Cholinergic properties of pinched-off synaptic nerve endings from the central nervous system of the cockroachPeriplaneta americana

Abstract

The preparation and cholinergic properties of a subcellular fraction, enriched in pinched-off nerve-endings (synaptosomes) from the central nervous system of the cockroach Periplaneta americana, are described. The endings retained their cytoplasmic components, as shown by the presence of marker enzymes and by ultrastructural examination. A carrier-mediated, high-affinity uptake of [3H] choline into the synaptosomes was demonstrated, and this uptake was saturable, depended on a Na+-gradient, and was inhibited by hemicholinium-3. It had an apparent Km value of 0.6 (±0.1) μM, and a Vmax of 20.5 (±2.5) pmol min−1 per mg of protein. The high-affinity [3H]choline uptake was associated with the synthesis of [3H]phosphocholine and [3H]O-acetylcholine. The rate of [3H]choline uptake in synaptosomes was increased by DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane] at 100 nM concentration, and this increase was inhibited by tetrodotoxin, while neostigmine appeared to be a potent inhibitor (I50 = 10 pM) of the DDT-activated uptake of [3H]choline. The site of action of the insecticides was specifically on the pre-synaptic nerve terminals because the synaptosomes preparation did not retain the post-synaptic membrane of the original nerve-endings. Cockroach synaptosomes provided a useful in-vitro preparation for studying the effects of insecticides on the pre-synaptic nerve endings.