IGE-mediated reaction to hydroxocobalamin injection in patient with pernicious anaemia.
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Aged↗
Biomedical subjects
C Hirth
Publications and source records attributed to C Hirth.
Photoaffinity labelling of cholinesterases. Discrimination between active and peripheral sites.
Two para-dialkylaminobenzenediazonium salts, the dimethylamino (A) and dibutylamino (B) derivatives, are presented as structural probes for acetylcholinesterase and butyrylcholinesterase. While being reversible competitive inhibitors in the dark, A and B behave, upon irradiation and through the formation of arylcation species, as irreversible labels of ammonium-binding sites of both enzymes. The observed variations of the different inactivation rate constants point to a different structural environment for acetylcholinesterase-binding and butyrylcholinesterase-binding sites. Moreover, in the case of acetylcholinesterase, protection experiments with specific ligands (edrophonium and propidium) showed that the dimethylamino salt A exclusively labels the hydrolytic anionic site, whereas the dibutylamino salt B also labels the peripheral site. Specificities and stoechiometries of the incorporations were determined and, in the case of acetylcholinesterase, the irradiated protein was submitted to chemical degradation. Peptide maps were obtained by gel-permeation chromatography and HPLC, giving access to labelled peptides which belong either to the active or to the peripheral site.
Acetylcholinesterase↗
m-Sulfonate benzene diazonium chloride: a powerful affinity label for the gamma-aminobutyric acid binding site from rat brain.
m-Sulfonate benzene diazonium chloride (MSBD) was used to affinity-label the gamma-aminobutyric acid (GABA) binding site from rat brain membranes. To assess the irreversibility of the labeling reaction, we used an efficient ligand dissociation procedure combined to a rapid [3H]muscimol binding assay, both steps being performed on filter-adsorbed membranes. Inactivation of specific [3H]-muscimol binding sites by MSBD and its prevention by GABA were both time- and concentration-dependent. The time course of MSBD labeling was shortened as the pH of the incubation medium was increased from 6.2 to 8. These data suggest that MSBD can efficiently label the GABA binding site through alkylation of a residue having an apparent dissociation constant around neutrality.
Affinity Labels↗
Bio-clinical prevalence or non-prevalence of drug addiction over psychiatric symptomatology.
It has been found that opiate abusers once detoxified are afflicted with personality disorders. An attempt was made to test the origin of drug abuse by comparing a group of drug abusers with personality disorders diagnosed as borderline or schizotypal with a group of people suffering from similar personality disorders but who did not use drugs. The results of the test are discussed.
Adult↗
Photoactivatable agonist of the nicotinic acetylcholine receptor: potential probe to characterize the structural transitions of the acetylcholine binding site in different states of the receptor.
The nicotinic acetylcholine receptor exhibits at least four different affinity states for agonists such as acetylcholine. In order to identify the structural changes occurring at or near the agonist binding site during the allosteric transitions, three photoactivatable compounds designed to display agonist activity were synthesized. Inhibition constants of these compounds for the cholinergic and the noncompetitive blocker binding sites were determined for the resting and the desensitized states of the receptor. Among these probes, two ligands, AC5 and AC7, displayed a high affinity for the agonist binding site and were poorly recognized by the binding site for noncompetitive blockers. Electrophysiological experiments revealed that these ligands behaved as agonists at low concentrations. We used these two compounds in photolabeling experiments and observed that they were able to inactivate the agonist binding site. Up to 50% of these sites were irreversibly inhibited, depending on the ligand, the irradiation conditions, and the selected receptor state. The compound with the most interesting properties (high affinity and selectivity for the acetylcholine binding site, as well as agonist activity and high photolabeling yield) is AC5, a structural analogue of the fluorescent agonist dansyl-C6-choline, which has been previously used to characterize the different states of the nicotinic receptor. After radioactive synthesis, [3H]AC5 was shown to label all four receptor subunits, in a protectable manner. This radioligand, thus, appears suitable for investigation of the dynamics of allosteric transitions occurring at the activated acetylcholine binding site.
Acetylcholine↗
Allosteric transitions of the acetylcholine receptor probed at the amino acid level with a photolabile cholinergic ligand.
