Kerstin Wirkner

P2X3 receptor involvement in pain states.

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X3 and P2X2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X3 receptor in different states of pain.

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 (ICa). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the ω-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 ICa by a pathway involving the βγ subunit of a Gq/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 Gα-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]i by inhibiting ICa via Gβγ. 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.

Ethanol-induced inhibition of NMDA receptor channels

Ethanol is a potent inhibitor of the N-methyl-D-aspartate (NMDA)-receptor subtype of glutamate receptor in a number of brain areas. The mechanism of ethanol action has been investigated by means of patch-clamp recording of ionic currents and fura-2 measurement of intracellular Ca2+ concentration in cell culture systems; the subunit composition of NMDA receptors and their influence on the effect of ethanol was determined by molecular biology methods. Ethanol does not appear to interact with NMDA either at the glutamate recognition site of the receptor, or at any of the hitherto known multiple modulatory sites, such as the glycine or polyamine site. Moreover, ethanol does not cause an open channel block by itself and fails to interact with Mg2+ at the site where it causes open channel block. The ability of ethanol to inhibit responses to NMDA is dependent on the subunit combination of NMDA receptors. The NR1/NR2A and NR1/NR2B combinations are preferentially sensitive to ethanol inhibition. Chronic treatment with ethanol leads to an increase of the NMDA receptor number at the transcriptional and posttranscriptional level; the receptor function is also facilitated. This causes withdrawal-type seizures after termination of chronic treatment with ethanol. The inhibition of NMDA receptors by ethanol leads to the depression of excitatory synaptic potentials mediated by this type of excitatory amino acid receptor. Ethanol-induced disturbances in certain regions of the brain, i.e. hippocampus, nucleus accumbens or locus coeruleus may lead to cognitive disorders or drug dependence. Brain slices containing the locus coeruleus may be used as an in vitro test system to investigate the addictive properties of ethanol.

The reaction of astrocytes and neurons in the hippocampus of adult rats during chronic ethanol treatment and correlations to behavioral impairments

Chronic ethanol treatment of Wistar rats to 10% (v/v) ethanol over a period of 4, 12, and 36 weeks produced distinct alterations of the glial fibrillary acidic protein immunoreactivity (GFAP-IR) of dorsal hippocampal astrocytes. Ethanol consumption over a period of 4 weeks caused an increase in the total GFAP-IR of the astrocytes. Down-regulation of the total GFAP-IR was measured in all examined brain regions after 36 weeks of ethanol treatment. Prolonged ethanol treatment induced a significant loss of the total number of hippocampal pyramidal and dentate gyrus granule cells. Regional differences in the vulnerability to the neurotoxic effects of chronic ethanol intake over 36 weeks were found: CA3 > CA1 + CA2 > > CA4 > GD. In agreement with the degree of neuronal cell loss, ethanol-induced behavioral impairments were found. The acquisition of maze performance using a complex elevated labyrinth was deteriorated after 36 weeks of ethanol treatment, suggesting a deficit in learning and memory. These findings illustrate the importance of time-response analysis when determining the structural and functional changes produced by chronic ethanol treatment.

Effect of adenosine and some of its structural analogues on the conductance of NMDA receptor channels in a subset of rat neostriatal neurones

