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Prevention of Mitochondrial Injury by Manganese Superoxide Dismutase Reveals a Primary Mechanism for Alkaline-induced Cell Death*

Open AccessPublished:April 03, 1998DOI:https://doi.org/10.1074/jbc.273.14.8217
      Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.
      Alkalosis, a process which raises the cellular pH, is associated with various clinical conditions. Alkalosis induced by alterations in the PCO2 is considered respiratory alkalosis since PCO2is regulated by respiration. The general symptoms produced by respiratory alkalosis are increased irritability of the central and peripheral nervous systems which are thought to be related to the ability of alkalosis to impair cerebral function (reviewed in Ref.
      • Rose B.D.
      ). Although it is well known that alkaline conditions are cytotoxic, the mechanisms leading to alkaline-induced cytotoxicity are unknown.
      In most aerobic cells, the respiratory processes which involve oxygen consumption generate reactive oxygen species (ROS)
      The abbreviations used are: ROS, reactive oxygen species; BCECF-AM, 2–7,bis-2-carboxyethyl-5-(and-6)-carboxyfluorescein acetoxymethyl ester; DCF, 2,7-dichlorofluorescein diacetate; dhRho, dihydrorhodamine 123; FSa-II, murine fibrosarcoma cell line; HNE, 4-hydroxy-2-nonenal; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide; SOD, superoxide dismutase; pHe, extracellular pH; pHi, intracellular pH; [Ca2+]i, intracellular Ca2+; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; TEMED,N,N,N′,N′-tetramethylethylenediamine.
      1The abbreviations used are: ROS, reactive oxygen species; BCECF-AM, 2–7,bis-2-carboxyethyl-5-(and-6)-carboxyfluorescein acetoxymethyl ester; DCF, 2,7-dichlorofluorescein diacetate; dhRho, dihydrorhodamine 123; FSa-II, murine fibrosarcoma cell line; HNE, 4-hydroxy-2-nonenal; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolycarbocyanine iodide; SOD, superoxide dismutase; pHe, extracellular pH; pHi, intracellular pH; [Ca2+]i, intracellular Ca2+; PBS, phosphate-buffered saline; HBSS, Hanks' balanced salt solution; TEMED,N,N,N′,N′-tetramethylethylenediamine.
      such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals. These reactive species can peroxidate membrane lipids and attack proteins or DNA (see Ref.
      • Halliwell B.
      • Gutteridge J.M.C.
      for a review). Superoxide radicals can also react with nitric oxide to form peroxynitrite, a potent biological oxidant which has been recently implicated in diverse forms of free radical-induced tissue injury (for a review, see Ref.
      • Beckman J.S.
      • Crow J.P.
      ).
      Among various potential intracellular targets for ROS-mediated tissue injury, mitochondria are proposed to be particularly prone to free radical-induced changes because most oxygen utilization by mammalian cells occurs in that organelle. Some components of the electron transport chain, such as the NADH-coenzyme Q reductase complex and the reduced form of coenzyme Q itself, leak electrons onto oxygen which produce a univalent reduction to generate superoxide radicals (
      • Oberley L.W.
      ,
      • Chance B.
      • Sies H.
      • Boveris A.
      ). Generation of hydroxyl radical and formation of 8-hydroxydeoxyguanosine in mitochondrial DNA secondary to mitochondrial electron transfer have been demonstrated (
      • Giulivi C.
      • Boveris A.
      • Cadenas E.
      ). Mitochondrial DNA is highly susceptible to mutation because mitochondrial DNA is not protected by histones. Mutations in any of the genes coding for cytochrome oxidase, cytochromebc1, NADH dehydrogenase, or ATPase complexes may lead to defective function of these enzymes. It has been shown that ROS generating agents can cause mitochondrial DNA damage (
      • Pettepher C.C.
      • LeDoux S.P.
      • Bohr V.A.
      • Wilson G.L.
      ). Furthermore, 4Fe-4S containing enzymes such as aconitases, are highly sensitive to redox-induced inactivation (
      • Gardner P.R.
      • Nguyen D.D.
      • White C.W.
      ). Thus oxidative stress in the mitochondria can cause mitochondrial defects and increased oxidative stress to the organism.
      Evidence has accumulated which supports a direct role for mitochondria in cell death. Mitochondrial respiration generates a proton gradient resulting from the mitochondrial transmembrane potential which is essential for mitochondrial function. It has been demonstrated that the mitochondrial permeability transition causes uncoupling of the respiratory chain with collapse of the mitochondrial membrane potential, calcium release, hypergeneration of superoxide radicals, and mitochondrial release of apoptotic proteins (
      • Lazebnik Y.A.
      • Kaugmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ,
      • Martin S.J.
      • Green D.R.
      ). Mitochondria release of cytochrome c has been shown to be a key step in staurosporin-induced apoptosis (
      • Yang J.
      • Liu X.
      • Balla K.
      • Kim C.N.
      • Ibrado A.M.
      • Cai J.
      • Peng T-I.
      • Jones D.P.
      • Wang X.
      ). In addition, activation of a member of the interleukin-1β converting enzyme family of cysteine proteases in tumor necrosis factor-induced apoptosis has been shown to be dependent on mitochondrial function (
      • Higuchi M.
      • Aggarwal B.B.
      • Yeh E.T.H.
      ). Furthermore, overexpression of bcl-2 prevented tumor necrosis factor-induced apoptosis and increased mitochondrial membrane potential, suggesting that mitochondria play an important role in apoptosis (
      • Zamzami N.
      • Susin S.A.
      • Marchetti P.
      • Hirsch T.
      • Gomez-Monterrey I.
      • Castedo M.
      • Kroemer G.
      ).
      Aerobic organisms possess antioxidant defense systems that deal with ROS produced as a consequence of aerobic respiration. One important family of enzymes are the superoxide dismutases. These enzymes dismutate superoxide radicals into hydrogen peroxide and molecular oxygen, which can then be acted upon by other antioxidant enzymes such as glutathione peroxidase and catalase. In humans there are three forms of superoxide dismutase: cytosolic Cu-Zn superoxide dismutase (Cu/Zn-SOD), mitochondrial manganese superoxide dismutase (Mn-SOD), and extracellular superoxide dismutase (EC-SOD). Mn-SOD is a nuclear-encoded primary antioxidant enzyme that functions to remove superoxide radicals in mitochondria (
      • Weisiger R.A.
      • Fridovich I.
      ). The biological importance of Mn-SOD is demonstrated by the following: 1) inactivation of Mn-SOD and iron containing SOD genes in Escherichia coli increased mutation frequency when grown under aerobic conditions (
      • Carlioz A.
      • Touati D.
      ,
      • Farr S.B.
      • D'Ari D.
      • Touati D.
      ). 2) Elimination of the Mn-SOD gene in Saccharomyces cerevisiaeincreased its sensitivity to oxygen (
      • van Loon A.P.G.M.
      • Pesold-Hurt B.
      • Schatz G.
      ). 3) Lack of Mn-SOD expression resulted in dilated cardiomyopathy and neonatal lethality in Mn-SOD knock-out mice (
      • Li Y.
      • Huang T.T.
      • Carlson E.J.
      • Melov S.
      • Ursell P.C.
      • Olson J.L.
      • Noble L.J.
      • Yoshimura M.P.
      • Berge C.
      • Chan P.H.
      • Wallace D.C.
      • Epstein C.J.
      ). 4) Tumor necrosis factor selectively induced Mn-SOD, but not Cu/Zn-SOD, catalase, or glutathione peroxidase mRNA in various mouse tissues and cultured cells (
      • Wong G.H.W.
      • Goeddel D.V.
      ). 5) Transfection of Mn-SOD cDNA into cultured cells rendered the cells resistant to paraquat-, tumor necrosis factor-, and adriamycin-induced cytotoxicity, and radiation induced-neoplastic transformation (
      • St. Clair D.K.
      • Oberley T.D.
      • Ho Y-S.
      ,
      • Wong G.H.W.
      • Elwell J.H.
      • Oberley L.W.
      • Goeddel D.V.
      ,
      • Hirose K.
      • Longo D.L.
      • Oppenheim J.J.
      • Matsushima K.
      ,
      • St. Clair D.K.
      • Wan X.S.
      • Oberley T.D.
      • Muse K.E.
      • St. Clair W.H.
      ). 6) Expression of human Mn-SOD genes in transgenic mice protected the mice against oxygen-induced pulmonary injury and adriamycin-induced cardiac toxicity (
      • Wispe J.R.
      • Warner B.B.
      • Clark J.C.
      • Dey C.R.
      • Neuman J.
      • Glasser S.W.
      • Crapo J.D.
      • Chang L-Y.
      • Whitsett J.A.
      ,
      • Yen H-C.
      • Oberley T.D.
      • Vichitbandha S.
      • Ho Y-S.
      • St. Clair D.K.
      ). Thus, the expression of Mn-SOD is essential for the survival of aerobic life and the development of cellular resistance to oxygen radical-mediated toxicity.
