Rat Liver Membranes Research Articles

Tauroursodeoxycholic acid inserts the bile salt export pump into canalicular membranes of cholestatic rat liver

Ursodeoxycholic acid exerts anticholestatic effects in chronic cholestatic liver disease in humans as well as in experimental animal models of cholestasis. Its taurine conjugate, TUDCA, was recently shown to stimulate insertion of the apical conjugate export pump, Mrp2 (ABCC2), into canalicular membranes of rat hepatocytes made cholestatic by exposure to taurolithocholic acid (TLCA). The aim of this immunoelectronmicroscopic study was to test whether TLCA and TUDCA modulate the canalicular density of the other key apical transporter, the bile salt export pump, Bsep (ABCB11), in a similar way. Immunoelectronmicroscopic analysis of Bsep density on canalicular membranes, microvilli, and pericanalicular area of hepatocytes was performed in rat liver tissue prepared after liver perfusion with bile acids or carrier medium only. TLCA (10?μmol/l for 50?min) decreased Bsep density in canalicular membranes to 31% of controls (P<0.05) when bile flow was reduced to 35% of controls (P<0.05). Concomitantly, Bsep density in a 1?μm pericanalicular zone increased to 202% (P<0.05) indicating effective retrieval of Bsep from the canalicular membrane induced by TLCA. Coadministration of TUDCA (25?μmol/l) led to a 3.2-fold increase of Bsep density in canalicular membranes equal to control liver (P<0.05 vs TLCA) in association with a 3.8-fold increase of bile flow (P<0.05 vs TLCA). Stimulation of apical membrane insertion of key transporters like the bile salt export pump, Bsep, and—as previously shown—the conjugate export pump, Mrp2, may contribute to the anticholestatic action of UDCA amides in cholestatic conditions.

Modulation of oxidative damage by natural products

Using rat liver membrane as a model system, modulation of oxidative damage induced by pathophysiological agents such as photosensitization and radiation was examined with two medicinal plant extracts, namely Andrographis paniculata (Ap) and Swertia chirata (Sc). Results showed that simultaneous addition of both the extracts (50 μg/ml) in independent experiments during generation of reactive oxygen species (ROS) could significantly prevent increased levels of products of lipid peroxidation, such as conjugated dienes, lipid hydroperoxide, TBARS and 4-hydroxylnonenals. The oxidative damage observed with depletion of major endogenous antioxidants, such as GSH, as well as enhanced formation of protein oxidation, was effectively reduced by these extracts. Similarly, degradation of mitochondrial proteins by ROS, induced during photosensitization, was effectively prevented by both the extracts (SDS–PAGE experiments). The antioxidative property of these extracts could be attributed to their scavenging ability with superoxide, hydroxyl radicals and singlet oxygen species, the major species generated during photosensitization and γ-radiation. The high scavenging ability of the extracts may be due to high phenolic contents, flavonoid constituents and considerable reducing equivalents. The pulse radiolysis studies showed high reactivity with ABTS −. The reaction of the extracts of Ap and Sc with dimethyl p-phenylene diamine dihydrochloride (DMPD), one of the important stable synthetic radicals gave >30% inhibition at 50 μg/ml. In view of these observations, termination of the free radical reaction, and quenching of reactive oxygen are suggested to be, in part, responsible for the antioxidant activity of Andrographis paniculata and Swertia chirata extracts. Therefore, Andrographis paniculata and Swertia chirata extracts may emerge as effective antioxidative agents, protecting cells from pathophysiological oxidants, generated during UV–vis photosensitization/radiation-induced injury, and may be useful in the food industry as effective synthetic antioxidants.

3H] N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)- yl)butyl]-2-methoxy-5-methylbenzamide: A novel sigma-2 receptor probe

N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)- yl)butyl]-2-methoxy-5-methyl-benzamide (RHM-1) and N-[2-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)- yl)ethyl]-2-methoxy-5-methylbenzamide (RHM-2), two conformationally flexible benzamide analogues, were radiolabeled with tritium (specific activity = 80 Ci/mmol) and the binding of [ 3H]RHM-1 and [ 3H]RHM-2 to sigma-2 (σ 2) receptors was evaluated in vitro. [ 3H]RHM-1 was found to have a higher affinity for σ 2 receptors compared to [ 3H]RHM-2 and [ 3H]1,3-di- o-tolylguanidine ([ 3H]DTG). [ 3H]RHM-1 had a dissociation constant ( K d) of 0.66 ± 0.12 nM in rat liver membrane homogenates, which was 30-fold higher than that of [ 3H]RHM-2 ( K d = 19.48 ± 0.51 nM). The lower affinity of [ 3H]RHM-2 can be attributed to its faster K off rate since both radioligands have similar K on rates. Competitive binding assays were also conducted using a panel of compounds with known affinity for σ 2 receptors. The pharmacologic profile of [ 3H]RHM-1 was in agreement with that of [ 3H]DTG. The results of this study indicate that [ 3H]RHM-1 is a useful ligand for studying σ 2 receptors in vitro.

