Intracellular uptake and degradation of extracellular tracers in mouse skeletal muscle in vitro: The effect of denervation

Abstract

Innervated and chronically denervated mouse skeletal muscles have been incubated under various conditions in a Ringer solution containing one of the three macromolecules: [ 3H]α-neurotoxin, [ 3H]inulin and horseradish peroxidase. Following extensive wash-out for 4 h of the extracellular compartment, the amount of each macromolecule retained intracellularly was obtained. Intracellular uptake of a [ 3H]monoacetylated α-neurotoxin in vitro at 37°C was found to be increased in denervated mouse extensor digitorum longus muscles compared to innervated control muscles. Similarly, the uptake in vitro at 37°C of [ 3H]inulin and horseradish peroxidase was also increased in denervated muscles. At 4°C the uptake of [ 3H]inulin and horseradish peroxidase was markedly reduced. Protamine was found to stimulate the uptake of [ 3H]inulin at 37°C but not at 4°C. Reduction in specific activity by addition of 50-fold excess of unlabelled inulin failed to affect the uptake of [ 3H]inulin suggesting that this uptake process obeyed bulk kinetics. Furthermore, the endocytized [ 3H]inulin was found to be strongly retained in the muscles since prolonged washing or addition of unlabelled inulin to the washing solution did not reduce the uptake. Characterization of [ 3H]inulin taken up by the muscles was performed by gel chromatography on Sephadex G-25. Using a purified [ 3H]inulin solution it was observed that about 45% of the total radioactivity remaining in the muscles was eluted as [ 3H]inulin. Additional radioactivity consisted of lower molecular weight compounds. These degradation products of [ 3H]inulin were only present in the muscle homogenate and were not detected in the incubation solution. The results suggest that intracellular uptake of different macromolecules by endocytosis in skeletal muscles increases following denervation, and that following uptake, degradation of the endocytized material may occur.