Immediate and delayed effects of a brief period of calcium loading, induced by the calcium channel agonist Bayer K 8644, on the ultrastructure and sarcolemmal integrity of murine diaphragm in vitro

Exposure of in vitro mouse diaphragm to the dihydropyridine Ca2+ channel agonist Bayer K 8644 (Bay) and partial depolarisation for 120 min induced severe structural damage within 30-60 min and membrane permeabilisation (60-120 min). Exposure to Bay and depolarisation for 30 min (brief Ca2+ loading) followed by washout for 90 min produced similar effects, even though significant membrane damage does not occur until the second hour of incubation. Development/expression of necrosis during the washout period was not inhibited by manoeuvres designed to combat the effects of any remaining Bay, or by use of a Ca(2+)-free, 1 mM EGTA medium for washout. Exposure to Bay for 15 min followed by washout did not induce damage. Electron microscopic examination revealed that the mitochondria of cells fixed after 30 or 120 min exposure to Bay and depolarisation were in the orthodox (swollen) configuration, but that mitochondria in muscles that had undergone washout with Ca(2+)-free-EGTA saline were electron dense. Further incubation of these muscles in Ca(2+)-containing medium caused readoption of the swollen configuration. We conclude that membrane permeabilisation can occur after removal of the Ca2+ load, and that mitochondrial Ca2+ overload is not necessary for Ca(2+)-induced damage/death to occur.