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Na+-glucose cotransport has been previously demonstrated to be inhibited by a decline in the cotransporter numbers in rabbits, while Na+-amino acid cotransport was inhibited by a reduction in the affinity for the amino acid. It is further established that Na+-bile acid cotransport decreases in villus cells from the chronically inflamed ileum. In villus cell brush-border membrane vesicles from the chronically inflamed ileum, Na+-bile acid cotransport was also decreased, indicating a direct consequence of the cotransporter itself.

Discusses a study into focal adhesion of leukocytes to the blood vessel lining, which plays a role in inflammation and certain vascular disease processes. Reports that endothelial leukocyte adhesion molecule-1 (ELAM-1), a glycoprotein, may be a member of a family of molecules that regulates these events on blood vessels.

Novel peptidic dual agonists of the glucagon-like peptide 1 (GLP-1) and glucagon receptor are reported to have enhanced efficacy over pure GLP-1 receptor agonists with regard to treatment of obesity and diabetes. We describe novel exendin-4 based dual agonists designed with an activity ratio favoring the GLP-1 versus the glucagon receptor. As result of an iterative optimization procedure that included molecular modeling, structural biological studies (X-ray, NMR), peptide design and synthesis, experimental activity, and solubility profiling, a candidate molecule was identified. Novel SAR points are reported that allowed us to fine-tune the desired receptor activity ratio and increased solubility in the presence of antimicrobial preservatives, findings that can be of general applicability for any peptide discovery project. The peptide was evaluated in chronic in vivo studies in obese diabetic monkeys as translational model for the human situation and demonstrated favorable blood glucose and body weight lowering effects.

Dual activation of the glucagon-like peptide 1 (GLP-1) and glucagon receptor has the potential to lead to a novel therapy principle for the treatment of diabesity. Here, we report a series of novel peptides with dual activity on these receptors that were discovered by rational design. On the basis of sequence analysis and structure-based design, structural elements of glucagon were engineered into the selective GLP-1 receptor agonist exendin-4, resulting in hybrid peptides with potent dual GLP-1/glucagon receptor activity. Detailed structure-activity relationship data are shown. Further modifications with unnatural and modified amino acids resulted in novel metabolically stable peptides that demonstrated a significant dose-dependent decrease in blood glucose in chronic studies in diabetic db/db mice and reduced body weight in diet-induced obese (DIO) mice. Structural analysis by NMR spectroscopy confirmed that the peptides maintain an exendin-4-like structure with its characteristic tryptophan-cage fold motif that is responsible for favorable chemical and physical stability.

Previous studies have shown that compound 1 displayed high affinity towards histamine H3 receptor (H3R), (human (h-H3R), K(i)=8.6 nM, rhesus monkey (rh-H3R), K(i)=1.2 nM, and rat (r-H3R), K(i)=16.5 nM), but exhibited high affinity for hERG channel. Herein, we report the discovery of a novel, potent, and highly selective H3R antagonist/inverse agonist 5a(SS) (SAR110068) with acceptable hERG channel selectivity and desirable pharmacological and pharmacokinetic properties through lead optimization sequence. The significant awakening effects of 5a(SS) on sleep-wake cycles studied by using EEG recording in rats during their light phase support its potential therapeutic utility in human sleep-wake disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H3 receptor (H3R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H3R FLIPR assays and rhesus monkey H3R binding assays., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The objective of the present study was to investigate in fed Wistar rats whether the cannabinoid-1 (CB1) receptor antagonist AVE1625 causes primary effects on metabolic blood and tissue parameters as well as metabolic rate, which are independent of reduced caloric intake. After single administration to rats postprandially, AVE1625 caused a slight dose-dependent increase in basal lipolysis. Six hours after single administration, liver glycogen content was dose-dependently reduced to approximately 60% of that of untreated controls. These findings demonstrate a primary acute effect of AVE1625 on induction of 1) lipolysis from fat tissue (increased FFA) and 2) glycogenolysis from the liver (reduced hepatic glycogen). Measured by indirect calorimetry, AVE1625 caused an immediate increase in total energy expenditure, a long-lasting increase of fat oxidation, and a transient increase of glucose oxidation, which were consistent with the acute findings on metabolic blood and tissue parameters. We conclude that, in addition to the well-investigated effects of CB1 receptor antagonists to reduce caloric intake and subsequently body weight, this pharmacological approach is additionally linked to inherently increased lipid oxidation. This oxidation is driven by persistently increased lipolysis from fat tissues, independently of reduced caloric intake, and might significantly contribute to the weight-reducing effect.

