open access

Vol 46, No 1 (2008)
ORIGINAL PAPERS
Published online: 2008-02-26
Submitted: 2011-12-19
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Ultrastructural visualization of the transmembranous and cytomatrix-related part of nicotinic acetylcholine receptor of frog motor endplate by means of an immunochemical avidity of IgG for d-tubocurarine.

Keiji Hirai, Yoshifumi Katayama, Gabriel Peltre, Shigeru Tsuji
DOI: 10.2478/v10042-008-0016-5
·
Folia Histochem Cytobiol 2008;46(1):111-116.

open access

Vol 46, No 1 (2008)
ORIGINAL PAPERS
Published online: 2008-02-26
Submitted: 2011-12-19

Abstract

In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR) was attempted, using d-tubocurarine (d-TC), a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG) for acetylcholine (ACh) and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA), a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC) to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB) precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest) of the postsynaptic membrane (between the junctional folds) which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm), while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex.

Abstract

In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR) was attempted, using d-tubocurarine (d-TC), a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG) for acetylcholine (ACh) and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA), a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC) to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB) precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest) of the postsynaptic membrane (between the junctional folds) which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm), while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex.
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About this article
Title

Ultrastructural visualization of the transmembranous and cytomatrix-related part of nicotinic acetylcholine receptor of frog motor endplate by means of an immunochemical avidity of IgG for d-tubocurarine.

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 46, No 1 (2008)

Pages

111-116

Published online

2008-02-26

DOI

10.2478/v10042-008-0016-5

Bibliographic record

Folia Histochem Cytobiol 2008;46(1):111-116.

Authors

Keiji Hirai
Yoshifumi Katayama
Gabriel Peltre
Shigeru Tsuji

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