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Calcium ions and oxidative cell injury.

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Calcium Ions and &dative Cell Injury
Sten Orrenius, MD, Mark J. Burkitt, PhD, George E. N. Kass, PhD,
Jeannette M. Dypbukt, DrMedSc, and Pierluigi Nicotera, M D
Exposure of mammalian cells to oxidative stress induced by oxidation-reduction-active quinones and other prooxidants
results in depletion of intracellular glutathione, followed by modification of protein thiols and loss of cell viability.
Protein thiol modification during oxidative stress is normally associated with impairment of various cell functions,
including inhibition of agonist-stimulated phosphoinositide metabolism, disruption of intracellular Ca2+homeostasis,
and perturbation of normal cytoskeletal organization. The latter effect appears to be responsible for formation of the
numerous plasma membrane blebs typically seen in cells exposed to cytotoxic concentrations of prooxidants. Following
disruption of thiol homeostasis in prooxidant-treated cells, there is impairment of Ca2+ transport and subsequent
perturbation of intracellular Ca2+homeostasis, resulting in a sustained increase in cytosolic Ca2 concentration. This
increase in Ca2 can cause activation of various Ca2+-dependentdegradative enzymes (e.g., phospholipases, proteases,
endonucleases),which may contribute to cell death. In contrast to the cytotoxic effects of excessive oxidative damage,
low levels of oxidative stress can lead to activation of enzymes involved in cell signaling. In particular, the activity of
protein kinase C is markedly increased by oxidation-reduction-cycling quinones through a thiolldisulfide exchange
mechanism, which may represent a mechanism by which prooxidants can modulate cell growth and differentiation.
Orrenius S, Burkitt MJ, Kass GEN, Dypbukt JM, Nicotera P. Calcium ions and
oxidative cell injury. Ann Neurol 1992;32:S33-S42
It has become clear over the past decade that oxidative
cell injury generated by chemicals during reoxygenation of hypoxic tissue or as a result of acute or chronic
inflammatory processes is associated with perturbation
of intracellular calcium ion homeostasis. This process
may be a consequence of impairment of the plasma
membrane Ca2 translocating systems, stimulation of
Ca2+ channels, or inhibition of Ca2+ sequestration by
the endoplasmic reticulum (ER) and mitochondria,
which results in an inability of the cell to maintain its
cytosolic free Ca2 concentration ({Ca2+li)within the
physiological range. A sustained elevation of {Ca2+li
has been causally linked to cell death caused by a wide
variety of compounds, such as metals (mercury and
lead), organotin compounds, hepatotoxins (acetaminophen, carbon tetrachloride, and diquat), reactive oxygen metabolites, excitatory amino acids, quinones, and
cyanide 11, 21. Ca2+ has also been implicated in the
mechanism of immune cell death caused by glucocorticoids and the environmental contaminant 2,3,7 ,a,tetrachlorodibenzo-p-dioxin (TCDD), as well as in the
killing of target cells by neutrophils, macrophages,
cytotoxic T lymphocytes, and natural killer cells. These
findings led us and others to propose the Ca2+hypothesis of cell injury, which suggests that the disruption
of intracellular Ca2+ homeostasis is a common step in
cell death caused by many toxic agents.
Ca2+-dependent degradative enzymes have been im-
plicated in Ca2+-mediatedcell death. In addition to this
proposed mechanism of acute lethal injury by these
catabolic processes, recent evidence has also accumulated that implicates signal transduction processes as an
important target for oxidant injury.
From the Department of Toxicology, Karolinska Instituter, Stockholm. Sweden.
Address correspondence to Dr Orrenius, Department of Toxicology,
Karolinska Instituter, Box 60400, S-104 01 Stockholm, Sweden.
Cellular Ca2+ Sequestration Processes
Studies using cell permeation indicators selective for
calcium ions have shown that the free Ca” concentration in the cytosol is maintained between 0.1 and 0.2
pmol/L { 3 , 4 ] .Thus, there is a concentration difference
of approximately four orders of magnitude between
the extracellular Ca’ + level (approximately 1.3 mmol/
L) and the cytosolic free Ca’ concentration, resulting
in a large electrochemical driving force in favor of net
Ca‘+ accumulation by the cells. This tendency to take
up Ca2+ is balanced primarily by active Ca2+ extrusion
systems located at the plasma membrane and by the
coordinated activities of Ca’ sequestering systems located in the endoplasmic reticular, mitochondrial, and
nuclear membranes (Fig 1).