Bile acids are generated in vivo from cholesterol in the liver, and they undergo an enterohepatic circulation involving the small intestine, liver, and kidney. To understand the molecular mechanism of this transportation, it is essential to gain insight into the three-dimensional (3D) structures of proteins involved in the bile acid recycling in free and complexed form and to compare them with homologous members of this protein family. Here we report the solution structure of the human ileal lipid-binding protein (ILBP) in free form and in complex with cholyltaurine. Both structures are compared with a previously published structure of the porcine ILBP-cholylglycine complex and with related lipid-binding proteins. Protein structures were determined in solution by using two-dimensional (2D)- and 3D-homo and heteronuclear NMR techniques, leading to an almost complete resonance assignment and a significant number of distance constraints for distance geometry and restrained molecular dynamics simulations. The identification of several intermolecular distance constraints unambiguously determines the cholyltaurine-binding site. The bile acid is deeply buried within ILBP with its flexible side-chain situated close to the fatty acid portal as entry region into the inner ILBP core. This binding mode differs significantly from the orientation of cholylglycine in porcine ILBP. A detailed analysis using the GRID/CPCA strategy reveals differences in favorable interactions between protein-binding sites and potential ligands. This characterization will allow for the rational design of potential inhibitors for this relevant system., (Copyright 2002 Wiley-Liss, Inc.)

Cyclosporin A (CsA) has been shown to inhibit synthesis and hepatobiliary transport of bile salts. However, effects of CsA on the enterohepatic circulation of bile salts in vivo are largely unknown. We characterized the effects of CsA on the enterohepatic circulation of cholate, with respect to synthesis rate, pool size, cycling time, intestinal absorption, and the expression of relevant transporters in liver and intestine in rats. CsA (1 mg. 100 g(-1). day(-1) s.c.) or its solvent was administered daily to male rats for 10 days. Cholate synthesis rate and pool size were determined by a 2H4-cholate dilution technique. Bile and feces were collected for determination of cholate and total bile salts, respectively. Cycling time and intestinal absorption of cholate were calculated. The mRNA levels and corresponding transporter protein levels in liver and intestine were assessed by real-time polymerase chain reaction and Western analysis, respectively. CsA treatment decreased cholate synthesis rate by 71%, but did not affect pool size or cycling time. CsA reduced the amount of cholate lost per enterohepatic cycle by approximately 70%. Protein levels of the apical sodium-dependent bile salt transporter (Asbt) were 2-fold increased in distal ileum of CsA-treated rats, due to post-transcriptional events. In conclusion, chronic CsA treatment markedly reduces cholate synthesis rate in rats, but does not affect cholate pool size or cycling time. Our results strongly suggest that CsA enhances efficacy of intestinal cholate reabsorption through increased Asbt protein expression in the distal ileum, which contributes to maintenance of cholate pool size in CsA-treated rats.