      Because alkaline conditions reduce the concentration of protons which could subsequently affect mitochondrial membrane potential, we examined the possible role of mitochondrial antioxidant defenses in alkaline-induced apoptotic cell death. We report here that increased expression of mitochondrial superoxide dismutase protects cells against alkaline-induced mitochondrial damage, suppresses ROS generation and calcium release, as well as prevents nuclear condensation, DNA fragmentation, and cell death. Our results demonstrate that mitochondrial-mediated intracellular ROS generation is an important mechanism by which alkaline conditions cause cellular lethality and suggest that removal of superoxide radicals in the mitochondria is a critical step in the prevention of alkaline-induced apoptosis.

      DISCUSSION

      Alkaline pH causes cell death although the mechanism leading to cell death is not presently understood. The present investigation was conducted to examine the link between alkaline conditions, mitochondrial antioxidant status, and cell death. This study is the first to demonstrate that oxidative stress is an early and critical event leading to cell death, and mitochondria are primary target organelles of injury in alkaline conditions. It has been demonstrated that alteration of mitochondrial function is an early feature of programmed cell death (
      • Zamzami N.
      • Marchetti P.
      • Castedo M.
      • Decaudin D.
      • Macho A.
      • Hirsch T.
      • Susin S.A.
      • Petit P.X.
      • Mignotte B.
      • Kroemer G.
      ). Our results show that under alkaline conditions, the mitochondrial transmembrane potential increased, suggesting an alteration of mitochondrial function as an early event in alkaline-induced cell death. The intracellular pH in the cells overexpressing Mn-SOD was not different from that in the control cells, and there was no significant change in the mitochondrial membrane potential of these cells, suggesting that the increased production of superoxide radicals in the control cells was associated with increased mitochondrial membrane potential.
      It is well established that mitochondrial respiration generates superoxide radicals (
      • Oberley L.W.
      ,
      • Chance B.
      • Sies H.
      • Boveris A.
      ). Under normal physiological conditions, a small percentage of the oxygen consumed for respiration is converted into superoxide radicals (
      • Halliwell B.
      • Gutteridge J.M.C.
      ). It has also been demonstrated that alkaline conditions are associated with an increase in oxygen consumption. Exposure of cultured French bean cells to an elicitor preparation from a fungal pathogen showed a rapid increase in oxygen uptake and a transient alkalinization of the apoplast (
      • Bolwell G.P.
      • Butt V.S.
      • Davies D.R.
      • Zimmerlin A.
      ). The oxygen uptake increased while the hydrogen ion concentration decreased in dogs and humans during hyperventilation (
      • Cain S.M.
      ,
      • Karetzky M.S.
      • Cain S.M.
      ,
      • Khambatta H.J.
      • Sullivan S.F.
      ). In anesthetized paralyzed patients, there was an increase in oxygen consumption with alkalosis (
      • Slater R.M.
      • Symreng T.
      • Sum Ping S.T.
      • Starr J.
      ). Thus, it is possible that under alkaline conditions, the generation of superoxide radicals from mitochondrial respiration is increased. Alkaline pH may act like a sink to draw protons away from the mitochondria, which normally maintains a proton gradient through respiration. Thus, under alkaline conditions, the mitochondria compensate for the loss of protons by increasing respiration which in turn will also produce more superoxide radicals. This hypothesis is supported by our findings that a significant increase in ROS levels was observed in control cells but not Mn-SOD-transfected cells. The identification of HNE in high amounts in parental but not in Mn-SOD-transfected cells suggests oxidative stress as a result of altering pH in the parental cells. It is interesting to note that in control cells, the increase in total ROS levels correlated with increased mitochondrial ROS. Furthermore, in Mn-SOD-transfected cells, there was no measurable increase in the levels of both mitochondrial ROS and total cytosolic ROS, suggesting that removal of superoxide radicals in the mitochondria reduced cellular oxidative stress at a distance from the mitochondria. Since SOD is specific for the removal of superoxide radicals (
      • Fridovich I.
      ), these results also suggest that superoxide is the initial radical that leads to the increased ROS detected. Why would removal of superoxide radicals in the mitochondria affect the levels of total cytosolic ROS? It has been hypothesized that in addition to its important role in respiration, mitochondrial respiration may also play a role in maintaining the cellular redox status by eliminating cytosolic superoxide radicals (
      • Guidot D.M.
      • Repine J.E.
      • Kitlowski A.D.
      • Flores S.C.
      • Nelson S.K.
      • Wright R.M.
      • McCord J.M.
      ). The cytosolic superoxide radical scavenging of mitochondria enhances the spontaneous dismutation of superoxide which diffuses into the mitochondrial intermembrane space. The mitochondrial intermembrane space has a localized proton-rich environment that can protonate superoxide radicals to form hydroperoxyl radicals, which then can diffuse into mitochondrial matrices and are dismuted by Mn-SOD. Thus, superoxide consumption in the mitochondria creates a gradient for superoxide radicals which favors diffusion from the cytosolic to the mitochondrial space (
      • Guidot D.M.