Ca2+ Transport by the Plasma Membrane
Ca” enters the cell mainly through specific Ca2+channels. One important group of Ca2 channels comprises
those activated by depolarization; these Ca2’ channels
are commonly referred to as voltage-operated channels
(VOCs) {53. At least four types of VOCs with different
Intrucellulur C d + Sequestration
Both the ER and mitochondria sequester Ca2+.Ca’+
sequestration in the ER involves a Ca’+-ATPase with
a high Ca2+ affinity and transport capacity, which translocates two moles Ca” for every mole of ATP that is
hydrolyzed. Liver ER Ca2+-ATPase has recently been
purified by Kraus-Friedmann [S]. This enzyme has a
molecular weight of 107 kd, a high affinity for Ca”
= 0.2-1.0 kmol/L), and catalyzes the sequestration of 10 to 20 nmol Ca2+/mg microsomal vesicle protein. The regulation of Ca2 sequestration by
the hepatic ER is still poorly understood. Although
involvement of calmodulin in this process has been
suggested, convincing evidence for this assumption is
still missing. More recently, a role for glucose-6phosphatase in regulation of Ca2+ sequestration by the
hepatic ER has been proposed in an attempt to couple
the hydrolysis of glucose-6-phosphate to the termination of the [Ca2+litransient by enhancing Ca’+ sequestration through the intraluminal accumulation of inorganic phosphate 191.
Ca2+ uptake and release by mitochondria occur via
different routes 14, lo]. The Ca2+ uptake process is
purely electrogenic, driven by the electrical component
(membrane potential) of the total proton-motive force.
Ca2+ is transported via a uniporter that has a relatively
low affinity for Ca2+;reported K , values range from
5 to 30 p,mol/L, depending on the mitochondrial tissue
of origin and the composition of the medium. Spermine and inorganic phosphate stimulate the rate and
extent of Ca2+uptake, whereas ruthenium red (a hexavalent ammonium complex of Ru), lanthanides, and
Mg2+ inhibit it.
Ca2+release from isolated mitochondria occurs in
exchange for Na+ or H’. For example, Ca2+ release
from heart and brain mitochondria can be stimulated
by Na’. The sodium-induced Car+ release is probably
electroneutral and occurs at a high transmembrane potential. In liver mitochondria, the Na+/Ca2+exchange
is quantitatively not important, and the existence of a
separate release pathway involving an electroneutral
Ca2+/H+ antiporter, also operating under high transmembrane potential conditions, is now generally accepted. Release of Ca’ + from isolated liver mitochondria is inhibited by adenosine diphosphate (ADP),
spermine, and m-iodo-benzylguanidine, whereas it is
stimulated by oxidants. Whether the latter phenomenon occurs through activation of the Ca2+/H+antiporter is currently unclear. Under conditions of high intramitochondrial Ca2+ concentrations, the Ca2+ efflux
pathway becomes saturated, and isolated mitochondria
act as efficient buffers of extramitochondrial Ca’+.
When isolated mitochondria contain little Ca2+ , as they
have been found to in situ [ll], the activity of the
efflux pathway increases with matrix [Ca2+], and under
these conditions the mitochondria will not act as Car+
Fig I. Intracellular C d + transport processes. ADP = adenosine diphosphate; ATP = adenosine triphosphate; ER = endoplasmic reticulum; M I T = mitochondrion; NUCL = nucleus;
ROC = receptor-operated channel; VOC = voltage-operated
pharmacological and electrophysiological properties
have been identified in most excitable cells and in many
nonexcitable cells. Recent studies in ours and other
laboratories have shown that in nonexcitable cells,
Ca” entry is also stimulated following the interaction
of Ca2 -mobilizing hormones and growth factors with
their respective receptors. The mechanism by which
Ca” influx through these receptor-operated channels
(ROCs) is regulated is still unclear; there is some evidence that G proteins or inositol phosphate turnover
and the content of an intracellular pool identified as
part of the agonist-sensitive Ca2+ store may be involved. A third physiological route for Ca” entry involves an antiporter that exchanges 3 Na+ ions for
every Ca2+ion translocated. In addition, it appears that
Ca2 enters through nonselective plasma membrane
leak channels.
