Phreatobacter oligotrophus gen. nov., sp. nov., an alphaproteobacterium isolated from ultrapure water of the water purification system of a power plant.

Strains of a novel alphaproteobacterium were isolated from ultrapure water of a Hungarian power plant on a newly developed medium. Phylogenetic analysis of the 16S rRNA gene sequences of the novel strains showed that these bacteria belong to a distinct lineage far from any known taxa. Based on the 16S rRNA gene sequences, strains PI_31, PI_25 and PI_21(T) exhibited the highest sequence similarity to Bosea minatitlanensis AMX51(T) (93.43 %) and Bosea thiooxidans DSM 9653(T) (93.36 %); similarity to all other taxa was less than 93.23 %. Fatty acid profiles, matrix-assisted laser-desorption/ionization time-of-flight mass spectra of cell extracts as well as physiological and biochemical characteristics indicated that our strains represent a novel genus and species within the class Alphaproteobacteria. The major isoprenoid quinone of the strains was Q-10, the major cellular fatty acids were C18 : 1ω7c and 11-methyl C18 : 1ω7c and the polar lipid profiles of the strains contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and several unknown phospholipids and other lipids. The characteristic diamino acid in their cell wall was meso-diaminopimelic acid. The G+C content of DNA of the proposed type strain PI_21(T) was 68.9 mol%. A new genus and species, Phreatobacter oligotrophus gen. nov., sp. nov., is proposed to accommodate the strains. Strain PI_21(T) ( = DSM 25521(T) = NCAIM B 02510(T)) is the type strain of Phreatobacter oligotrophus.

Aquipuribacter hungaricus gen. nov., sp. nov., an actinobacterium isolated from the ultrapure water system of a power plant.

A Gram-positive actinobacterium, strain IV-75(T), was isolated by using R2A agar from the ultrapure water system of a power plant in Hungary. The strain exhibited a rod-coccus cell cycle, and was strictly aerobic, non-motile, catalase-positive and oxidase-negative. 16S rRNA gene sequence analysis revealed that strain IV-75(T) belonged to the suborder Micrococcineae and clustered with members of the family Intrasporangiaceae. Its closest phylogenetic neighbour was Arsenicicoccus bolidensis CCUG 47306(T) (94.3% 16S rRNA gene sequence similarity). The peptidoglycan of strain IV-75(T) contained meso-diaminopimelic acid and MK-10(H(4)) was the major menaquinone. The polar lipid pattern contained phosphatidylglycerol, two unidentified phospholipids, one glycolipid and several other lipid components. The major fatty acids were anteiso-C(15:0), C(18:1)ω9c and C(16:0). Based on the moderate levels of 16S rRNA gene sequence similarity to all members of the family Intrasporangiaceae and the unique combination of chemotaxonomic characteristics, strain IV-75(T) is considered to represent a novel species of a new genus, for which the name Aquipuribacter hungaricus gen. nov., sp. nov. is proposed. The type strain of Aquipuribacter hungaricus is IV-75(T) (=DSM 21674(T)=NCAIM B 02333(T)).

Nociceptin inhibits uterine contractions in term-pregnant rats by signaling through multiple pathways.

The actions of the endogenous peptide nociceptin (PNOC; previously abbreviated as N/OFQ) on the myometrium have not been investigated previously. Our aim was to study the presence and functional role of PNOC in the modulation of uterine contractility in pregnant rats at term. The presence of PNOC and its receptors (OPRL1; previously called NOP) in the uterus were detected by radioimmunoassay and radioligand-binding experiments. The PNOC-stimulated G protein activation was assessed by a [(35)S]GTPgammaS-binding technique. The effects of PNOC in uterine rings precontracted with KCl or oxytocin were also tested in vitro. Uterine levels of cAMP were measured by enzyme immunoassay. The K(+) channel blockers tetraethylammonium and paxilline were used to study the role of K(+) channels in mediating the uterine effects of PNOC. Both PNOC and OPRL1 were present in the uterus. PNOC revealed a maximum contraction inhibition of approximately 30%, which was increased to 40% by naloxone. Naloxone and pertussis toxin significantly attenuated the G protein-stimulating effect of PNOC. The uterine cAMP levels were elevated by PNOC and naloxone and after preincubation with pertussis toxin. Tetraethylammonium and paxilline reduced the contraction-inhibiting effect of PNOC and naloxone to approximately 10% and 15%, respectively. We presume that PNOC plays a role in regulating uterine contractility at term. Its effect is mediated partly by stimulatory heterotrimeric G (G(s)) proteins coupled to OPRL1 receptors and elevated cAMP levels, and also by Ca(2+)-dependent K(+) channels. Our results demonstrate a novel action and signaling pathway for PNOC that might be a potential drug target.

