Judit Farkas

Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and nonopioid sites in rat brain.

Receptor binding properties of the naturally occurring opioid heptapeptide MERF were studied in rat brain membrane preparations using tritium‐labeled derivative of the peptide with 40 Ci/mmol specific radioactivity. Binding assays were performed in the presence of broad‐spectrum peptidase inhibitors at 0°C. Under these conditions, the equilibrium binding was achieved in 30–40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis. The apparent affinity (Kd value) of [3H]Met‐enkephalin‐Arg6‐Phe7, calculated from saturation binding data, was 10.2 ± 2.5 nM, and the maximal number (Bmax) of the heptapeptide binding sites was found to be 468 ± 43 fmol/mg protein. About half the sites represent nonopioid sites because the Bmax was only 255 ± 30 fmol/mg, when the nonspecific binding was measured with 1 μM naloxone. The rank order potencies of the examined compounds revealed that the opioid component of [3H]Metenkephalin‐Arg6‐Phe7 recognition sites are probably not μ and certainly not κ1 sites, whereas these sites are characterized by a κ2‐like binding profile. Considering the discrepancies between rat and frog brain found in the affinity of some compounds, including naltrindole and norbinaltorphimine, the presence of a novel, MERF‐selective “heptapeptide” binding site in rat brain membranes is also suggested. A number of the heterologous competition curves could be described by a high‐affinity stereospecific component and a substantially lower‐affinity binding element, which could completely be displaced with several peptide ligands such as Met5‐enkephalin, dynorphin(1–13), and unlabeled MERF but not by other compounds such as [D‐Ala2‐(Me)Phe4‐Gly5‐ol]enkephalin, morphine, or naloxone. [3H] Met‐enkephalin‐Arg6‐Phe7 binding can also be inhibited by FMRF‐amide analogs and sigma receptor ligands, such as (+)N‐allyl‐normetazocine and haloperidol, although with moderate affinity. It is concluded that the stereo‐specific high‐affinity binding is of opioid in character, whereas the residual sites characterized with their lower affinity are naloxone‐insensitive nonopioid sites. J. Neurosci. Res. 48:249–258, 1997.

High and low affinity receptors mediate growth effects of gastrin and gastrin-Gly on DLD-1 human colonic carcinoma cells.

Gastrin (G17) and N‐carboxymethylgastrin (G17‐Gly) have been shown to stimulate the growth of colon cancer cells both in vivo and in vitro. The identity of the receptor mediating these effects is controversial. A recent study demonstrated the presence of a low affinity binding site for G17 and G17‐Gly on the DLD‐1 human colon cancer cell line. The goal of the current study was to further investigate the role of this receptor in mediating the growth‐promoting effects of gastrin peptides. Binding of [Leu15]G17 and [Leu15]G17‐Gly to DLD‐1 cell membranes in competition with [3H]G17‐Gly was examined. Binding of [3H]cholecystokinin‐8 (CCK8) to DLD‐1 cell membranes was also assessed. Whole cell binding experiments were carried out using [125I‐Tyr12,Leu15]G17‐Gly. In addition, the ability of [Leu15]G17 and [Leu15]G17‐Gly to stimulate cell growth, as determined by cell counting, was tested. [Leu15]G17 and [Leu15]G17‐Gly competed with [3H]G17‐Gly at both a high and a low affinity site on DLD‐1 membranes. The IC50 values for [Leu15]G17 were 6.0×10−8 M and 6.9×10−6 M while those for [Leu15]G17‐Gly were 3.2×10−9 M and 4.9×10−6 M. [3H]CCK8 did not bind to either site. [Leu15]G17‐Gly also competed with [125I‐Tyr12,Leu15]G17‐Gly at both a high and a low affinity site on DLD‐1 cells with similar affinities as observed with membranes. [Leu15]G17 and [Leu15]G17‐Gly significantly stimulated the growth of DLD‐1 cells in a dose‐dependent and biphasic manner. The binding profiles of the peptides tested suggest that these sites are different from previously identified wild‐type and mutant CCK1 or CCK2 receptors.

Characterization of [3H]Met-Enkephalin-Arg6-Phe7 Binding to Opioid Receptors in Frog Brain Membrane Preparations

Abstract: A tritiated heptapeptide, [3H]Tyr‐Gly‐Gly‐Phe‐Met‐Arg‐Phe ([3H]Met‐enkephalin‐Arg6‐Phe7), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent KD value of the radioligand calculated from homologous displacement experiments was 3.4 nM, and the maximal number of specific binding sites was 630 fmol/mg of protein. The KD determined from equilibrium saturation binding studies was found to be 3.6 nM. However, the Hill coefficient was far below unity (nH = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3H]Met‐enkephalin‐Arg6‐Phe7 binding reflects a κ2‐ and/or δ‐subtype specificity of the heptapeptide. Binding to a κ1 and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met‐enkephalin‐Arg6‐Phe7 in frogs cannot be ruled out. The [3H]Met‐enkephalin‐Arg6‐Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.

