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N J Birdsall

Publications and source records attributed to N J Birdsall.

At least 37 records · Page 2Linked to original sources
PubMed1995

The role of the cholinergic system in the development of the human cerebellum.

High affinity (-)nicotine ([3H]nicotine), alpha-bungarotoxin ([125I]alpha-bungarotoxin) and muscarinic binding ([3H]N-methyl scopolamine) in the human cerebellum were compared between the foetal period (23-39 weeks gestation) and young adulthood (14-34 years) in an autoradiographic study. To estimate proportions of muscarinic receptor subtypes variable wash times and displacement with pirenzepine were employed. [3H]Nicotine binding and total muscarinic binding in foetuses exceeded that in young adults by a factor of 6 and 2 respectively in the dentate nucleus, and by a factor of 3 in white matter. [3H]Nicotine and muscarinic binding was also higher in the foetal external granule cell layer than in the internal granule cell layer of adult, [125I]alpha-Bungarotoxin binding was raised in the dentate nucleus of the foetus compared with the adult. The M2 subtype appeared to be the predominant muscarinic receptor in the cerebellum, however it tended to represent a lower proportion of the muscarinic binding in the foetus than the adult. All 3 receptor types were highest in the foetal brainstem where the M3 + M4 muscarinic subtypes appeared to predominate. The p75 nerve growth factor receptor, measured by immunocytochemistry, in common with cholinergic receptors, paralleled choline acetyltransferase activity which has previously been reported to be high in the cerebellum during late foetal development and to fall in adulthood.

Acetylcholine↗
PubMed1995

Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors.

Novel methods of detecting and quantitating cooperative interactions between an agent and both a tritiated (muscarinic) antagonist and the endogenous agonist (acetylcholine), acting at a common (muscarinic) receptor, have been devised. In a semiquantitative protocol, binding data are transformed into affinity ratios (the ratios of the apparent affinity of the ligand in the presence of the agent to the affinity of the ligand alone), which allow estimates to be made of the potency of the agent and its cooperativity with the tritiated antagonist and with the unlabeled ligand. These parameters have been quantitated by detailed binding assays or guanosine-5'-O-(3-[35S]thio)triphosphate functional assays. The kinetic phenomena associated with the allosteric interactions have been exploited in two non-equilibrium binding assays, from which the affinity constants describing the allosteric interactions can be extracted. The different assay methods give quantitatively similar and internally consistent estimates of the parameters describing the cooperative interactions. Using these assays, strychnine has been found to act allosterically at muscarinic receptors. Strychnine has an affinity of approximately 10(5) M-1 at the unliganded m1, m2, and m4 receptors but is 5-10-fold weaker at m3 receptors. It is positively cooperative with N-methylscopolamine at m2 and m4 receptors and exhibits neutral and negative cooperativity with m1 and m3 receptors, respectively. With acetylcholine, it is negatively cooperative but the degree of cooperativity is relatively low (2-7-fold), particularly at m1 and m4 receptors. The methods and equations described should be useful in detecting and quantitating allosteric interactions of agents with the endogenous neurotransmitter at G protein-coupled receptors.

Acetylcholine↗
PubMed1995

Probing of the location of the allosteric site on m1 muscarinic receptors by site-directed mutagenesis.

In an attempt to locate the allosteric site on muscarinic receptors to which gallamine binds, 21 residues in the putative external loops and loop/transmembrane helix interfaces have been mutated to alanine. These residues are conserved in mammalian m1-m5 receptors. All mutant receptors can be expressed in COS-7 cells at high levels and appear to be functional, in that acetylcholine binding is sensitive to GTP. The gallamine binding site does not appear to involve the first, second, and most of the third extracellular loops. Tryptophan-400 and -101 inhibit gallamine binding when mutated to alanine or to phenylalanine and may form part of the allosteric site. Several mutations also affect antagonist binding. Surprisingly, tryptophan-91, a residue conserved in monoamine and peptide receptors, is important for antagonist binding. This residue, present in the middle of the first extracellular loop, may have a structural role in many G protein-coupled receptors. Antagonist binding is also affected by mutations of tryptophan-101 and tyrosine-404 to alanine or phenylalanine. In a helical wheel model, trytophan-101 and tyrosine-404, in conjunction with serine-78, aspartate-105, and tyrosine-408, form a cluster of residues that have been reported to affect antagonist binding when mutated, and they may therefore be part of the antagonist binding site. It is suggested that the allosteric site may be located close to and just extracellular to the antagonist binding site. The binding of methoctramine, an antagonist with allosteric properties, is not substantially affected by mutations at tryptophan-91, -101, and -400 and tyrosine-404, and thus these amino acids are not important for its binding. The binding of himbacine, another antagonist with allosteric properties, is affected by these mutations but in a manner different from that of gallamine or competitive antagonists. It has not been possible to determine whether methoctramine and himbacine bind exclusively to the allosteric site or to both the competitive site and the allosteric site.