Binding studies of novel, non-mammalian enkephalins, structures predicted from frog and lungfish brain cDNA sequences.

Leu- and Met-enkephalin were the first endogenous opioid peptides identified in different mammalian species including the human. Comparative biochemical and bioinformatic evidence indicates that enkephalins are not limited to mammals. Various prodynorphin (PDYN) sequences in lower vertebrates revealed the presence of other enkephalin fingerprints in these precursor polypeptides. Among the novel enkephalins Ile-enkephalin (Tyr-Gly-Gly-Phe-Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs, while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by analyzing brain cDNA sequences encoding a PDYN of the African lungfish (Protopterus annectens). Ile-enkephalin can also be found in the PDYNs of four other fish species including the eel, bichir, zebrafish and tilapia, but no further occurrence for the Phe-enkephalin motif is available as yet. Based on sequencing data, the biological relevance of Phe- and Ile-enkephalin is suggested, because both of them can arise by regular posttranslational enzymatic processing of the respective neuropeptide precursors. In various receptor binding assays performed on rat brain membrane preparations both of the new peptides turned out to be moderate affinity opioids with a weak preference for the delta-opioid receptor (DOP) sites. Phe-enkephalin of the lungfish displayed rather unexpectedly low affinities toward the mu-opioid receptor (MOP) and DOP, while exhibiting moderate affinity toward the kappa-opioid receptor (KOP). In receptor-mediated G-protein activation assays measured by the stimulation of [(35)S]GTPgammaS binding, Met-enkephalin produced the highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin, whereas the least efficacious among these endogenous peptides was still more effective than the prototype opiate agonist morphine in these functional tests.

The effect of increased NaCl intake on rat brain endogenous µ-opioid receptor signalling.

Numerous studies demonstrate the significant role of central ß-endorphin and its receptor, the µ-opioid receptor (MOR), in sodium intake regulation. The present study aimed to investigate the possible relationship between chronic high-NaCl intake and brain endogenous MOR functioning. We examined whether short-term (4 days) obligatory salt intake (2% NaCl solution) in rats induces changes in MOR mRNA expression, G-protein activity and MOR binding capacity in brain regions involved in salt intake regulation. Plasma osmolality and electrolyte concentrations after sodium overload and the initial and final body weight of the animals were also examined. After 4 days of obligatory hypertonic sodium chloride intake, there was clearly no difference in MOR mRNA expression and G-protein activity in the median preoptic nucleus (MnPO). In the brainstem, MOR binding capacity also remained unaltered, although the maximal efficacy of MOR G-protein significantly increased. Finally, no significant alterations were observed in plasma osmolality and electrolyte concentrations. Interestingly, animals that received sodium gained significantly less weight than control animals. In conclusion, we found no significant alterations in the MnPO and brainstem in the number of available cell surface MORs or de novo syntheses of MOR after hypertonic sodium intake. The increased MOR G-protein activity following acute sodium overconsumption may participate in the maintenance of normal blood pressure levels and/or in enhancing sodium taste aversion and sodium overload-induced anorexia.

In vitro and in vivo pharmacological profile of UFP-512, a novel selective delta-opioid receptor agonist; correlations between desensitization and tolerance.

BACKGROUND AND PURPOSE: Delta-opioid receptors (DOP receptors) could represent a novel target in the treatment of depressive disorders. To explore this new field of interest, the development of highly selective DOP receptor agonists is essential. UFP-512 [H-Dmt-Tic-NH-CH(CH2-COOH)-Bid], was recently shown to behave in vitro as a selective and potent DOP receptor agonist and to promote antidepressant- and anxiolytic-like effects in vivo (Vergura et al., 2007). Here, we have characterized the pharmacological properties of UFP-512 and established a link between desensitization and tolerance. EXPERIMENTAL APPROACH: Studies were performed in the human neuroblastoma SK-N-BE cells to establish i) binding parameters for UFP-512 ii) signalling pathways activated after acute and chronic treatment iii) regulation (phosphorylation and trafficking) of human DOP (hDOP) receptors after sustained activation by UFP-512. In vivo, we studied UFP-512-induced antidepressant-like effects after acute or chronic treatment in the mouse forced swimming test. KEY RESULTS: In vitro, UFP-512 was a high affinity agonist for DOP receptors. While UFP-512 induced marked phosphorylation of DOP receptors on Ser363, we observed a low desensitization of the cAMP pathway, associated with receptor endocytosis and recycling without any reduction on extracellular signal-regulated protein kinase 1/2 activation. In vivo, acute administration of UFP-512 produced an antidepressant-like effect, without any sign of tolerance after chronic administration. CONCLUSIONS AND IMPLICATIONS: There was a correlation between weak desensitization, significant internalization and recycling of the human DOP receptors and lack of tolerance to UFP-512. This suggests that this compound would be a promising drug prototype for exploring innovative treatments for mood disorders.

