[3H]benzylpempidine, a new radioligand for probing a putative channel site on nicotinic cholinergic receptors.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.

Expression and characterization of the rat alpha 4 beta 2 neuronal nicotinic cholinergic receptor in baculovirus-infected insect cells.

Baculovirus expression systems have been developed to generate 1) a neuronal nicotinic cholinergic receptor comprising both the alpha 4 and beta 2 subunits and 2) the alpha 4 and beta 2 subunits individually. The presence of the alpha 4 and beta 2 genes in the various baculovirus-infected Sf9 cells was confirmed following polymerase chain reaction (PCR) of the extracted viral DNAs, gel electrophoresis, and double strand sequencing. Autofluorography, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of infected cell lysates radiolabeled with 35S-methionine and immunoprecipated with mAb 270 (specific for the beta 2 subunit), revealed the presence of characteristic 52-kD bands in beta 2- and alpha 4 beta 2 recombinant viral-infected cells, but not in control cells or cells infected with wild-type virus or recombinant virus containing alpha 4 alone. The 52-kD protein, which is specific for mAb 270, is known to be the beta 2 subunit of neuronal nAChRs. Specific [3H]methylcarbamylcholine binding was observed in cells infected with both alpha 4 or beta 2 but not with the alpha 4 or beta 2 genes alone. Scatchard analysis revealed a Bmax = 5.50 pmol/mg and a Kd = 1 nM. The degree of [3H]methylcarbamylcholine binding/mg membrane protein was 180-fold greater than that found in rat brain. The study demonstrates that the major neuronal nAChR, which comprises alpha 4 and beta 2 subunits and is present in very low abundance in mammalian brain, can be prepared by a baculovirus expression system in sufficient quantities for chemical and crystallographic structural analysis.

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique.

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.

Antagonism of nicotine's action by cocaine analogs.

Structure-activity relationships of a number of synthetic cocaine analogs are described comparing their effectiveness in antagonizing the behavioral effects of nicotine in mice with their ability to compete for [3H]mecamylamine, [3H]nicotine, and [3H]3-quinuclidinylbenzilate ([3H]QNB) binding to calf brain membranes. Within a series of phenyltropane carboxylic acid methyl esters the most potent analogues were the 4-I and 4-F-phenyl analogs, while replacement of F by Cl or alkyl groups diminished potency. The isopropyl and phenylcarboxylic acid esters were comparable in potency to the methyl esters. There appeared to be a relationship between the potency of the analogs in inhibiting the dopamine transporter and nicotine antagonism. A good correlation was observed between pharmacologic potency and [3H]mecamylamine binding to brain membranes. It was concluded that the antagonistic action of the cocaine analogs involved an ion channel site on the neuronal nicotinic cholinergic receptors.

Differences in ligand binding and phosphoinositide turnover between M1 muscarinic receptor gene transfected cells and mouse and rat brain membranes.

The present study describes some unexpected receptor mediated effects of N-methylcarbamylcholine on mouse M1 muscarinic receptor gene transfected cell line (M1Y1) that were not evident from biochemical studies with mouse and rat brain tissue where N-methylcarbamylcholine exhibited only nicotinic properties. Although N-methylcarbamycholine was devoid of muscarinic properties in mouse and rat brain preparations, as determined by phosphoinositide turnover and inhibition of [3H]QNB binding, it exhibited significant muscarinic characteristics in the transfected M1Y1 cell line. At a concentration of 10(-6) M or greater, N-methylcarbamycholine caused a transient increase in intracellular Ca2+ of 50 s duration that was reversible by atropine or pirezepine. The Ca(2+)-transient was not elicited by other nicotinic agents such as nicotine and N,N-dimethylcarbamylcholine, a close analogue of N-methylcarbamylcholine, with comparable affinity for nicotinic receptors and devoid of muscarinic activity. N-Methylcarbamylcholine also stimulated phosphoinositide turnover in M1Y1 cells with an estimated EC50 value 10 times greater than that of carbachol, and the effect was blocked by atropine. Both carbachol and N-methylcarbamycholine inhibited [3H]QNB binding in a concentration-dependent manner; however, the IC50 for carbachol was over two orders of magnitude greater than that observed in mouse and rat brain membranes. In considering possible explanations for the differential characteristics of N-methylcarbamylcholine in mouse and rat brain as compared to the transfected M1Y1 cells, it was concluded that the difference may be attributable to differences in the receptor-transduction coupling efficiency and the microenvironment of the muscarinic receptors.

5-Isothiocyanonicotine: a high-affinity irreversible ligand for brain nicotinic receptors.

