Ion transporters and ischemic mitochondrial dysfunction

Abstract

Ischemia-induced ionic imbalance leads to the activation of numerous events including mitochondrial dysfunction and eventual cell death. Dysregulation of mitochondrial Ca²⁺ (Ca²⁺_m) plays a critical role in cell damage under pathological conditions including traumatic brain injury and stroke. High Ca²⁺_m levels can induce the persistent opening of the mitochondrial permeability transition pore and trigger mitochondrial membrane depolarization, Ca²⁺ release, cessation of oxidative phosphorylation, matrix swelling and eventually outer membrane rupture with release of cytochrome c and other apoptogenic proteins. Thus, the dysregulation of mitochondrial Ca²⁺ homeostasis is now recognized to play a crucial role in triggering mitochondrial dysfunction and subsequent apoptosis. Recent studies show that some secondary active transport proteins, such as Na⁺-dependent chloride transporter and Na⁺/Ca²⁺ exchanger, contribute to ischemia-induced dissipation of ion homeostasis including Ca²⁺_m.

Introduction

Ionic imbalance plays a critical role in pathogenesis of ischemic cell damage. Ischemia-induced perturbation of ion homeostasis leads to intracellular accumulation of Ca²⁺ and Na⁺ which results in the subsequent activation of numerous events including mitochondrial dysfunction and eventual cell death. Structural, biochemical and functional abnormalities of mitochondria are widely believed to contribute to ischemic or hypoxic cell injury.¹ Many lines of evidences suggest that Ca²⁺ is a key regulator of mitochondrial function.^2,3 Mitochondrial Ca²⁺ overload triggers the mitochondrial death pathway, which features the loss of mitochondrial membrane potential (Ψ_m), the opening of the mitochondrial permeability transition pore (PTP), release of Cytochrome c (Cyt. C), and enhanced generation of reactive oxygen species.^4–6

This review will highlight recent studies reflecting the role of secondary active transport proteins in ischemia-induced dissipation of ion homeostasis and subsequent mitochondrial dysfunction. Secondary active ion transport proteins such as the Na⁺-dependent chloride transporter (NKCC) and the Na⁺/Ca²⁺ exchanger (NCX) do not use energy stored in ATP directly, but derive their energy from the combined electrochemical gradients generated by Na⁺/K⁺-ATPase and Ca²⁺-ATPase. Secondary active ion transport proteins are important in maintaining steady-state intracellular ionic concentrations. Results from both in vitro and in vivo experimental studies suggest that these ion transport proteins are involved in ischemia-mediated loss of ion homeostasis, leading to mitochondrial dysfunction.^7–11 Therefore, they may be important targets for therapeutic intervention.

Mitochondrial Ca²⁺ Imbalance and Ischemic Mitochondrial Dysfunction

Mitochondrial Ca²⁺ imbalance.

Calcium is one of the most prominent intracellular messenger molecules, and plays a vital role in the physiology and biochemistry of cells, particularly in signal transduction pathways.¹² Cytosolic free Ca²⁺ (Ca²⁺_cyt) is maintained at roughly 100 nM while the extracellular milieu generally has a [Ca²⁺] of over 1 mM under physiological conditions. Loss of Ca²⁺ homeostasis, often in the form of an increase in cytoplasmic Ca²⁺, leads to multiple destructive processes such as the activation of proteases, lipases, nucleases, nitric oxide synthases, protein kinases and eventual cell death.^4,12

