Báo cáo khoa học: Ca2+ rise within a narrow window of concentration prevents functional injury of mitochondria exposed to hypoxia ⁄reoxygenation by increasing antioxidative defence

Giới thiệu đề tài

Nghiên cứu này khám phá vai trò của nồng độ Ca²⁺ ngoại bào ở mức micromolar thấp trong việc bảo vệ chức năng ty thể gan khỏi tổn thương do thiếu oxy/tái oxy hóa (hypoxia/reoxygenation). Kết quả cho thấy một ngưỡng nồng độ Ca²⁺ hẹp, khoảng 2 µM, có khả năng bảo vệ ty thể hiệu quả bằng cách tăng cường hệ thống phòng thủ chống oxy hóa.

Ngành nghiên cứu

• Hóa sinh
• Sinh lý học

Tóm tắt nội dung

Nghiên cứu đã điều tra ảnh hưởng của các nồng độ Ca²⁺ ngoại bào khác nhau (từ 0.2 đến 4.4 µM) lên ty thể gan khi tiếp xúc với điều kiện thiếu oxy/tái oxy hóa. Phát hiện chính cho thấy nồng độ Ca²⁺ khoảng 2 µM mang lại sự bảo vệ gần như hoàn toàn cho chức năng ty thể, được đo lường bằng tỷ lệ kiểm soát hô hấp (RCR) và tốc độ hô hấp trạng thái 3 (state 3 respiration). Ngược lại, nồng độ Ca²⁺ thấp hơn hoặc cao hơn ngưỡng này đều dẫn đến suy giảm chức năng ty thể. Cụ thể, nồng độ Ca²⁺ cao hơn 2 µM gây ra sự thấm màng ty thể, trong khi nồng độ thấp hơn có thể được bảo vệ bởi chất ức chế nitric oxide synthase. Quan trọng hơn, nghiên cứu chỉ ra rằng cơ chế bảo vệ này liên quan đến việc tăng cường hoạt động của các enzyme chống oxy hóa như superoxide dismutase (Mn-SOD) và glutathione peroxidase (GPx) ở nồng độ Ca²⁺ tối ưu. Sự gia tăng hoạt động của Mn-SOD được quan sát rõ rệt dưới điều kiện thiếu oxy/tái oxy hóa kết hợp với 2 µM Ca²⁺, cho thấy sự phối hợp giữa Ca²⁺ và stress oxy hóa trong việc điều hòa enzyme này. Nghiên cứu kết luận rằng Ca²⁺ đóng vai trò quan trọng trong việc điều chỉnh sự cân bằng giữa quá trình oxy hóa và chống oxy hóa trong ty thể dưới điều kiện thiếu oxy/tái oxy hóa, với một cửa sổ nồng độ hẹp mang lại hiệu quả bảo vệ tối ưu.

Mục lục chi tiết

• Ca²⁺ rise within a narrow window of concentration prevents functional injury of mitochondria exposed to hypoxia/reoxygenation by increasing antioxidative defence
• Keywords
• Correspondence
• Abbreviations
• Injury of liver by ischaemia crucially involves mitochondrial damage. The role of Ca²⁺ in mitochondrial damage is still unclear.
• We investigated the effect of low micromolar Ca²⁺ concentrations on respiration, membrane permeability, and antioxidative defence in liver mitochondria exposed to hypoxia/reoxygenation.
• Hypoxia/reoxygenation caused decrease in state 3 respiration and in the respiratory control ratio.
• Liver mitochondria were almost completely protected at about 2 µm Ca²⁺.
• Below and above 2 µm Ca²⁺, mitochondrial function was deteriorated, as indicated by the decrease in respiratory control ratio.
• Above 2 µm Ca²⁺, the mitochondrial membrane was permeabilized, as demonstrated by the sensitivity of state 3 respiration to NADH.
• Below 2 µm Ca²⁺, the nitric oxide synthase inhibitor nitro-L-arginine methylester had a protective effect.
