic transmission in the CA1 area of the hippocampus. VIP (0.1-100 nM) increased [(3)H]GABA release from hippocampal synaptosomes (maximum effect at 1 nM VIP; 63.8 +/- 4.0%) but did not change [(3)H]glutamate release. VIP (0.3-30 nM) enhanced synaptic transmission in hippocampal slices (maximum effect at 1 nM VIP; field excitatory postsynaptic potentials (epsp) slope: 23.7 +/- 1.1%; population spike amplitude: 20.3 +/- 1.7%). The action on field epsp slope was fully dependent on GABAergic transmission since it was absent in the presence of picrotoxin (50 microM) plus CGP55845 (1 microM). VIP (1 nM) did not change paired-pulse facilitation but increased paired-pulse inhibition in CA1 pyramidal cells (16.0 +/- 0.9%), reinforcing the involvement of GABAergic transmission in the action of VIP. VIP (1 nM) increased muscimol-evoked inhibitory currents by 36.4 +/- 8.7% in eight out of ten CA1 interneurones in the stratum radiatum. This suggests that VIP promotes increased inhibition of interneurones that control pyramidal cells, leading to disinhibition of synaptic transmission to pyramidal cell dendrites. In conclusion, concerted pre- and postsynaptic actions of VIP lead to disinhibition of pyramidal cell dendrites causing an enhancement of synaptic transmission.

Animals↗

PubMed2004

Inhibition of N-type voltage-activated calcium channels in rat dorsal root ganglion neurons by P2Y receptors is a possible mechanism of ADP-induced analgesia.

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca2+ currents (I(Ca)). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the omega-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium, two P2Y1 receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X3 and P2Y1 receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5'-O-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y1 receptors appear to block I(Ca) by a pathway involving the betagamma subunit of a G(q/11) protein. Less efficient buffering of the intracellular Ca2+ concentration ([Ca2+]i) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca2+]i by a Galpha-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]i by inhibiting I(Ca) via Gbetagamma. Adenosine 5'-O-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP.

Adenosine Diphosphate↗

PubMed2004

Adenosine A2A receptor-induced inhibition of NMDA and GABAA receptor-mediated synaptic currents in a subpopulation of rat striatal neurons.

The function of adenosine A(2A) receptors, localized at the enkephalin-containing GABAergic medium spiny neurons of the striatum, has been discussed controversially. Here we show that, in the absence of external Mg(2+), the adenosine A(2A) receptor agonist CGS 21680 postsynaptically depressed the NMDA, but not the non-NMDA (AMPA/kainate) receptor-mediated fraction of the electrically evoked EPSCs in a subpopulation of striatal neurons. Current responses to locally applied NMDA but not AMPA were also inhibited by CGS 21680. However, in the presence of external Mg(2+), the inhibition by CGS 21680 of the GABA(A) receptor-mediated IPSCs led to a depression of the EPSC/IPSC complexes. The current response to the locally applied GABA(A) receptor agonist muscimol was unaltered by CGS 21680. Whereas, the frequency of spontaneous (s)IPSCs was inhibited by CGS 21680, their amplitude was not changed. Hence, it is suggested that under these conditions the release rather than the postsynaptic effect of GABA was affected by CGS 21680. In conclusion, under Mg(2+)-free conditions, CGS 21680 appeared to postsynaptically inhibit the NMDA receptor-mediated component of the EPSC, while in the presence of external Mg(2+) this effect turned into a presynaptic inhibition of the GABA(A) receptor-mediated IPSC.

Adenosine↗

PubMed2004

P2X2 and P2Y1 immunofluorescence in rat neostriatal medium-spiny projection neurones and cholinergic interneurones is not linked to respective purinergic receptor function.