A non-radioactive method for angiotensin II receptor binding studies using the rat liver

Introduction: A new non-radioactive method based on competitive ELISA has been developed for binding studies on angiotensin II (Ang II) receptors. Method: Rat liver membrane was used as the source of angiotensin receptors and FITC-angiotensin II (FITC-Ang II) was used as the labeled ligand with an affinity similar to unlabeled Ang II. The effects of different concentrations of Ang II, losartan, CGP-42112A and saralasin were studied on FITC-Ang II binding. Results: The Ki values for Ang II, losartan and CGP-42112A were calculated as 0.52 ± 0.22 nM, 6 ± 3 nM and 0.15 ± 0.07 nM, respectively. Saralasin inhibited the binding of labeled ligand biphasically, revealing two different populations of Ang receptor with different affinities for saralasin. About 74% of the binding sites were more sensitive to saralasin with a Ki value of 0.32 ± 0.04 nM while saralasin showed a Ki value of 2.7 ± 0.8 nM for the remaining binding sites. Discussion: The competitive ELISA method developed in this work yields Ki values for angiotensin antagonists similar to those obtained by others using radiolabeled ligands. The simplicity of this method makes it a suitable alternative to radioligand studies for routine analysis of interaction of drugs with angiotensin receptors.

Regulation of SR-BI protein levels by phosphorylation of its associated protein, PDZK1

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor that mediates the selective uptake of HDL cholesterol and cholesterol secretion into bile in the liver. Previously, we identified an SR-BI-associated protein, termed PDZK1, from rat liver membrane extracts. PDZK1 contains four PSD-95/Dlg/ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PDZK1 controls hepatic SR-BI expression in a posttranscriptional fashion both in cell culture and in vivo. In this study, we demonstrated that the C-terminal region of PDZK1 is crucial for up-regulating SR-BI protein expression. Metabolic labeling experiments and phosphoamino acid analysis revealed that PDZK1 is phosphorylated at Ser residues within this region. Point-mutation analysis demonstrated that PDZK1 is phosphorylated at Ser-509. Interestingly, a mutant PDZK1, in which Ser-509 was replaced with Ala, lost the ability to up-regulate SR-BI protein. We identified Ser-509 of PDZK1 as the residue that is phosphorylated by the cAMP-dependent PKA in vitro as well as in cell culture. Ser-509 of PDZK1 in rat liver was also phosphorylated, as shown by an Ab that specifically detects phosphorylated Ser-509. Administration of glucagon to Wistar rats increased PDZK1 phosphorylation as well as hepatic SR-BI and PDZK1 expression while it decreased plasma HDL levels, indicating that PDZK1 phosphorylation is hormonally regulated. These findings suggest that phosphorylation of PDZK1 has an important role in the regulation of hepatic SR-BI expression and, thus, influences plasma HDL levels.

Effect of Ubiquinone Q10 and Antioxidant Vitamins on Free Radical Oxidation of Phospholipids in Biological Membranes of Rat Liver

We studied the effects of 30-day peroral treatment with beta-carotene, a complex of antioxidant vitamins (vitamins C and E and provitamin A) and selenium, and solubilized ubiquinone Q(10) on the antioxidant potential in rat liver (ascorbate-dependent free radical oxidation of unsaturated membrane phospholipids). beta-Carotene irrespective of the administration route increased antioxidant potential of the liver by 2-3.5 times. The complex of antioxidant vitamins and selenium increased this parameter by more than 15 times. Antiradical activity in rat liver was extremely high after administration of solubilized ubiquinone Q(10) (increase by more than by 36 times). It can be expected that reduced ubiquinone Q(10) in vivo should produce a more pronounced protective effect due to activity of the system for bioregeneration of this natural antioxidant.

Interaction with PDZK1 Is Required for Expression of Organic Anion Transporting Protein 1A1 on the Hepatocyte Surface

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.