The two transport systems that can extrude Ca2+
against its concentration gradient to balance the basal
as well as the hormone-stimulated influx of Ca2’ are
the Ca2+-adenosine triphosphatase (Ca’ +-ATPase) and
the Na+/Ca2+ exchanger. The plasma membrane
Ca2+-ATPase,of which the enzyme found in erythrocytes is best characterized, utilizes the free energy liberated by hydrolysis of adenosine triphosphate (ATP)
to electroneutrally expel Ca2+ in exchange for protons
161. The general kinetic properties of Ca2+-ATPase
include a high affinity for Ca2+ but a low capacity for
transport; however, these parameters may be modulated by calmodulin- and protein kinase C-dependent
phosphorylation to favor Ca” extrusion [7].
The Na+/Ca2’ exchanger is most active in heart and
brain cells and catalyzes the electrogenic exchange of
3 Na+ ion for every Ca2+ ion. Consequently, the direction of the exchange depends on the transmembrane potential; in the presence of a resting membrane
potential, Ca2+is extruded from the cell, whereas Ca’+
influx occurs during depolarization [4].
S34 Annals of Neurology
Supplement to Volume 32, 1992
buffers, but matrix [Ca2+} will reflect the extramitochondrial [Ca2+]. This is probably the mechanism by
which the mitochondrial Ca2 -regulated enzymes, such
as pyruvate dehydrogenase, are regulated by {Ca2+1,
The existence of pores in the nuclear envelope has
generally been considered evidence of free diffusion
of ions and small molecules in and out of the nucleus;
however, it is becoming increasingly clear that permeability barriers for Ca2+ and other ions exist in the
nucleus. Using suspensions of isolated liver nuclei, we
recently identified and partially characterized a Ca”
uptake system that apparently mediates an increase in
intranuclear Ca2+1131. The Ca2+ uptake is coupled to
ATP hydrolysis and appears to be regulated by calmodd n . Additional studies have shown that liver nuclei
can sequester approximately 2 nmol Ca2+/mgprotein
and have suggested that an agonist-sensitive Ca2+store
is associated with the nuclear envelope 1141. Furthermore, recent work using patch clamping indicated that
ion channels may be located within the nuclear pore
complex 1151.
Control of Cellular CL?+Homeostasis
As described, the low resting {Ca2+], of 0.1 to 0.2
pmoYL is achieved by concerted action of the plasma
membrane Ca2+ pump and active Ca2+ sequestration
by mitochondria, the ER, and the nucleus (see Fig 1).
Isolated mitochondria can accumulate large amounts of
Ca2+;however, the affinity of the uniport carrier for
Ca2+ is low. Therefore, mitochondria have been suggested to have a minor role in buffering cytosolic Ca2+.
Electron probe radiographic microanalysis of rapidly
frozen liver sections also demonstrated that mitochondria contain little Ca2+ in situ (approximately 1 nmol
Ca2+/mgprotein), whereas the ER represents the major intracellular Ca2+store [ll]. Estimates of the mitochondria’s ability to lower [Ca2+},, however, have
mainly been derived from experiments performed in
the absence of possible natural modulators of Ca2+
uptake and release. With this point in mind, Rottenberg and colleagues 1161 recently showed that mitochondria isolated from brain tissue can lower the Ca2+
concentration of the incubation medium to 0.1 pmoY
L in the presence of physiological concentrations of
spermine and ADP. It is generally believed, however,
that the ER is the main regulator of [Ca2+1,in most
tissues. Our recent finding that the nucleus has a high
Ca2 buffering capacity suggests the possibility that
this compartment may also be involved in the regulation of (Ca2’], within cells.
Disruption of Intracellular Ca2+ Homeostasis
by Toxic Agents
With the knowledge that Ca2+can operate as an intracellular signal for several hormones and growth factors,
it has also’become clear that disturbances of intracellular Ca2+ homeostasis and the resulting Ca2+overload
may result in alteration of cell function and ultimately
in cell death. Cellular Ca2+ overload can be the result
of either an enhanced influx of extracellular Ca2+ or
an impairment of Ca2 extrusion from the cell. In addition, interference with individual Ca2 translocases can
compromise the ability of the cell to buffer cytosolic
Ca” changes and contribute to an increase in the cytosolic Ca2+ level.
