Calcium mediated proteolysis enhances calcium release in skinned L6 myotubes.
The mechanism for calcium (Ca2+) release in heart and skeletal muscle during excitation-contraction coupling is currently unknown. A widely held hypothesis is that a small amount of Ca2+ enters the cell and elicits a larger intracellular release of Ca2+ from the sarcoplasmic reticulum (SR), termed "Ca2+-induced Ca2+-release" (CICR). In addition to its role in excitation-contraction coupling, Ca2+ is also known to activate the cysteine protease calpain, which has been recently found to specifically cleave the ryanodine receptor in vitro. The authors investigated the question of whether Ca2+ sensitive protease activation could account for an apparent CICR. The authors first reproduced the phenomenon of CICR using detergent treated L6 myotubes ("skinned cells"). Leupeptin, a cysteine protease inhibitor, reduced the initial velocity and extent of Ca2+ release from the SR; a similar result was obtained when skinned cells were treated with iodoacetate, a sulfhydryl alkylating agent. Dithiothreitol enhanced both the rate and extent of Ca2+ release. Caffeine-induced Ca2+-release was unaffected by the thiol protease inhibitors or activators. This suggests that a cysteine protease may be responsible, in part, for CICR in vitro. The authors also found that vesicles exposed to Ca2+ to induce CICR were unable to fully reaccumulate Ca2+ a second time. Yet, when caffeine released comparable amounts of Ca2+, the initial Ca2+ level was fully restored. Similarly, leupeptin protected the vesicles from the reaccumulation deficit induced by Ca2+. The authors' findings suggest that proteolysis activated by a Ca2+-sensitive protease may account for the direct in vitro demonstration of CICR; such an effect may more likely reflect a role in apoptosis than excitation-contraction coupling.
Wingertzahn
M A
MA
Department of Pharmacy and Allied Health, Saint Johns University Jamaica, NY, USA.
Ochs
R S
RS
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D002460:Cell Line; D002999:Clone Cells; D004229:Dithiothreitol / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D007976:Leupeptins / Q000187:drug effects; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction
D007976:Leupeptins; D011480:Protease Inhibitors; D002110:Caffeine; D010450:Endopeptidases; C032854:leupeptin; D002118:Calcium; D004229:Dithiothreitol