3H]linopirdine binding to rat brain membranes is not relevant for M-channel interaction

Linopirdine was developed as a cognitive enhancing molecule and demonstrated to specifically block the potassium current generated by the brain specific KCNQ2–KCNQ3 proteins (M-channel). In this study we investigated the relevance of [ 3H]linopirdine binding in rat brain extracts to the interaction with the M-channel proteins. Our results confirm the presence of a high affinity site for [ 3H]linopirdine in rat brain tissues ( K D = 10 nM) but we also identified a high affinity binding site for [ 3H]linopirdine in rat liver tissues ( K D = 9 nM). Competition experiments showed that [ 3H]linopirdine is displaced by unlabelled linopirdine with comparable affinities from its binding sites on rat brain and rat liver membranes. [ 3H]linopirdine was completely displaced by a set of cytochrome P450 (CYP450) ligands suggesting that [ 3H]linopirdine binding to rat brain and liver membranes is linked to CYP450 interaction. The testing of CYP450 ligands on the M-channel activity, using a Rb + efflux assay on cells expressing the KCNQ2–KCNQ3 proteins, demonstrated that [ 3H]linopirdine binding results cannot be correlated to M-channel inhibition. The results obtained in this study demonstrate that [ 3H]linopirdine binding to rat brain and rat liver membranes is representative for CYP450 interaction and not relevant for the binding to the M-channel proteins.

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.

Immunodetection of the expression of microsomal proteins encoded by the glucose 6-phosphate transporter gene

Glucose 6-phosphate transport has been well characterized in liver microsomes. The transport is required for the functioning of the glucose-6-phosphatase enzyme that is situated in the lumen of the hepatic endoplasmic reticulum. The genetic deficiency of the glucose 6-phosphate transport activity causes a severe metabolic disease termed type 1b glycogen storage disease. The cDNA encoding a liver transporter for glucose 6-phosphate was cloned and was found to be mutated in patients suffering from glycogen storage disease 1b. While related mRNAs have been described in liver and other tissues, the encoded protein(s) has not been immunologically characterized yet. In the present study, we report (using antibodies against three different peptides of the predicted amino acid sequence) that a major protein encoded by the glucose 6-phosphate transporter gene is expressed in the endoplasmic reticulum membranes of rat and human liver. The protein has an apparent molecular mass of approx. 33 kDa using SDS/PAGE, but several lines of evidence indicate that its real molecular mass is 46 kDa, as expected. The glucose 6-phosphate transporter protein was also immunodetected in kidney microsomes, but not in microsomes derived from human fibrocytes, rat spleen and lung, and a variety of cell lines. Moreover, little or no expression of the glucose 6-phosphate transporter protein was found in liver microsomes obtained from three glycogen storage disease 1b patients, even bearing mutations that do not directly interfere with protein translation, which can be explained by a (proteasome-mediated) degradation of the mutated transporter.

The Inner Mitochondrial Membrane Has Aquaporin-8 Water Channels and Is Highly Permeable to Water

Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (Pf) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM Pf. AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg2+, while the related activation energy remained low, suggesting the presence of a Hg2+-insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals.

Size of vasopressin receptors from rat liver and kidney.

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al., Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974) J. Biol. Chem. 249, 6390-6400; Butlen et al. (1978) Mol. Pharmacol. 14, 1006-1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopresin receptors. For this purpose, rat liver and kidney membranes were incubated with [3H]vasopressin and solubilized with Triton X-100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30-37 degrees C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone-receptor complexes were estimated from Utrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml x g-1; molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size.

Synthesis and AT2 receptor-binding properties of angiotensin II analogues.

The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.

The F 1-ATPase β-subunit is the putative enterostatin receptor

It has been suggested that the F 1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin 1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin 1–7 weakly binds to the rat F 1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin 1–7 binding to the F 1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin 1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin 1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F 1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F 1-ATPase complex. Western blot analysis showed the F 1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F 1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin 1–7 bind to distinct sites on the protein.

Chiral, nonracemic (piperazin-2-yl)methanol derivatives with $sigma;-receptor affinity

Starting with the proteinogenic amino acid (S)-serine a series of chiral nonracemic (piperazin-2-yl)methanols 3 with various N-4 substituents is described. The key step in the synthesis of 3 is the reaction of the chloroacetamide 5 with various primary amines to yield the diastereomeric bicyclic piperazinediones cis-6 and trans-6. The scope and limitation of this transformation is thoroughly investigated. The σ1- and σ2-receptor affinities of the piperazines 3 are determined in receptor binding studies with guinea pig brain and rat liver membrane preparations using [3H]-labeled (+)-pentazocine and ditolylguanidine, respectively. It was found, that an additional phenyl residue in the N-4 substituent is favorable to high σ1-receptor affinity. In this series the p-methoxybenzyl substituted piperazine 3d reveals the highest σ1-receptor affinity (Ki=12.4 nM) with selectivity toward σ2-, NMDA-, κ-opioid, and μ-opioid receptors.