C d + Sequestration by the ER and Its RoIe in
Oxidant Injtlry
More than a decade ago, Moore and co-workers [ 171
showed that Ca2+ sequestration by liver microsomes
isolated from carbon tetrachloride-intoxicated rats was
substantially inhibited. Since then, a number of chemical toxins, including the oxidants tert-butyl hydroperoxide, diamide, and cystamine, have been found to impair
Ca2 sequestration by isolated microsomal fractions
118, 191. The thiol-reducing agents, dithiothreitol and
reduced glutathione (GSH), protected against inhibition by the oxidants; therefore, oxidation of essential
sulfhydryl groups of the Ca2+-ATPase molecule may
be involved in the mechanism of oxidative inactivation
of the ER Ca2+ translocase. Recent work also suggests,
however, that the inhibitory effect on Ca” sequestration observed with low concentrations of the oxidant
may be due to the stimulation of a specific Ca2+release
pathway [201.
The findings of Moore and co-workers 1171 also suggest that an impairment of Ca2+ sequestration may be
the mechanism by which many chemical toxins cause
liver cell death. Recent work in our laboratory, however, has shown that the selective inhibitor of the
microsomal (ER) Ca2+-ATPase, 2,5-di-(tert-butyl)1,4-benzohydroquinone (tBuBHQ), rapidly releases
endoplasmic reticular Ca2+ {213 without producing
hepatotoxicity in isolated hepatocytes (Kass GEN. Unpublished observations) or in the isolated perfused rat
liver 1221. Hence, at least short-term interference with
Ca2+sequestration by the ER does not appear to have
a major role in the development of acute hepatotoxicity.
CL?+ Seqtlestration by Mitochondria and Its Role in
Oxidant lnjtlry
Mitochondria contain little Ca2 under physiological
conditions, although they have the capacity to sequester large quantities of Ca2+ and could therefore act as
efficient buffers of [Ca2’l, under toxic conditions. This
potentially important line of defense, however, may
not be operational under conditions of oxidative stress
because many oxidants, such as tert-butyl hydroperoxide 1231, menadione 124J, and 3,5-dimethyl-N-acetylp-bentoquinone imine (3,5-Me2-NAPQI) 1251, have
Orrenius et a1 Ca2+ in Oxidative Injury S35
been found to stimulate rapid release of Ca2+ from
isolated liver mitochondria.
The mechanism by which oxidants cause mitochondrial Ca2+ efflux has been a matter of intense debate
over the past 15 years {lo}. Several groups have suggested that oxidants, in the presence of Ca2+,stimulate
the reversible opening of a pore (26) that shows many
of the characteristics of the adenine nucleotide carrier
of the mitochrondrial inner membrane {27, 28). Other
investigators have implicated nonselective damage to
the inner membrane, which results in loss of the transmembrane potential and reversal of the uniport Ca”
uptake route in the mechanism of oxidant-induced
Ca2’ release {29}. In apparent contrast, work from
Richter and associates’ {30) and our laboratory has
demonstrated that (1) the initial phase of Ca2+ release
occurs from intact mitochondria under conditions of
high transmembrane potential, and the observed loss
of transmembrane potential is the result of continuous
re-uptake of Ca2+ by the uniporter; and (2) Ca2+ release following exposure to oxidants seems to be regulated by mitochondrial pyridine nucleotides. Addition
of oxidants such as menadione, tert-butyl hydroperoxide, or 3,5-Me2-NAPQI results in rapid oxidation of
reduced nicotinamide-adenine dinucleotide (NADH)
and NADPH followed by their hydrolysis to nicotinamide and ADP-ribose. Pyridine nucleotide oxidation
is necessary, although not sufficient, to cause Ca” release and requires further hydrolysis to nicotinamide
and ADP-ribose. bchter and Kass {lo} postulated that
the oxidant-sensitive Ca2+ release mechanism involves
mono- ADP-ribosylation of a target protein (possibly
the Ca2+/H+antiporter) regulating Ca2+ efflux. Evidence in support of this mechanism is the prevention
of oxidant-induced Ca2+ release by cyclosporin A, an
inhibitor of mitochondrial pyridine nucleotide hydrolysis (30, 3 11, and by rn-iodo-benzylguanidine,a competitive inhibitor of protein mono- ADP-ribose formation
C31, 32). Furthermore, we presented evidence that
during oxidant-induced Ca” cycling (i.e., in the presence of Ca2’), there is no release of ATP from the
mitochondrial matrix. This finding demonstrates that in
liver mitochondria, the mechanism of oxidant-induced
Ca2+ efflux does not initially involve opening of a pore
[26) or modification of the adenine nucleotide carrier
into a nonselective channel (28, 33).