Objectives: Percutaneous transhepatic biliary drainage (PTBD) has been believed to reduce hyperbilirubinemia in patients with obstructive cholestasis and to lessen liver injury through bile acid retention. The efficacy may be closely related to the capability of cholestatic liver to produce and secrete bile, which in turn depends on the expressions and functional activities of plasma membrane transporters in the liver. The aim of the present study was to determine the expression levels of these transporters in the cholestatic liver of patients undergoing PTBD., Methods: A total of 24 patients who had experienced obstructive cholestasis and had undergone preoperative PTBD were included in the study. Liver biopsy specimens were analyzed to determine the expression levels of the multidrug resistance-associated proteins (MRP) MRP2 and MRP3 and the canalicular bile salt export pump BSEP in the liver., Results: The messenger RNA (mRNA) levels of MRP2, the canalicular bilirubin conjugate export pump, and bile salt export pump (BSEP) were unchanged in liver specimens from the 14 patients well drained by PTBD but were reduced in specimens from the 10 patients poorly drained, compared to the levels of control subjects. Immunostainings of MRP2 and BSEP outlined the canalicular membrane domain but seemed fuzzy to varying degrees in specimens obtained from cholestatic liver, especially in specimens from liver that had been poorly drained, in contrast to the linear and intense localization in the liver of control subjects, correlating with the impaired bilirubin conjugate and bile acid secretion. The mRNA of MRP3, functioning as an inducible export pump for bilirubin conjugate and bile acid, was expressed not only in the cholestatic liver but also in the liver of control subjects, and the mRNA level was increased in specimens from both the cholestatic liver that had been well drained and from the liver that had been poorly drained. Immunostaining of MRP3 was observed in the epithelia of intrahepatic bile ducts in the liver of both control subjects and cholestatic patients, and in the epithelia of proliferated bile ductules and the hepatocytes surrounding the portal tracts in the cholestatic liver., Conclusions: From the results of the present study, it is concluded that 1) the mRNA and immunohistochemical expression levels of MRP2 and BSEP may be altered in the cholestatic liver of patients undergoing PTBD; 2) both the decreased mRNA levels and the diminished canalicular membrane localization may be associated with the impairment of bile formation and secretion, i.e., the efficacy of PTBD; and 3) upregulated MRP3 in the cholangiocytes and hepatocytes may play a significant role in bile acid transport in the cholestatic hepatobiliary system.

Reabsorption of bile acids occurs in the terminal ileum by a Na(+)-dependent transport system composed of several subunits of the ileal bile acid transporter (IBAT) and the ileal lipid-binding protein. To identify the bile acid-binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7-azi-derivative of cholyltaurine followed by enrichment of IBAT protein by preparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypsin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. With epitope-specific antibodies generated against the C terminus a peptide of molecular mass of 6.6-7 kDa was identified as the smallest peptide fragment carrying both the C terminus and the covalently attached radiolabeled bile acid derivative. This clearly indicates that the ileal Na(+)/bile acid cotransporting protein IBAT contains a bile acid-binding site within the C-terminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the seventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site is localized to the ninth transmembrane domain and the C terminus.

The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His(100)-Thr(101)-Ser(102) using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112-128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.

Background/aims: Accumulation of toxic bile acids in cholestasis contributes to liver injury and depends on their synthesis, secretion and intestinal absorption. In the present study, we investigated the effect of cholestasis on the active ileal absorption of bile acids in vivo and the adaptation of transporters involved in ileal bile acid absorption., Methods: Male Wistar rats underwent ligation of the common bile duct or biliary diversion. Sham-operated rats served as controls. Active ileal bile acid absorption of taurocholate was measured by an intestinal perfusion technique. Transporter mRNA levels of the Na+/bile acid cotransporting protein (IBAT), ileal lipid binding protein (ILBP) and organic anion transporter subtype 3 (Oatp3) and protein expression of IBAT and ILBP were determined in the distal ileum., Results: After bile duct ligation the intestinal absorption rates of taurocholate were lower (p<0.05) and after biliary diversion absorption rates were higher compared to sham-operated animals (p<0.05). The absorption rates were inversely correlated to serum bile acid concentrations. Levels of IBAT-, ILBP- and Oatp3- mRNA were not different between the groups. However, in cholestatic rats, the expression of the 99-kDa dimer of IBAT was decreased compared to controls (p<0.05), whereas the 46-kDa monomeric protein of IBAT and the expression of ILBP was unchanged. After biliary diversion a similar pattern of protein expression was observed, despite an increased absorption rate., Conclusions: Cholestasis leads to a decreased active ileal absorption of taurocholate. The changes in protein expression may not account for the different absorption rates. The intestinal absorption of bile acids seems to be regulated by their systemic concentration.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.

