A Study of the Muscarinic Receptor by Gel Electrophoresis

Birdsall, N. J. M.; Burgen, A. S. V.; Hulme, Edward C.

doi:10.1111/j.1476-5381.1979.tb13685.x

Cited by 112 publications

(33 citation statements)

References 11 publications

(9 reference statements)

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“…Lysophosphatidylcholine exhibited intermediate properties. cholate/NaCl/PC) with molecular weights which agree with those estimated by SDS-polyacrylamide gel electrophoresis (Birdsall et al, 1979;Ruess & Lieflander, 1979;Amitai etal., 1982;Venter, 1983). In general, however, the soluble receptor-detergent complexes exhibited heterogeneity which was manifest both in gel filtration and sedimentation studies.…”

Section: Solubilization Of Muscarinic Receptorssupporting

confidence: 65%

“…When muscarinic receptors from the forebrain, smooth muscle and heart are labelled with irreversible ligands and then solubilized by sodium dodecylsulphate (SDS), they migrate as a single band, Mr= -83, 000, on SDS-polyacrylamide gel electrophoresis (Birdsall et al, 1979;Ruess & Lieflander, 1979;Amitai etal., 1982;Venter, 1983 SDS gel electrophoresis, suggesting that the receptors may be monomers in this detergent (Haga, 1980a). However, only a small fraction of muscarinic receptor sites was solubilized by this procedure and it is possible that the insoluble or denatured binding sites represent receptor aggregates or specific receptor-protein complexes.…”

Section: Introductionmentioning

confidence: 99%

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Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Berrie¹,

Birdsall²,

Haga³

et al. 1984

British J Pharmacology

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Section: Solubilization Of Muscarinic Receptorssupporting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Berrie¹,

Birdsall²,

Haga³

et al. 1984

British J Pharmacology

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“…With regard to this lack of effect of GTP, it should be noted that the ileal smooth muscle homogenates exhibit a considerable degree of proteolytic activity (Birdsall et al, 1979) and that the circular ileal smooth muscle homogenate used in the present study was able to degrade extensively adiphenine and hexahydroadiphenine. It is therefore possible that the failure of GTP to modify binding of [3H]-NMS to circular ileal smooth muscle muscarinic receptors may have been due to degradation of GTP by some enzymatic activity of the tissue preparation.…”

Section: Discussionmentioning

confidence: 67%

Direct binding studies on ileal and cardiac muscarinic receptors

Michel¹,

Whiting²

1987

British J Pharmacology

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1 Functional studies have indicated that muscarinic receptors in cardiac tissue differ from those in the ileum. In the present study ileal and cardiac muscarinic receptors identified by [3H]-N-methyl scopolamine ([3H]-NMS) were characterized and the selectivity of currently available ileal and atrial selective antagonists determined. 2 In terms of the current functional classification of muscarinic receptors both ileal and cardiac muscarinic receptors were of the M2 subtype based upon their low affinity for pirenzepine. 3 Cyclohexylphenyl(2-piperidinoethyl)silanol (CPPS), a highly ileal selective antagonist in functional studies, was unable to distinguish between ileal and atrial muscarinic receptors identified in binding studies. Furthermore, although AF-DX 116 and dicyclomine were able to differentiate atrial and ileal muscarinic receptors, neither compound was more than 2 fold selective. These data indicate that it is not possible to subclassify ileal and atrial muscarinic receptors using direct ligand binding studies with these antagonists. 4 In circular ileal smooth muscle, apparent heterogeneity of the M2 muscarinic receptor population was observed. Thus AF-DX 116 identified two populations of sites with affinities differing by 30 fold. These two populations of M2 muscarinic receptors may represent the typical M2 muscarinic receptors identified in cardiac tissue and the more recently discovered 'gland type' M2 muscarinic receptors. 5 The circular ileal smooth muscle tissue homogenate was able to decrease dramatically the apparent affinity of adiphenine. This activity, which appeared to result from a phenylmethylsulphonylfluoride (PMSF) sensitive protease effect, should be considered when conducting studies using this tissue preparation and compounds of similar structure to adiphenine.

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“…The protocol was adapted from that of Birdsall et al (14). Retinas from embryonic and hatched chickens were dissected and homogenized in 50 mM sodium phosphate (pH 7.4), containing the protease inhibitors phenylmethylsulfonyl fluoride (0.5 mM), phenanthroline (0.5 mM), EGTA (1.0 mM), benzamidine (1.0 mM), pepstatin A (0.1 A&M), and soybean trypsin inhibitor (10 ,ug/ml), and placed on ice for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

Large¹,

Rauh²,

Mello³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Muscarinic acetylcholine receptors from the avian central nervous system were examined for developmental changes that correlated with the differentiation of cholinergic synapses. In contrast to previous studies that showed a single molecular weight form of muscarinic receptors in the mature central nervous system, the current study of receptors from embryonic and newly hatched chick retina showed the presence of two electrophoretic forms having apparent molecular weights of 86,200 ± 400 and 72,200 ± 300. Two receptor forms also were observed in embryonic cerebrum, optic tectum, and cerebellum. Each form was present, although decreased in molecular weight by 6000, after treatment with deglycosylating enzymes, consistent with molecular differences occurring in the protein portions, rather than the carbohydrate portions, of the molecules. The relative proportions of the high and low molecular weight receptors in retina showed a striking inversion during development. Before synaptogenesis, receptors were mainly of Mr 86,000, whereas after synaptogenesis, receptors were mainly of Mr 72,000. Development of a predominantly low molecular weight receptor population also occurred in aggregate, but not monolayer, cell culture, suggesting a possible role for cell-cell interactions in triggering the change. Pulse-chase labeling of receptors on cultured cells indicated that both forms were present on the cell surface; the labeled Mr 86,000 population had a half-life of 5 hr, whereas the labeled Mr 72,000 population had a half-life of 19 hr. The change in size of muscarinic receptors during development may reflect the action of regulatory mechanisms critical to the proper assembly and function of synapses in the central nervous system. An important goal in neurobiology is to characterize the molecular events associated with synaptic development and plasticity. Considerable progress towards this goal has come from studies of nicotinic acetylcholine receptors during formation of the neuromuscular junction. Soon (11), whereas in chicken heart, newly synthesized receptors only gradually become coupled to inhibition of adenylate cyclase (12). Recent studies of the developing avian retina have shown that the muscarinic receptor system, although functional well before synapses appear, continues to undergo biochemical differentiation during the period when synapses form. Agonist binding affinity increases and the isoelectric point of the receptor increases from 4.25 to 4.50, consistent with the possibility that molecular alteration may be involved in maturation of the system (13).Results presented here extend these studies of muscarinic receptors in the developing central nervous system. They show that muscarinic receptors have two molecular forms, Mr 86,000 and Mr 72,000, and that the predominant form changes with development. The possibilities that the high molecular weight form represents a proreceptor or, alternatively, that the two receptors are products of different genes or differentially processed RNA are discussed....

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A Study of the Muscarinic Receptor by Gel Electrophoresis

Cited by 112 publications

References 11 publications

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Hydrodynamic properties of muscarinic acetylcholine receptors solubilized from rat forebrain

Direct binding studies on ileal and cardiac muscarinic receptors

Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system: switch in predominant form during differentiation of synapses.

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