Mitochondria play a central role in cellular metabolism and are responsible for cellular respiration that generates ATP from ADP and inorganic phosphate. Under physiological conditions, Ca²⁺_m increases to buffer the amplitude of the Ca²⁺_cyt rise. Ca²⁺_m is typically in the range of 0.2–3 mM, which is ideal for the activation of Ca²⁺-dependent enzymes of the Krebs cycle. Increases of Ca²⁺_m can be compensated by mitochondrial Ca²⁺ efflux mechanisms such as NCX and matrix Ca²⁺ buffering.³ Thus mitochondria accumulate Ca²⁺ and efficiently control the spatial and temporal shape of cellular Ca²⁺ signals, yet this situation exposes them to the hazards of Ca²⁺ overload.^3,13 During anoxia/ischemia, oxidative phosphorylation is inhibited, triggering a rapid decline in ATP production and initiating multiple destructive processes.¹⁴ These changes include the compromising of ionic homeostasis, activation of glycolysis and intracellular acidosis, degradation of phospholipids and an increase in the plasma membrane permeability to Na⁺ and Ca²⁺.^14,15 Elevated cytoplasmic Na⁺ (Na⁺_cyt) during ischemia will favor the reverse-mode operation of NCX (NCX_rev), causing Ca²⁺_cyt levels to further increase.¹⁶ In the presence of ATP, Mg²⁺ and inorganic phosphate, respiring mitochondria are able to accumulate large amounts of Ca²⁺ via the mitochondrial Ca²⁺ uniporter and/or the rapid uptake mode.¹⁷ However, during ischemia when ATP is decreased, mitochondria would not be able to accumulate Ca²⁺. Upon reperfusion, increases in Ca²⁺_cyt or the release of Ca²⁺ from the endoplasmic reticulum invariably induce Ca²⁺_m uptake which helps in reestablishing physiological Ca²⁺_cyt levels.^2,15

Sustained increases in Ca²⁺_m will initiate several death factors. The rapid uptake of Ca²⁺ by mitochondria stimulates the Ca²⁺-sensitive matrix dehydrogenases, which are key sites of NADH production for the respiratory chain and thereby for stimulation of mitochondrial energy metabolism.² A high concentration of mitochondria Ca²⁺ can also induce the opening of the PTP, a high conductance inner membrane channel which consists of the voltage-dependent anion channel, the adenine nucleotide translocator and the cyclophilin D, as well as several other proteins.^18–20 Activation of PTP triggers a cascade of events during cellular damage.

Ischemic mitochondrial dysfunction.

PTP opening contributes significantly to mitochondrial dysfunction. In particularly it can dramatically increase the mitochondrial membrane permeability, allowing passage of solutes with molecular masses up to 1,500 Da. Persistent PTP opening is followed by depolarization of mitochondria leading to Ca²⁺ release, cessation of oxidative phosphorylation, matrix swelling with inner membrane remodeling and eventual outer membrane rupture with release of Cyt. C and other apoptogenic proteins.^3,21,22 Released Cyt. C then binds to apoptotic protease activating factor-1, which recruits and activates caspase-9 to form the apoptosome. Caspase-9 activation results in the cleavage and activation of caspases-3 and -7, initiating a proteolytic cascade that ultimately results in cell death.²³ There are a number of factors that lower the threshold for Ca²⁺ induced mitochondrial permeability transition, including inorganic phosphate, prooxidants that oxidize membrane SH-groups, oxidation of NAD(P)H and GSH.¹⁵ On the other hand, a protective effect is exerted by Mg²⁺, ADP (and ATP), some antioxidants, carnitine, decrease in pH and cyclosporin A that binds to cyclophilin.¹⁵

Members of the Bcl-2 family of proteins, notably Bax and Bak, can also trigger the permeabilization of the outer mitochondrial membrane by integrating into it as oligomers. This process is stimulated by t-Bid and other BH3-only proteins,²⁴ while inhibited by anti-apoptotic family members, Bcl-2 and Bcl-X_L.²⁵ Mice deficient in both Bax and Bak are resistant to most apoptotic stimuli, providing a strong support for the role of the mitochondrial pathway in apoptosis signaling.^2,26

The release of Cyt. C to cytoplasm is one of the key events of mitochondrial dysfunction. In healthy cells, Cyt. C is normally bound to the inner mitochondrial membrane by its association with the anionic phospholipid cardiolipin, where it is predominantly located. The dissociation of Cyt. C from cardiolipin leads to the peroxidation of cardiolipin^27,28 and decreases its binding affinity to haemoprotein. This is a crucial first step for Cyt. C release into the cytosol and for the induction of apoptosis.^2,27 In addition, Ca²⁺ can bind to cardiolipin in the inner mitochondrial membrane, leading to decreased lipid mobility, formation of cardiolipin-enriched domains and protein aggregation.²⁹ In turn, this rearrangement leads to increased production of reactive oxygen species by the respiratory chain, which promotes the oxidation of membrane phospholipids and proteins resulting in an increase in membrane permeability. In fact, the Ca²⁺-cardiolipin interaction might be an early and crucial step in the sequence of events by which Ca²⁺ triggers mitochondrial membrane permeabilization.²

