Anna Borsodi - Academia.edu (original) (raw)
Papers by Anna Borsodi
Neuropeptides, 1988
A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synth... more A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH&I were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).
Neurosignals, 2006
were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high af... more were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine resi-
Neuropeptides, 1986
The role of microtubules in opioid receptor binding was studied by using microtubule assembly inh... more The role of microtubules in opioid receptor binding was studied by using microtubule assembly inhibitors.
Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from t... more Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from tritiated Tyr-D-Ala*-Me-Phe4-Gly-o15 enkephalin (DAGO) and Tyr-D-Ala*-L-Leu5-enkephalin (DALA) binding (Jand h-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (sZo,J for the K receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas p-and d-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the K subtype represents a separate molecular entity from the p and 6 receptor sites.
The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH 2 ) and endomorphin 2 (Tyr-Pro-Phe-Phe-... more The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH 2 ) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH 2 ) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the m opioid receptor, an effect reversed by the m receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 ), but they had no in¯uence on cells stably expressing the d opioid receptor. To further establish the selectivity of these peptides for the m opioid receptor, brain preparations of mice lacking the m opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a speci®c radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a K d value of 1.77 nM and a B max of 63.33 fmol/mg protein. In membranes of mice lacking the mreceptor gene, no binding was observed, and both endomorphins failed to stimulate [ 35 S]guanosine-5¢-O-(3-thio)triphosphate ([ 35 S]GTPgS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the m opioid receptors.
Regulatory Peptides, 1994
ABSTRACT
Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively ... more Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.
ACS Medicinal Chemistry Letters, 2014
Two novel opioid analogues have been designed by substituting the native D-Ala residues in positi... more Two novel opioid analogues have been designed by substituting the native D-Ala residues in position 2,2′ of biphalin with two residues of D-penicillamine or L-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing D-penicillamines showed excellent μ/δ mixed receptor affinities (K i δ = 5.2 nM; K i μ = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.
Life Sciences, 1995
The pseudopeptide H-Tyr-Tic psi [CH2-NH]Phe-Phe-OH (TIPP[psi]) is a delta opioid antagonist with ... more The pseudopeptide H-Tyr-Tic psi [CH2-NH]Phe-Phe-OH (TIPP[psi]) is a delta opioid antagonist with high delta receptor affinity and unprecedented delta selectivity. TIPP[psi] was radiolabelled by catalytic tritiation of its precursor [Tyr(3',5'-I2)1]TIPP[psi]. The resulting radioligand, [3H]TIPP[psi], had a specific activity of 1.77 TBq/mmol (47.9 Ci/mmol) and showed high stability against enzymatic degradation. [3H]TIPP[psi] binding to rat brain membranes was saturable and Scatchard analysis indicated a single binding site with a Kd of 0.98 nM and a Bmax of 105.4 fmol/mg. A study of [3H]TIPP[psi] binding displacement by various receptor-selective opioids showed the expected rank order of potency (delta > mu > kappa). [3H]TIPP[psi] represents an excellent new radioligand for delta receptor labelling studies in vitro and in vivo.
Life Sciences - LIFE SCI, 1991
C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones ... more C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the (³H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na{sup +}-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (³H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.
![Research paper thumbnail of [3H]dynorphin1–8 binding sites in frog (Rana esculenta) brain membranes](https://attachments.academia-assets.com/45213869/thumbnails/1.jpg)
](Neuropeptides, 1994
Opioid binding sites specific for [3Hldynorphin,., were characterized in the particulate membrane... more Opioid binding sites specific for [3Hldynorphin,., were characterized in the particulate membrane fraction of frog (Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [3H]dynorphinl., to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin,.,,, bremazocine, levorphanol and naloxone. The specific binding of 13Hldynorphin,., can be significantly inhibited by Na' ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a K, z 7.5 nM is present in the membranes. The maximum density of binding sites (B,,, M 1.1 pmol [3Hldynorphin,., per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the p-and d-types of opioid receptor showed that in the frog brain most of the sites labelled by 13Hldynorphin,, are k--sites and that this is a rich source of such sites.