1 In order to investigate the modulatory effects of adenosine on excitatory amino acid projections onto striatal medium spiny neurones, whole‐cell patch clamp experiments were carried out in rat brain slices. The effects of various agonists for P1 (adenosine) and P2 (ATP) purinoceptors and their antagonists were investigated. The A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680; 0.1 μM), the A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA; 10 μM) and the non‐selective P1 purinoceptor antagonist 8‐(p‐sulphophenyl)‐theophylline (8‐SPT; 100 μM) did not alter the resting membrane potential, the threshold current necessary to elicit an action potential, the amplitude of spikes, their rise time, the amplitude of the afterhyperpolarization (AHP) and the time to peak of the AHP. 2 N‐methyl‐D‐aspartate (NMDA; 1–1000 μM) caused a concentration‐dependent inward current which was larger in the absence than in the presence of Mg2+ (1.3 mM). In a subset of striatal neurones, the current response to NMDA (10 μM) and the accompanying increase in conductance were both inhibited by CGS 21680 (0.01–1 μM). The effect of CGS 21680 (0.1 μM) persisted in the presence of tetrodotoxin (0.5 μM) or in a Ca2+‐free medium, under conditions when synaptically mediated influences may be negligible. 3 The A3 receptor agonist N6‐2‐(4‐aminophenyl)ethyladenosine (APNEA; 0.1–10 μM) also diminished the effect of NMDA (10 μM), while the A1 receptor agonists CCPA (0.01–10 μM) and (2S)‐N6‐[2‐endo‐norbornyl]adenosine [S(−)‐ENBA; 10 μM] as well as the endogenous, non‐selective P1 purinoceptor agonist adenosine (100 μM) were inactive. The endogenous non‐selective P2 purinoceptor agonist ATP (1000 μM) also failed to alter the current response to NMDA (10 μM). Adenosine (100 μM), but not ATP (1000 μM) became inhibitory after blockade of nucleoside uptake by S(4‐nitrobenzyl)‐6‐thioguanosine (NBTG; 30 μM). 4 8‐(p‐Sulphophenyl)‐theophylline (8‐SPT; 100 μM), as well as the A2A receptor antagonist 8‐(3‐chlorostyryl)caffeine (CSC; 1 μM) and the A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) at 0.03, but not 0.003 μM abolished the inhibitory action of CGS 21680 (0.1 μM). None of these compounds altered the effect of NMDA (10 μM) by itself. DPCPX (0.03 μM) prevented the inhibition by APNEA (10 μM). 5 There was no effect of CGS 21680 (0.1 μM), when guanosine 5′‐O‐(3‐thiodiphosphate (GDP‐β‐S; 300 μM) was included in the pipette solution in order to block G protein‐mediated reactions. 6 In conclusion, adenosine receptors, probably of the A2A‐subtype, inhibit the conductance of NMDA receptor channels in a subset of medium spiny neurones of the rat striatum by a transduction mechanism which involves a G protein.

Supersensitivity of P2X7 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.

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.

Inhibition by adenosine A2A receptors of NMDA but not AMPA currents in rat neostriatal neurons

Whole‐cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A2A receptors in modulating N‐methyl‐D‐aspartate (NMDA)‐induced currents in rat striatal brain slices. The A2A receptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) current in a subset of striatal neurons. Lucifer yellow‐filled pipettes in combination with immunostaining of A2A receptors were used to identify CGS 21680‐sensitive cells as typical medium spiny striatal neurons. Dibutyryl cyclic AMP and the protein kinase A activator Sp‐cyclic AMPs, but not the protein kinase A inhibitors Rp‐cyclic AMPS or PKI(14–24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U‐73122, but not the inactive structural analogue U‐73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12‐myristate 13‐acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1,4,5‐trisphosphate (InsP3) and a more efficient buffering of intracellular Ca2+ by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680‐induced inhibition. The calmodulin antagonist W‐7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281–309) and KN‐93 but not the inactive structural analogue KN‐92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680. It is suggested that the transduction mechanism of A2A receptors to inhibit NMDA receptor channels is the phospholipase C/InsP3/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

Neuroreceptors and ion channels as targets of alcohol

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Kinya Kuriyama. The presentations were (1) Modulation of neuroreceptors and ion channels by alcohol, by T. Narahashi; (2) Inhibition by ethanol of NMDA and AMPA receptor-channels, by P. Illes, K. Wirkner, W. Fischer, K. Mühlberg, P. Scheibler, and C. Allgaier; (3) Effects of ethanol on metabotropic glutamate receptors, by K. Minami; (4) Acute alcohol actions on the 5-HT3 ligand-gated ion channel, by D. Lovinger; (5) Inhibition of NMDA receptors by MK801 attenuates ethanol-induced taurine release from the hippocampus, by F. Lallemand, R.J. Ward, and P. DeWitte; and (6) Effect of ethanol on voltage-operated Ca2+ channels in hepatic stellate cells, by T. Itatsu, Y. Takei, H. Oide, M. Hirose, X. E. Wang, S. Watanabe, M. Tateyama, R. Ochi, and N. Sato.

Interaction of P2 purinergic receptors with cellular macromolecules

Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via Gs, Gq/11 or Gi/o protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Gα-dependent signalling with other G protein-coupled receptors and via Gβγ (or indirectly Gα)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects.