PubMed Health⌕ Search

Biomedical subjects

M Szücs

Publications and source records attributed to M Szücs.

At least 37 records · Page 2Linked to original sources
PubMed1990

Sigma and opioid receptors in human brain tumors.

Human brain tumors (obtained as surgical specimens) and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [3H]1,3-di-o-tolylguanidine (DTG), whereas opoid receptor subtypes were measured with tritiated forms of the following: mu, [D-ala2,mePhe4,gly-ol5]enkephalin (DAMGE); kappa, ethylketocyclazocine (EKC) or U69,593; delta, [D-pen2,D-pen5]enkephalin (DPDPE) or [D-ala2,D-leu5]enkephalin (DADLE) with mu suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels (pmol/mg protein) found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. kappa opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed.

Adenocarcinoma↗
PubMed1990

Evidence for delta-opioid binding and GTP-regulatory proteins in 5-day-old rat brain membranes.

The availability of the bispenicillamine enkephalin [3H] [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for delta-opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5-day-old neonatal (P-5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P-5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The delta-specificity of the sites was further proven by competition experiments with mu- and delta-selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 microM 5'-guanylylimidodiphosphate inhibited it by decreasing the total number of high-affinity binding sites in both P-5 animals and adults. Pertussis toxin-catalyzed ADP ribosylation experiments revealed the presence of 40-kDa proteins, with a molecular mass corresponding to G protein subunits alpha i/alpha o, as early as 1 h after birth. There was a low, but detectable, basal low-Km GTPase activity in P-5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high-affinity [3H]DPDPE binding as well as GTP-regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.

Animals↗
PubMed1989

Cation and guanine nucleotide effects on ligand binding properties of mu and delta opioid receptors in rat brain membranes.

Monovalent cations such as Na+, K+ and Li+ at 100 mM concentration inhibit the binding of mu and delta opioid agonists (e.g. (3H) dihydromorphine and (3H) D-Ala2-Leu5-enkephalin), enhance opioid antagonist [(3H) naloxone] binding in rat brain membranes. Divalent cations have an opposite effect: Mg2+ or Mn2+ (2mM) increase both mu and delta agonist binding, and decrease the antagonist binding. The effect of guanosine 5'-triphosphate and its non-hydrolysable analogue 5'-guanylyl-imidodiphosphate at micromolar concentrations is similar to that of sodium ion. In kinetic experiments, guanine nucleotides promote the dissociation of radiolabelled agonists from their binding sites by increasing the rate of dissociation. Equilibrium saturation binding studies show that only binding of the opioid agonist ligands are significantly inhibited by 5'-guanylyl-imidodiphosphate.

Animals↗
PubMed1987

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. I. Binding characteristics in rat brain membranes.

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin was synthesized in a radioactive form, and the resulting compound (3H-DALECK) was used to label opioid receptors. 3H-DALECK binds with high affinity, specificity and saturability to rat brain membranes. The number of sites labeled is 130 fmoles/mg protein. Unlabeled opioids inhibited the binding of 3H-DALECK; etorphine and DAGO being most potent. A 10-fold preference for mu sites over delta was seen in site-specific competition experiments; while DALECK displayed low affinity for kappa sites of rat brain. DALECK irreversibly blocked a certain population of sites. Approximately 40% of 3H-DALECK binding at 15 min, and 60% at 60 min association time did not dissociate in the presence of a large excess of unlabeled DALECK and was resistant to washing. Autoradiography performed after SDS-PAGE revealed specific alkylation of proteins with molecular weight of 74, 65, 56, 43 and 34 kD. These results demonstrate the applicability of using 3H-DALECK to covalently label opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes.

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.

Amino Acid Chloromethyl Ketones↗
PubMed1987

Detailed analysis of heterogeneity of [3H]naloxone binding sites in rat brain synaptosomes.