A newly synthesized affinity ligand, (R,S)-5-isothiocyanonicotine (ISCN-N) was found to inhibit irreversibly the binding of [3H]methylcarbamylcholine (a specific nicotinic receptor ligand) to brain membranes. Plots of percent inhibition versus ligand concentration yielded an IC50 of 7 x 10(-8) M for SCN-N and Ki values of 6 x 10(-9) and 2 x 10(-9) M for (R,S)-5-aminonicotine and (S)-nicotine, respectively. The IC50 value for irreversible inhibition of [3H]methylcarbamylcholine by SCN-N was 2 x 10(-7) M. The affinity ligand irreversibly inhibited brain nicotinic receptors in vivo in a dose-dependent manner, the inhibition being 49% at a dose of 20 mumol/kg. Behavioral studies in mice revealed that SCN-N had less than one-fifth the potency of nicotine in producing muscle weakness and seizures, whereas 5-aminonicotine was without significant behavioral effects at doses up to 20 mumol/kg.

Affinity ligands and related agents for brain muscarinic and nicotinic cholinergic receptors.

This study describes the chemical synthesis and receptor binding characteristics of various affinity ligands and related ligands for brain muscarinic and nicotinic cholinergic receptors, including the 4-bromoacetamidobenzoic acid esters of dimethylaminoethanol (DMBAB) and choline (BABC) and 4-iodoacetamidobenzoylcholine (IABC). The reversible binding of [3H]3-quinuclidinylbenzilate ([3H]QNB) to calf brain membranes was inhibited in a concentration-dependent and saturable manner by DMBAB, BABC, and IABC with Ki values of 8 x 10(-7), 3 x 10(-7) and 8 x 10(-7) M, respectively; the Ki values for inhibition of reversible binding of the nicotinic ligand, [3H]methylcarbamylcholine ([3H]-MCC), were 1 x 10(-6), 6 x 10(-8), and 1 x 10(-6) M, respectively. The Ki values for irreversible inhibition of [3H]QNB binding were 8 x 10(-7), 1 x 10(-7), and 2 x 10(-7) M for DMBAB, BABC, and IABC, respectively, and for [3H]MCC binding, 8 x 10(-5), 1 x 10(-5), and 2 x 10(-5) M, respectively. Although DMBAB was found to inhibit the QNB-induced hyperactivity in mice, it did not antagonize the toxic or other pharmacologic effects of oxotremorine. Structure-activity studies with various non-affinity analogues of the 4-aminobenzoate ester of dimethylaminoethanol and choline revealed that removal of the NH2 moiety from the phenyl group increased affinity for the muscarinic but not the nicotinic cholinergic site, and quaternization of the ester side chain greatly increased affinity for the muscarinic site. Dimethylation of NH2 in 4-aminobenzoylcholine decreased the affinity for both cholinergic sites. Replacement of NH2 by NO2 increased affinity for the muscarinic but not the nicotinic site, whereas quaternization of the 4-nitrobenzoyl ester markedly increased affinity for the nicotinic site while diminishing affinity for the muscarinic site. The findings indicate that DMBAB and its analogues are useful affinity ligands for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nanomolar concentration range.

Opioid receptor antagonist affinity ligands: 6 beta-bromoacetamido-6-desoxynaltrexone and 6 beta-thioglycolamido-6-desoxynaltrexone.

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6 beta-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6 beta-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [3H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had Ki values of 1 x 10(-9) and 1 x 10(-10) M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5 x 10(-8) and 1 x 10(-8) M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [3H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1 x 10(-7) M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED50 dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.

4-Bromoacetamidoprocaine: an affinity ligand for brain muscarinic and nicotinic cholinergic receptors.

This study describes the synthesis, receptor binding characteristics, and some behavioral effects of p-bromoacetamidoprocaine (BAP), a new affinity ligand for brain muscarinic and nicotinic cholinergic receptors. The reversible binding of [3H]QNB to rat brain membranes was inhibited in a concentration dependent and saturable manner by both procaine and BAP, with Ki values of 4 x 10(-6) and 3 x 10(-7) M, respectively, and complete inhibition at 1 x 10(-5) M. Both procaine and BAP, although at much concentrations, inhibited the binding of [3H]methylcarbamylcholine in a concentration dependent manner, with Ki values of 5 x 10(-5) and 1 x 10(-5) M, respectively, and complete inhibition for both at 1 x 10(-3) M. Plots of the % irreversible inhibition of [3H]QNB, [3H]nicotine, and [3H]MCC vs [BAP] yielded Ki values of 7 x 10(-8), 1 x 10(-4), and 6 x 10(-5) M, respectively. In behavioral studies BAP was able to antagonize the QNB-induced hyperactivity in mice; however, BAP did not appear to alter nicotine-induced seizure activity or other behavioral effects in mice. A plot of the time course of inhibition by BAP for [3H]QNB binding revealed that the inhibition was almost complete within 10 min exposure at 37 degrees. The findings indicate that BAP is a useful affinity ligand for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nM concentration range.