Acetylcholine↗
PubMed1994

Acetylcholine mustard labels the binding site aspartate in muscarinic acetylcholine receptors.

Acetylcholine mustard (AChM) is an analogue of acetylcholine (ACh) in which the onium headgroup is replaced by a chemically reactive aziridinium moiety. AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site. Purified mAChRs from rat forebrain have been specifically labeled with [3H]AChM. The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecular masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine. Edman degradation reveals a site of label attachment 26 residues C-terminal to a CNBr cleavage site. As in the case of the alkylating antagonist analogue [3H]propylbenzilylcholine mustard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the mAChRs, corresponding to Asp-105 (m1 sequence), is the site of label attachment.

Acetylcholine↗
PubMed1993

Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1-m4.

We have studied muscarinic agonist stimulated [35S]GTP gamma S binding and [gamma 32P]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1-m4 receptors. 'Full' agonists were at least 10-fold more potent at m2 & m4 receptors than at m1 & m3. This pattern was less marked with 'partial' agonists, which had a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors. Antagonist affinity constants were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectivity for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype; m2 & m4 receptors activated only pertussis (PTX) sensitive G-proteins, while m1 & m3 coupled to both PTX sensitive and insensitive G-proteins. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX-treated m1 cells than in controls.

Animals↗
PubMed1993

Estimation of antagonist Kb from inhibition curves in functional experiments: alternatives to the Cheng-Prusoff equation.

Recent Principles articles have considered procedures for the functional estimation of an antagonist's dissociation constant from the results of an antagonist inhibition curve in the presence of a fixed concentration of agonist. Leff and Dougall (TiPS 14, 110-112) have derived an equation, analogous to the (generally invalid) Cheng-Prusoff equation, which requires that the agonist concentration--effect curves have the form of a logistic function. In the final article of the series, Sebastian Lazareno and Nigel Birdsall consider this equation in the context of two related methods for analysing functional inhibition curves-a null method that makes no assumptions about the shapes of curves, and a curve-fitting method that provides an estimate of the antagonist Schild slope.

Drug Antagonism↗
PubMed1993

Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations.

1. The estimation of antagonist affinity from functional experiments in which the effect of a fixed agonist concentration is reduced by a range of antagonist concentrations ('functional inhibition curves') has been considered from both a theoretical and experimental viewpoint. 2. Theoretical predictions are compared with results obtained from the stimulation of [35S]-GTP gamma S binding by acetylcholine to membranes of Chinese hamster ovary (CHO) cells stably transfected with human m1-m4 muscarinic receptors, and inhibition of the stimulated binding by pirenzepine and AQ-RA 741. 3. The usual procedure of applying the Cheng-Prusoff correction is shown to be theoretically invalid, and predictions are made of the size and distribution of errors associated with this procedure. 4. A different procedure for estimating antagonist affinity, using the principles of dose-ratio analysis and analogous to use of the Gaddum equation, is found to be accurate and theoretically valid. 5. A novel method of analysis allows accurate estimation of both antagonist affinity and Schild slope, by fitting the combined data from an antagonist inhibition curve and an agonist activation curve directly to a form of the Schild equation (derived by Waud) using non-linear regression analysis. 6. It is shown that the conventional Schild analysis can be enhanced by treating part of the data as a family of inhibition curves and including in the Schild plot dose-ratios estimated from the inhibition curves.

Acetylcholine↗
PubMed1993

Pharmacological characterization of acetylcholine-stimulated [35S]-GTP gamma S binding mediated by human muscarinic m1-m4 receptors: antagonist studies.

1. We have used dose-ratio analysis to estimate functionally the affinity constants (pKb) and Schild slope factors of a range of selective or atypical antagonists at human muscarinic m1-m4 receptors. 2. The functional response was the stimulation by acetylcholine of [35S]-GTP gamma S binding to membranes from Chinese hamster ovary (CHO) cells stably expressing individual receptor subtypes. 3. A novel experimental design and analysis was used which allowed the estimation of affinity and Schild slope factor from a single antagonist inhibition curve, and the results were compared with other methods of analysis, both theoretically valid and invalid. 4. In general, the affinity estimates were very similar to previously reported values obtained in binding studies with animal tissues and cloned human receptors and the Schild slope factors were close to unity. 5. These results demonstrate the validity of the assay and provide no evidence for species differences in antagonist affinity for muscarinic receptor subtypes. 6. The results confirm both the utility of himbacine in distinguishing between m1 and m4 receptors and a previously reported modest m4-selectivity for tropicamide and secoverine. 7. The cholinesterase inhibitor, tacrine (THA), had a potency profile similar to that of gallamine but with less selectivity. Its affinity could not be determined since it had Schild slope factors of about 2 at all subtypes. 8. o-Methoxy-sila-hexocyclium had only a modest selectivity for the m1 subtype.