Recent data suggests that Cyt. C release follows a biphasic kinetics rather than in an all-or-nothing manner as previously believed. An in vitro study identifies Cyt. C as a messenger that coordinates mitochondrial-endoplasmatic reticulum interactions that drive apoptosis.³⁰ Early in apoptosis, Cyt. C translocates to the endoplasmic reticulum where it selectively binds to inositol 1,4,5-triphosphate receptor (InsP(3)R), resulting in sustained, oscillatory cytosolic Ca²⁺ increases.³⁰ These Ca²⁺ signaling events are linked to the coordinate release of Cyt. C from all mitochondria. These findings identify a feed-forward mechanism whereby early Cyt. C release increases InsP(3)R function, resulting in augmented Cyt. C release then amplifies the apoptotic signal.^30,31 Cyt. C also interacts with InsP(3) R type 1 and ryanodine receptor type 2 in gerbil hippocampus subjected to transient brain ischemia with a short reperfusion, and contributes to postischemic neuronal death.³² These findings illustrate the importance of intracellular Ca²⁺ stores in apoptosis, and ischemic cell damage.

Secondary Active Transport Proteins and Ca²⁺ Overload

Loss of Ca²⁺ homeostasis in the cell plays a critical role in pathogenesis of ischemic mitochondrial dysfunction and subsequent cell damage. Recent studies show that the important secondary active transport proteins, NKCC and NCX, contribute to ischemia-induced dissipation of ion homeostasis.

NKCC.

The electroneutral NKCC protein belongs to the cation-chloride cotransporter family, which transports Na⁺, K⁺ and Cl⁻ into cells under physiological conditions with a stoichiometry of 1Na⁺:1K⁺:2Cl⁻.^33,34 The transmembrane chemical gradients for Na⁺, K⁺ and Cl⁻ determine net inward ion movement via NKCC.³⁴ In most cells, the driving force for ion influx is in part supplied by the inward Na⁺ gradient, maintained by Na⁺/K⁺-ATPase. However, in Cl⁻ secretory epithelial cells, the Cl⁻ gradient is also an important driving force for ion influx.³⁴ NKCC is characteristically inhibited by the sulfamoybenzoic acid loop diuretics, such as bumetanide and furosemide.³⁵ To date, only two distinct isoforms, NKCC1 and NKCC2, have been identified. NKCC1 has a broad tissue distribution, while the NKCC2 isoform is only found in vertebrate kidney.^33,36

NKCC serves multiple functions, including ion and fluid movements in secreting or reabsorbing epithelia and cell volume regulation.^33,34 However, understanding of the role of NKCC1 in the central nervous system just began. NKCC1 is important in the maintenance of intracellular Cl⁻ in neurons and contributes to GABA-mediated depolarization in immature neurons. Thus, NKCC1 may affect neuronal excitability through regulation of intracellular Cl⁻ concentration.^37,38 NKCC1 may also contribute to K⁺ clearance and maintenance of intracellular Na⁺ in astrocytes and oligodendrocytes. Recent studies suggest that high [K⁺]_o activation of NKCC1 leads to astrocyte swelling and glutamate release, as well as neuronal Na⁺, and Cl⁻ influx during acute excitotoxicity.³⁹ In addition, inhibition of NKCC1 activity significantly reduces infarct volume and cerebral edema following cerebral focal ischemia.³⁹

NCX.

NCX belongs to the Ca²⁺/cation antiporter superfamily. It is a ubiquitous transporter that plays an important role in regulating and maintaining cellular Ca²⁺ balance in various tissues. At present, three isoforms in the NCX family have been identified: NCX1, NCX2 and NCX3.⁴⁰ They share biophysical and biochemical properties but exhibit differences in expression during development and in various adult tissues. NCX1 is present in most tissues and is highly expressed in heart, brain and kidney, while NCX2 and NCX3 are expressed mainly in the brain and skeletal muscles.^40–42 Immunogold EM studies reveal that neuronal NCXs are preferentially located in dendrites and dendritic spines, while glial NCXs are prominently expressed in astrocytic processes ensheathing excitatory synapses.⁴³ The specific localization indicates that NCXs are favourably located for buffering Ca²⁺_cyt in excitatory postsynaptic sites and may participate in shaping astrocytic Ca²⁺_cyt transients evoked by ongoing synaptic activity.⁴³