• The activities of the manganese superoxide dismutase and glutathione peroxidase after hypoxia showed maximal values at about 2 µm Ca²⁺.
• We conclude that Ca²⁺ exerts a protective effect on mitochondria within a narrow concentration window, by increasing the antioxidative defence.
• It has been shown in animal models that transient ischaemia in liver results in mitochondrial damage.
• The involvement of oxidative stress in the impairment of the organelle was demonstrated by the finding that glutathione (GSH) exerts a protective role.
• A hallmark of ischaemia/reperfusion in liver is a significant increase in cytosolic and mitochondrial Ca²⁺ concentration.
• Oxidative stress and increase in the cytosolic Ca²⁺ concentration favour opening of the mitochondrial permeability transition pore (MPTP) mediating mitochondrial damage.
• In fact, cyclosporin A (CSA), a specific inhibitor of MPTP, has been demonstrated to prevent mitochondrial and liver dysfunction in the reperfusion phase.
• Long-lasting ischaemia in liver was shown to induce cytochrome c release and necrosis, whereas short ischaemia with reperfusion results in the release of cytochrome c and apoptosis.
• A further factor determining the outcome after liver ischaemia/reperfusion is nitric oxide (NO).
• However, reports about the effect of NO on mitochondrial and tissue damage are still controversial.
• Using either exogenous NO donors, or endogenous NO precursors or inhibitors of NO synthesis, protective as well as harmful effects have been found with in vivo models of liver ischaemia.
• Investigations on isolated liver mitochondria have clearly shown that extramitochondrial Ca²⁺, reactive oxygen species (ROS), and NO, which are known to change in concentration during ischaemia/reperfusion, affect mitochondria.
• Elevation of Ca²⁺ concentrations
• Ca²⁺ protects mitochondria during hypoxia
• Results
• Ca²⁺ affects respiration and membrane permeability upon hypoxia/reoxygenation
• In liver ischaemia/reperfusion the cytosolic Ca²⁺ concentration in hepatic cells is elevated into the low micromolar range.
• In order to investigate the influence of extramitochondrial Ca²⁺ on the impairment of mitochondria by ischaemia/reperfusion, isolated rat liver mitochondria were subjected to 5 min hypoxia followed by 10 min reoxygenation in the continuous presence of Ca²⁺ at concentrations varying from 0.2 up to 4.4 µm.
• Rates of respiration were determined after hypoxia/reoxygenation with 5 mm glutamate and 5 mm malate as substrates.
• Transient hypoxia in the presence of 0.2 µm Ca²⁺ caused decrease in state 3 respiration to 45% of the normoxic control value (incubation in air saturated medium).
• The influence of Ca²⁺ on state 3 respiration obtained with hypoxia/reoxygenation was characterized by a bell-shaped concentration dependence.
• Increasing the Ca²⁺ concentration improved state 3 respiration.
• Almost complete protection was seen at 2 µm extramitochondrial Ca²⁺ (91% of normoxic mitochondria).
• This was not observed when Ca²⁺ uptake was inhibited by 10 µm ruthenium red.
• Further increase in extramitochondrial Ca²⁺ concentration resulted in decreased rates of state 3 respiration measured after hypoxia/reoxygenation.
• At the maximally used concentration of 4.4 µm Ca²⁺, no stimulation of oxygen consumption by ADP could be reached.
• The respiration determined in the absence of ADP (state 4) had no clear Ca²⁺ dependence.
• In order to test whether the effect of hypoxia/reoxygenation and elevated Ca²⁺ on state 3 respiration was due to opening of the MPTP, isolated rat liver mitochondria were exposed to hypoxia/reoxygenation and Ca²⁺ in the additional presence of 2 µm of the MPTP inhibitor CSA.
• At this concentration, CSA completely prevented Ca²⁺-induced swelling of liver mitochondria.
• CSA partially protected liver mitochondria against decrease in state 3 respiration at Ca²⁺ concentrations below and above 2 µm.