Cd’ Fluxes Across the FEasmd Membrane
There is compelling evidence that many oxidants interfere with Ca2+ uptake and extrusion mechanisms at
the plasma membrane level. Inhibition of Ca2+ efflux
will result in the net accumulation of Ca2+ and in a
pathological elevation of {Ca2+),. In addition, it has
become clear that chemical toxins can stimulate Ca”
entry by interacting with existing Car+ channels or by
increasing plasma membrane permeability to Ca2’. For
example, we recently observed that tributyltin, a highly
S36 Annals of Neurology Supplement
immunotoxic environmental pollutant, stimulates Ca2
influx in immature rat thymocytes, in addition to inhibiting their plasma membrane Ca2+ translocase { 341.
Tributyltin also releases intracellular Ca2+ stores, including the agonist-sensitive pool located within the
ER (Chow SC, Kass GEN, McCabe MJ, Orrenius S.
Unpublished observations). Hence, stimulation of
Ca2+ entry by tributyltin may involve a capacitative
type of mechanism similar to that suggested for Ca2+mobilizing hormones and growth factors.
Nuclear C d and Cytotoxicity
As a result of the recent finding that permeability barriers appear to exist between the nucleus and the cytoplasm, it has become clear that compartmentalized increases in Ca2+ within the nucleus may be involved in
physiological or pathological responses. We recently
showed that the toxicity of tumor necrosis factor a in
a human adenocarcinoma cell line (BT-20) is preceded
by a compartmentalized increase in Ca2 in the nuclear
region, which is associated with onset of widespread
DNA fragmentation 1353. Studies in other laboratories
have indicated that selective Ca2+ increases in the nucleus are involved in the physiological breakdown of
chromatin during erythropoiesis (36). The possibility
that nuclear Ca” increases may also occur during oxidative cell injury is currently under investigation.
Mechanisms of Ca2+-mediatedCell Death
The main evidence for the importance of Ca2+ overload in cell death comes from experiments in which
removal of extracellular Ca2+ or loading of cells with
intracellular Ca2+ chelators, such as 2-{{2-bis(carboxymethyl) amino-5-methyl-phenoxyl)-methyl}-6-methoxy8-bis-(carboxymethyl)-aminoquinoline (quin-2) or bis(o-aminophenoxy)-ethane-N,N,N’,N’-tetraaetic
(BAPTA), have been found to prevent or delay cell
death induced by various agents 11). In addition to
Ca2+ chelators, Ca2+ channel blockers have been used
to prevent Ca2+ overload and cell death in several experimental systems.
Both the duration and the extent of the increase in
{Ca”), appear to be critical for development of cytotoxicity. Even moderate increases in cytosolic Ca2+ can
impair the ability of the cell to respond correctly to
agonist stimulation and thereby interfere with cell control by hormones and growth factors. Another early
effect of sustained elevation of {Ca2+),is impairment
of mitochondrial function. In addition, more prolonged
and intense increases in cytosolic Ca2+ will result in
disruption of cytoskeletal organization and in activation
of a number of Ca2+-stimulated catabolic processes,
such as proteolysis, membrane degradation, and chromatin fragmentation (Fig 2).
Alterations of Cell Signaling Processes by Oxidunts
Calcium ions are required for many physiological functions, including control of metabolic processes, cell dif-
Volume 32, 1992
Active oxygen species
Oxidation of cellular thiols
inhibition of Ca'+-tmnslocasss
Sustained [Cd+li elevation
Activation of Cd+-dependent
catabolic pmcesses
Loss ofmembrane
Disruption of
Fig 2. Molecular events involved in the development of oxidative
cell injuy.
ferentiation and proliferation, and secretory functions
131. These Ca2+-dependent processes are tightly controlled by hormones and growth factors. Loss of the
ability to respond to such hormones and growth factors
will not only deprive the cell of a trophic stimulus
but may also, as recent evidence 1371 clearly indicates,
result in activation of a suicide process characteristic of
apoptotic cell death.
The inability of cells to respond to Ca2+-mobilizing
hormones can be the consequence of selective depletion of the intracellular agonist-sensitive Ca2+ pool by
compounds such as tBuBHQ 12 11 or bromotrichloromethane C381. Also, G,, which is the transducing G
protein for inositol 174,5-trisphosphate-generatingreceptors, has been reported to be susceptible to inactivation by oxidants 1391. Finally, prolonged increases in
[Ca2+1, may obliterate the Ca2 transients normally
evoked by physiological agonists, thereby resulting in
impairment of cell signaling.