Seizure, 2007
Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with... more Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with intractable mesial temporal lobe epilepsy (MTLE) with and without subacute high frequency electrical stimulation (HFS) in this brain area. Mu, delta and nociceptin receptor binding was determined by autoradiography in PHC of five patients (ESAE group) with MTLE history of 14.8 AE 2.5 years and seizure frequency of 11 AE 2.9 per month, two of them (40%) with mesial sclerosis. This group demonstrated antiepileptic effects following subacute HFS (130 Hz, 450 ms, 200-400 mA), applied continuously during 16-20 days in PHC. Values were compared with those obtained from patients with severe MTLE (history of 21.7 AE 2.8 years and seizure frequency of 28.2 AE 14 per month) in whom electrical stimulation did not induce antiepileptic effects (ESWAE group, n = 4), patients with MTLE in whom no electrical stimulation was applied (MTLE group, n = 4) and autopsy material acquired from subjects without epilepsy (n = 4 obtained from three subjects). Enhanced 3H-DAMGO (MTLE, 755%; ESAE, 375%; ESWAE, 693%), 3H-DPDPE (MTLE, 242%; ESAE, 80%; ESWAE, 346%) and 3H-nociceptin (MTLE, 424%; ESAE, 217%; ESWAE, 451%) binding was detected in the PHC of all epileptic groups. However, tissue obtained from ESAE group demonstrated lower opioid receptor binding (3H-DAMGO, 44.5%, p < 0.05; 3H-DPDPE,
Reproduction, 2005
The effectiveness of b 2 -agonists in preterm delivery is reduced by several factors. The aim of ... more The effectiveness of b 2 -agonists in preterm delivery is reduced by several factors. The aim of this study was to determine the influence of late pregnancy in the uterus-relaxing effect of terbutaline in the rat in vitro. Rat uterine tissues from late pregnancy (days 15, 18, 20 and 22) were used. In vitro electrical field-stimulation (EFS) was used to evoke contractions. The radioligand-binding technique, reverse transcription-polymerase chain reaction and radioimmunoassay technique were used to determine the b-adrenergic receptor density and mRNA level and the plasma sex hormone level, respectively. The activated G-protein level of the b-adrenergic receptors was investigated by a radiolabelled GTP binding assay. EFS-induced contractions were inhibited by terbutaline. This effect decreased towards term with respect to both the EC 50 and maximal inhibition values. A drop in plasma progesterone level was also detected. Binding studies revealed an increase in b-adrenergic receptor number on the last day of pregnancy, which correlated with the change in receptor mRNA level. The G-protein-activating effect of terbutaline decreased continuously between days 15 and 20. Surprisingly, terbutaline decreased the G-protein activation to below the basal level on day 22. However, progesterone pretreatment set back the uterine action of terbutaline, increased the density of the b 2 -adrenergic receptors and their mRNA level and increased the G-proteinactivating property of terbutaline. These data provide evidence of a pregnancy-induced decrease in activated G-protein level after b 2 -agonist stimulation. The decrease in plasma progesterone level has a crucial role in this process. The effects of b 2 -adrenergic receptor agonists in tocolytic therapy may possibly be potentiated with progesterone. Reproduction (2005) 130 113-122 q 2005 Society for Reproduction and Fertility
Regulatory Peptides, 2004
In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-ac... more In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra-and pentapeptides selective for the n-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [ 35 S]GTPgS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K e ) of 2.44 nM, and no agonist effect at 10 AM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the y-opioid receptor agonist [D-Ala 2 ,D-Leu 5 ]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [ 3 H]nociceptin-NH 2 binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [ 35 S]GTPgS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N-and the C-terminal region of the molecule. D
Pure and Applied Chemistry, 2000
ABSTRACT
Pharmacology, 1994
To find a delta-opioid receptor preferring peptide structure containing an Asp residue in a poten... more To find a delta-opioid receptor preferring peptide structure containing an Asp residue in a potentially interacting position, Tyr-Pro-Phe-Asp, Tyr-D-Ala-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp alpha- and beta-methyl ester and Tyr-Gly-Gly-Phe-Asp peptides were synthesized and their biological activities were analyzed in vitro in mouse vas deferens and longitudinal muscle strip of guinea pig ileum. Changing the beta-methyl ester for an alkylating chloromethyl ketone moiety in the delta-receptor-selective agonist Tyr-D-Ala-Gly-Phe-Asp-beta-methyl ester enhanced further the delta-receptor preference. The delta-receptor selective chloromethyl ketone but not the beta-methyl ester gave a very slow washout after prolonged incubation in the mouse vas deferens bioassay; however, it was still readily displaceable by naloxone. The washout pattern of mu-specific Tyr-D-Ala-Gly-(Me)Phe chloromethyl ketone did not differ in the bioassays from that of the corresponding Gly5-ol derivative. Both chloromethyl ketones gave irreversible characteristics in the receptor binding assay.
