Elsevier

Neuroscience

Volume 93, Issue 4, August 1999, Pages 1427-1436
Neuroscience

Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-α(181–192) peptide antibodies

https://doi.org/10.1016/S0306-4522(99)00160-8Get rights and content

Abstract

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different α subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the α(181–192) region of α3, α4, α5, and α7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among α3, α4, α5, and α7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the α3, α5, and α7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly α4-positive. In contrast, only about one-third of the intracardiac neurons were α3-positive, about 50% were α4-positive, one-seventh were α5-positive, and one-fifth were α7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell.

It is concluded that α3, α5, and α7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas α4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.

Section snippets

Materials

Peroxidase-conjugated goat anti-mouse and anti-rabbit IgG, peroxidase–antiperoxidase complex, collagenase from Clostridium histolyticum, type IA, Freund's adjuvants and culture media were from Sigma, U.S.A.; AH-Sepharose 4B was from Pharmacia Fine Chemicals, Sweden; fetal calf serum was either from Seromed, Germany, or from Sigma, U.S.A.; fluorescein isothiocyanate (FITC)-labelled goat anti-mouse or goat anti-rabbit IgG were from Coulter Clone, France. BALB/c mice were bred in the Palladin

Antibodies to α(181–192) peptides discriminate among the peptides of different α subunits

The sera of immunized rabbits obtained after two rounds of immunization strongly preferred to bind α(181–192) peptides used for immunization and poorly bound corresponding peptides of other subunits, as studied by the enzyme-linked immunosorbent assay. The potential cross-reactivity was minimized to negligible values following the antibody affinity purification. Monoclonal antibody (MAb) 1D6-3-8 (IgG2a), obtained against α3 peptide, did not bind other peptides (Table 2). MAb 5C7 (IgM) obtained

Characterization of the antibodies used

Affinity purified α(181–192)-specific antibodies discriminated among the peptides of different α-subunits in enzyme-linked immunosorbent assay. Flow cytometry and immunocytochemical experiments performed with PC-12 cells and cultured autonomic ganglia neurons revealed that the antibodies bound native AChRs. Relative numbers of PC-12 cells, SCG or ICG cells stained with α(181–192)-specific antibodies were different for different antibodies (Fig. 2). Therefore, the antibodies discriminated not

Conclusions

(1) Antibodies directed against (181–192) fragments of α3, α4, α5 and α7 subunits of neuronal AChR bind native AChRs and block ACh-induced membrane currents in autonomic ganglia. (2) Single SCG neurons expresses heteromeric AChRs; the neurons differ in relative amounts of AChR subtypes expressed. (3) The neurons of SCG and ICG differ markedly in the α subunit composition of their AChRs.

Acknowledgements

The work was supported with PECO grant ERBCIPDCT 940244 and with INTAS-Ukraine grant 95-0056.

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