Selective and high affinity labeling of neuronal and recombinant nociceptin receptors with the hexapeptide radioprobe [3H]Ac-RYYRIK-ol

The synthetic hexapeptide Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-ol (Ac-RYYRIK-ol) represents a highly potent and selective partial agonist ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (nociceptin receptor, NOPr). Ac-RYYRIK-ol has been labeled with tritium yielding [(3)H]Ac-RYYRIK-ol with exceptionally high specific radioactivity of 94Ci/mmol. The radioprobe is chemically stable even at 24 degrees C in ethanol solution for at least 4 days. No significant decomposition of the [(3)H]ligand occurred under the condition of the binding experiments indicating a fine enzymatic stability of the peptide. Radioreceptor binding studies were conducted using native neuronal NOPr preparation of rat brain membrane fractions and recombinant human nociceptin receptor ((h)NOPr) preparations from cultured Chinese Hamster Ovary (CHO) cells stably expressing (h)NOPr. Specific binding of the compound was reversible, saturable and of high affinity. No cross-reaction with the opioid receptors was observed suggesting superior NOPr selectivity of the ligand. Monophasic isotherm curves obtained in radioligand binding saturation and homologous displacement experiments indicated the presence of single binding sites in both preparations. Average densities of the [(3)H]Ac-RYYRIK-ol recognition sites were 237 and 749fmol/mg protein in rat brain and transfected cells, respectively. Equilibrium affinity values (K(d)s) were determined by three independent way providing identical results. In rat brain membranes K(d)s of 0.3-1.3nM were found depending upon the assay type. In homologous competition studies performed on (h)NOP-CHO cell membranes almost the same binding affinities were measured for Ac-RYYRIK-ol either with [(3)H]Ac-RYYRIK-ol (K(i) 2.8nM) or with [(3)H](Leu(14))nociceptin (2.3nM). A number of NOPr and opioid ligands were screened in heterologous displacement experiments and displayed a rank order of affinity profile being consistent with fairly good NOPr selectivity of the sites labeled by [(3)H]Ac-RYYRIK-ol. Taken together, the high molar activity, improved chemical and biological stability and the capability of the selective and high affinity labeling make this novel radioprobe available for further exploring the biochemical pharmacology and receptor-ligand interaction of the NOP receptor.

Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to multiple sites in rat and guinea pig cerebellum.

[3H]Met-enkephalin-Arg6-Phe7 (MERF) has been shown to label opioid (kappa2 and delta) and sigma2 sites in rat and frog brain membrane preparations, and no specific binding to kappa1 opioid receptors could be established (refs. 6 and 8). In this study the binding was examined in rat cerebellar membranes which are relatively rich in kappa2-sites, and in guinea pig cerebellar preparations where kappa1 opioid receptors are almost exclusively present. In accordance with our previous results, [3H]MERF binding could not be displaced in guinea pig cerebellar membranes neither with U-69,593 nor with naloxone or levorphanol suggesting no interaction with opioid sites, nevertheless a Kd of 2.8 nM was calculated in cold saturation experiments. In rat cerebellar membrane fractions about the half of the specific [3H]MERF binding sites was inhibited by opiate alkaloids such as naloxone, ethylketocyclazocine, or bremazocine. This portion of the heptapeptide binding sites was stereoselective as demonstrated by the difference in the affinities of the enantiomeric compounds levorphanol and dextrorphan, therefore it would represent an opioid site. In both tissues (-)N-allyl-normetazocine (SKF-10,047), which is also considered as sigma2 ligand, displayed the highest affinities. Among opioid peptides beta-endorphin and dynorphin(1-13) showed the highest potencies, displacing [3H]MERF also from its non-opioid sites. It was concluded therefore that [3H]MERF does not bind to kappa1 sites, and besides kappa2-opioid sites substantial binding to peptide preferring non-opioid sites, and/or sigma2 receptors also occurs.

Synthesis and binding characteristics of a novel enkephalin analogue, [3H]Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe.

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.

Biological half-life and organ distribution of [3H]8-arginine vasopressin following administration of vasopressin receptor antagonist OPC-31260.