The experiments reported in this paper address the question of heterogeneity of [3H]naloxone binding sites in rat brainstem synaptosomal preparations at 23 degrees C in the presence of 100 mM sodium chloride. Kinetic analysis in the presence of 0.4, 4 and 10 nM [3H]naloxone gave pseudo-first order association rate of 0.9 +/- 0.04, 1.23 +/- 0.08 and 1.06 +/- 0.08 min-1, respectively. The dissociation of a 1 nM [3H]naloxone receptor complex was biphasic with dissociation rate constants of 1.8 and 0.4 min-1. On the other hand, dissociation of 10 nM [3H]naloxone was monophasic with a kd of 1.1 min-1. Two subpopulations of binding sites were also observed by steady state binding studies, with Kd values of 0.5 and 3.4 nM and a ratio of high to low affinity sites of 1:9. Heterogeneity of [3H]naloxone binding sites could be seen by displacement studies performed with opioid peptides and alkaloids. We suggest that our data best fits a model with two independent naloxone binding sites wherein either one or both undergo a multi-step interaction with ligand.

Animals↗
PubMed1987

Purification of a kappa-opioid receptor subtype from frog brain.

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1987

Differential ontogeny of divalent cation effects on rat brain delta-, mu-, and kappa-opioid receptor binding.

The effect of the divalent cations, Mn2+, Mg2+ and Ca2+ on rat forebrain delta-, mu- and kappa-receptor binding was examined during postnatal development. It was found that delta-receptor binding, assessed with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) (+ 10 nM D-Ala2- MePhe4-Gly-ol5-enkephalin (DAMGE)), was stimulated by the 3 cations in a dose- and developmental time-dependent manner. delta-Binding was most sensitive to the cations during the first week postnatal, prior to the appearance of high-affinity delta-binding. In contrast, inhibition of mu-receptor binding ([3H]DAMGE) by divalent cations appeared early in development and remained constant throughout the postnatal period. Divalent cation inhibition of kappa-binding ([3H]ethylketocyclazocine ([3H]EKC) + 100 nM DAMGE and 100 nM DADLE) appeared after the second week postnatal. These results demonstrate that the characteristics and postnatal development of divalent cation modulation of mu-, delta- and kappa-binding is distinctly different. Thus, the neonate may be a good model system to examine the binding properties and functions of delta- and kappa-receptor subtypes.

Animals↗
PubMed1987

Guanine nucleotide and cation regulation of mu, delta, and kappa opioid receptor binding: evidence for differential postnatal development in rat brain.

A study of the onset of cation and guanine nucleotide regulation of delta, mu, and kappa rat brain opioid receptors during postnatal development was undertaken. Site-specific binding assays were utilized for each receptor type and the effects of 0.5 mM MnCl2, 100 mM NaCl, and/or 50 microM guanosine-5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] were assessed. The most pronounced changes of opioid binding were seen in the presence of Mn2+. In adults, agonist binding to delta sites was stimulated by Mn2+, whereas that to mu sites was not affected and kappa binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on delta sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of kappa site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of delta binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of mu and delta receptor binding by Na+ was greater, and the Mn2+ reversal of this effect was smaller, in the first 2 postnatal weeks than in adults. Gpp(NH)p + Na+ regulation did not change appreciably during the postnatal period. However, Mn2+ reversal of the considerable inhibition elicited by the combination of Na+ and Gpp(HN)p was developmental time-dependent. The data are discussed in terms of multiple sites of interaction for guanine nucleotides and cations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Kinetics and physical parameters of rat brain opioid receptors solubilized by digitonin and CHAPS.

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.

Animals↗
PubMed1986

Multiple sites of divalent cation modulation of delta opioid receptor binding.