NCX works bidirectionally depending on cytosolic Na⁺ concentration ([Na⁺]_cyt), cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and plasma membrane potential. The stoichiometry of NCX1 is generally accepted to be 3 Na⁺ ions/1 Ca²⁺ ion. Thus, NCX1 transport is electrogenic, and dependent on plasma membrane potential.⁴⁴ Under physiological conditions, NCX is thought to primarily pump Ca²⁺ to the outside of the cell using the Na⁺ concentration gradient across the cell membrane. In contrast, under conditions in which Na⁺ accumulates inside the cell such as during ischemia, NCX may work in reverse mode-operation and conduct Ca²⁺ influx.

Roles of NKCC1 and NCX in ischemic mitochondrial dysfunction.

Loss of Na⁺ homeostasis in cells following in vitro hypoxia/ischemia. A steep inwardly directed Na⁺ gradient is essential for cell functions, such as glutamate reuptake and regulation of intracellular ion concentrations by other secondary ion transporters.^46,47 Dissipation of the Na⁺ gradient is one of the key elements in promoting cellular damage in cells during energy failure.^47,48

A recent study shows that one hour oxygen-glucose deprivation (OGD) triggers an approximately four-fold increase in [Na⁺]_cyt in cultured cortical neurons.¹¹ Additionally, 60 min reoxygenation (REOX) following two hour OGD can induce a seven-fold increase in [Na⁺]_cyt in neurons.⁴⁸ An ∼3.6-fold increase in [Na⁺]_cyt after OGD is also reported in cultured astrocytes.⁷ Additionally, Kintner DB, et al. reported that the onset of REOX following either of two hypoxic conditions, OGD and HAIR (a hypoxic, acidic, ion-shifted ringer buffer), triggers a rapid Na⁺_cyt overload in astrocytes.⁸ This finding is consistent with reports on [Na⁺]_cyt accumulation in rat spinal cord astrocytes,⁴⁹ rat cortical astrocytes⁴⁷ and mouse cortical astrocytes,⁵⁰ when these cells are exposed to glucose deprivation, NaN₃-mediated chemical hypoxia and simulated ischemia, respectively.

Lenart B, et al. reported that NKCC1 activity in cortical astrocytes is increased during reoxygenation (REOX) via protein phosphorylation, which is a major regulatory mechanism for stimulation of NKCC1 activity.^7,51 It also has been reported that loss of NKCC1 activity either by treatment with its inhibitor bumetanide or by genetic ablation blocks ∼64% of the rise in Na⁺_cyt in astrocytes,⁸ and results in ∼50% less Na⁺_cyt accumulation in neurons.¹¹ The remaining Na⁺_cyt accumulation could be the result of other Na⁺ influx pathways (such as voltage-gated Na⁺ channels, Na⁺/H⁺ exchanger, etc.) or a decrease in Na⁺ extrusion via Na⁺/K⁺ ATPase during ischemia.^7,11 In addition, NKCC1 also plays a role during glutamate-mediated excitotoxicity. [Na⁺]_cyt accumulation is found in cultured oligodendrocytes exposed to alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and cyclothiazide (CTZ),¹⁰ and in cortical neurons exposure to 60 µM N-methyl-D-aspartic acid (NMDA).³⁸ Blocking NKCC1 activity with bumetanide significantly reduces the AMPA/CTZ-induced Na⁺ influx in oligodendrocytes¹⁰ as well as the accumulation of Na⁺_cyt content after NMDA receptor activation in neurons.³⁸ Thus, these results imply that stimulation of NKCC1 activity evokes excessive Na⁺ entry and disrupts Na⁺ homeostasis following hypoxia/ischemia.

