The activity of the muscarinic cholinergic system (acetylcholine, ACh; acetylcholinesterase, AChE; choline acetyltransferase, ChAT; muscarinic acetylcholine receptors) was studied in the carp brain. The ACh content (13.9 ± 1.1 nmol/g wet tissue) was estimated by gas chromatography after microwave irradiation focused to the head. The AChE and ChAT activities were 153 ± 13 nmol/min/mg protein and 817 ± 50 pmol/min/mg protein, respectively. The characteristics of [3H](-)quinuclidinyl benzilate ([3H](-)QNB) and [3H]pirenzepine ([3H]PZ) binding were also studied in brain membranes. Their specific binding was linearly dependent on the protein content and they appeared to bind with high affinity to a single, saturable binding site. A dissociation constant (Kd) of 47 ± 6.3 pM and a maximum number of binding sites (Bmax) of 627 ± 65 fmol/mg protein were obtained for [3H](-)QNB, with a Kd value of 3.85 ± 0.67 nM and a Bmax value of 95.3 ± 6.25 fmol/mg protein for [3H]PZ binding. The [3H]PZ binding amounted to only 15% of the [3H](-)QNB-labeled sites, as estimated from the ratio of the Bmax values of [3H](-)QNB and [3H]PZ, suggesting a low density of M1 subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (nH) close to unity. The nH value of AF-DX 116 was close to 1 against [3H](-)QNB binding, while it was 0.75 against [3H]PZ binding. The displacement curves of oxotremorine and carbachol were shallow for the binding of both radioligands. The rank order of potency of muscarinic ligands against [3H](-)QNB binding (Ki nM) was atropine sulfate (0.55) > atropine methylnitrate (1.61) > PZ (61.19) > oxotremorine (156.3) > AF-DX 116 (307) > carbachol (1301), while in the case of [3H]PZ binding it was atropine sulfate (0.24) > atropine methylnitrate (0.34) > PZ (10.38) > AF-DX 116 (55.87) > oxotremorine (62.79) > carbachol (1696). The results indicate the presence of a well-developed muscarinic cholinergic system with predominantly M2 receptors in the carp brain.
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience
- Cell Biology