Regulation of protein degradation in muscle by calcium. Evidence for enhanced nonlysosomal proteolysis associated with elevated cytosolic calcium.

Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. A23187, KC1, sucrose, and 8-(diethylamino)octyl-3,4, 5-trimethoxybenzoate hydrochloride increase proteolysis while tetracaine, verapamil, and low extracellular calcium caused significant decreases. Additionally, dantrolene decreases proteolysis in the presence of depolarizing levels of potassium, while it has no effect on degradation in normal media. The dose dependence of calcium ionophore A23187 on proteolysis and contracture tension are parallel. Furthermore, excess KC1 and hypertonic solutions increased protein degradation at doses reported to cause tension. Thus, the parallel increase in proteolysis and tension in response to various agents supports the hypothesis that protein degradation in muscle is regulated by calcium. To determine the responsible proteolytic systems involved in calcium-dependent degradation, the effect of different classes of protease inhibitors was tested. Addition of the inhibitors leupeptin and E-64-c blocked the A23187-induced increase in degradation. Since proteases sensitive to these agents are present in both the sarcoplasm and lysosomes, known lysosomotropic agents, methylamine and chloroquine, as well as 3-methyladenine, a specific autophagy inhibitor, were used in combination with A23187. These agents did not inhibit calcium ionophore-induced proteolysis, although these three agents selectively inhibited enhanced degradation seen in the absence of insulin, demonstrating an autophagic/lysosomal pathway in these muscles. Thus, our results suggest that nonlysosomal leupeptin- and E-64-c-sensitive proteases are responsible for calcium-dependent proteolysis in muscle.