• Within the narrow concentration range at around 2 µm extramitochondrial Ca²⁺, no effect of CSA was observed.
• The rates of state 4 respiration were slightly higher in CSA-containing incubations in comparison to CSA-free incubations.
• At 2 µm extramitochondrial Ca²⁺ both values were equal.
• In order to evaluate precisely the functional injury of mitochondria, the coupling of oxidative phosphorylation was quantified by calculating respiratory control ratios (RCR), which are given by the ratios of state 3 and state 4 respiration.
• Highest RCR values were found between 1 and 2 µm extramitochondrial Ca²⁺ indicating a protective Ca²⁺ concentration range.
• Below and above this concentration range, loss of mitochondrial coupling was observed.
• Inhibition of pore opening by CSA had no significant effect on RCR over the whole Ca²⁺ concentration range investigated.
• The RCR of freshly isolated mitochondria was 6.4 ± 0.7 (n = 12).
• To investigate the possibility of a CSA-insensitive permeabilization of the mitochondrial membrane upon hypoxia/reoxygenation and Ca²⁺, we used a different approach.
• The membrane impermeable pyridine nucleotide NADH (5 mm) and cytochrome c (10 µm) were added to mitochondria respiring under state 3 conditions.
• Both compounds have no effect on state 3 respiration in intact mitochondria.
• However, permeabilization of the membrane allows access of NADH and cytochrome c to the respiratory chain resulting in stimulation of state 3 respiration.
• The relative changes of state 3 respiration without and with NADH addition plus cytochrome c, measured after exposure of mitochondria to hypoxia/reoxygenation and various Ca²⁺ concentrations, is depicted in Fig. 1C.
• Up to 2 µm extramitochondrial Ca²⁺, no stimulation of state 3 respiration by NADH plus cytochrome c was observed, documenting the tightness of the mitochondrial membrane.
• Even in the presence of 2 µm CSA, elevation of the Ca²⁺ concentration from 2 µm to 4.4 µm was paralleled by an increase in the ratio of state 3 respiration without and with NADH addition plus cytochrome c clearly.
• This indicates permeabilization of the mitochondrial membrane.
• The permeabilization (Fig. 1C) was associated with loss of mitochondrial function (Fig. 1B).
• Fig. 1. Influence of cyclosporin A on the Ca²⁺ sensitivity of respiration upon hypoxia/reoxygenation.
• Rat liver mitochondria (1 mg·mL-1) were subjected to 5 min hypoxia and 10 min reoxygenation with and without 2 µm CSA in the presence of various Ca²⁺ concentrations at 30 °C.
• Afterwards, respiration was determined in the presence of 5 mm glutamate, 5 mm malate either without (state 4) or with 200 μμ ADP (state 3).
• Subsequently 5 mm NADH and 10 µm cytochrome c were added to demonstrate permeabilization of the mitochondrial membrane.
• The rates of respiration (A), RCR (B) and the ratio of the rates of state 3 respiration before and after the addition of NADH and cytochrome c (C) are presented.
• The rate of state 3 respiration of freshly isolated mitochondria was 82.3 ± 6.8 nmol O2 mg 1 min 1.
• Data are presented as mean ± SEM of five mitochondrial preparations.
• Ca²⁺ and hypoxia/reoxygenation regulate antioxidative activity in liver mitochondria
• In our previous work we have shown that hypoxia/reoxygenation induces oxidative stress indicated by the formation of protein carbonyls (marker of oxidative protein modification) which depends on the Ca²⁺ concentration.
• To investigate how Ca²⁺ modulates oxidative stress during hypoxia/reoxygenation, we measured the activity of the Mn-SOD in normoxic incubation and after hypoxia/reoxygenation.
• The enzyme activity in the normoxic incubation was maximal in the presence of 2 µm extramitochondrial Ca²⁺.