1. The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K(+) currents in cultured neostriatal neurones, was investigated in medium-spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5-26-day-old rats. 2. In these cells, adenosine-5'-triphosphate (ATP) (100-1000 microm), 2-methylthioadenosine-5'-triphosphate (2MeSATP), alpha,beta-methyleneadenosine-5'-triphosphate (alpha,betameATP, 30-300 microm, each) and adenosine-5'-O-(3-thiotriphosphate (ATPgammaS) (100 microm) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microm), apyrase (10 U ml(-1)) or intracellular Cs(+) was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K(+) channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 microm) evoked GABA(A) receptor-mediated currents under all these conditions. 3. When used as a control, locus coeruleus neurones responded with P2X receptor-mediated currents to ATP (300 microm), 2MeSATP and alpha,betameATP (100 microm, each). 4. ATP and adenosine-5'-diphosphate (ADP) (100 microm, each) did not activate K(+) currents in the neostriatal neurones. 5. Despite the observed lack of function, P2X(2) and P2Y(1) immunofluorescence was found in roughly 50% of the medium-spiny neurones and cholinergic interneurones. 6. A role of ATP in synaptic transmission to striatal medium-spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions.

Adenosine Triphosphate↗

PubMed2003

Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor-type.

1. N-type Ca(2+) channel modulation by an endogenous P2Y receptor was investigated by the whole-cell patch-clamp method in HEK 293 cells transfected with the functional rabbit N-type calcium channel. 2. The current responses (I(Ca(N))) to depolarizing voltage steps were depressed by ATP in a concentration-dependent manner. Inclusion of either guanosine 5'-O-(3-thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. 3. In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration-response curves of ADP and adenosine 5'-O-(2-thiodiphosphate) indicated a higher potency of these agonists than that of ATP, alpha,beta-methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 2'deoxy-N(6)-methyladenosine-3',5'-diphosphate or 2-methylthio AMP. 4. Using reverse transcription and polymerase chain reaction, mRNA for the P2Y(1), P2Y(4), P2Y(6), P2Y(11) and P2Y(13) receptor subtypes, but not the P2Y(2), and P2Y(12) subtypes, was detected in HEK 293 cells. 5. Immunocytochemistry confirmed the presence of P2Y(1), and to a minor extent that of P2Y(4), but not of P2Y(2) receptors. 6. Hence, it is tempting to speculate that P2Y(13) receptors may inhibit N-type Ca(2+) channels via the betagamma subunits of the activated G(i) protein.

Adenine Nucleotides↗

PubMed2003

Subsensitivity of P2X but not vanilloid 1 receptors in dorsal root ganglia of rats caused by cyclophosphamide cystitis.

The application of cyclophosphamide to rats was used to induce interstitial cystitis. Behavioural studies indicated a strong pain reaction that developed within 2 h and levelled off thereafter causing a constant pain during the following 18 h. Neurons prepared from L6/S1 dorsal root ganglia innervating the urinary bladder responded to the application of capsaicin or alpha,beta-methylene ATP (alpha,beta-meATP) with an increase of intracellular Ca2+ ([Ca2+]i). The [Ca2+]i responses to capsaicin were identical in the dorsal root ganglion cells of cyclophosphamide- and saline-treated rats, whereas alpha,beta-meATP induced less increase in [Ca2+]i in the cyclophosphamide-treated animals than in their saline-treated counterparts. Hence, alpha,beta-meATP-sensitive P2X3 and/or P2X2/3 receptors of L6/S1 dorsal root ganglion neurons were functionally downregulated during subacute pain caused by experimental cystitis. In contrast, capsaicin-sensitive vanilloid 1 receptors did not react to the same procedure. Thoracal dorsal root ganglia, not innervating the urinary bladder, were also unaltered in their responsiveness to alpha,beta-meATP by cyclophosphamide treatment.

Animals↗

PubMed2003

P2Y(1) receptor activation inhibits NMDA receptor-channels in layer V pyramidal neurons of the rat prefrontal and parietal cortex.