A novel non-opioid binding site for endomorphin-1.

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors, such as rodent cerebellum, or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.

Inhibition of esterase and amidase activities of alpha- and beta-thrombin in the presence of antithrombin III and heparin.

Inhibition of the esterase and amidase activities of bovine alpha- and beta-thrombin in the presence of antithrombin III and heparin has been studied. It was found that both the esterase and amidase activities of alpha-thrombin were inhibited by antithrombin III and the reactions were accelerated by heparin. The inhibition of amidase and esterase activities of beta-thrombin by antithrombin III has also been demonstrated. Heparin however did not increase the rate of inactivation of the enzyme.

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.

Deltorphin II analogues with 6-hydroxy-2-aminotetralin-2-carboxylic acid in position 1.

Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties. Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead of Tyr(1) in the message domain. In the radioreceptor-binding studies, in which type-specific tritiated opioid ligands were used, (R)- and (S)-Hat-deltorphins exhibited similar K(i) values, revealing high delta selectivity. The peptides displayed agonist properties in the in vitro bioassay, with IC(50) values in the subnanomolar range in the mouse vas deferens assay and in the micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity toward delta receptors. The agonist property of the new ligands was confirmed by means of [(35)S]GTPgammaS-binding experiments in membranes of the rat frontal cortex.

Conformationally constrained deltorphin analogs with 2-aminotetralin-2-carboxylic acid in position 3.

Two approaches to the design of very active and highly selective delta opioid peptides were used to obtain new deltorpin analogs with altered hydrophobic and stereoelectronic properties. Deltorphin I and II analogs were synthesized involving the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 2-aminotetralin-2-carboxylic acid (Atc) instead of Phe in the message domain. The peptides were agonists in the subnanomolar range in the MVD assay and in the micromolar or higher range in the GPI assay, showing a very high selectivity for delta receptors. A very similar trend could be observed in radioreceptor binding assays in which selective tritiated opioid ligands were used. (R)- and (S)-Atc-deltoriphins exhibited similar Ki values in the binding assay, with almost complete loss of the stereospecificity of the binding. Conformational studies provided evidence for the little disturbance of the backbone conformational equilibrium when Phe3 is replaced by (S)- or (R)-Atc. The use of the Atc constraint gives additional evidence that, during its interaction with the delta receptor, the side chain of residue 3 adopts the trans conformation at chi 1.

The effect of repeated administration of morphine, cocaine and ethanol on mu and delta opioid receptor density in the nucleus accumbens and striatum of the rat.

