Mária Szücs

Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Abstract: Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3‐[(3‐cholamido‐propyl)‐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 μ, δ, and κ opioid receptor binding sites were similar in membrane and digitonin‐solubilized fractions (48–50%μ, 35–37%κ, and 13–17%δ 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 45Å and 65Å for the digitonin extract and 31Å and 76Å for the CHAPS‐solubilized preparation.

Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain

Abstract: Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, and Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane‐bound and the solubilized receptors showed weak binding of enkephalin and μ‐specific drugs, suggesting that they are predominantly of the k‐type. The membrane‐bound and the soluble receptors showed the same distribution of subtypes, i.e., 70%k, 13%μ, and 17%δ for the membrane‐bound and 71%k, 17%μ, and 12%δ for the soluble receptors

New endomorphin analogues containing alicyclic β-amino acids: Influence on bioactive conformation and pharmacological profile

Endomorphins were subjected to a number of structural modifications in a search for their bioactive conformations. The alicyclic beta-amino acids cis-(1 S,2 R)ACPC/ACHC, cis-(1 R,2 S)ACPC/ACHC, trans-(1 S,2 S)ACPC/ACHC, and trans-(1 R,2 R)ACPC/ACHC were introduced into endomorphins to examine the conformational effects on the bioactivity. Use of a combination of receptor binding techniques, (1)H NMR, and molecular modeling allowed the conclusion that Pro (2) substitution by these residues causes changes in structure, proteolytic stability, and pharmacological activity. It seems that the size of the alicyclic beta-amino acids does not have marked influence on the receptor binding affinities and/or selectivities. Among the new analogues, the cis-(1 S,2 R)ACPC (2) and cis-(1 S,2 R)ACHC (2)-containing derivatives displayed the highest binding potencies and efficacies in receptor binding and ligand-stimulated [ (35)S]GTPgammaS functional experiments. Molecular dynamic simulations and (1)H NMR studies of the cis-ACPC/ACHC-containing analogues revealed that many conformations are accessible, though it is most likely that these peptides bind to the mu-opioid receptor in a compact, folded structure rather than extended.

CB1 receptor-independent actions of SR141716 on G-protein signaling: Coapplication with the μ-opioid agonist tyr-D-ala-gly-(NMe)phe- gly-ol unmasks novel, pertussis toxin-insensitive opioid signaling in μ-opioid receptor-Chinese hamster ovary cells

The CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716) has been shown by many investigators to inhibit basal G-protein activity, i.e., to display inverse agonism at high concentrations. However, it is not clear whether this effect is cannabinoid CB1 receptor-mediated. Using the ligand-stimulated [35S]guanosine 5′-3-O-(thio)triphosphate (GTPγS) assay, we have found that 10 μM SR141716 slightly but significantly decreases the basal [35S]GTPγS binding in membranes of the wild-type and CB1 receptor knockout mouse cortex, parental Chinese hamster ovary (CHO) cells, and CHO cells stably transfected with μ-opioid receptors, MOR-CHO. Accordingly, we conclude that the inverse agonism of SR141716 is CB1 receptor-independent. Although the specific MOR agonist Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) saturably and concentration-dependently stimulated [35S]GTPγS binding, SR141716 (10 μM) inhibited the basal by 25% and competitively inhibited DAMGO stimulation in the mouse cortex. In MOR-CHO membranes, DAMGO caused a 501 ± 29% stimulation of the basal activity, which was inhibited to 456 ± 22% by 10 μM SR141716. The inverse agonism of SR141716 was abolished, and DAMGO alone displayed weak, naloxone-insensitive stimulation, whereas the combination of DAMGO and SR141716 (10 μM each) resulted in a 169 ± 22% stimulation of the basal activity (that was completely inhibited by the prototypic opioid antagonist naloxone) because of pertussis toxin (PTX) treatment to uncouple MORs from Gi/Go proteins. SR141716 proved to bind directly to MORs with low affinity (IC50 = 5.7 μM). These results suggest the emergence of novel, PTX-insensitive G-protein signaling that is blocked by naloxone when MORs are activated by the combination of DAMGO and SR141716.