Having established differential ontogeny for rat brain mu, delta, and kappa opioid receptors (Spain et al. 1985) we have examined the effect of divalent cations on delta receptors during postnatal development. The pronounced inhibition of the binding of 1 nM [3H]D-Ala2-D-Leu5-enkephalin (DADLE) by 100 mM Na+ was reversed by Mn++ to the levels seen in the absence of ions both in adults and in 10-day-old neonates. Mg++ and Ca++ on the other hand were much less potent in adults, and ineffective in neonates. These results suggest that, in the presence of Na+, regulation of delta binding with Mg++ or Ca++ emerges subsequent to Mn++ modulation in postnatal development. Dose-dependency studies in adults performed with each ion in the presence of fixed concentrations of either of the other ions revealed that while Mg++ or Ca++ antagonized stimulation of delta binding by Mn++ and vice versa, Mg++ and Ca++ displayed no antagonism toward each other. Our data suggest the existence of at least two divalent cation regulatory sites for delta opioid receptors, one of which appears to be specific for Mn++ and can readily be detected in the presence of Na+.

Animals↗
PubMed1986

[Amiodarone-induced hyperpigmentation].

Five patients are reported in whom brownish grey discoloration occurred on the light-exposed parts of the dermis after long-term amiodarone-medication. Histological investigation shows that the deposition of lipofuscin in the small veins and perivasal superior part of the corium is responsible for the occurrence of this discoloration. With reference to recent data in the literature, the pathomechanism and prevention of this pigmentation are discussed.

Aged↗
PubMed1985

Separation of kappa-opioid receptor subtype from frog brain.

Complete separation of the [3H]ethylketocyclazocine [( 3H]EKC) specific binding (kappa subtype) from tritiated Tyr-D-Ala2-Me-Phe4-Gly-ol5 enkephalin (DAGO) and Tyr-D-Ala2-L-Leu5-enkephalin (DALA) binding (mu-and delta-subtypes, respectively) was achieved by Sepharose-6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (s20.w) for the kappa 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 mu- and delta-specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular to compete for the [3H]EKC binding sites, indicating that the kappa subtype represents a separate molecular entity from the mu and delta receptor sites.

Animals↗
PubMed1985

Melphalan potently substitutes the N-terminal Tyr of D-Ala2-Leu5-enkephalin methyl ester.

In search of an affinity label of the opioid receptor, the nitrogen mustard melphalan, Mel, was built into the peptide chain of D-Ala2-Leu5-enkephalin (DALE) methyl ester in different positions. We report now that in contrast to the previous observations that an intact Tyr in position 1 is essential for opioid activity [(1980) Annu. Rev. Pharmacol. Toxicol. 20, 81-110], substitution of Tyr by Mel did not result in a loss of the binding affinity. Mel1, Leu5-enkephalin-OMe competed for the binding sites of [3H]naloxone as potently as DALE did; IC50 values for both compounds were 50 nM. Mel substitution has led to one order potency decrease in binding to the delta-sites. 0.5-1 microM of the compound irreversibly inactivates 50% of the binding sites of [3H]naloxone, and 5-10 microM of that of [3H]DALE. These results shed new light on the structural requirements established for opioid peptides. In addition, the new derivative can be used as an affinity label of the opioid receptor.

Animals↗
PubMed1985

Characterization of rat brain opioid receptors by [Tyr-3,5-3H]1, D-Ala2, Leu5-enkephalin binding.

[Tyr-3,5-3H]1, D-Ala2, Leu5-enkephalin [( 3H]DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from [Tyr-3,5-diiodo]1, D-Ala2, Leu5-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting [Tyr-3,5-3H]1, D-Ala2, Leu5-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24 degrees C within 45 min. The pseudo first order association rate constant was 0.1 min-1. The dissociation of the receptor-ligand complex was biphasic with k-1-s of 0.009 and 0.025 min-1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed a KD = 0.7 nM and Bmax = 60 fmol/mg protein; the low affinity site had a KD = 5 nM and Bmax = 160 fmol/mg protein. A series of opioid peptides inhibited [3H]DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds preferentially to the delta subtype of opioid receptors. Modification of the original peptides either at the C or N terminal ends of the molecules resulted in a decrease in their affinity.

Animals↗