We have investigated in detail the role of intra-organelle Ca
2+ content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca
2+ 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 Ca
2+ responses because of the release of Ca
2+ from both ER and acidic stores. Ca
2+ responses to menadione were accompanied by elevation of Ca
2+ in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca
2+ stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca
2+ at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca
2+ with 10 μ
m RU360, then caspase-9 activation did not occur irrespective of the content of other Ca
2+ stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca
2+ with pericam. We conclude that elevated Ca
2+ in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.Apoptosis, a mechanism of programmed cell death, usually occurs through intrinsic or extrinsic apoptotic pathways. The caspases involved in apoptosis can be split into two groups, the initiator caspases such as caspase-9 and effector caspases such as caspase-3. Effector caspases are activated by initiator caspases and mediate many of the morphological cellular changes associated with apoptosis (
1).Calcium is an important signaling ion involved in the regulation of many physiological as well as pathological cellular responses (
2). In the pancreas, we have shown that Ca
2+ signals elicit enzyme secretion (
3), apoptosis (
4–
6), and pathological intracellular activation of digestive enzymes (
7). As such, there must be mechanisms in place by which the cell can differentiate between apoptotic and non-apoptotic Ca
2+ signals.The spatiotemporal pattern of calcium signaling is crucial for the specificity of cellular responses. For example, repetitive cytosolic calcium spikes confined to the apical region of the pancreatic acinar cell are elicited by physiological stimulation with acetylcholine (ACh) or cholecystokinin (CCK) and result in physiological secretion of zymogen granules (
8,
9). However, a sustained global increase in free cytosolic Ca
2+ induced by supramaximal stimulation with CCK, which resembles prolonged hyperstimulation of pancreatic acinar cells in the pathophysiology of acute pancreatitis, can lead to premature activation of digestive enzymes and vacuole formation within the cell (
10–
12). Alternatively, global repetitive calcium spikes induced in the pancreatic acinar cell in response to oxidant stress can lead to induction of the mitochondrial permeability transition pore (mPTP)
4 and apoptosis (
4,
5,
13).To understand the role of calcium in apoptosis, several investigators have examined the influence of intracellular stores on the molding of calcium signals that lead to cell death (
14–
16). It has been well established in a range of cell types that the endoplasmic reticulum (ER) is the major intracellular calcium store required for induction of apoptosis. Pinton
et al. (
17) have shown that decreasing ER Ca
2+ concentration with tBuBHQ increased HeLa cell survival in response to oxidant stress induced by ceramide. Scorrano and Korsmeyer (
18) also observed that double knock-out Bax and Bak (pro-apoptotic proteins) mouse fibroblasts displayed a reduced resting concentration of ER Ca
2+ compared with wild type and were resistant to induction of apoptosis by various stimulants, including ceramide. These important studies strongly suggest that the concentration of Ca
2+ in the ER is a critical determinant of cellular susceptibility to apoptotic stimuli in the cell types studied.A key event in early apoptosis is permeabilization of the mitochondrial membrane. The mPTP is a pore whose molecular composition is still debated (
19). Activation of an open pore state can result in swelling of the mitochondrial matrix and release of the apoptogenic proteins from the intermembrane space (
20).One important activator of the mPTP is Ca
2+ (
20–
22), a function which implicates Ca
2+ in the initiation of apoptosis (
23,
24). Once Ca
2+ is released from the ER into the cytoplasm, mitochondria take up part of the released Ca
2+ to prevent propagation of large calcium waves (
25–
27). This influx is followed by calcium efflux from the mitochondria back into the cytosol (
28,
29). An increase in mitochondrial Ca
2+ concentration in response to physiological stimuli induces increased activity of the mitochondrial respiratory chain and the synthesis of ATP to meet with increasing energy demands on the cell. When mitochondria are exposed to a pathological overload of calcium, opening of the mPTP is triggered, leading to mitochondrial dysfunction and eventually cell death. The mechanism through which calcium can trigger mPTP opening is still unclear and may involve cyclophilin D (
30) and voltage-dependent anion channel (
31). The mitochondria are endowed with selective and efficient calcium uptake (a calcium-selective uniporter) and release mechanisms (Ca
2+/Na exchanger, Ca
2+/H
+ exchanger, and mPTP) (
16,
29,
32,
33).Oxidant stress is a well known inducer of apoptosis in several cell types (
34) and is thought to play an important role in the pathogenesis of acute pancreatitis (
35). We have used the quinone compound menadione to induce oxidative stress in the pancreatic acinar cell. Menadione is metabolized by flavoprotein reductase to semiquinone and then is oxidized back to quinone, resulting in generation of superoxide anion radicals, hydrogen peroxide, and other reactive oxygen species (ROS) (
36).
In vivo, menadione causes depolarization and swelling of the mitochondria (
37). In pancreatic acinar cells, treatment with menadione not only produces an increase in ROS, but has also been found to evoke cytosolic Ca
2+ responses, mPTP opening, activation of caspases and apoptotic cell death (
4,
5). When cells were pretreated with the calcium chelator BAPTA-AM, menadione was unable to induce apoptosis, indicating that oxidant stress-induced apoptosis in the pancreatic acinar cell is highly calcium-dependent. Here we show that in pancreatic acinar cells, oxidative stress-induced apoptosis is strongly dependent on the Ca
2+ concentration within mitochondria at the time of ROS production.
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