Calcium Elevation in Mitochondria Is the Main Ca2+ Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

摘要

We have investigated in detail the role of intra-organelle Ca²⁺ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca²⁺ load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca²⁺ responses because of the release of Ca²⁺ from both ER and acidic stores. Ca²⁺ responses to menadione were accompanied by elevation of Ca²⁺ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca²⁺ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca²⁺ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca²⁺ with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca²⁺ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca²⁺ with pericam. We conclude that elevated Ca²⁺ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.