The present study was carried out to evaluate the effect of morphine, cocaine and ethanol on the density of opioid receptors in the nucleus accumbens and striatum of rat brain. The animals were injected i.p. with morphine in a single dose 20 mg/kg, or twice daily for 10 days in increasing doses of 20-100 mg/kg. Cocaine was administered in a dose of 60 mg/kg/day following the "binge" paradigm, every hour for 3 h, one day (single treatment) or five days (chronic treatment). Ethanol was administered in drinking water at increasing concentrations of 1-6% v/v, for one month. As shown by receptor autoradiography, single morphine and cocaine administration did not influence the binding density of the selective ligand of delta2 receptors [3H]Ile5,6deltorphin b, but single administration of cocaine decreased binding density of a highly selective antagonist of delta receptors, [3H]H-Tyr-Tic psi[CH2-NH]Phe-Phe-OH. Repeated morphine administration decreased the receptor density after both ligands of the delta receptor in the nucleus accumbens after 3, 24 and 48 h, and in the striatum after 24 and 48 h. The density of [3H]Ile5,6deltorphin b binding remained unchanged in both structures following repeated cocaine administration. After repeated cocaine administration either no changes (3 h) or a decrease in the binding of [3H]H-Tyr-Tic psi[CH2-NH]Phe-Phe-OH in the nucleus accumbens and striatum were observed after 24 and 48 h. Ethanol did not influence the binding density of [3H]H-Tyr-Tic psi[CH2-NH]Phe-Phe-OH and [3H]Ile5,6deltorphin b in the nucleus accumbens and striatum at any time-point studied. In the nucleus accumbens and striatum, no changes were found in the binding density of [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol following single or repeated morphine administration. At 3 h after single or repeated "binge" cocaine administration, the binding of [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol was not changed in either structure, but after 24 h the density of mu opioid receptors was decreased in both structures. Ethanol given to rats in drinking water decreased the binding of [3H]Tyr-D-Ala-Gly-MePhe-Gly-ol at the time of exposure to ethanol, yet in the nucleus accumbens only. Ethanol withdrawal decreased the density of the mu receptor in both structures after 24, 48 and 96 h. The above data indicate that repeated administration of morphine evokes a long-lasting down-regulation of the density of delta1 and delta2 opioid receptors, whereas cocaine affects in a similar way only the delta1 subtype in the nucleus accumbens, and to a lesser extent in the striatum. A long-term intake of ethanol solution down-regulates mu opioid receptors in both structures, but has no effect on any type of delta receptors. Thus changes in the particular opioid receptor depend on the type of drug used. Furthermore, the most profound changes are observed after late withdrawal, which may play some role in maintaining the state of dependence.

Inactivation of alpha- and beta- thrombin by antithrombin-III and heparin.

Inactivation of alpha- and beta-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b). It was found that the inactivation rates of alpha- and beta-thrombin by antithrombin were different, namely alpha-thrombin was more sensitive to antithrombin than beta-thrombin. Heparin facilitated the complex formation between alpha-thrombin and antithrombin-III, whereas beta-thrombin inactivation was only slightly affected. Furthermore, heparin protected alpha-thrombin against the inactivating effect of heat, while beta-thrombin lost its activity during the heat treatment. These findings suggest that the formation of beta-thrombin in blood circulation may have an important role in thrombosis predisposition.

Isolation of antithrombin III from normal and alpha1-antitrypsin-deficient human plasma.

The plasma of individuals, hetero- or homozygous for alpha1-antitrypsin deficiency, contains greatly decreased amounts of antithrombin activity as assayed against factor Xa. However, heparin stimulation of the residual antithrombin activity is observed, which is comparable to that of normal plasma. Antithrombins isolated from both normal and alpha1-antitrypsin deficient plasma by a simplified procedure are indistinguishable in both properties and yields. The microheterogeneity observed on isoelectric focusing of both preparations can be eliminated by treatment with neuraminidase. Neither purified human antithrombin nor alpha1-antitrypsin, when assayed against bovine trypsin, is stimulated by heparin. These results clearly establish the unique natures of antithrombin and alpha1-antitrypsin and show that about 75% of the antithrombin activity measured in normal plasma is due to alpha1-antitrypsin. Estimates of antithrombin III activity in normal plasma by assays dependent on enzymatic activity can probably be obtained only in the presence of heparin.

Weaning-induced development of delta-opioid receptors in rat brain: differential effects of guanine nucleotides and sodium upon ligand-receptor recognition.

1. We have previously shown that weaning at day 21 increases delta-opioid receptor binding in the brain at day 25, which might be due to stimulation of the development of a delta-opioid receptor subtype or activation of G-protein coupling processes. 2. We have addressed the possibility that weaning stimulates coupling of the delta-receptor by homogenate binding studies with four agonist and one antagonist radioligand in the presence of a GTP analogue and Na+ in brain tissue from weaned and non-weaned animals. 3. Saturation studies with three agonist ligands ([3H]-deltorphin I, [3H]-S-Atc-Ile(5,6)deltorphin I and [3H]-R-Atc-Ile(5,6)deltorphin II) showed higher levels of maximal binding in brains from 25-day weaned than in brains from non-weaned rats. The magnitude of the effects of GMPPNP and Na+ in decreasing this binding was ligand dependent and in each case was significantly more marked in brains from weaned animals. GMPPNP and Na+ were completely without effect on Bmax for, [3H]-S-Atc-Ile(5,6)deltorphin I and [3H]-R-Atc-Ile(5,6)deltorphin II in brains from non-weaned rats. 4. [3H]-Ile(5,6)deltorphin II and [3H]-naltrindole showed no differences in labelling between weaned and non-weaned groups and both groups responded similarly to the effects of GMPPNP and Na+ treatment. 5. GMPPNP and Na+ had small effects on binding affinity (K(D)) for some of the agonist radioligands which were similar in both weaned and non-weaned groups. 6. Weaning induced increases in binding of delta-receptors in 25-day rats can be explained in terms of the way delta-agonist radioligands recognize the receptor environment.

