Muscarinic cholinergic components in the carp brain

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 [(3)H](?)quinuclidinyl benzilate ([(3)H](?)QNB) and [(3)H]pirenzepine ([(3)H]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 (K(d)) of 47 +/- 6.3 pM and a maximum number of binding sites (B(max)) of 627 +/- 65 fmol/mg protein were obtained for [(3)H](?)QNB, with a K(d) value of 3.85 +/- 0.67 nM and a B(max) value of 95.3 +/- 6.25 fmol/mg protein for [(3)H]PZ binding. The [(3)H]PZ binding amounted to only 15% of the [(3)H](?)QNB-labeled sites, as estimated from the ratio of the B(max) values of [(3)H](?)QNB and [(3)H]PZ, suggesting a low density of M(1) subtype. Atropine sulfate, atropine methylnitrate and PZ inhibited the binding of both radioligands with Hill slopes (n(H)) close to unity. The n(H) value of AF-DX 116 was close to 1 against [(3)H](?)QNB binding, while it was 0.75 against [(3)H]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 [(3)H](?)QNB binding (K(i) 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 [(3)H]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 M(2) receptors in the carp brain.