Neuropharmacology, 2005
Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been sugge... more Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been suggested to act at a novel kappa3 receptor but also appears to bind to classical opioid receptors, and possibly the ORL1 receptor. We have used opioid receptor triple knockout mice, deficient in genes coding for the mu, delta and kappa-receptor, to characterise the relative contributions of opioid and ORL1 activity to the binding of this ligand, by carrying out receptor autoradiography with [3H]NalBzoH. As competing ligands we have used diprenorphine and nociceptin at 1 microM, alone or in combination, to determine the contribution of opioid and ORL1 receptor binding. At 4 nM [3H]NalBzoH showed labelling in wild-type brains indicative of broad spectrum classical opioid receptor binding. In the triple knockout brains all labelling was completely absent, suggesting that at this concentration there is no binding to ORL1 sites. However at 50 nM [3H]NalBzoH showed labelling in triple knockout brains with a distribution pattern indicative of ORL1 labelling. Quantitative analysis showed that nociceptin displaced typically 30% of the residual labelling in knockout brains whilst diprenorphine had relatively little effect. The data show that at 50 nM NalBzoH no binding was detected other than to classical opioid receptors or to ORL1 in an approximate ratio of 2:1.
Neuropeptides, 1986
ke,$one derivatives of leucine ertkephalfn (LE), D-Ala -Leu -enkephalin (DALE) and D-Ala -D-Leu -
Neuropeptides, 1988
A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synth... more A radioactive enkephalin affinity reagent, selective for the b opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH&I were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).
Neurosignals, 2006
were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high af... more were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine resi-
Neuropeptides, 1986
The role of microtubules in opioid receptor binding was studied by using microtubule assembly inh... more The role of microtubules in opioid receptor binding was studied by using microtubule assembly inhibitors.
Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from t... more Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (K subtype) from tritiated Tyr-D-Ala*-Me-Phe4-Gly-o15 enkephalin (DAGO) and Tyr-D-Ala*-L-Leu5-enkephalin (DALA) binding (Jand h-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (sZo,J for the K receptor-detergent complex was 13.1 S and the corresponding Stokes radius 64 A. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas p-and d-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the K subtype represents a separate molecular entity from the p and 6 receptor sites.
The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH 2 ) and endomorphin 2 (Tyr-Pro-Phe-Phe-... more The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH 2 ) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH 2 ) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the m opioid receptor, an effect reversed by the m receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH 2 ), but they had no in¯uence on cells stably expressing the d opioid receptor. To further establish the selectivity of these peptides for the m opioid receptor, brain preparations of mice lacking the m opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a speci®c radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a K d value of 1.77 nM and a B max of 63.33 fmol/mg protein. In membranes of mice lacking the mreceptor gene, no binding was observed, and both endomorphins failed to stimulate [ 35 S]guanosine-5¢-O-(3-thio)triphosphate ([ 35 S]GTPgS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the m opioid receptors.
Regulatory Peptides, 1994
ABSTRACT
Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively ... more Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.
ACS Medicinal Chemistry Letters, 2014
Two novel opioid analogues have been designed by substituting the native D-Ala residues in positi... more Two novel opioid analogues have been designed by substituting the native D-Ala residues in position 2,2′ of biphalin with two residues of D-penicillamine or L-penicillamine and by forming a disulfide bond between the thiol groups. The so-obtained compound 9 containing D-penicillamines showed excellent μ/δ mixed receptor affinities (K i δ = 5.2 nM; K i μ = 1.9 nM), together with an efficacious capacity to trigger the second messenger and a very good in vivo antinociceptive activity, whereas product 10 was scarcely active. An explanation of the two different pharmacological behaviors of products 9 and 10 was found by studying their conformational properties.