Structural changes occurring upon desensitization of the Torpedo marmorata acetylcholine receptor were monitored with tritiated p-(N,N-dimethyl)aminobenzenediazonium fluoroborate, a reversible competitive antagonist in the dark, which may serve as a photoaffinity probe of the area of the receptor molecule with which cholinergic ligands interact. Addition of meproadifen, an allosteric effector that stabilizes the high-affinity desensitized state of the receptor upon binding to a site topographically distinct from the cholinergic ligand-binding domains, caused a major increase in labeling of the alpha subunit, a smaller increase in the delta subunit, and decreased labeling in the gamma subunit, thus revealing changes in the alpha and non-alpha subunits' contribution to cholinergic ligand binding. Also, in agreement with the tighter binding of cholinergic ligands to the desensitized receptor, differential labeling of three peptide loops of the alpha subunit was detected: while Tyr-190, Cys-192, and Cys-193 were labeled in a roughly identical manner in both resting and desensitized conformations, the labeling of Tyr-93 and Trp-149 increased up to 6-fold in the desensitized state. Tyr-93 and Trp-149 belong to separate regions containing strictly conserved "canonical" amino acids, common to all nicotinic, gamma-aminobutyrate, and glycine receptor subunits. These regions are thus likely to play a critical role in the regulation of ligand-gated ion channels.
Affinity Labels↗
2-Diazohistidinyl thyrotropin-releasing hormone (TRH): a photosensitive TRH analog.
We have synthesized a new thyrotropin releasing hormone (TRH) analog, pGlu-2-diazo-His-Pro-NH2 5, which is a potential photoaffinity label for the TRH receptor. Its precursor, pGlu-2-amino-His-Pro-NH2 4, has been synthesized through successive coupling of a 2-aminohistidine derivative 1 with proline and pyroglutamic acid derivatives. Diazotization of the fully deprotected tripeptide 4 gave the photoactivatable TRH analog 5. Compounds 4 and 5 exhibit IC50 values of 2 and 8 microM respectively, to be compared to the 0.02 microM value for native TRH.
Amino Acid Sequence↗
Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites.
The native, membrane-bound, acetylcholine receptor from Torpedo marmorata was photolabeled by the competitive antagonist p-[3H]dimethylaminobenzene-diazonium fluoroborate (DDF) in the presence of the noncompetitive blocker phencyclidine and under energy transfer conditions. The isolated alpha-subunits were treated with cyanogen bromide and fractionation of the resulting fragments yielded three radiolabeled peptides, at the level of which, incorporation of [3H]DDF (i) was equally inhibited by the agonist carbamoylcholine and the competitive antagonist alpha-bungarotoxin and (ii) was insensitive to "scavenging" reagents. Subfragmentation of cyanogen bromide peptide III with omicron-iodosobenzoic acid or trypsin and sequence analysis of the fragments led to the identification of a novel amino acid alpha-Tyr-93 (and possibly Trp-86) as labeled by [3H]DDF in a carbamoylcholine-sensitive manner. alpha-Tyr-93 is conserved in the muscle and neuronal alpha-subunits but not in the other subunits of muscle receptor. This result provides evidence for a site involving at least a third loop of the alpha-subunit amino-terminal hydrophilic domain, in addition to the ones previously identified (Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, J. Y., Lazure, C., Chretien, M., and Changeux, J. P. (1988) Biochemistry 27, 2346-2357). Possible contribution of tyrosine side-chains to the complexation of the quaternary ammonium group of cholinergic ligands is discussed.
Affinity Labels↗
Photoaffinity labelling of the mu-opioid receptor is dependent on the nature of the photosensitive group of carfentanil derivatives.
Arylazido and aryldiazonium derivatives of carfentanil, bearing their photoactivatable function at the same position on the molecule, were synthesized. In the dark both of them exhibited similar binding affinity profiles and behaved as mu-selective and reversible ligands of the opioid receptor sites. Following irradiation, only the azido analog displayed the properties of an efficient, irreversible and selective label of the mu-opioid receptor, allowing physicochemical requirements for alkylation of this receptor subtype to be examined. Evidence is presented to consider this azido compound a promising tool for characterizing the mu-opioid receptor protein.
Affinity Labels↗
Photoactivatable opiate derivatives as irreversible probes of the mu-opioid receptor.
