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.     

Metaphit inhibits dopamine transport and binding of [3H]methylphenidate, a proposed marker for the dopamine transport complex

Metaphit, an acylating derivative of phencyclidine, was shown to interact with components of the dopamine nerve terminal in rat striatal tissue. This compound, previously demonstrated to be an irreversible inhibitor at the phencyclidine receptor, was shown in these experiments to irreversibly inhibit synaptosomal [3H]dopamine uptake. It also inhibited binding of [3H]methylphenidate to its recognition site, which is thought to be a subunit of the dopamine transporter. Although the inhibition was due primarily to a reduction in the binding and transport capacity of the systems studied, increases in the apparent KD of [3H]methylphenidate and the Km of [3H]dopamine were also observed. Differences in the behavior of Metaphit and phencyclidine in these dopaminergic systems compared to their effects on the NMDA receptor-linked phencyclidine receptor suggest that Metaphit may be interacting with two distinct molecular sites in the rat striatum.     

Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles

Noncompetitive inhibition of acetylcholine receptor-controlled ion translocation was studied in membrane vesicles prepared from both Torpedo californica and Electrophorus electricus electroplax. Ion flux was measured in the millisecond time region by using a spectrophotometric stopped-flow method, based on fluorescence quenching of entrapped anthracene-1,5-disulfonic acid by Cs+, and a quench-flow technique using 86Rb+. The rate coefficient of ion flux prior to receptor inactivation (desensitization), JA, was measured at different acetylcholine and inhibitor concentrations, in order to assess which active (nondesensitized) receptor forms bind noncompetitive inhibitors. The degree of inhibition of JA by the inhibitors studied (cocaine, procaine, and phencyclidine) was found to be independent of acetylcholine concentration. The results are consistent with a mechanism in which each compound inhibits by binding to a single site that exists with equal affinity on all active receptor forms. Mechanisms in which the inhibitors bind exclusively to the open-channel form of the receptor are excluded by the data. The same conclusions were reached in cocaine experiments at 0-mV and procaine experiments at -25-mV transmembrane voltage in T. californica vesicles. It had been previously shown that phencyclidine, in addition to decreasing JA (by binding to active receptors), also increases the rate of rapid receptor inactivation (desensitization) and changes the equilibrium between active and inactive receptors (by binding better to inactivated receptor than to active receptor in the closed or open conformations). These effects were not observed with cocaine or procaine. Here it is shown that despite these differential effects on inactivation, cocaine and phencyclidine bind to the same inhibitory site on active receptors (in E. electricus vesicles).(ABSTRACT TRUNCATED AT 250 WORDS)     

Etoxadrol-meta-isothiocyanate: a potent, enantioselective, electrophilic affinity ligand for the phencyclidine-binding site

Etoxadrol-meta-isothiocyanate (2S,4S,6S-2-ethyl-2-(3-isothiocyanatophenyl)-2-piperidyl)1,3-dioxolane, 4a) has been synthesized and characterized as an irreversible ligand for the phencyclidine (PCP)-binding site. It is the first chiral electrophilic affinity ligand for this site to have been described. This affinity ligand is based upon etoxadrol, a 1,3-dioxolane known to have PCP-like effects in vivo and in vitro. Etoxadrol-meta-isothiocyanate was found to be four-five times more potent in vitro than metaphit (1-[1-(3- isothiocyanatophenyl)cyclohexyl]piperidine), the only previously known electrophilic affinity ligand for the PCP-binding site. The binding was shown to be highly enantioselective for etoxadrol-meta-isothiocyanate (4a). The 2R,4R,6R-enantiomer of 4a was essentially inactive. The ability of the 2S,4S,6S-enantiomer (4a) to interact with the benzodiazepine, muscarinic, and mu opioid receptor systems was also examined, and it was found not to interact with these receptor systems. It seems likely that 4a will prove to be a valuable tool in the study of structure and function of the PCP-binding site.     

Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding

The spectroscopic properties and specificity of binding of a fluorescent quaternary amine, ethidium, with acetylcholine receptor-enriched membranes from Torpedo californica have been examined. Competition binding with [3H]phencyclidine in the presence of carbamylcholine showed that ethidium binds with high affinity to a noncompetitive inhibitor site (KD = 3.6 X 10(-7) M). However, in the presence of alpha-toxin, ethidium's affinity is substantially lower (KD approximately 1 X 10(-3) M). Ethidium was also found to enhance [3H]acetylcholine binding with a KD characteristic of ethidium binding to a high-affinity noncompetitive inhibitor site. These findings indicate that ethidium binds to an allosteric site which is regulated by agonist binding and can convert the agonist sites from low to high affinity. Fluorescence titrations of ethidium in the presence of carbamylcholine yielded a similar KD (2.5 X 10(-7) M) and showed an ethidium stoichiometry of one site/acetylcholine receptor monomer. Ethidium was completely displaced by noncompetitive inhibitors such as phencyclidine, histrionicotoxin, and dibucaine. The enhanced fluorescence lifetime of the bound species showed that the increased fluorescence intensity reflects a 13-fold increase in quantum yield for the complex compared to ethidium in buffer. Fractional dissociation of ethidium with phencyclidine produced a double-exponential fluorescence decay rate with lifetime components characteristic of ethidium free in solution and bound to the receptor. These data argue that the alterations in ethidium fluorescence elicited by other ligands is due to a change in the fraction of specifically bound ethidium rather than a change in quantum yield of a pre-existing ethidium-acetylcholine receptor complex. The extent of polarization indicates that bound ethidium is strongly immobilized. The magnitude of the quantum yield enhancement and the shifts of excitation and emission maxima of bound ethidium suggest that its binding site is within a hydrophobic domain with limited accessibility to the aqueous phase.     

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.     

A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.     

Decidium. A novel fluorescent probe of the agonist/antagonist and noncompetitive inhibitor sites on the nicotinic acetylcholine receptor

We have examined the interaction of the nicotinic acetylcholine receptor with decidium diiodide, a bisquaternary analogue of ethidium containing 10 methylene groups between the endocyclic and trimethylamino quaternary nitrogens. Decidium inhibits mono-[125I]iodo-alpha-toxin binding, inhibits agonist-elicited 22Na+ influx in intact cells, augments agonist competition with mono-[125I]iodo-alpha-toxin binding, and enhances [3H]phencyclidine (PCP) binding to a noncompetitive inhibitor site. These effects occur over similar concentration ranges (half-maximum effects between 0.1 and 0.4 microM). Thus, decidium binds to the agonist site and converts the receptor to a desensitized state exhibiting increased affinity for agonist and heterotropic inhibitors. These properties are similar to metaphilic antagonists characterized in classical pharmacology. At higher concentrations decidium associates directly with the noncompetitive inhibitor site identified by [3H]phencyclidine binding. Dissociation constants of decidium at this site in the resting and desensitized states are determined to be 29 and 1.2 microM, respectively. Analysis of fluorescence excitation and emission maxima reveal that binding to both the agonist and noncompetitive inhibitor sites is associated with approximately 2-fold enhancement of fluorescence. The excitation maximum for decidium bound at the agonist site appears at 490 nm while that for decidium bound at the noncompetitive inhibitor site appears at 530 compared to 480 nm in buffer. These results suggest that decidium experiences a more hydrophobic environment upon binding to the nicotinic acetylcholine receptor sites, particularly to the noncompetitive inhibitor site. Fluorescence energy transfer between N'-fluorescein isothiocyanate-lysine-23 alpha-toxin (FITC-toxin), and decidium is not detected when each is bound to one of the two agonist sites on the receptor. This allows a minimal distance to be estimated between fluorophores. In contrast, energy transfer is observed between decidium nonspecifically associated with the membrane or with nonspecific sites and the FITC-toxin at the agonist sites.     

Identification of enzyme coupling sites with aromatic diazonium salts-a resonance raman study.

The coupling reaction of diazonium salts of aromatic compounds with the aromatic residues of proteins results in chromophoric covalent derivatives which yield strong resonance enhanced Raman spectra. The protein residues modified by these coupling reactions have been identified using the ν(NN) and ν(N-φ) vibrational bands in the resonance Raman spectra. Previous studies have established that diazoarsanilic acid couples with carboxypeptidase at tyrosine 248. The resonance Raman spectrum of arsanilazocarboxypeptidase was compared with spectra of arsanilazotyrosine and arsanilazohistidine model compounds; the results are consistent only with coupling at a tyrosine residue. This confirmation of the previously established site of modification establishes the utility of resonance Raman spectroscopy as a tool for identification of the site of covalent modification. To further investigate this approach, the diazonium salt of sulfanilamide (a site-specific reagent) was used to prepare a covalent coupling derivative of bovine carbonic anhydrase. The coupling reaction appears to have a stoichiometry of 1:1 and results in nearly complete loss of sulfanilamide binding capability and esterase activity. Comparison of the pH dependence of the resonance Raman spectra of sulfanilazocarbonic anhydrase with the spectra of sulfanilazotyrosine, sulfanilazohistidine, and sulfanilazotryptophan suggests that histidine is the site of modification of this new carbonic anhydrase derivative.     