Life Sciences, 1995
The pseudopeptide H-Tyr-Tic psi [CH2-NH]Phe-Phe-OH (TIPP[psi]) is a delta opioid antagonist with ... more The pseudopeptide H-Tyr-Tic psi [CH2-NH]Phe-Phe-OH (TIPP[psi]) is a delta opioid antagonist with high delta receptor affinity and unprecedented delta selectivity. TIPP[psi] was radiolabelled by catalytic tritiation of its precursor [Tyr(3',5'-I2)1]TIPP[psi]. The resulting radioligand, [3H]TIPP[psi], had a specific activity of 1.77 TBq/mmol (47.9 Ci/mmol) and showed high stability against enzymatic degradation. [3H]TIPP[psi] binding to rat brain membranes was saturable and Scatchard analysis indicated a single binding site with a Kd of 0.98 nM and a Bmax of 105.4 fmol/mg. A study of [3H]TIPP[psi] binding displacement by various receptor-selective opioids showed the expected rank order of potency (delta > mu > kappa). [3H]TIPP[psi] represents an excellent new radioligand for delta receptor labelling studies in vitro and in vivo.
Life Sciences - LIFE SCI, 1991
C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones ... more C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the (³H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na{sup +}-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of (³H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.
Neuropeptides, 1994
Opioid binding sites specific for [3Hldynorphin,., were characterized in the particulate membrane... more Opioid binding sites specific for [3Hldynorphin,., were characterized in the particulate membrane fraction of frog (Rana esculenta) brain. The degradation of the radioligand during the assay was prevented by the use of a broad spectrum of peptidase inhibitors. The binding of [3H]dynorphinl., to frog brain membranes was stereoselective, reversible, saturable, and displaceable by a series of opioid ligands including dynorphin,.,,, bremazocine, levorphanol and naloxone. The specific binding of 13Hldynorphin,., can be significantly inhibited by Na' ions and/or guanine nucleotides confirming the agonist property of the ligand in vitro. A single set of high affinity opioid binding sites with a K, z 7.5 nM is present in the membranes. The maximum density of binding sites (B,,, M 1.1 pmol [3Hldynorphin,., per mg protein) was considerably higher than such sites in guinea-pig brain. In addition, comparison with binding of tritiated opioid peptides selective for the p-and d-types of opioid receptor showed that in the frog brain most of the sites labelled by 13Hldynorphin,, are k--sites and that this is a rich source of such sites.
Seizure, 2007
Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with... more Opioid receptor binding was evaluated in parahippocampal cortex (PHC) obtained from patients with intractable mesial temporal lobe epilepsy (MTLE) with and without subacute high frequency electrical stimulation (HFS) in this brain area. Mu, delta and nociceptin receptor binding was determined by autoradiography in PHC of five patients (ESAE group) with MTLE history of 14.8 AE 2.5 years and seizure frequency of 11 AE 2.9 per month, two of them (40%) with mesial sclerosis. This group demonstrated antiepileptic effects following subacute HFS (130 Hz, 450 ms, 200-400 mA), applied continuously during 16-20 days in PHC. Values were compared with those obtained from patients with severe MTLE (history of 21.7 AE 2.8 years and seizure frequency of 28.2 AE 14 per month) in whom electrical stimulation did not induce antiepileptic effects (ESWAE group, n = 4), patients with MTLE in whom no electrical stimulation was applied (MTLE group, n = 4) and autopsy material acquired from subjects without epilepsy (n = 4 obtained from three subjects). Enhanced 3H-DAMGO (MTLE, 755%; ESAE, 375%; ESWAE, 693%), 3H-DPDPE (MTLE, 242%; ESAE, 80%; ESWAE, 346%) and 3H-nociceptin (MTLE, 424%; ESAE, 217%; ESWAE, 451%) binding was detected in the PHC of all epileptic groups. However, tissue obtained from ESAE group demonstrated lower opioid receptor binding (3H-DAMGO, 44.5%, p < 0.05; 3H-DPDPE,
Reproduction, 2005
The effectiveness of b 2 -agonists in preterm delivery is reduced by several factors. The aim of ... more The effectiveness of b 2 -agonists in preterm delivery is reduced by several factors. The aim of this study was to determine the influence of late pregnancy in the uterus-relaxing effect of terbutaline in the rat in vitro. Rat uterine tissues from late pregnancy (days 15, 18, 20 and 22) were used. In vitro electrical field-stimulation (EFS) was used to evoke contractions. The radioligand-binding technique, reverse transcription-polymerase chain reaction and radioimmunoassay technique were used to determine the b-adrenergic receptor density and mRNA level and the plasma sex hormone level, respectively. The activated G-protein level of the b-adrenergic