Erzsébet Kató

Amino Acid Residues Constituting the Agonist Binding Site of the Human P2X3 Receptor

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca2+ imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca2+]i responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.

Astrocyte–neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor-mediated release of glutamate and reactive oxygen species

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole‐cell patch clamp recordings from SG spinal cord slices documented that in a low Ca2+/no Mg2+ (low X2+) external medium adenosine triphosphate (ATP)/dibenzoyl‐ATP, Bz‐ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X2+‐containing bath medium. The effect of Bz‐ATP was antagonized by the selective P2X7 receptor antagonist A‐438079. Neuronal, but not astrocytic Bz‐ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP‐5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H2O2 potentiated the effect of Bz‐ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP‐5 and CNQX almost abolished the effect of Bz‐ATP, H2O2 was inactive. A Bz‐ATP‐dependent and A‐438079‐antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7‐immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA‐ and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions. GLIA 2014;62:1671–1686

Modulation of excitatory neurotransmission by neuronal/glial signalling molecules: interplay between purinergic and glutamatergic systems.

Glutamate is the main excitatory neurotransmitter of the central nervous system (CNS), released both from neurons and glial cells. Acting via ionotropic (NMDA, AMPA, kainate) and metabotropic glutamate receptors, it is critically involved in essential regulatory functions. Disturbances of glutamatergic neurotransmission can be detected in cognitive and neurodegenerative disorders. This paper summarizes the present knowledge on the modulation of glutamate-mediated responses in the CNS. Emphasis will be put on NMDA receptor channels, which are essential executive and integrative elements of the glutamatergic system. This receptor is crucial for proper functioning of neuronal circuits; its hypofunction or overactivation can result in neuronal disturbances and neurotoxicity. Somewhat surprisingly, NMDA receptors are not widely targeted by pharmacotherapy in clinics; their robust activation or inhibition seems to be desirable only in exceptional cases. However, their fine-tuning might provide a promising manipulation to optimize the activity of the glutamatergic system and to restore proper CNS function. This orchestration utilizes several neuromodulators. Besides the classical ones such as dopamine, novel candidates emerged in the last two decades. The purinergic system is a promising possibility to optimize the activity of the glutamatergic system. It exerts not only direct and indirect influences on NMDA receptors but, by modulating glutamatergic transmission, also plays an important role in glia-neuron communication. These purinergic functions will be illustrated mostly by depicting the modulatory role of the purinergic system on glutamatergic transmission in the prefrontal cortex, a CNS area important for attention, memory and learning.

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide-receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or (111)In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1-17)-NH2 at the epsilon-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1-17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In (115)In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPgammaS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.

The role of P2X7 receptors in a rodent PCP-induced schizophrenia model

P2X7 receptors (P2X7Rs) are ligand-gated ion channels sensitive to extracellular ATP. Here we examined for the first time the role of P2X7R in an animal model of schizophrenia. Using the PCP induced schizophrenia model we show that both genetic deletion and pharmacological inhibition of P2X7Rs alleviate schizophrenia-like behavioral alterations. In P2rx7+/+ mice, PCP induced hyperlocomotion, stereotype behavior, ataxia and social withdrawal. In P2X7 receptor deficient mice (P2rx7−/−), the social interactions were increased, whereas the PCP induced hyperlocomotion and stereotype behavior were alleviated. The selective P2X7 receptor antagonist JNJ-47965567 partly replicated the effect of gene deficiency on PCP-induced behavioral changes and counteracted PCP-induced social withdrawal. We also show that PCP treatment upregulates and increases the functional responsiveness of P2X7Rs in the prefrontal cortex of young adult animals. The amplitude of NMDA evoked currents recorded from layer V pyramidal neurons of cortical slices were slightly decreased by both genetic deletion of P2rx7 and by JNJ-47965567. PCP induced alterations in mRNA expression encoding schizophrenia-related genes, such as NR2A, NR2B, neuregulin 1, NR1 and GABA α1 subunit were absent in the PFC of young adult P2rx7−/− animals. Our findings point to P2X7R as a potential therapeutic target in schizophrenia.

Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5+/-7.5 pg/tube (n=7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, beta-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6+/-40.0 (n=3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1+/-30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a mu-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA&GTP gamma S binding and mouse vas deferens), whereas the latter was a weak mu-opioid receptor agonist with a significant delta-opioid receptorial action as well and a definite indication of partial agonism.

A new potent analgesic agent with reduced liability to produce morphine tolerance.

The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 μmol/kg morphine or with 14-O-MeM6SU (12 μmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 μM) and morphine (1 μM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.