In contrast to the situations in which oxidants appear
to block cell signaling machinery at lethal cell concentrations, it has recently become clear that low levels of
oxidants can have quite the opposite effect. For example, it has been reported that low levels of oxidants
can stimulate cell proliferation 1401. Moreover, noncytotoxic levels of prooxidants can lead to tumor promotion, as evidenced by the work from Cerutti's laboratory 1411. Tumor promotional activity has been
demonstrated for active oxygen species and organic
peroxides such as benzoyl peroxide. Because the
Ca2+-stimulated, phospholipid-dependent protein kinase (protein kmase C) has a crucial role in cell proliferation and because activation of this enzyme by phorbol esters has been associated with tumor promotion,
we investigated the effects of oxidants on protein kinase C. Exposure of rat hepatocytes to low levels of
oxidants resulted in a rapid 2- to 3-fold increase in
the specific activity of protein kinase C 1421. Only the
cytosolic form of protein kinase C displayed an increase in specific activity, whereas activity of the mem+
brane-bound form remained unchanged. This increase
in protein kinase C activity was due to oxidative modification of the protein, most likely through modification of the thioUdisulfide balance of the enzyme.
Involvement of thiol residues in the activation phenomenon was confirmed when we found that partially
purified protein kinase C from rat brain could be activated using low concentrations of oxidized glutathione
in a glutathione oxidation-reduction (redox) buffer.
Thus, it is conceivable that selective mechanisms may
be involved in low-oxidant toxicity and in carcinogenesis, whereas multiple mechanisms may be recruited in
a dose-dependent fashion and cause cytotoxicity under
conditions of pronounced oxidative stress.
The selective dose-dependent recruitment of different mechanisms for proliferation and cell death caused
by oxidants is exemplified in our recent studies on the
effects of the redox-cycling quinone, 2,3-dimethoxy1,4-naphthoquinone (DMNQ), in RINm5F cells, a cell
line of pancreatic origin: At a concentration of 10
p.mol/L, DMNQ stimulated RINmSF cell proliferation, whereas at only marginally higher concentrations
of DMNQ (30 FrnollL), cell growth was inhibited and
a portion of the cell population underwent apoptosis.
Finally, at concentrations of 100 pmollL, DMNQ
caused GSH and ATP depletion, Ca2+ overload, and
acute necrosis (Dypbukt JM, Nicotera P. Unpublished
results). In our studies with DMNQ, we observed an
induction of ornithine decarboxylase (ODC) activity in
connection with stimulation of cell proliferation. Because it is well established that protein kinase C stimulation by growth factors mediates ODC induction, it
is conceivable that the effects of the lowest DMNQ
concentrations described may be the result of protein
kinase C activation caused by oxidative stress. These
cellular effects of different levels of oxidative stress are
illustrated in Figure 3.
In addition to protein lunase C, other protein kinases have been shown to become activated under conditions of oxidative stress. For example, oxidants have
been found to activate the rat liver insulin receptor
tyrosine kinase 143, 441. More recently, exposure of
mouse epidermal cells to oxidants has been found to
stimulate phosphorylation of the ribosomal protein S6
1451 as the result of an apparently Ca2+-dependent
event. Analysis of the primary structure of a number
of protein kinases (e.g., serine, threonine, and tyrosine
kinases) has revealed substantial sequence homology
in the catalytic (lunase) domains 1461. Thus, a common
structural feature may predispose different protein kinases to activation through alterations of their thiol or
disulfide status. Consequently, modulation of kinase
activities involved in signal transduction and cellular
metabolism could constitute a mechanism by which a
noncytotoxic state of oxidative stress can have profound implications on cell division and differentiation.
Orrenius et al: Ca"
in Oxidative Injury
PKC activation
ODC activation
cell proliferation
PKC inhibition (7)
ODC inhibition
DNA fragmentation
cell death
Fig 3. Cellular effects at different levels of oxidative stress. PKC
= protein Rinase C; ODC = ornithine decarboxylase.
Cytoskeletal Alterations
One of the early signs of cell injury caused by oxidants
and a variety of other toxic agents is the appearance of
multiple protrusions (blebs) on the cell surface 1471.