In the 1st part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex (PFC) were evoked by electrical stimulation of layer I. Recordings with intracellular sharp, microelectrodes showed a concentration-dependent inhibition of the EPSP by adenosine 5'-O-(2-thiodiphosphate) (ADP-beta-S). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), when given alone depressed the EPSP and in addition antagonized the effect of ADP-beta-S. Exclusion of the N-methyl-D-aspartate (NMDA) component of the EPSP by D(.)-amino-5-phosphonopentanoic acid (AP-5) abolished the ADP-beta-S-induced depression. The pressure-application of both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused reproducible depolarizations. ADP-beta-S inhibited the effect of NMDA, but did not alter that of AMPA. PPADS was also under these conditions antagonistic with ADP-beta-S. In the 2nd part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ADP-beta-S caused a concentration-dependent inhibition of the responses to NMDA. PPADS alone did not alter the NMDA-currents but again antagonized the action of ADP-beta-S; 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS 2179) also abolished the NMDA effect. The ADP-beta-S-induced inhibition persisted in the presence of tetrodotoxin (TTX) or guanosine 5'-O-(3-thiodiphosphate) (GDP-beta-S) applied to the external medium and the pipette solution, respectively. The 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) moderately decreased the ADP-beta-S effect. The inhibitory function of ADP-beta-S on EPSPs and the interaction with PPADS was observed also in layer V pyramidal neurons of the parietal somatosensory cortex. In conclusion, metabotropic P2Y(1) receptors appear to exert a new modulatory influence on fast excitatory amino acid transmission in the cerebral cortex.

Adenosine Diphosphate↗

PubMed2003

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol.

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.

Adenosine Diphosphate↗

PubMed2002

Adenosine A2A receptors inhibit the N-methyl-D-aspartate component of excitatory synaptic currents in rat striatal neurons.

The effects of the adenosine A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on currents mediated by excitatory amino acid receptors were examined in rat striatal brain slices. In a Mg(2+)-free superfusion medium, CGS 21680 decreased the amplitude of excitatory postsynaptic currents (EPSCs) in about 70% of striatal neurons. The inhibitory effect of CGS 21680 disappeared both in the presence of the adenosine A(2A) receptor antagonist 8-(3-chlorostyryl) caffeine and the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5). NMDA-induced currents were also depressed by CGS 21680 in a subset of striatal cells, whereas alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-induced currents were not affected. The results suggest that adenosine A(2A) receptor agonists inhibit the NMDA component of the EPSC.

Animals↗

PubMed2002

Interaction between P2Y and NMDA receptors in layer V pyramidal neurons of the rat prefrontal cortex.

In the first part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex were evoked by electrical stimulation of layer I. Recordings by intracellular sharp microelectrodes showed that EPSPs were concentration-dependently facilitated by the P2 receptor antagonistic ATP analogue 2-methylthio ATP (2-MeSATP), while ATP itself depressed the synaptic potentials. The inhibitory effect of ATP turned into facilitation in the presence of the adenosine A(1) receptor antagonist DPCPX. The 2-MeSATP-induced potentiation of EPSP amplitudes were prevented by the P2 receptor antagonists PPADS and Suramin. The EPSP was almost abolished by coapplication of the NMDA receptor antagonist AP-5 and the AMPA/kainate receptor antagonist CNQX. After blockade of the NMDA receptor-mediated part of the EPSP by AP-5, the stimulatory effect of 2-MeSATP disappeared. When NMDA or AMPA were pressure-applied onto pyramidal cells, only the NMDA-induced depolarization was potentiated by 2-MeSATP. In the second part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ATP, 2-MeSATP, UDP and UTP potentiated the response to NMDA, while ADP-beta-S was inactive. PPADS antagonized the effect of ATP. Synaptic isolation of pyramidal neurons by a Ca(2+)-free medium or tetrodotoxin did not alter the effect of ATP which, however, was markedly depressed when GTP in the micropipette was replaced by GDP-beta-S. These observations suggest that in layer V pyramidal neurons of the prefrontal cortex postsynaptically localized P2Y receptors interact with NMDA receptor-channels.

Adenosine Triphosphate↗