Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation.
Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors. When the whole nerve-muscle preparation (110-180 mg) was incubated with [3H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow. Incubation of the end-plate preparation (16-29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [3H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [3H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [3H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [3H]acetylcholine tissue content per pulse. It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [3H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used.(ABSTRACT TRUNCATED AT 250 WORDS)
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002794:Choline / Q000378:metabolism; D003964:Diaphragm / Q000294:innervation / Q000378:metabolism; D006426:Hemicholinium 3 / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D009121:Muscle Denervation; D009469:Neuromuscular Junction / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D010791:Phrenic Nerve / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D018377:Neurotransmitter Agents; D006426:Hemicholinium 3; D002794:Choline; D000109:Acetylcholine