Expression of G-protein subtypes in cultured cerebral endothelial cells

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Identification of synaptic plasma membrane proteins co‐precipitated with fibrillar β‐amyloid peptide

The β‐amyloid peptide that is overproduced in Alzheimers disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar β‐amyloid (fAβ) 1–42. Triton X‐100‐soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co‐precipitation with fAβ, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one‐dimensional reversed‐phase HPLC and analysing them using an ion‐trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in‐gel tryptic digestion and to matrix‐assisted laser desorption/ionization time‐of‐flight mass measurements and post‐source decay analysis. Six different synaptosomal proteins co‐precipitated with fAβ were identified by both methods: vacuolar proton‐pump ATP synthase, glyceraldehyde‐3‐phosphate dehydrogenase, synapsins I and II, β‐tubulin and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase. Most of these proteins have already been associated with Alzheimers disease, and the biological and pathophysiological significance of their interaction with fAβ is discussed.

Binding characteristics and analgesic activity of D-Ala2-Leu5-enkephalin chloromethyl ketone

The chloromethyl ketone derivative of D-Ala2-Leu5-enkephalin (DALECK) was synthesized and its potency was tested in competing for 3H-naloxone binding sites and inducing analgesia. It was established that the compound is a potent affinity reagent at alkaline pH, blocking selectively and irreversibly the high-affinity (KD less than 1 nM) binding site. Intracisternally given DALECK showed a long-lasting, dose-dependent antinociceptive effect in the rat tail-withdrawal test. This could be completely antagonized by naloxone administration showing the reversible nature of DALECK in this in vivo assay. It is suggested that DALECK binds reversibly to the morphine receptor which mediates analgesia but irreversibly to the enkephalin receptor, the function of which remains to be elucidated.

Synthesis and opioid binding activity of dermorphin analogues containing cyclic β-amino acids

In the present work, eight conformationally constrained analogues of the mu specific opioid peptide dermorphin were synthesized by replacing D-Ala2 with stereoisomers of beta-amino-cycloalkane or cycloalkene carboxylic acids. The resulting peptides were tested for their potency to mu and delta opioid binding sites of rat brain membranes labelled with [3H]Tyr1-D-Ala2-MePhe4-Gly-ol, [3H]DAMGO and [3H]Ile5,6deltorphin, respectively. All of the new derivatives displayed highly attenuated binding to both receptor types, albeit the decrease in their potency seemed to be less in the case of delta binding. Trans position of the beta-amino groups resulted in higher binding affinities than that of the corresponding cis isomers, the latter being more flexible than the former. It is concluded that conformational constraints caused either by a rigid ring structure or cis isomers instead of D-Ala2 in dermorphin-derived peptides are unfavourable for binding activity to either opioid receptors. We propose that interaction of the larger heptapeptide derivatives of dermorphins with the mu receptor is distinct from that of the tetrapeptide morphiceptin.

Separation of κ-opioid receptor subtype from frog brain

Complete separation of the [3H]ethylketocyclazocine ([3H]EKC) specific binding (k subtype) from tritiated Tyr‐D‐Ala2‐Me‐Phe4‐Gly‐ol5 enkephalin (DAGO) and Tyr‐D‐Ala2‐L‐Leu5‐enkephalin (DALA) binding (μ‐and δ‐subtypes, respectively) was achieved by Sepharose‐6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient (S 20,w) for the k receptor‐detergent complex was 13.1 S and the corresponding Stokes radius 64 Å. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas μ‐ and δ‐specific ligands were unable to compete for the [3H]EKC binding sites, indicating that the κ subtype represents a separate molecular entity from the μ and δ receptor sites.

Species differences in the stereoselectivity of kappa opioid binding sites for [3H]U-69593 and [3H]ethylketocyclazocine.

Stereoselectivity of the binding sites for the specific kappa-opioid agonist [3H]U-69593, a benzeneacetamido based ligand was investigated in membrane suspension prepared from frog and rat brain, as well as guinea pig cerebellum, using the pure chiral forms of different unlabelled opiates. The ligand binding sites showed stereospecificity with at least three orders of magnitude differences in the affinities (measured as Ki values) of the opioid stereoisomer pairs both in rat and guinea pig membrane fractions. However, in frog brain membranes there was no substantial difference in potencies of the (-) and (+) isomers competing for the [3H]U-69593 binding sites. Another type of the kappa-site preferring opioid ligand, [3H]ethylketocyclazocine, a benzomorphan derivative was able to discriminate between (-) and (+) forms of the same compounds even in frog brain membrane preparation. Our data concerning binding profile of [3H]U-69593 in frog brain membranes are consistent with the observation that kappa opioid binding sites in frog (Rana esculenta) brain differ from those kappa-sites found in mammalian brains.