• At 0.2 µm Ca²⁺, lower activity of Mn-SOD was found (73 at 0.2 µm Ca²⁺ vs. 118 units·mg⁻¹ at 2 µm Ca²⁺).
• Similar results were obtained with Mn-SOD from bovine erythrocytes.
• In the presence of 2 µm Ca²⁺, the enzyme activity was increased from 0.134 ± 0.021 units·mg⁻¹ (at 0.2 µm Ca²⁺) to 1.851 ± 0.056 units·mg⁻¹.
• This stimulation could be reversed by the addition of 2 mm EGTA.
• The activity of the enzyme determined after hypoxia/reoxygenation also reached a maximum value in the presence of 2 µm Ca²⁺, but was significantly higher than in a normoxic incubation (157 units·mg⁻¹ after hypoxia/reoxygenation vs. 118 units·mg⁻¹ without hypoxia/reoxygenation).
• Increase in the extramitochondrial Ca²⁺ concentration from 0.2 to 2 µm resulted in a 2.6-fold increase in the activity of Mn-SOD, whereas in normoxic incubations the elevation was only 1.6-fold.
• Thus, the combination of hypoxia/reoxygenation and 2 µm extramitochondrial Ca²⁺ caused a considerable increase in the activity of this antioxidative defence enzyme.
• In a further series of experiments we investigated whether the activity of a second antioxidative enzyme, that is GPx, is sensitive to hypoxia/reoxygenation and extramitochondrial Ca²⁺.
• The activity of this enzyme did not depend on the extramitochondrial Ca²⁺ concentration in the low micromolar range under normoxic conditions.
• In Ca²⁺-free incubations, the activity of GPx was double after 5 min hypoxia followed by 10 min reoxygenation (819 vs. 405 units·mg⁻¹ at 0.2 µm extramitochondrial Ca²⁺).
• The activity determined after hypoxia/reoxygenation was slightly
• Fig. 2. Change of Ca²⁺-sensitivity of Mn-SOD activity by hypoxia/reoxygenation.
• Rat liver mitochondria (1 mg·mL⁻¹) were either incubated at various Ca²⁺ concentrations and 5 mm glutamate plus 5 mm malate in the incubation medium or were subjected to 5 min hypoxia and 10 min reoxygenation in the presence of various Ca²⁺ concentrations at 30 °C.
• After the reoxygenation period 5 mm glutamate and 5 mm malate were added.
• For the determination of Mn-SOD activity, 500 µL samples were withdrawn from the incubations.
• The data are presented as mean ± SEM from five preparations of mitochondria.
• Additional student's t-test analysis gave a significant difference in Mn-SOD activities between the values at 0.1 and 2.0 µм Ca²⁺ (P < 0.01), both without and with hypoxia/reoxygenation.
• *Differences in Mn-SOD activities of incubations with and without hypoxia/reoxygenation were significant with P < 0.05.
• Fig. 3. Influence of hypoxia/reoxygenation and Ca²+ on the activity of glutathione peroxidase (GPx).
• Rat liver mitochondria (1 mg·mL⁻¹) were either incubated at various Ca²⁺ concentrations and 5 mm glutamate plus 5 mm malate in the incubation medium or were subjected to 5 min hypoxia and 10 min reoxygenation in the presence of various Ca²⁺ concentrations at 30 °C.
• After the reoxygenation period 5 mm glutamate and 5 mm malate were added.
• For the determination of GPx activity, 500 µL samples were withdrawn from the incubations.
• The data are presented as mean ± SEM from five preparations of mitochondria.
• The differences between GPx activities of incubations with and without hypoxia/reoxygenation in the presence of similar Ca²⁺ concentrations were significant with P < 0.01.
• Fig. 4. Modulation of the effect of NO on state 3 respiration after hypoxia/reoxygenation by extramitochondrial Ca²⁺.
• Rat liver mitochondria (1 mg·mL⁻¹) were subjected to 5 min hypoxia and 10 min reoxygenation with and without 10 mM L-NAME in the continuous presence of either 0.2 μμ, 2 μμ or 4.4 µm extramitochondrial Ca²⁺ at 30 °C.
