Abstract
Treatment of isolated rat skeletal muscles with the Ca2+ ionophores, A23187 or ionomycin, increased overall protein degradation 45-140%. Removal of extracellular Ca2+ reduced overall proteolysis and most of the stimulation by A23187. Treatment of the muscles with the sulfhydryl inhibitor, mersalyl, completely inactivated the Ca2+-activated protease without altering overall protein breakdown or the stimulation by A23187. This agent did not inhibit the lysosomal protease, cathepsin B, in the muscle; however, leupeptin and Ep-475, which inhibit this enzyme in intact cells, decreased the stimulation of proteolysis by Ca2+. Thus, this effect does not require the Ca2+-activated enzyme, but seems to involve lysosomal proteases. Prostaglandin E2 (PGE2) and its precursor arachidonic acid, were previously shown to stimulate protein degradation in rat muscle through an effect on lysosomal function. We tested whether the enhancement of muscle proteolysis by Ca2+ ionophores may result from increased synthesis of PGE2. A23187 increased release of PGE2 and PGF2 alpha by the muscles 3-4-fold. High extracellular potassium also markedly promotes muscle proteolysis, apparently by increasing intracellular Ca2+, and this treatment also stimulates prostaglandin production. Indomethacin and aspirin, which inhibit the cyclooxygenase, and mepacrine, which inhibits the Ca2+-activated phospholipase A2, markedly reduced the increase in prostaglandin production. These agents also reduced the enhancement of protein degradation by Ca2+ or high K+. Thus, Ca2+ appears to promote protein breakdown by stimulating synthesis of PGE2, which in turn activates the lysosomal apparatus.
Highlights
Lular Ca2+reduced overall proteolysis and most of the stimulation by A23187
We tested whether the enhancemeonft protease (3),was found to reduce the effect of the ionophore muscle proteolysisby CaZ+ ionophores mareysult from on protein degradation (I), b u t this agent inhibits lysoincreased synthesis o f PGE2
Because no significant change in protein synthesiswas observed with A23187 (Table I), in subsequent experiments with this agent, only tyrosine release was used as a measure of protein breakdown
Summary
Effects of Ca'+ Ionophores and High K' on MuscleProtein Turnover-Upon the addition of the Cat' ionophore A23187 to the incubation medium, rates of tyrosine release from the diaphragm, red soleus, and white extensor digitorum longus muscles increased between 63 and 116% (Table I) in accord with earlier reports (1). Theoverall rates of protein degradation increased in all muscle types tested between 42% and 63% ( p < 0.001) (Table I).A similar increase in tyrosine production has been obtained when absolute rates of proteindegradation were measuredin the presence of cycloheximide (data nosthown). Because no significant change in protein synthesiswas observed with A23187 (Table I), in subsequent experiments with this agent, only tyrosine release was used as a measure of protein breakdown. These results with A23187 are supported by findings obtained with another specific Ca2+ionophore, ionomycin. Thesedata were obtained in the presence of cycloheximide;in relatedstudies;' no significant change in protein synthesis was observed in accord with the findings with A23187 (Table I). After 2 h, net protein balance and rates of protein synthesis were measured directly and rates of protein degradation were calculated as described under "Methods." When ionomycin was added to the medium, protein degradation was directly measured in the presence of 0.5 mM cycloheximide
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