The effects of the antidiuretic (V(2)) non-peptide receptor antagonist OPC-31260 on the plasma vasopressin level and the biological half-life and organ distribution of radiochemically pure, biologically active [(3)H]8-arginine vasopressin [spec. act.: 15.9 mCi/mmol (588 GBq/mmol)] were studied in Wistar rats. The plasma vasopressin level increased significantly throughout the whole experimental period (24 h). There was no change in the fast phase of the curves of total radioactivity disappearance from the plasma after the administration of [(3)H]arginine vasopressin (control: 1.51+/-0.17 min, OPC-31260-treated: 1.42+/-0.12 min, n=10). The fast phase of the disappearance curves of intact [(3)H]arginine vasopressin did not change either following the administration of OPC-31260 in a dose of 30 mg/kg p.o. (control: 1.06+/-0.19 min, OPC-31260-treated: 1.00+/-0.15 min, n=6). The slow phase of the biological half-life, which is characteristic for the examined compound, proved to be significantly longer (total radioactivity control: 9.29+/-0.61 min, OPC-31260-treated: 12.33+/-0.42 min, P<0.05, n=10; [(3)H]arginine vasopressin radioactivity: control: 5.96+/-0.58 min, OPC-31260-treated: 8.90+/-0.37 min, P<0.05, n=6). In the control rats, the radioactivity was accumulated to the greatest extent in the neurohypophysis, adenohypophysis and kidney. Following OPC-31260 administration, significantly more radioactive compounds accumulated in the kidney (control: 0.30+/-0.052 total radioactivity %/100 mg organ weight, OPC-31260-treated: 0.50+/-0.133 total radioactivity %/100 mg organ weight, P<0.05, n=10) and neurohypophysis (control: 0.37+/-0.053 total radioactivity %/100 mg organ weight, OPC-31260-treated: 0.52+/-0.076 total radioactivity %/100 mg organ weight, P<0.05, n=10). Our results permit the conclusion that the antidiuretic antagonist OPC-31260 not only blocks the V(2) receptors, but also increases the biological half-life of vasopressin. The longer biological half-life of vasopressin following OPC-31260 administration may play a role in the elevation of the plasma vasopressin level.

Pharmacological and functional biochemical properties of D-Ala2-D-Nle5-enkephalin-Arg-Phe

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.

A kontrasztív topik inverzhatóköri rejtélye – ami nincs is

Kiindulopontunk a hatokori elv, amely szerint a magyarban az ige előtti bővitmenyek hatokori sorrendje megfelel a szorendnek. Ezen elv alol viszont mintha kivetelt kepezne a kontrasztiv topik specifikalojaban allo kvantor- vagy mas ω operatorkifejezes, amelynek mintha inverz hatokore lenne a kontrasztiv topikot kotelezően kovető fokuszhoz kepest: Foc > ω, a szorend elleneben. Az inverzhatokori rejtelyre kidolgozott Gyuris-fele megoldasi javaslat egyik eleme a kovetkező – megmagyarazatlan – megfigyeles: csak azok a kontrasztiv topikot tartalmazo magyar mondatok jol formaltak, amelyeknek van egy olyan jol formalt megfelelőjuk, amelyben a kontrasztivan topikalizalt ω kifejezes posztverbalisan helyezkedik el. Azt allitjuk, hogy ebből a „jol formalt megfelelőből” egy szamos magyar szintaktikai jelensegre sikerrel alkalmazott maradvanymozgatasos technikaval ugy hozhatjuk letre a „problematikus” szorendi valtozatot, hogy inverzhatokori rejtelyről beszelni egyszerűen okafogyotta valik, mert az Foc > ω egyenes hatokori sorrenden az elvegzett maradvanymozgatas nem valtoztat. Our point of departure is that in Hungarian the scope order of preverbal (non-in-situ) constituents corresponds to their surface order; however, quantifiers and other operator expressions ω in a contrastive topic position give the impression of having inverse scope (Foc>ω) relative to the Focus expression following the given contrastive topic, apparently violating this generalization. The solution to this “scope-inversion puzzle” in Hungarian proposed by Gyuris (2009:150) rests upon this, unexplained, observation: “only those Hungarian sentences containing a contrastive topic are well-formed that have well-formed counterparts with the contrastive topic expression [ω] in postverbal position.” We claim that there is a remnant-movement-based technique, successfully applied to numerous Hungarian syntactic phenomena, which can also be applied to such “well-formed counterparts”, providing a syntactic structure for the “puzzling” sentences in which the relevant scope order is straight, and hence the inverse scope problem will simply not emerge at all.

HATNÉK-nominalization: Two subtypes of a highly verbal Hungarian deverbal nominalization

The paper discusses two subtypes of a special kind of Hungarian deverbal nominalization, “HATNEK-nominalization”, whose derivational suffix -hAtnek coincides with a sequence of the following three verbal suffixes: (i) the permissive modal suffix -hAt ‘can’, (ii) the conditional suffix -ne-, and (iii) a number-person suffix -k. Within the system of Hungarian deverbal nominalizations, a very high degree of verbalness is typical of both HATNEK-noun subtypes, of which we attribute a Giusti-style split-DP structure to the basic type, while the other, special, subtype is argued to have an exceptional structure with an “unboundedly expandable” (Spec,NP) position, capable of hosting huge verbal “inclusions”.