To design a reliable 3D QSAR model of the intestinal Na(+)/bile acid cotransporter, we have used a training set of 17 inhibitors of the rabbit ileal Na(+)/bile acid cotransporter. The IC(50) values of the training set of compounds covered a range of four orders of magnitude for inhibition of [(3)H]cholyltaurine uptake by CHO cells expressing the rabbit ileal Na(+)/bile acid cotransporter allowing the generation of a pharmacophore using the CATALYST algorithm. After thorough conformational analysis of each molecule, CATALYST generated a pharmacophore model characterized by five chemical features: one hydrogen bond donor, one hydrogen bond acceptor, and three hydrophobic features. The 3D pharmacophore was enantiospecific and correctly estimated the activities of the members of the training set. The predicted interactions of natural bile acids with the pharmacophore model of the ileal Na(+)/bile acid cotransporter explain exactly the experimentally found structure;-activity relationships for the interaction of bile acids with the ileal Na(+)/bile acid cotransporter (Kramer et al. 1999. J. Lipid. Res. 40: 1604;-1617). The natural bile acid analogues cholyltaurine, chenodeoxycholyltaurine, or deoxycholyltaurine were able to map four of the five features of the pharmacophore model: a) the five-membered ring D and the methyl group at position 18 map one hydrophobic site and the 21-methyl group of the side chain maps a second hydrophobic site; b) one of the alpha-oriented hydroxyl groups at position 7 or 12 fits the hydrogen bond donor feature; c) the negatively charged side chain acts as hydrogen bond acceptor; and d) the hydroxy group at position 3 does not specifically map any of the five binding features of the pharmacophore model. The 3-hydroxy group of natural bile acids is not essential for interactions with ileal or hepatic Na(+)/bile acid cotransporters. A modification of the 3-position of a natural bile acid molecule is therefore the preferred position for drug targeting strategies using bile acid transport pathways.

Background & Aims: Reduced activity of ferrochelatase in erythropoietic protoporphyria (EPP) results in protoporphyrin (PP) accumulation in erythrocytes and liver. Liver disease may occur in patients with EPP, some of whom develop progressive liver failure that necessitates transplantation. We investigated the mechanisms underlying EPP-associated liver disease in a mouse model of EPP., Methods: Liver histology, indicators of lipid peroxidation, plasma parameters of liver function, and bile composition were studied in mice homozygous (fch/fch) for a point mutation in the ferrochelatase gene and in heterozygous (fch/+) and wild-type (+/+) mice., Results: Microscopic examination showed bile duct proliferation and biliary fibrosis with portoportal bridging in fch/fch mice. PP content was 130-fold increased, and thiobarbituric acid-reactive substances (+30%) and conjugated dienes (+75%) were slightly higher in fch/fch than in fch/+ and +/+ livers. Levels of hepatic thiols (-12%) and iron (-52%) were reduced in fch/fch livers. Liver enzymes and plasma bilirubin were markedly increased in the homozygotes. Plasma bile salt levels were 80 times higher in fch/fch than in fch/+ and +/+ mice, probably related to the absence of the Na(+)-taurocholate cotransporting protein (Ntcp) in fch/fch liver. Paradoxically, bile flow was not impaired and biliary bile salt secretion was 4 times higher in fch/fch mice than in controls. Up-regulation of the intestinal Na(+)-dependent bile salt transport system in fch/fch mice may enhance efficiency of bile salt reabsorption. The bile salt/lipid ratio and PP content of fch/fch bile were increased 2-fold and 85-fold, respectively, compared with +/+, whereas biliary glutathione was reduced by 90%. Similar effects on bile formation were caused by griseofulvin-induced inhibition of ferrochelatase activity in control mice., Conclusions: Bile formation is strongly affected in mice with impaired ferrochelatase activity. Rather than peroxidative processes, formation of cytotoxic bile with high concentrations of bile salts and PP may cause biliary fibrosis in fch/fch mice by damaging bile duct epithelium.