Sigma-2 receptors are specifically localized to lipid rafts in rat liver membranes

We have previously shown that sigma-2 receptors are relatively difficult to solubilize (Eur. J. Pharmacol. 304 (1996) 201), suggesting possible localization in detergent-resistant lipid raft domains. Rat liver membranes were treated on ice with 1% Triton X-100 or 20 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and the extract subjected to centrifugation on a discontinuous gradient of 5%, 38%, and 40% sucrose. Gradient fractions were analyzed for sigma-1 receptors using [ 3H](+)-pentazocine and for sigma-2 receptors using [ 3H]1,3-di- o-tolylguanidine ([ 3H]DTG), in the presence of dextrallorphan. Flotillin-2 was assessed by immunoblotting as a marker for lipid rafts. Sigma-2 receptors were found to discretely co-localize with flotillin-2 in lipid raft fractions. However, sigma-1 receptors were found throughout the gradient. Rafts prepared in CHAPS had sigma-2 receptors with normal pharmacological characteristics, whereas those in Triton X-100-prepared rafts had about seven-fold lower affinity for [ 3H]DTG and other ligands. Thus, sigma-2 receptors are resident in membrane lipid rafts, whereas sigma-1 receptors appear in both raft and non-raft membrane domains. Lipid rafts may play an important role in the mechanism of sigma-2 receptor-induced apoptosis.

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events.

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.

Chiral, nonracemic (piperazin-2-yl)methanol derivatives with σ-receptor affinity

Starting with the proteinogenic amino acid ( S)-serine a series of chiral nonracemic (piperazin-2-yl)methanols 3 with various N-4 substituents is described. The key step in the synthesis of 3 is the reaction of the chloroacetamide 5 with various primary amines to yield the diastereomeric bicyclic piperazinediones cis- 6 and trans- 6. The scope and limitation of this transformation is thoroughly investigated. The σ 1- and σ 2-receptor affinities of the piperazines 3 are determined in receptor binding studies with guinea pig brain and rat liver membrane preparations using [ 3H]-labeled (+)-pentazocine and ditolylguanidine, respectively. It was found, that an additional phenyl residue in the N-4 substituent is favorable to high σ 1-receptor affinity. In this series the p-methoxybenzyl substituted piperazine 3d reveals the highest σ 1-receptor affinity ( K i=12.4 nM) with selectivity toward σ 2-, NMDA-, κ-opioid, and μ-opioid receptors.

Characteristics of non-opioid beta-endorphin receptor.

Tritium-labeled selective agonist of non-opioid beta-endorphin receptor, the decapeptide immunorphine ([3H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid beta-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid beta-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [3H]immunorphine-receptor complex was 2.4 +/- 0.1 nM) and beta-endorphin (Ki of the [3H]immunorphine specific binding was 2.9 +/- 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro3]pentarphine (VKPFY) (Ki values were 0.0060 +/- 0.0004, 2.7 +/- 0.2, 2.6 +/- 0.2, and 2.8 +/- 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 microM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [3H]immunorphine. Values of the specific binding of 8.4 nM [3H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 +/- 44.7, 698.6 +/- 28.1, 279.1 +/- 15.4, and 172.2 +/- 1.8 fmol/mg protein, respectively. Unlabeled beta-endorphin, pentarphine, [Pro3]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [3H]immunorphine to membranes from these organs. No specific binding of [3H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Synthesis of novel 4- and 5-substituted benzyl ether derivatives of vesamicol and in vitro evaluation of their binding properties to the vesicular acetylcholine transporter site

Detection of the central cholinergic deficits, a consistent feature of Alzheimer's disease, is essential to allow preventive measures and/or symptomatic treatment already at a very early stage of the disease. The vesicular acetylcholine transporter (VAChT) represents an appropriate target to establish PET radiotracer that are adequate for brain imaging the loss of cholinergic terminals. Here we describe the synthesis and binding characteristics of novel derivatives of vesamicol, known to represent a specific antagonist of VAChT sites. Novel benzyl ether derivatives of vesamicol either 4- or 5-substituted at the cyclohexylring have been synthesized by different regioselective ring opening reactions of a same epoxide precursor. The affinity and selectivity of the novel compounds to VAChT sites were analyzed by competitive radioligand binding studies in rat brain and liver membrane preparations using tritium labeled radioligands. The 4-substituted fluorobenzylether of vesamicol 10b was shown to exhibit a high affinity to VAChT sites ( K i-value 10b=10.7±1.7 nM), but demonstrated also binding capacities to sigma receptors (K i-value 10b=18.5±6.9 nM, [ 3H]DTG; K i-value 10b=30.6±9.6 nM, [ 3H]haloperidol). The data suggest the potential of vesamicol derivatives to design appropriate radiotracer for PET imaging of central cholinergic deficits.