(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] but not (-)-3-PPP produces (+)-N-allylnormetazocine-like (SKF 10,047) discriminative stimuli

In rats trained to discriminate the prototypic sigma receptor agonist, (+)-N-Allylnormetazocine [(+)-N-Allylnormetazocine [(+)-NANM/SKF 10,047], from saline, the (+)- but not the (-)-isomer of 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) produced (+)-NANM-like discriminative stimuli. (+)-3-PPP binds stereo selectively to the (+)-NANM binding site, but not to the phencyclidine binding site. Additionally, phencyclidine was found to produce (+)-NANM-like discriminative stimuli. Although the 3-PPP isomers were shown to produce changes in central dopaminergic activity (Hjorth et al. Life Sci 37, 673, 1985), the discriminative stimulus properties of (+)-3-PPP are apparently not mediated via the dopaminergic system. This hypothesis is supported by the fact that apomorphine did not produce (+)-NANM-like discriminative stimuli. These stimuli are thus non-dopaminergic and may be due to the (+)-3-PPP actions at the sigma binding site. However, it is possible that (+)-NANM, PCP, and (+)-3-PPP may have common non-sigma pharmacologic properties that account for the similar discriminative stimulus properties of these compounds.     

Metaphit Irreversibly Inhibits [3H]threo- (±)-Methylphenidate Binding to Rat Striatal Tissue

Metaphit (1-[1-(3-isothiocyanatophenyl)cyclohexyl]-piperidine), a derivative of phencyclidine that contains an isothiocyanate group on the meta position of the aromatic ring, resembles its parent compound (phencyclidine) in its ability to inhibit the binding of the stimulant drug [3H]threo-(+/-)-methylphenidate to crude synaptosomal membranes from rat striatal tissue (IC50 = 1.4 and 6.2 microM for phencyclidine and Metaphit, respectively). Unlike phencyclidine, however, Metaphit appears to inhibit binding of the radiolabeled stimulant in an irreversible manner, as the degree of inhibition of binding of the stimulant does not diminish when the Metaphit-treated tissue is subjected to repeated washings before determination of the binding of [3H]threo-(+/-)-methylphenidate. This finding suggests that Metaphit may be a useful tool in the study of the molecular basis of stimulant action.     

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

Summary Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits⁸⁶Rb flux (IC₅₀=6.6m), and [³H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC₅₀=17 m and 0.22 m, respectively). From an indirect Hill plot of the inhibition of [³H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in -bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.     

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

Synthesis and NMDA-receptor affinity of 4-oxo-dexoxadrol derivatives

A synthesis of novel dexoxadrol analogues is described, which allows modifications of the piperidine substructure. The key step of the synthesis is a hetero Diels-Alder reaction of the imine 12 with Danishefsky's diene 6. After separation of the diastereomeric piperidones 14a and 14b, the relative configuration of the unlike configured piperidone 15b was determined by X-ray crystal structure analysis. In receptor binding studies the like configured secondary amine 15a (racemate) showed considerable affinity toward the phencyclidine binding site of the NMDA receptor (Ki=470 nM).     

Binding of phencyclidine to the detergent solubilized acetylcholine receptor from Torpedo marmorata

The binding of phencyclidine to the acetylcholine receptor from Torpedo marmorata electroplaque was measured following solubilization of the receptor in sodium cholate followed by the exchange of cholate for Tween 80. In both the membrane-bound and solubilized AChR, the addition of cholinergic agonists simultaneously with the addition of PCP results in a 100 to 1000 fold increase in the PCP association rate and a 5 to 10 fold increase in the dissociation rate as compared to the unliganded AChR or AChR equilibrated with agonist prior to PCP addition. In addition, the number of binding sites and the pharmacological properties of the binding are not markedly changed in the soluble receptor. These results suggest that the acetylcholine receptor can undergo similar conformational transitions in the membrane-bound and the Tween 80 solubilized form and that phencyclidine can monitor these transitions in both cases.     

A novel photoaffinity ligand for the phencyclidine site of the N-methyl-D-aspartate receptor labels a Mr 120,000 polypeptide

A radiolabeled photoaffinity ligand has been developed for the N-methyl-D-aspartate (NMDA)-preferring excitatory amino acid receptor complex. [3H]3-Azido-(5S, 10R)(+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine [3H]3-azido-MK-801 demonstrated nearly identical affinity, density of binding sites, selectivity, pH sensitivity, and pharmacological profile in reversible binding assays with guinea pig brain homogenates to those displayed by its parent compound, MK-801. When employed in a photo-labeling protocol designed to optimize specific incorporation, [3H]3-azido-MK-801 labeled a single protein band which migrated in sodium dodecyl sulfate-polyacrylamide gels with Mr = 120,000. Incorporation of tritium into this band was completely inhibited when homogenates and [3H]3-azido-MK-801 were coincubated with 10 microM phencyclidine. These data suggest that the phencyclidine site of the NMDA receptor complex is at least in part comprised of a Mr = 120,000 polypeptide.     