The synthesis of aryldiazonium and arylazido derivatives of carfentanil, etonitazene, and naltrexone and of a triazaspirodecane derivative is described. The chemical stability and the spectral characteristics of these compounds were verified, and their binding affinity constants for the different opioid receptor classes were determined, in the absence of light, from competition experiments. With the exception of the naltrexyl derivatives, which remained nonselective, all compounds tested displayed a pronounced mu-binding selectivity with mu/delta and mu/kappa ratios ranging from 12 to 1000. After irradiation, only the arylazido probes led to an irreversible mu-binding-site inactivation. This inactivation fulfilled the criteria for photoaffinity labeling such as protection against inactivation by other opiate ligands and absence of an effect of scavengers on the extent of the inactivation. Most of the photoactivatable probes formed long-lasting reversible complexes with the opioid binding sites: an efficient dissociation procedure was thus required to discriminate between pseudoirreversible and covalent complexes. The marked differences in labeling efficacy between aryldiazonium salts and their corresponding arylazido derivatives are discussed.
Affinity Labels↗
Vasodilatory action of endogenous atrial natriuretic factor in a rat model of chronic heart failure as determined by monoclonal ANF antibody.
Elucidation of the role of (elevated) endogenous atrial natriuretic factor (ANF) in chronic heart failure has been hampered by a lack of specific inhibitors. We used a newly developed monoclonal antibody that has been shown to specifically block both exogenously and endogenously released ANF in vivo. For assessment of the vasodilatory action of ANF in chronic heart failure, either this antibody against ANF or ascites (control serum) was injected in rats with myocardial infarction and failure and in sham animals. Ascites did not alter central hemodynamics in either the sham or infarcted group. Antibody significantly increased right atrial pressure, left ventricular end-diastolic pressure, and systemic vascular resistance (SVR) in the infarction group but did not affect these variables in the sham group. Because renal blood flow, as measured by radioactive microspheres, decreased significantly in all four groups, probably due to nonspecific renal vasoconstrictor effects of the ascites, a separate group of infarcted animals was treated with purified ANF antibody (devoid of nonspecific effects) or mouse IgG as a control injection. In these animals, right atrial pressure increased from 1.1 +/- 0.7 to 2.6 +/- 0.7 mm Hg (p less than 0.001). Although SVR, renal blood flow velocity (measured by Doppler probe), and renal vascular resistance did not change in the infarcted animals after administration of purified ANF antibody, a significant correlation was found between baseline plasma ANF values and the change in SVR exerted by purified ANF antibody (r = 0.758, p less than 0.02, n = 9); that is, SVR increased in rats with high baseline plasma ANF (greater than 350 pg/ml), but decreased in animals with plasma ANF less than 200 pg/ml. These results suggest that moderately elevated endogenous plasma ANF levels in chronic heart failure do affect central hemodynamics, primarily by reducing venous pressure (e.g., by decreasing intravascular volume or by venous dilation). Arterial vasodilation, however, appears to emerge when plasma ANF is greatly increased.
Animals↗
Non peptide photoaffinity probes of the mu-opioid receptor.
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Affinity Labels↗
New photoaffinity labels for rat brain muscarinic acetylcholine receptors.
Localization of the ligand binding site on muscarinic acetylcholine receptors is one of the new fields of interest opened by the recent determination of their primary structures. Owing to their interesting photochemical properties, aryldiazonium salts may be considered as appropriate tools for "tagging" the agonist/antagonist binding domain and to get precise identification and positioning of covalently labelled residues along the primary sequence of these receptors. A series of aryldiazonium derivatives and some of their azido-analogs were synthesized and their reversible muscarinic binding component was assessed through competition experiments involving either the whole population of receptor sites [( 3H]QNB assay) or the super high affinity of their agonist binding sites [( 3H]OXO-M assay). Three compounds fulfilled the criteria for efficient photolabels, allowing substantial and irreversible occupation of the receptor sites to be obtained. Interestingly, the two diazonium derivatives which were selected have been previously described as potent photoprobes of the peripheral nicotinic receptor and of acetylcholinesterase, though displaying lower binding affinities for these acetylcholine binding proteins than for the muscarinic receptors. These findings, together with the all-to-none photolabelling efficiency observed for a quinuclidine derivative, substituted either by an azido or a diazonium group, are discussed. Finally, the apparent lack of binding selectivity of these new photo-affinity probes towards muscarinic receptor affinity states or subtypes should allow comparative studies of the acetylcholine binding site on different muscarinic receptor proteins, obtained either through purification procedures or expression of separate gene products.
Affinity Labels↗
Direct and energy-transfer photolabelling of brain muscarinic acetylcholine receptors.