The substrate specificity of the ileal and the hepatic Na(+)/bile acid cotransporters was determined using brush border membrane vesicles and CHO cell lines permanently expressing the Na(+)/bile acid cotransporters from rabbit ileum or rabbit liver. The hepatic transporter showed a remarkably broad specificity for interaction with cholephilic compounds in contrast to the ileal system. The anion transport inhibitor diisothiocyanostilbene disulfonate (DIDS) is a strong inhibitor of the hepatic Na(+)/bile acid cotransporter, but does not show any affinity to its ileal counterpart. Inhibition studies and uptake measurements with about 40 different bile acid analogues differing in the number, position, and stereochemistry of the hydroxyl groups at the steroid nucleus resulted in clear structure;-activity relationships for the ileal and hepatic bile acid transporters. The affinity to the ileal and hepatic Na(+)/bile acid cotransport systems and the uptake rates by cell lines expressing those transporters as well as rabbit ileal brush border membrane vesicles is primarily determined by the substituents on the steroid nucleus. Two hydroxy groups at position 3, 7, or 12 are optimal whereas the presence of three hydroxy groups decreased affinity. Vicinal hydroxy groups at positions 6 and 7 or a shift of the 7-hydroxy group to the 6-position significantly decreased the affinity to the ileal transporter in contrast to the hepatic system. 6-Hydroxylated bile acid derivatives are preferred substrates of the hepatic Na(+)/bile acid cotransporter. Surprisingly, the 3alpha-hydroxy group being present in all natural bile acids is not essential for high affinity interaction with the ileal and the hepatic bile acid transporter. The 3alpha-hydroxy group seems to be necessary for optimal transport of a bile acid across the hepatocyte canalicular membrane. A modification of bile acids at the 3-position therefore conserves the bile acid character thus determining the 3-position of bile acids as the ideal position for drug targeting strategies using bile acid transport pathways.

A sodium dependent bile acid carrier has recently been cloned and characterized in rat ileum. The present study demonstrates the presence of a mRNA species specific for the rat ileal bile acid carrier (r-IBAT) in rat biliary epithelial cells. Moreover, immunohistochemistry with a peptide specific antibody demonstrates protein expression in biliary epithelial cells from normal and bile duct ligated rat livers. Besides a cytoplasmic staining a predominant staining of the apical membrane could be observed. These observations indicate that biliary epithelial cells are involved in bile acid transport across the biliary tree. In addition the carrier could also play a role in the signal transduction of bile acid induced ductular secretion.

Photoaffinity labelling of brush border membrane vesicles from rabbit ileum with radiolabelled 3,3-azo and 7,7-azo derivatives of taurocholate identified integral membrane proteins of molecular masses 93 and 46 kDa, as well as a 14 kDa peripheral membrane protein, as components of the ileal Na+/bile acid transport system [Kramer, Girbig, Gutjahr, Kowalewski, Jouvenal, Müller, Tripier and Wess (1993) J. Biol. Chem. 268, 18035-18046]. Differential photoaffinity labelling in the presence of non-radiolabelled bile acid derivatives led, as expected, to a concentration-dependent decrease in the extent of labelling of the 93 and 46 kDa transmembrane proteins, which are the monomeric and dimeric forms of the ileal bile acid transporter protein. The extent of labelling of the 14 kDa ileal lipid-binding protein (ILBP), however, increased on the addition of unlabelled bile acids, the increase being dependent on the structure of the bile acid added. The possibility of artifacts was excluded by photoaffinity labelling experiments in the frozen state as well as by model calculations. The experimental results suggest that the binding of bile acids to ILBP can increase the affinity of ILBP for bile acids. These results would be in accordance with a substrate-load modification of transport activity and a positive-feedback regulation mechanism for active uptake of bile acid in the ileum.