Psychotomimetics moderately affect dopamine receptor binding in the rat brain

The hypothesis that psychotomimetics induce a rapid dopamine receptor regulation that could participate in the expression of the brain dopaminergic overactivation and in the early signs of psychotic-like behaviour, was checked by radioligand binding on rat brain cryosections. For this purpose, subchronic 7-day-d-amphetamine pretreatment was combined with acute amphetamine, phencyclidine or LSD challenge. Acute application of psychotomimetics affected only striatal and accumbens but not nigral and olfactory dopamine receptor binding after 40 min, while subchronic amphetamine expressed no effect, as revealed by two-way ANOVA. Post-hoc statistical analysis showed that only striatal and accumbens[3H]SCH 23390 binding decrease (10-12%) following phencyclidine and striatal [3H]spiperone binding increase (11%) after acute amphetamine were significant. It is assumed that such moderate dopamine receptor binding changes probably reflect the fast receptor regulation responses without important influence on a proposed drug-induced dopaminergic overactivity. The registered alterations of D1 receptor binding after phencyclidine are suggested to be capable to modify the activity of some other neural pathways in the basal ganglia and thus participate in a psychotic-like behaviour.     

The effects of ketamine, phencyclidine and lidocaine on catecholamine secretion from cultured bovine adrenal chromaffin cells

The ability of ketamine, phencyclidine and analogues to alter catecholamine secretion from cultured bovine adrenal chromaffin cells was investigated. Both ketamine and phencyclidine specifically inhibited nicotinic agonist-induced secretion at concentrations which did not alter secretion induced by elevated K+ depolarization. The inhibition of nicotinic agonist-induced secretion was not overcome by increasing concentrations of nicotinic agonist. The effects of stereoisomer pairs of phencyclidine-like drugs - dexoxadrol, levoxadrol and (+)PCMP, (-)PCMP - did not reveal stereospecificity for the inhibition, in contrast to the stereospecific behavioral effects of the drugs. The local anesthetic lidocaine (0.3 mM) also noncompetitively inhibited nicotinic agonist-induced secretion without inhibiting elevated K+-induced secretion. The data indicate that ketamine and phencyclidine at clinically relevant concentrations specifically inhibit the adrenal chromaffin cell nicotinic receptor at a site similar to or identical with the site of action of local anesthetic. Although the nicotinic receptor inhibition is probably not related to the anesthetic and behavioral effects of ketamine and phencyclidine, it is likely that the centrally mediated increase in sympathetic nervous system activity which is characteristic of these drugs is moderated by the peripheral blocking effects on catecholamine secretion from the adrenal medulla.     

A specific acylating agent for the [3H]phencyclidine receptors in rat brain

A derivative of phencyclidine (PCP, 1 in fig. 1) bearing an isothiocyanate moiety on the meta position of the aromatic ring (Metaphit, 3 in fig. 1) has been synthesized and identified as a rapid and specific site-directed acylating agent of the [3H]phencyclidine binding site in rat brain homogenates. The percentage of sites irreversibly inactivated by Metaphit was found to be the same in the hippocampus and striatum and the remaining sites were unaffected by Metaphit treatment under any conditions, suggesting that at least two distinct binding sites are present. An isomeric isothiocyanate derivative did not irreversibly inhibit [3H]phencyclidine receptors, indicating structural specificity for Metaphit in the inhibition of these receptors. The availability of Metaphit should greatly facilitate study of the structure and function of the phencyclidine receptors.     

Pseudoallosteric modulation by (+)-MK801 of NMDA-coupled phencyclidine binding sites

Two high affinity phencyclidine (PCP) binding sites, labelled by [3H] 1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP), have been identified in guinea pig brain, with one site coupled to the N-methyl-D-aspartate (NMDA) receptor (site 1) and the other site associated with the dopamine reuptake carrier complex (site 2). In this study, PCP enhanced the dissociation of [3H]TCP from PCP site 1 and site 2, while (+)-MK801 only enhanced dissociation of [3H]TCP from PCP site 1. Although additional studies will be required to determine the exact mechanism(s) of these effects, these data demonstrate that the interactions of PCP with both site 1 and site 2 are more complex than previously appreciated.