[3H]mecamylamine binding to rat brain membranes. Studies with mecamylamine and nicotine analogues.

Mecamylamine, an antagonist to nicotine, does not compete at the nicotinic recognition site, but is believed to block the ion channel of the nicotinic receptor. The present study demonstrates specific, saturable [3H]mecamylamine binding in rat brain membranes. [3H]Mecamylamine binding was destroyed by heating at 100 degrees and trypsin. Scatchard analysis revealed the presence of two sites with Kd values of 9.6 x 10(-8) and 1.1 x 10(-6) M and Bmax values of 7 x 10(-12) and 3 x 10(-11) mol/mg protein respectively. A good correlation was observed between the Ki values for [3H]mecamylamine binding of a number of mecamylamine and related analogues and their ability to block nicotine-induced prostration in rats and seizures in mice. Inorganic cations, particularly divalent, and various ion channel blockers, such as phencyclidine and verapamil, exhibited a high affinity for the [3H]mecamylamine site. Although mecamylamine did not block nicotine binding, nicotine and its analogues exhibited a high affinity for the [3H]mecamylamine site, a finding which suggests that nicotine acts directly on ion channels as well as the nicotinic cholinergic recognition sites. The data are consistent with the notion that mecamylamine interacts with the open ion channel of the nicotinic receptor.

Arginine vasopressin-anti-idiotypic immunostaining of human brain cells.

Abstract Polyclonal anti-idiotypic antibodies, generated against the IgG fraction of antisera to arginine vasopressin (AVP), were shown to recognize two proteins in rat brain and bovine pituitary associated with [(3) H]AVP binding. Immunochemical analyses with these antisera revealed reactivity in paraventricular and supraoptic nucleus neuronal elements and in terminals of the posterior pituitary in the human central nervous system. With the use of a dual immunocytochemical staining technique employing both the anti-idiotype and idiotype for AVP it was possible to demonstrate a pattern of AVP-anti-idiotypic-immunoreactivity on AVP neuronal elements which suggests the existence of autoreceptors.

Differential desensitization of muscarinic receptor-stimulated phosphoinositide turnover in the rat brain.

Desensitization of the muscarinic receptor-mediated phosphoinositide (PI) turnover response in the striatum and cortex of the rat brain was examined. The rate of accumulation of inositol phosphates during carbachol-stimulated PI turnover was constant for at least 60 min in the cortex, but decreased with time in the striatum. The effects of preincubation in the presence of the muscarinic agonist carbachol on muscarinic receptor-mediated PI turnover in slices and subsequent receptor binding in cell aggregates prepared from the slices were measured. Preincubation of striatal tissue produced a significant and sustained loss of responsiveness, which reached a minimum after 2 h; whereas, a transient and much weaker loss of responsiveness was observed in the cortex. Preincubation did not affect the number of muscarinic receptors as measured by the binding of [ (3)H ]l- quinuclidinyl benzilate or [ (3)H ]N- methylscopolamine in either cortex or striatum. The data suggest that the muscarinic receptor-stimulated PI turnover response can be more readily desensitized in striatum compared with cortex.

Sites, mechanisms, and structural characteristics of the brain's nicotine receptor.

Studies are described dealing with the molecular features of nicotine, the receptor binding and psychotropic properties of nicotine agonists and antagonists, and the neuroanatomical locus of action of nicotine associated with its psychotropic action. Bridged analogues of nicotine have been developed to define the optimal conformation of the molecule for maximal receptor affinity and psychotropic action in rats. With another series of analogues, it was demonstrated that contraction of the pyrrolidine ring to a 4-member azetidine enhances potency while expansion diminishes it. A major site for nicotine's central action is the vestibular cerebellum as demonstrated by kainic acid lesioning studies and direct administration of nicotine into this region. Included among the antagonists to nicotine was alpha-lobeline, which appeared to be a mixed agonist-antagonist.

Interaction of putative vasopressin receptor proteins of rat brain and bovine pituitary gland with an antibody against a nanopeptide encoded by the reverse message of the complementary mRNA to vasopressin.

An antibody directed against the reverse message of the complementary mRNA for arginine vasopressin was demonstrated to be immunoreactive with 62 and 55 kdalton proteins, obtained by affinity chromatography of rat brain and bovine posterior pituitary extracts and believed to be associated with the vasopressin binding site. A similar pattern of immunoreactivity was seen with an anti-idiotypic antibody for arginine vasopressin.