Intracellular Na⁺ overload leads to reversal of NCX. Reduction in the Na⁺ gradient affects Ca²⁺ homeostasis in cells. Ca²⁺ entry pathways involve L-type voltage-gated Ca²⁺ channels, ionotropic glutamate receptors, and NCX_rev.⁵² Li et al. reported that elevation of [Na⁺]_cyt increases Ca²⁺_cyt via NCX_rev in neurons and causes irreversible injury during anoxia and ischemia.⁵³ An increase in Ca²⁺_cyt via NCX_rev occurs in rat astrocytes when [Na⁺]_cyt is raised by inhibition of Na⁺/K⁺-ATPase activity or activation of AMPA channels.^54,55 Furthermore, Ca²⁺ entry is detected in astrocytes after OGD. Inhibition of NCX_rev with 3 µM KB-R7943 abolishes the OGD-mediated increase in releasable Ca²⁺ in bradykinin-sensitive Ca²⁺ stores.⁷ These findings further suggest that NCX_rev plays a role in Ca²⁺ entry and Ca²⁺ signaling in cells after OGD. So far, more benzyloxyphenyl derivatives (SEA0400, SN-6 and YM-244769) have been developed as selective NCX_rev inhibitors.⁴⁵ They effectively prevent ischemia-reperfusion injury in several animal models.⁴⁵ Thus, NCX inhibitors may have therapeutic potential as novel drugs for reperfusion injury.

In addition to a significant rise in [Ca²⁺]_cyt during HAIR, REOX induces a secondary increase in [Ca²⁺]_cyt.⁸ Approximately 70% of the REOX-triggered Ca²⁺ rise following 15 min HAIR is abolished with 10 µM KB-R7943. Interestingly, NKCC1^-/- astrocytes exhibit a much lower level of REOX-triggered Ca²⁺ rise.⁸ The data suggest that Ca²⁺entry during REOX is largely via NKCC1-mediated reversal of NCX in astrocytes. This finding is consistent with thermodynamic modeling that predicts NCX will operate in the reverse mode and mediate Ca²⁺ influx when [Na⁺]_cyt in astrocytes is increased ∼25 mM.⁵⁶ Maximum values of [Na⁺]_cyt reached 70–80 mM during REOX following either OGD or HAIR, a condition that favors NCX_rev. This view is supported by several recent reports. For example, inhibition of NCX with 100 nM KB-R7943 significantly attenuates the rise in [Ca²⁺]_cyt in response to severe mechanical strain injuries in rat cortical astrocytes. The strain injury leads to a rapid rise in [Na⁺]_cyt in astrocytes that is sustained for ∼50 min.⁵⁷

Moreover, a transient elevation of [Ca²⁺]_cyt following 25–30 min HAIR is blocked by NCX_rev inhibitor KB-R7943.⁵⁸ The effects of KB-R7943 imply that REOX evokes Na⁺ entry, which subsequently triggers the reversal of NCX. The elevation of [Na⁺]_cyt and the reversal of NCX have been suggested to play a role in spinal cord astrocyte ischemic damage during the reperfusion period.⁴⁹ NCX inhibitors bepridil and KB-R7943 improve functional recovery of white matter tracts after anoxic and traumatic injury.⁵³ In addition, Ca²⁺ influx via NCX_rev is decreased by approximately 70% in NCX^+/− neurons, which exhibit reduced NCX1 protein expression compared to NCX^+/+ neurons.⁵⁹ KB-R7943 treatment attenuates the transient Ca²⁺ influx by 60% and sustained Ca²⁺ influx by 70% in oligodendrocytes.¹⁰ Moreover, inhibition of NKCC1 activity with bumetanide reduced the sustained Ca²⁺ rise by 50%.¹⁰ These data suggest that NKCC1 and NCX_rev are involved in AMPA-triggered Ca²⁺ signaling in oligodendrocytes. The protective effects, via inhibition of NKCC1 and NCX_rev on Ca²⁺_cyt overload, may result from reducing Na⁺_cyt overload and inhibition of NCX_rev.¹⁰