The events leading to bleb formation have not yet been
fully elucidated, and several mechanisms may independently contribute to their formation. It is generally
accepted, however, that perturbation of cytoskeletal
organization and of the interaction between the cytoskeleton and the plasma membrane has an important
role. The finding that Ca2+ ionophores induce similar
blebbing, and that this process is prevented by the
omission of Ca2+ from the incubation medium, led to
the proposal that Ca2+ is involved in the cytoskeletal
alterations associated with formation of surface blebs
during cell injury 1471. Many cytoskeletal constituents,
including actin-binding proteins such as caldesmon,
gelsolin, and villin, require Ca2 to interact with other
cytoskeletal constituents. Moreover, Ca2 regulates
the function of three other actin-binding proteins directly involved in the association of microfilaments
with the plasma membrane. Among these proteins,
alpha-actinin is involved in the normal organization of
actin filaments into regular, parallel arrays; however,
in the presence of micromolar Ca2+ concentrations,
alpha-actinin dissociates from the actin filaments {48).
The two other actin-binding proteins, vinculin and
actin-binding protein, are substrates for Ca2+-dependent proteases C49J. Thus, an increase in {Ca2+},
to micromolar levels will result in the proteolysis of
these two polypeptides.
Recent work has provided evidence for involvement
of Ca2+in the toxic alterations of actin microfilaments
and actin-binding proteins. For example, incubation of
human platelets with the redox-active quinone menadione resulted in dissociation of alpha-actinin from the
whole cytoskeleton and in proteolysis of actin-binding
protein 1501. These changes were largely prevented
in cells preloaded with the intracellular Ca” chelator,
quin-2. Furthermore, immunocytochemical investigations revealed that dissociation of alpha-actinin from
the actin filaments may be responsible for bleb formation during oxidant injury {5l). Other studies of canine
heart during development of ischemia and reperfusion
injury revealed a progressive loss of vinculin staining
S38 Annals of Neurology
along the lateral margin of myocytes {52]. This loss
was associated with the appearance of subsarcolemmal
blebs and breaks in the plasma membrane. Because
vinculin is a substrate for CaZt-dependent proteases,
and because the cytosolic Ca2+ concentration during
ischemia and reperfusion increases well above the level
necessary for protease activation, it appears that Ca2+activated proteases may be responsible for the loss of
Microtubule structure and distribution are also controlled by Ca2+. In addition, turnover and distribution
of microtubules are controlled by microtubule-associated proteins (MAPS), whose activities are modulated
by Ca2+- and calmodulin-dependent protein kinase
1531. Other studies demonstrated that microin jection
of Ca”/calmodulin complexes in 3T3 fibroblasts results in complete depolymerization of microtubules
that is spatially limited to the site of injection 1541.
Although depolymerization of microtubules during oxidant injury has been reported {55}, possible involvement of Ca2’ in this process has not been clarified.
Little is also known about the role of Ca” in toxic
modifications of intermediate filaments. In fact, our
knowledge of the composition of intermediate filaments and of the role of these structures in cell physiology is still fragmentary. It has been shown that several
intermediate filament proteins, including vimentin and
cytokeratins, are substrates for a Ca2+-activated protease; however, direct evidence is still missing regarding
a physiological role for proteolysis in the control of
intermediate filament function, and for a possible contribution of intermediate filament cleavage to the cytoskeletal alterations occurring during cell injury.
Cd +-dependentDegradative Enzymes
Catabolism of phospholipids, proteins, and nucleic
acids involves enzymes, most of which require Calf
for activity. Ca2+overload can result in sustained activation of these enzymes and in degradation of cell constituents, which may ultimately lead to cell death.
Phospholipases catalyze hydrolysis
of membrane phospholipids. They are widely distributed in biological membranes and generally require
Ca2+ for activation. A specific subset of phospholipases, collectively known as phospholipase A,, have
been proposed to participate in detoxication of phospholipid hydroperoxides by releasing fatty acids from
peroxidized membranes 1561. Phospholipase activation, however, can also mediate pathophysiological reactions by stimulating membrane breakdown or by
generating toxic metabolites. One of the best known
examples for phospholipase-mediated toxicity is probably that caused by phospholipase-based snake and bee
venoms. Therefore, phospholipase activation has been
proposed as an important mechanism of cell death.