Characterization of N,N(Me)2-Dmt-Tic-OH, a delta selective opioid dipeptide antagonist.

N,N(Me)2-Dimethyl-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-OH (N,N(Me)2-Dmt-Tic-OH) is a very selective delta opioid dipeptide with elevated antagonist activity. We have radiolabelled this compound by catalytic tritiation of the N,N(Me)2-Dmt(3',5'-I2)-Tic-OH precursor. The ligand labelled rat brain membranes with a Kd value of 0.42 nM and a Bmax of 63.12 fmol/mg protein. The new tritiated ligand showed high affinity for the delta opioid receptor whereas its binding at mu and kappa opioid receptors was weak. N,N(Me)2-Dmt-Tic-OH was able to inhibit the agonist-stimulated binding of the non-hydrolysable GTP analogue ¿35SGTPgammaS, thus attenuating the activation of G proteins via opioid receptors. This simple opioid dipeptide in both normal and labelled form may serve as a useful tool to study delta opioid receptors in vitro and in vivo.

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.

Interaction of agonist peptide [3H]Tyr-D-Ala-Phe-Phe-NH2 with mu-opioid receptor in rat brain and CHO-mu/1 cell line.

Opioid receptor binding properties of [3H]Tyr-D-Ala-Phe-Phe-NH2 (TAPP) were characterized in rat brain and Chinese hamster ovary (CHO) cells expressing the rat mu-receptor. In rat brain, [3H]TAPP labeled a single class of opioid sites with a dissociation constant (Kd) of 0.31 nM and maximal number of binding sites (Bmax) of 119 fmol/mg protein. In CHO-mu/1 cell membranes, the Kd and Bmax values were 0.78 nM and 1806 fmol/mg protein, respectively. Binding to rat brain was demonstrated to be pharmacologically identical to that obtained with CHO-mu/1 cell membranes and modulated by Na+ ions and guanine nucleotides. The high affinity and selectivity of [3H]TAPP together with its low non-specific binding make this radioligand a useful tool for labeling the native and cloned mu-opioid receptor.

Microtubule disassembly increases the number of opioid receptor binding sites in rat cerebrum membranes.

The role of microtubules in opioid receptor binding was studied by using microtubule assembly inhibitors. Preincubation of rat cerebrum membranes with podophyllotoxin or colchicine provoked a marked increase in the number of binding sites as judged by [3H]-naloxone, [3H]-morphine and [3H]-D-Ala2-Leu5-enkephalin binding experiments. These results indicate microtubule involvement in regulation of opioid receptor expression.

Synthesis and binding characteristics of the highly selective radiolabelled deltorphin analogues containing 2-aminotetralin-2-carboxylic acid in position 3.

Following the description of [3H]Ile5,6deltorphin II, when it was reported that changes in hydrophobicity at positions 5 and 6 give rise to analogues with increased delta-receptor affinity and selectivity, new conformationally restricted deltorphin analogues were designed. A synthetic amino acid, 2-aminotetralin-2-carboxylic acid (Atc), was introduced at position 3 instead of Phe in Ile5,6deltorphin I and II, and the resultant compounds were prepared in tritiated form. Opioid binding sites specific for [3H]S-Atc3,Ile5,6deltorphin I and [3H]R-Atc3,Ile5,6deltorphin II were characterized in rat brain membranes. Their binding was saturable, stereoselective and inhibited by delta-selective ligands with high potency. They labelled single class of opioid sites at 35 degrees C with high affinity (Kd approximately 0.3 nM), Bmax values of 130 fmol/mg protein, and very low non-specific binding was observed. Both tritiated deltorphin analogues showed delta-receptor specificity in rat brain, therefore they could represent excellent new radioligands for investigating the complexity of the opioid receptor systems.