Efficient photolabelling of muscarinic acetylcholine receptors was obtained using either two aryldiazonium salts or an azido derivative. These probes did not discriminate between muscarinic binding subtypes or affinity states and became irreversibly bound to the receptor sites, in an entirely atropine-protectable manner, upon ultraviolet irradiation. The extent of labelling was dependent both on probe concentration and on time of irradiation and reached up to 80% of the receptor population, under optimal alkylating conditions. In contrast to the azido derivative, both diazonium salts behave as potent irreversible labels of muscarinic receptors, provided energy-transfer photolabelling conditions were followed. Such an indirect activation of diazonium ligands, through an energy transfer from photoexcited tryptophan residues, has been previously found to increase the site-specificity and the rate of labelling of other acetylcholine binding proteins. Analogies in the photolabelling process of acetylcholinesterase or of nicotinic and muscarinic receptors by the two diazonium salts are discussed. Altogether, these findings suggest that these new probes may be promising tools to investigate the location and the topography of the agonist-antagonist binding domain on purified muscarinic receptors, through amino acid and/or sequence analyses of radioactive, photolabelled residues.
Affinity Labels↗
Dynorphin stimulates the release of ANP from isolated rat atria.
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Animals↗
A mu-opioid receptor-filter assay. Rapid estimation of binding affinity of ligands and reversibility of long-lasting ligand-receptor complexes.
A filter-associated binding technique, originally described by Leysen and Gommeren [J. Receptor Res. 4, 817 (1984); Drug Dev. Res. 8, 119 (1986)], was focused on the study of mu-opioid receptor sites. The interesting binding features of 3H-Sufentanil, a mu-selective radioligand, permitted such a filter procedure to be performed. Its application was 3-fold. A 5-min binding assay allowed us to verify that specific 3H-Sufentanil binding to filter-absorbed rat brain membranes was endowed with the known kinetic and equilibrium binding properties of membrane-bound mu-opioid receptor sites. Moreover, the filtration technique allowed us to get rapid information about the dissociation rates of unlabelled compounds from the receptor sites. In practice, membranes, preincubated with high concentrations of cold drugs, were absorbed to filters. Dissociation was achieved by repeatedly applying buffer samples on the filter, and monitored by the recovery in free specific 3H-Sufentanil binding sites. Finally, such a dissociation procedure, improved by a washing buffer at high ionic strength, was found to be much more efficient and attractive than the classical dilution-centrifugation procedure, especially for slowly-dissociating compounds. Special attention was paid to the discrimination between pseudo-irreversible binding of drugs and stable covalent labelling of mu-opioid receptor sites (either by affinity or photoaffinity probes), particularly when unlabelled ligands were the only tools available.
Animals↗
Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative.
p-(Dimethylamino)benzenediazonium fluoroborate (DDF) behaves, in the dark, as a reversible competitive antagonist of the electrical response of Electrophorus electricus electroplaque to acetylcholine and of the acetylcholine-gated single-channel currents recorded in the C2 mouse cell line. This chemically stable but highly photoreactive compound binds irreversibly to the acetylcholine receptor when irradiated by visible light. In vivo, it irreversibly blocks the postsynaptic response of E. electricus electroplaque to agonists. In vitro, it reduces the alpha-bungarotoxin-binding capacity of acetylcholine receptor rich membrane fragments prepared from Torpedo marmorata electric organ. Once reversibly bound to the T. marmorata acetylcholine receptor, this ligand can be selectively photodecomposed by an energy-transfer reaction involving a tryptophan residue(s) of the protein. By use of reagent concentrations that are below the dissociation constant at equilibrium, up to 60% of the agonist-binding sites are covalently labeled. Under these conditions the alpha subunit of the acetylcholine receptor is preferentially labeled, and this labeling is partially prevented by agonists or competitive antagonists. This protective effect is substantially increased by prior incubation with phencyclidine, a compound known to prevent the binding of DDF at the level of the high-affinity site for noncompetitive blockers [Kotzyba-Hibert, F., Langenbuch-Cachat, J., Jaganathen, J., Goeldner, M. P., & Hirth, C. G. (1985) FEBS Lett. 182, 297-301]. The incorporation of about one molecule of label in an agonist/competitive antagonist protectable manner per alpha-bungarotoxin-binding site suffices to fully block alpha-bungarotoxin binding to the membrane-bound receptor. Thus, DDF behaves as a monovalent photoaffinity label of the acetylcholine-binding site.
Acetylcholine↗
Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site.
The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.
Acetylcholine↗