receptors was investigated by a radiolabelled GTP binding assay. EFS-induced contractions were inhibited by terbutaline. This effect decreased towards term with respect to both the EC 50 and maximal inhibition values. A drop in plasma progesterone level was also detected. Binding studies revealed an increase in b-adrenergic receptor number on the last day of pregnancy, which correlated with the change in receptor mRNA level. The G-protein-activating effect of terbutaline decreased continuously between days 15 and 20. Surprisingly, terbutaline decreased the G-protein activation to below the basal level on day 22. However, progesterone pretreatment set back the uterine action of terbutaline, increased the density of the b 2 -adrenergic receptors and their mRNA level and increased the G-proteinactivating property of terbutaline. These data provide evidence of a pregnancy-induced decrease in activated G-protein level after b 2 -agonist stimulation. The decrease in plasma progesterone level has a crucial role in this process. The effects of b 2 -adrenergic receptor agonists in tocolytic therapy may possibly be potentiated with progesterone. Reproduction (2005) 130 113-122 q 2005 Society for Reproduction and Fertility
Regulatory Peptides, 2004
In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-ac... more In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra-and pentapeptides selective for the n-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [ 35 S]GTPgS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile-lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (K e ) of 2.44 nM, and no agonist effect at 10 AM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the y-opioid receptor agonist [D-Ala 2 ,D-Leu 5 ]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [ 3 H]nociceptin-NH 2 binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [ 35 S]GTPgS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N-and the C-terminal region of the molecule. D
Pure and Applied Chemistry, 2000
ABSTRACT
Pharmacology, 1994
To find a delta-opioid receptor preferring peptide structure containing an Asp residue in a poten... more To find a delta-opioid receptor preferring peptide structure containing an Asp residue in a potentially interacting position, Tyr-Pro-Phe-Asp, Tyr-D-Ala-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp, Tyr-D-Ala-Gly-Phe-Asp alpha- and beta-methyl ester and Tyr-Gly-Gly-Phe-Asp peptides were synthesized and their biological activities were analyzed in vitro in mouse vas deferens and longitudinal muscle strip of guinea pig ileum. Changing the beta-methyl ester for an alkylating chloromethyl ketone moiety in the delta-receptor-selective agonist Tyr-D-Ala-Gly-Phe-Asp-beta-methyl ester enhanced further the delta-receptor preference. The delta-receptor selective chloromethyl ketone but not the beta-methyl ester gave a very slow washout after prolonged incubation in the mouse vas deferens bioassay; however, it was still readily displaceable by naloxone. The washout pattern of mu-specific Tyr-D-Ala-Gly-(Me)Phe chloromethyl ketone did not differ in the bioassays from that of the corresponding Gly5-ol derivative. Both chloromethyl ketones gave irreversible characteristics in the receptor binding assay.
Neuropharmacology, 2005
Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been sugge... more Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been suggested to act at a novel kappa3 receptor but also appears to bind to classical opioid receptors, and possibly the ORL1 receptor. We have used opioid receptor triple knockout mice, deficient in genes coding for the mu, delta and kappa-receptor, to characterise the relative contributions of opioid and ORL1 activity to the binding of this ligand, by carrying out receptor autoradiography with [3H]NalBzoH. As competing ligands we have used diprenorphine and nociceptin at 1 microM, alone or in combination, to determine the contribution of opioid and ORL1 receptor binding. At 4 nM [3H]NalBzoH showed labelling in wild-type brains indicative of broad spectrum classical opioid receptor binding. In the triple knockout brains all labelling was completely absent, suggesting that at this concentration there is no binding to ORL1 sites. However at 50 nM [3H]NalBzoH showed labelling in triple knockout brains with a distribution pattern indicative of ORL1 labelling. Quantitative analysis showed that nociceptin displaced typically 30% of the residual labelling in knockout brains whilst diprenorphine had relatively little effect. The data show that at 50 nM NalBzoH no binding was detected other than to classical opioid receptors or to ORL1 in an approximate ratio of 2:1.
Neuropeptides, 1986
ke,$one derivatives of leucine ertkephalfn (LE), D-Ala -Leu -enkephalin (DALE) and D-Ala -D-Leu -