      • Repine J.E.
      • Kitlowski A.D.
      • Flores S.C.
      • Nelson S.K.
      • Wright R.M.
      • McCord J.M.
      ).
      Mitochondria are believed to participate in the intracellular calcium network (
      • Babcock D.F.
      • Herrington J.
      • Goodwin P.C.
      • Park Y.B.
      • Hille B.
      ). The relationship between mitochondrial damage and increased intracellular free calcium has been extensively studied (for a review, see Ref.
      • Mattson M.P.
      • Burger S.W.
      • Begley J.G.
      • Mark R.J.
      ). It has been demonstrated that perturbation of calcium homeostasis is tightly associated with cell death (see Ref.
      • Nicotera P.
      • Bellomo G.
      • Orrenius S.
      for a further review). Increased intracellular calcium can lead to increase ROS generation through activation of calcium-dependent proteases (
      • Friedl H.P.
      • Till T.O.
      • Ryan U.S.
      • Ward P.A.
      ). On the other hand, increased intracellular ROS can cause disturbances in calcium homeostasis. Thus, it is not yet clear whether generation of ROS or increase intracellular calcium is the earlier event leading to cell death. Our results which indicate that removal of ROS prevents intracellular calcium increases suggest that ROS production precedes the increased calcium levels observed under alkaline conditions. However, our results do not exclude the possibility that ROS-independent pathways of cell death can occur under certain circumstances. In fact, it has been shown that cells cultured under a near anaerobic atmosphere, where ROS generation is reduced, inhibit apoptotic cell death induced by oxygen generating agents but not by antibodies to Fas/APO or interleukin 3 (
      • Jacobson M.D.
      • Raff M.C.
      ).
      Several potential mediators responsible for mitochondria-mediated cell death have recently been identified. Activation of specific members of the interleukin-1β converting enzyme cysteine protease family (
      • Lazebnik Y.A.
      • Kaugmann S.H.
      • Desnoyers S.
      • Poirier G.G.
      • Earnshaw W.C.
      ,
      • Martin S.J.
      • Green D.R.
      ) and cytochrome c release from mitochondria (
      • Yang J.
      • Liu X.
      • Balla K.
      • Kim C.N.
      • Ibrado A.M.
      • Cai J.
      • Peng T-I.
      • Jones D.P.
      • Wang X.
      ) have been shown to be key participants of apoptotic cell death. More recently, it has been shown that mitochondrial function is necessary for the activation of the CPP-32 like protease by tumor necrosis factor but not staurosporin (
      • Higuchi M.
      • Aggarwal B.B.
      • Yeh E.T.H.
      ). Our results clearly indicate that mitochondrial damage is a critical event in alkaline-induced cell death. Thus, it is possible that damaged mitochondria release cytochrome c and/or activate these proteases in the alkaline-induced cell death process. Although the relative time course for each event to occur after alkaline treatment remains to be established, the finding that the mitochondria were taken up by lysosomes by 6 h after treatment indicate that mitochondrial damage occurred much earlier. Furthermore, the facts that the function of Mn-SOD is to remove superoxide radicals in mitochondria and mitochondria damage and cell death are preventable by overexpression of Mn-SOD, places oxidative stress and mitochondrial damage as early steps leading to cell death under alkaline conditions.
      The finding that expression of Mn-SOD prevents alkaline-induced cell death may also have practical implications. Increased Mn-SOD expression in response to oxidative stress has been demonstrated in cultured cells and in animals. We have previously shown that ionizing radiation induced Mn-SOD activity in mouse heart (
      • Oberley L.W.
      • St. Clair D.K.
      • Autor A.P.
      • Oberley T.D.
      ) and increased Mn-SOD activity protected transgenic mouse against adriamycin-induced cardiac toxicity (
      • Yen H-C.
      • Oberley T.D.
      • Vichitbandha S.
      • Ho Y-S.
      • St. Clair D.K.
      ). Production of ROS by paraquat, ionizing radiation, and adriamycin, have been well documented (
      • Krall J.
      • Bagley A.C.
      • Mullenbach G.T.
      • Hallewell R.A.
      • Lynch R.E.
      ,
      • Sinha B.K.
      • Mimnaugh E.G.
      ). The finding that alkaline conditions is accompanied by increasing oxidative stress adds to the growing list of pathological conditions where oxidative stress plays an important role in determining the fate of a cell and suggests that alkaline-induced cell death may be preventable by increasing levels of mitochondrial antioxidants.

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