Acetylcholine↗
PubMed1991

Modulation of the structure-binding relationships of antagonists for muscarinic acetylcholine receptor subtypes.

1. Membranes from rat cerebral cortex, myocardium and extraorbital lacrimal gland were used as sources of M1, M2 and M3 muscarinic acetylcholine receptors respectively and the affinities of seven antagonists for the three subtypes were examined under different experimental conditions. 2. The affinities for the membrane-bound receptors were measured at different ionic strengths and temperatures and compared with those determined on the receptor solubilised in the neutral detergent digitonin or the zwitterionic detergent, CHAPSO. 3. The range of measured affinity constants of a given antagonist for a specific subtype varied from 2 (atropine at M1 receptors) to 1000 (AF-DX 116 at M2 receptors). 4. As a consequence of these changes in affinity, which were dependent on the drug, the subtype and the experimental conditions, both the structure-binding relationships of a given subtype can be markedly changed as well as the selectivity of a drug for the different subtypes. For example it is possible to change the relative affinities of AF-DX 116 and gallamine at membrane-bound M1 receptors from 50:1 to 1:60. 5. Experimental conditions for the observation of high selectivity of pirenzepine, AF-DX 116, gallamine and hexahydrosiladiphenidol for the three subtypes are given. 6. When the receptors are removed from their membrane environment by solubilisation in detergent, antagonist affinities are changed but the subtypes still retain different structure-binding relationships. 7. In general, AF-DX 116 and the allosteric antagonist, gallamine, behave differently from the other antagonists, suggesting that they bind in different ways to muscarinic receptors. Careful attention should therefore be paid to the experimental conditions in binding assays used to assess the affinities and selectivities of new muscarinic antagonists in order to avoid misleading results. 9. The ability to produce enhanced or attenuated affinities and selectivities of antagonists, resulting from the induction of different conformations of the receptor by a variety of physical, chemical or molecular biological perturbations may lead to a better understanding of the structural basis of drug receptor interactions.

Animals↗
PubMed1989

The affinity, selectivity and biological activity of telenzepine enantiomers.

The binding of the enantiomers of telenzepine to muscarinic receptor subtypes present in guinea-pig cerebral cortex, myocardium and salivary glands has been examined. The (+) enantiomer is more potent in all assays and exhibits a greater selectivity than the (-) enantiomer for the different receptor subtypes. As a consequence, the enantiomeric potency ratio varies from ca. 400 (cortical 'M1' receptors) to ca. 50 (cardiac receptors). In functional assays in vitro in the rabbit vas deferens and rat atria, the affinity constants and enantiomeric potency ratios for the two isomers agree with those found for the appropriate muscarinic receptor subtype in binding assays. A high enantiomeric potency ratio, 180, is found in vivo for the ability of the telenzepine enantiomers to inhibit the production of lesions in the modified Shay rat preparation. The data are compatible with the blockade of M1 receptors by (+)-telenzepine being responsible for this action of telenzepine and would tend to exclude the possibility that the anti-ulcer action of telenzepine is mediated via a muscarinic or non-muscarinic action of the (-) enantiomer.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1989

Propylbenzilylcholine mustard labels an acidic residue in transmembrane helix 3 of the muscarinic receptor.

Muscarinic acetylcholine receptors were purified from rat forebrain and labeled with [3H]N-(2-chloroethyl)N-(2',3'-[3H2]propyl)-2-aminoethylbenzilate. Cleavage of the labeled muscarinic acetylcholine receptors with a lysine-specific protease yielded labeled, glycosylated peptides about 130 and 200 residues in length, which came from different receptor sequences. The probable cleavage sites are in the second intracellular loop and in the second extracellular or third intracellular loop. The N-terminal 130 residues are disulfide-bonded to another part of the receptor structure, supporting the presence of a link between the second and third extracellular loops. The [3H]propylbenzilylcholine mustard-receptor link is cleaved by nucleophiles, acids, and bases under denaturing conditions, suggesting modification of an acidic residue. Cyanogen bromide cleavage points to transmembrane helix 3 as the site of label attachment.

Animals↗