Fanni Tóth

Hungarian Academy of Sciences, Budapest, Budapest fovaros, Hungary

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Publications (7)15.41 Total impact

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    ABSTRACT: The purpose of the present study was to elucidate the role of protein kinase A and C in the mechanism of capsaicin inhibition on mu-opiate receptors. H89, a protein kinase A inhibitor and BIM (bisindolylmaleimide), a protein kinase C inhibitor were used for this purpose. BIM suspended the inhibition of capsaicin in endomorphin-1 competition binding. The addition of BIM alone had no effect itself on this reaction. H89 however, exerted a strong inhibitory effect on the endomorphin-1 binding. We can conclude that protein kinase C certainly plays a role in the inhibition of capsaicin. The role of protein kinase A in this reaction could not be established, owing to the blocking effect of H89 on the mu-opioid receptors.
    Brain research bulletin 11/2012; · 2.18 Impact Factor
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    ABSTRACT: Endogenously occurring opioid peptides are rapidly metabolized by different ectopeptidases. Human opiorphin is a recently discovered natural inhibitor of the enkephalin-inactivating neutral endopeptidase (NEP) and aminopeptidase-N (AP-N) (Wisner et al., 2006). To date, in vitro receptor binding experiments must be performed either in the presence of a mixture of peptidase inhibitors and/or at low temperatures, to block peptidase activity. Here we demonstrate that, compared to classic inhibitor cocktails, opiorphin dramatically increases the binding of [(3)H]MERF and [(3)H]MEGY ligands to rat brain membrane preparations. We found that at 0 °C the increase in specific binding is as high as 40-60% and at 24 °C this rise was even higher. In contrast, the binding of the control [(3)H]endomorphin-1, which is relatively slowly degraded in rat brain membrane preparations, was not enhanced by opiorphin compared to other inhibitors. In addition, in homologous binding displacement experiments, the IC(50) affinity values measured at 24 °C were also significantly improved using opiorphin compared to the inhibitor cocktail. In heterologous binding experiments the differences were less obvious, but still pronounced using [(3)H]MERF and MEGY compared to dynorphin(1-11), or naloxone and DAGO competitor ligands.
    Regulatory Peptides 07/2012; 178(1-3):71-5. · 2.06 Impact Factor
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    ABSTRACT: Dermorphin analogues, containing a (S)- and (R)-4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one scaffold (Aba) and the α-methylated analogues as conformationally constrained phenylalanines, were prepared. Asymmetric phase-transfer catalysis was unable to provide the (S)-α-Me-o-cyanophenylalanine precursor for (S)-α-MeAba in acceptable enantiomeric purity. However, by using a Schöllkopf chiral auxiliary, this intermediate was obtained in 88 % ee. [(S)-Aba 3-Gly 4]dermorphin retained μ-opioid affinity but displayed an increased δ-affinity. The corresponding R epimer was considerably less potent. In contrast, the [(R)-α-MeAba 3-Gly 4]dermorphin isomer was more potent than its S epimer. Tar-MD simulations of both non-methylated [Aba 3-Gly 4]dermorphin analogues showed a degree of folding at the C-terminal residues toward the N terminus of the peptide, without however, adopting a stabilized β-turn conformation. The α-methylated analogues, on the other hand, exhibited a type I/I' β-turn conformation over the α-MeAba 3 and Gly 4 residues, which was stabilized by a hydrogen bond involving Tyr 5-HN and D-Ala 2-CO.
    ChemMedChem 11/2011; 6(11):2035-47. · 2.84 Impact Factor
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    ABSTRACT: Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.
    Regulatory Peptides 11/2004; 122(2):139-46. · 2.06 Impact Factor
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    ABSTRACT: The endogenous opioid heptapeptide (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF) has been shown to interact with multiple opioid as well as non-opioid sites in mammalian brain membranes. To increase the stability and bioavailability of MERF, new synthetic derivatives with D-amino acid substitutions were prepared and studied. One of the new compounds in this series, Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN), had only moderate affinity in competing with [3H]MERF, whereas it displayed the highest potency in producing antinociception following intrathecal administration. DADN was radiolabeled with 41Ci/mmol specific activity. Specific binding of [3H]DADN was saturable, stereoselective and of high affinity. Chemical stability, increased micro-receptor selectivity, and hydrophobicity of the peptide all contribute to the effectiveness observed in biochemical and pharmacological studies.
    Peptides 10/2003; 24(9):1433-40. · 2.52 Impact Factor
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    ABSTRACT: The highly potent micro -opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level.
    European Journal of Neuroscience 07/2003; 18(2):290-5. · 3.75 Impact Factor
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    ABSTRACT: Az antinocicepcióban kulcsszerepet játszó mu opioid receptor endogén ligandumának tekintett endomorfinok (1 és 2) kötőhelyeit karakterizáltuk biokémiai és autoradiográfiás módszerekkel. Megállapítottuk, hogy az endomorfin 1 két kötőhelyet ismer fel, amelyek közül a kis affinitású a klasszikus mu receptortól részben eltérő lokalizációval, ligandspecificitással bír és regulációja nem követi az ismert reakció utakat. Új, konformációsan gátolt endomorfin analógok kifejlesztésével módosítható volt a peptidek affinitása, szelektivitása. Világossá vált, hogy akár kismértékű szerkezeti módosítások is jelentős funkcionális eltéréseket eredményezhetnek. Az opioid és kannabinoid rendszer összefüggéseit vizsgáltuk molekuláris biológiai és biokémiai módszerekkel in vitro és in vivo. A legmarkánsabb változásokat a mu opioid receptorok esetében kaptunk, legfőképpen az előagyi és az agytörzsi területeken. Mind endogén CB1 agonista, mind CB2 antagonista befolyásolta a mu opioid receptor expresszióját és regulációját. A kannabisz és opioid rendszer közötti interakciók pontos mechanizmusainak feltárásával új kapuk nyílhatnak meg a fájdalomcsillapítás terápiás alkalmazásában a távolabbi jvőben | The endomorphins (1 and 2) are putative endogenous ligands for mu opioid receptors, which play a major role in antinociception. It was found, that endomorphin1 labels two distinct sites with partially different localization, ligand selectivity profiles. The regulation of its binding is not identical to that of the classical opioid ligands. The recently developed endomorphin analogs with constrained structure show changes in affinity and selectivy. Relatively small chemical modifications might lead to major changes in functional consequences. We investigated the interaction of the opioid system with several others related to nociception/antinociception. Among them, the occurrence of possible changes in mRNA expression and in functional activity of opioid receptors after acute in vivo and in vitro treatments with cannabinoids were studied. Wild-type, CB1 knockout mice and CB2 receptor deficient animals were among the the subjects of the study. We examined the changes of opioid receptor?s mRNA levels by using real-time PCR, analyzed the capability of mu-, delta and kappa opioid agonists to activate G-proteins and investigated mu-opioid receptors binding properties by using competition assays. Our data show changes in the expression and functional integrity of mu opioid receptors in forebrain, cerebellum and brainstem after different cannabinoid treatments. A better knowledge of the observed interactions may lead to exciting therapeutic possibilities in a long term.