For the investigation of the topology of the rabbit ileal Na+/bile-salt-cotransport system, composed of a 93-kDa integral membrane protein and a peripheral 14-kDa bile-acid-binding protein (ILBP), we have synthesized photolabile dimeric bile-salt-transport inhibitors (photoblockers), G1-X-G2, where two bile acid moieties (G1 and G2) are tethered together via a spacer, X, and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na+/bile-salt-cotransport system as demonstrated by a concentration-dependent inhibition of [3H]cholyltaurine uptake by rabbit ileal brush-border membrane vesicles and by inhibition of photolabeling of the 93-kDa and 14-kDa bile-salt-binding proteins by 7,7-azo and 3,3-azo derivatives of cholyltaurine. Ileal bile-salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion. Dependent on the photoblocker used several polypeptides in the molecular-mass range of 14-130 kDa were labeled. The cytoplasmically attached 14-kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety G1, suggesting that during binding and translocation of a bile-salt molecule by the ileal bile-salt-transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush-border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3-azo and 7,7-azo derivatives of cholyltaurine revealed a time-dependent increase in the extent of labeling of the 14-kDa and 93-kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush-border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time-dependent changes in the extent of photoaffinity labeling of bile-salt-binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na+/bile-salt-cotransport system.

A bile-acid-binding protein of Mr 14000 has been previously identified by photoaffinity labeling in rabbit ileal brush border membrane vesicles [Kramer et al. (1993) J. Biol. Chem. 268, 18035-18046]. This peripheral membrane-associated protein was purified and identified as an ileal lipid-binding protein. It was further shown to be identical to the cytosolic 14-kDa bile-acid-binding protein from the same tissue. Starting with sequence information from tryptic fragments, we cloned and sequenced the gene and its transcript. It has four exons (123, 176, 90, 115 bp) and three introns (1372, 2291, 3137 bp) and a similar structure as the genes from other members of the fatty-acid-binding protein family. The deduced protein has 128 amino acid residues and a calculated molecular mass of 14404 Da. It exhibits high similarity to its human (83%), mouse (77%), rat (76%) and porcine (72%) counterparts. Furthermore, the recombinant protein was produced in Escherichia coli and shown to be identical to native protein from ileal tissue. Functionality of the recombinant protein was demonstrated by labeling with various photoaffinity derivatives of bile acids. Ranking of the photolabeling efficiency of these probes towards the recombinant protein was comparable to the respective ranking towards the native protein. Polyclonal antibodies that were raised in hens against the recombinant protein, specifically recognized the ileal lipid-binding protein in the brush border membrane and cytosol from rabbit ileum. In contrast, no labeling was observed with jejunal tissue. Our results suggest a specific role of the membrane-associated ileal lipid-binding protein for the process of ileal bile acid uptake.

1. Radioactive derivatives of neurotoxin I (ATX I) from Anemonia sulcata have been synthesized: Iodination of ATX I with 125I yielded a mixture of reaction products from which monoiodo and diiodo ATX I were isolated. 2. 125I-ATX I was shown to bind to the axonal membrane from Astacus leptodactylus main walking nerve. Specificity of binding was shown by saturability of the binding sites and by competitive binding of native and radioactive toxin. 3. Astacus nerve bound 44 fmol of 125I-ATX I/mg nerve (wet weight). The axonal membrane surface of the nerve was determined to be 7800 cm2/g nerve. This amounts to a binding site density of around 35/mu2 axonal surface. Binding was not inhibited by tetrodotoxin, the blocker of the selectivity filter of voltage-dependent sodium channels. 125I-ATX I therefore may bind to the sodium channel-inactivating gate. 4. The affinity of the nerve membrane receptors for 125I-ATX I appears to be voltage-dependent: KD = 5 nM was found with whole crayfish nerves in the presence of tetrodotoxin, KD = 40nM in the absence of tetrodotoxin and an even lower affinity was obtained with axonal membrane fragments isolated from the nerve. Drugs destabilizing the membrane potential, e.g. veratridine, ouabain and sodium azide lowered the affinity or abolished binding completely.