• Afterwards, 5 mm glutamate, 5 mm malate and 200 μμ ADP were added to stimulate state 3 respiration.
• Data are presented as mean ± SEM of five preparations of mitochondria.
• *State 3 respiration with and without L-NAME is different with P < 0.05 according to Student's t-test.
• Discussion
• Extramitochondrial Ca²⁺ can amplify or attenuate the impairment of liver mitochondria by hypoxia/reoxygenation
• Isolated mitochondria have been successfully used to study the effect of distinct factors impairing mitochondria which are relevant in pathophysiological situations such as ischaemia/reperfusion.
• Both the in vivo studies of ischaemia and the cell culture investigation on hypoxia/reoxygenation require a relatively long period of hypoxia to achieve significant injury.
• However, in isolated mitochondria a few minutes of hypoxia are sufficient to cause dramatic damage.
• Differences in local oxygen concentration may be the reason for this different time required to reach injury either in vivo or in isolated mitochondria.
• We have found that at elevated extramitochondrial Ca²⁺ concentrations, ADP at physiological concentration protects mitochondria from hypoxia/reoxygenation-induced damage.
• Only when all the ADP is converted into AMP, mitochondrial damage occurs.
• This finding may contribute to the fact that longer periods of ischaemia are required to achieve damage in tissue, in comparison with results obtained with isolated mitochondria, which have to be exposed only for a short period of time to hypoxia in order to induce damage.
• In previous papers we reported that hypoxia/reoxygenation reduces state 3 respiration in isolated rat liver mitochondria and that extramitochondrial Ca²⁺ in the low micromolar range modulates mitochondrial damage and oxidative stress.
• Now, by testing the action of CSA and measuring RCR we show that opening of the MPTP is not significantly involved in functional impairment of liver mitochondria exposed to hypoxia/reoxygenation and Ca²⁺.
• This is surprising as increases in extramitochondrial Ca²⁺ concentration and oxidative stress are known to be major factors for increasing the probability for pore opening.
• Reasons for the CSA-independent injury of mitochondrial function might be the increase in oxidative stress below and above 2 µm Ca²⁺ as demonstrated earlier, possibly causing damage to respiratory chain complexes, and/or CSA-insensitive permeabilization of the mitochondrial membrane.
• In fact, CSA-insensitive permeabilization of the mitochondrial membrane was found after hypoxia/reoxygenation in the presence of Ca²⁺ concentrations higher than 2 μμ (Fig. 1C).
• Ca²⁺ affects the balance between oxidative and antioxidative processes during hypoxia/ reoxygenation
• As we have demonstrated earlier, hydrogen peroxide which is formed from superoxide anion radicals accumulates during reoxygenation in the presence of high Ca²⁺ concentration.
• However, the protection of mitochondria from hypoxia/reoxygenation-induced damage and the low amount of protein carbonyls at 2 µm Ca²⁺ suggest relatively low superoxide radical concentration.
• This is consistent with our finding of considerably increased activity of the Mn-SOD at this Ca²⁺ concentration.
• At 2 µm Ca²⁺, no protection of state 3 respiration was seen during hypoxia/reoxygenation in the presence of ruthenium red (data not shown).
• Therefore, it can be concluded that Ca²⁺ has to enter the mitochondrial matrix in order to cause increase in Mn-SOD activity.
• Both Ca²⁺ and hypoxia/reoxygenation synergistically contribute to this effect.
• Under these conditions, protein levels of the Mn-SOD remained unchanged as determined by western blot analysis (data not shown).
• This is not surprising, as protein synthesis of this enzyme takes place within the cytosolic compartment.
• Thus chemical modification is responsible for the change in the activity of the enzyme.
• It has been shown that inactivation of the enzyme may result from tyrosine nitration by peroxynitrite.
• In the in vitro model of ischaemia/reperfusion applied here