Radioactive Choline Uptake in the Isolated Rat Phrenic Nerve‐Hemidiaphragm Preparation. A Biochemical and Autoradiographic Study

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Abstract
When hemidiaphragms are stimulated via the phrenic nerve in the presence of 10 .mu.M radioactive choline (Ch), the rate of radioactive Ch uptake in the endpiate-rich area (EPA) is greater than that in the endplate-poor muscle (M). Ch uptake in the EPA is temperature-dependent, with a Q10 of 2.9 and an activation energy of 19.5 kcal/mol. It is inhibited in a Na+-depleted medium, in the absence of Ca2+, and by 10-20 .mu.M hemicholinium-3 (HC-3) and it is not inhibited by .alpha.-bungarotoxin even when the muscle is completely paralyzed. In the absence of stimulation the rate of uptake in the EPA is slightly, but not significantly, greater than in M. Using autoradiography, an enhanced amount of isotope is found in the nerve terminals and their immediate vicinities compared with the muscle fibers, in both stimulated and unstimulated hemidiaphragms. There is no enhanced uptake of isotope into the nerve terminals in stimulated tissues in the presence of 26 .mu.M HC-3. The uptake of isotope into the muscle is not altered by any of these treatments. There is a positive correlation between the initial rate of radioactive Ch uptake in the EPA and the amount of isotope in the nerve terminals (the mean corrected grain density above the nerve terminals). Without correcting for the large amount of diffusion that occurs, the ratio of the grain density above the synapses to that above the muscle fibers is 1.66 in tissue stimulated at 1 Hz, 1.04 in stimulated tissues in the presence of 26 .mu.M HC-3, and 1.31 in unstimulated tissues. The increase in the rate of radioactive Ch uptake in the EPA of diaphragms caused by stimulation is probably due to an enhanced uptake of isotope into the nerve terminals and not into the muscle fibers. This rate is directly proportional to the stimulation frequency (up to 2 Hz), and the slope of the line is equal to 0.84 pmol radioactive Ch/impulse/whole diaphragm.