The synthesis of diazidopropidium and diazidoethidium is described. The applicability of these compounds as photoaffinity labels for cholinergic proteins has been investigated: diazidopropidium inhibits neuromuscular transmission. This inhibition is reversible if the compound is applied in the dark but becomes irreversible after irradiation with white light. Inhibition is accompanied by a disappearance of miniature endplate potentials. Electrophysiological analysis of this effect indicates that diazidopropidium acts postsynaptically by blocking the acetylcholine receptors. At the molecular level the action of diazidopropidium and diazidoethidium on acetylcholinesterase has been investigated: both compounds appear to bind to a peripheral acetylcholine binding site of this enzyme. Binding of 125I-labeled alpha-neurotoxin from Naja naja siamensis to purified membranes from Torpedo californica electric tissue rich in acetylcholine receptors is diminished after incubation and irradiation with diazidopropidium. About half of the toxin binding sites appear to be blocked by the photoaffinity label.

Palytoxin, a toxin isolated from the Caribean corrall Palythoa caribaeorum, increases the cation permeability of excitable membranes in vitro. Three membrane systems have been investigated: axonal membranes from crayfish walking leg nerves, membranes rich in nicotinic acetylcholine receptor isolated from Torpedo californica electric tissue and, for control, artificial liposomes. Ion permeability of the latter was not affected by palytoxin, but with both biological membranes an increase in cation permeability was observed at a palytoxin concentration of 0.14 microM. Palytoxin-induced cation flow through the axonal membrane was not inhibited by tetrodotoxin, indicating that the voltage-dependent sodium channels were not involved. The effect of palytoxin on the receptor-rich membranes was not blocked by alpha-bungarotoxin, a competitive antagonist of the nicotinic acetylcholine receptor, nor by triphenylmethylphosphonium, a blocker of the receptor-ion channel. But with both the axonal and the receptor-rich membranes ouabain was an inhibitor of the palytoxin-induced cation flow. Evidence is presented that it is not the (Na+ + K+)-ATPase which is affected by palytoxin as has been postulated for similar observations with non-neuronal membranes (Chhatwal, G.S., Hessler, H.-J. and Habermann, E. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 323, 261-268).

cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage lambda gammagt10) vectors with poly (A+) RNA isolated from the nonfusing mouse muscle cell line BC3H-1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor gamma subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor gamma subunits from Torpedo californica, chick, calf, and human. One isolate, lambda BMG419, has 88 nucleotides of 5'-untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor gamma subunit, and 144 nucleotides of 3'-untranslated sequence. Alignment of the lambda BMG419-deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse gamma subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled lambda BMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A+) RNA prepared from BC3H-1 cells and denervated mouse limb muscle. gamma Subunit-coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of the gamma subunit is exerted through regulation of the amount of its mRNA.

The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size-fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5'-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3'-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.

Two cDNA clones encoding different but related receptors for immunoglobulin G constant domains were isolated from cDNA expression libraries by a ligand-mediated selection procedure ('affinity cloning'). Because both of the receptors encoded by the cDNAs react with CDw32 monoclonal antibodies, and both show the appropriate IgG binding affinity, both appear to be forms of the receptor formerly thought to be a single species called FcRII. The extracellular domains encoded by the isolated clones are closely related to the murine IgG2b/1 beta receptor extracellular domains, but the intracellular domains are unrelated. The receptors expressed in COS cells show a preference for IgG1 among IgG subtypes and no affinity for IgM, IgA or IgE. Abundant expression of the RNAs was detected in myeloid cell lines and placenta.