Ca_m²⁺ overload, mitochondrial dysfunction and cell death. The mechanisms of ischemic injury include Ca²⁺ entry and Ca²⁺-mediated cell death. Ca²⁺_m overload leads to mitochondrial dysfunction via many pathways, including inhibition of oxidative phosphorylation, oxygen free radical formation or formation of the mitochondrial PTP, which subsequently releases proapoptotic molecules such as Cyt. C.⁵ OGD/REOX can cause a dramatic increase in Ca²⁺_m accumulation and Cyt. C release in cultured neurons.⁴⁸ An approximately five-fold increase of Ca²⁺_m accompanies an ∼58% depolarization of Ψ_m and subsequent Cyt. C release in cortical astrocytes at 1 h REOX following 2 h OGD (Figs. 1 and 2).⁸ In addition, REOX following 15 min HAIR can cause significant dissipation of Ψ_m and Ca²⁺_m increase in NKCC1^+/+ astrocytes. Pharmacological inhibition or genetic ablation of NKCC1 not only attenuates REOX-induced Ca²⁺_m overload and Ψ_m depolarization, but it also blocks Cyt. C release from mitochondria and results in less cell death in culture astrocytes (Figs. 1 and 2).⁸ Moreover, in NCX1^+/−/NKCC1^+/− double heterozygous neurons, with lower NCX1 and NKCC1 protein expression, NCX-mediated Ca²⁺ influx is nearly abolished. These neurons exhibit approximately 65% less neuronal death and increased tolerance to ischemic damage.⁹ Studies in glutamate-mediated excitotoxicity in oligodendrocyte damage shows the AMPA/CTZ-elicited Ca²⁺_cyt increase, Ca²⁺_m overload, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX_rev.¹⁰ In summary, these findings suggest that NKCC1 in conjunction with NCX1 mediate dysregulation of cellular Na⁺ and Ca²⁺ homeostasis and can seriously impair cell function and survival.⁸

Figure 1.

Changes in mitochondrial Ca²⁺ (Ca²⁺_m) in astrocytes following OGD/REOX. (A) Mitotracker (green in a) and Rhod-2 (red in b) images were shown in NKCC1^+/+ astrocytes at the end of 2 h OGD and at 60 min REOX (c and d). Scale bar = 12.3 µm. (B) Average tracings of relative change in Rhod-2 fluorescence during 60 min REOX following 2 h OGD in NKCC1^+/+ and NKCC1^−/− astrocytes. Arrow: response of Rhod-2 fluorescence to FCCP. (C) Summary data. Data are means ± SEM; n = 4–5. * p < 0.05 vs. control; # p < 0.05 vs. NKCC1^+/+ OGD/REOX. Reprinted with permission from Kinter et al.⁸

Figure 2.

Changes in membrane potential (Ψ_m) in astrocytes following OGD/REOX. (A) JC-1 aggregate (a.) and monomer (b.) fluorescence in NKCC1^+/+ cells under normoxic control conditions. Insets in (A): changes of JC-1 fluorescence following 3 min of FCCP (10 µM). Scale bar = 12.3 µm. (B) A representative tracing of changes of the JC-1 ratios. n = 4–5. Reprinted with permission from Kinter et al.⁸

Conclusion

Loss of ionic homeostasis plays an important role in ischemic brain damage. Recent findings suggest that NKCC and NCX_rev are involved in dissipation of ion homeostasis and mitochondrial dysfunction. The concerted activities of multiple ion transport proteins not only perturbate intracellular Na⁺ and Ca²⁺ homeostasis in response to hypoxia/ischemia, but also affect mitochondrial Ca²⁺ and Cyt. C release. Therefore, these ion transport proteins may be potential therapeutic targets to reduce or prevent ischemia-mediated loss of ion homeostasis.

Abbreviation

AMPA

alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

Ca²⁺_m

mitochondrial Ca²⁺

Ca²⁺_cyt

cytosolic free Ca²⁺

[Ca²⁺]_cyt

cytosolic Ca²⁺ concentration

Cyt. C

cytochrome c

CTZ

cyclothiazide

HAIR

a hypoxic, acidic, ion-shifted ringer buffer

InsP(3) R

inositol 1,4,5-triphosphate receptor

Na⁺_cyt

cytoplasmic Na⁺

[Na⁺]_cyt

cytosolic Na⁺ concentration

NCX

Na⁺/Ca²⁺ exchanger

NCX_rev

reverse-mode operation of NCX

NKCC

Na⁺-dependent chloride transporter

NMDA

N-methyl-D-aspartic acid

OGD

oxygen-glucose deprivation

PTP

permeability transition pore

REOX

reoxygenation

Ψ_m

mitochondrial membrane potential