Supplement to Volume 32, 1992
Phospholipase A, is Ca2+- and calmodulin-dependent
and thus is susceptible to activation following an increase in cytosolic Ca” concentrations. Hence, it has
been suggested that a sustained increase in cytosolic
Ca2+can result in enhanced breakdown of membrane
phospholipids and, in turn, in mitochondrial and cell
damage. However, although a number of studies indicated that accelerated phospholipid turnover occurs
during anoxia or toxic cell injury [5?1, the importance
of phospholipase activation in development of cell
damage by oxidants remains to be established.
overload can trigger endonuclease activation. The Ca2+
ionophore A23 187 stimulates DNA fragmentation in
thymocytes, and characteristic endonuclease activity in
isolated nuclei is dependent on Ca2+E6ll. In addition,
Ca2 -mediated endonuclease activation appears to be
involved in the cytotoxicity of TCDD and tributyltin
in thymocytes 134, 62). Although Ca2+-dependent endonuclease activation has been most extensively studied in thymocytes, it appears that this process may also
be important in a variety of other tissues. For example,
we identified a constitutive endonuclease in liver nuclei
activated by submicromolar Ca2 concentrations when
intact nuclei are incubated in the presence of ATP to
stimulate Ca2+ uptake 16 1). Endonuclease activation
has also been implicated in damage to macrophages
caused by oxidative stress [631. Although the responsible endonuclease requires Ca” for activity, its regulation appears to be more complex and probably involves
additional signals. Recent work in ours and other laboratories indeed indicates that the ability of the endonuclease to cleave DNA is dependent on the chromatin
superstructure {641.
Ca2+ overload may also stimulate other enzymatic
processes that result in DNA damage. Elevated Ca2+
levels can lock topoisomerase I1 in a form that cleaves,
but does not religate, DNA, and topoisomerase IImediated DNA fragmentation has been implicated in
the cytotoxic action of some anticancer drugs {65). In
addition, generation of DNA single-strand breaks in
cells exposed to oxidative stress can also involve a
Caz+-dependent mechanism [bbl.
The chemical mechanisms by which active oxygen
species, such as the hydroxyl radical (‘OH), can cause
DNA strand breaks are relatively well established. For
example, electron spin resonance spectroscopy, coupled to continuous flow systems, has allowed direct
observation of radicals formed on DNA model substrates during oxidative fragmentation of the nucleic
acid sugar-phosphate backbone by ‘OH 167). However, although it might be expected that similar mechanisms of DNA strand breakage are responsible for the
DNA fragmentation observed in cells exposed to oxidative stress, there exists a growing body of evidence
to suggest that during oxidative stress, DNA fragmentation can also be brought about via mechanisms not
invo!ving direct attack by ‘OH, but via activation of
calcium-dependent mechanisms. For example, studies
by Cantoni and colleagues E6Sl and Dypbukt and associates 166) demonstrate that DNA single-strand breakage can be inhibited with quin-2, an intracellular calcium chelator.
The role of Ca2+ in DNA damage during oxidative
stress and activation of endonuclease-mediated cleavage has recently been the subject of intense investigation. The currently accepted hallmark of endonuclease-mediated DNA fragmentation, and hence
During the past 10 years, involvement of
nonlysosomal proteolysis in several cell processes has
become progressively clear. Proteases that have a neutral pH optimum include the ATP- and ubiquitindependent proteases and the calcium-dependent proteases, or calpains. Calpains are present in virtually all
mammalian cells and appear to be IargeIy associated
with membranes in conjunction with a specific inhibitory protein (calpastatin). The extralysosomal localization of this proteolytic system allows the proteases to
participate in several specialized cell functions, including cytoskeletal and cell membrane remodeling, receptor cleavage and turnover, enzyme activation, and
modulation of cell mitosis.
Cellular targets for these enzymes include cytoskeletal elements and membrane integral proteins [Sol.
Thus, activation of Ca” proteases has been shown to
cause modification of microfilaments in platelets and
to be involved in cell degeneration during muscle dystrophy, as well as in development of ischemic injury in
nervous tissue. Studies from our laboratory suggested
involvement of Ca2+-activated proteases in oxidant injury in liver {%, 591. Although the substrates for protease activity during cell injury remain largely unidentified, it appears that cytoskeletal proteins may be a
major target for Ca2+-activated proteases during chemical toxicity.
During physiological cell death, a
suicide process is activated in affected cells, which is
known as apoptosis, or programmed cell death. Several
early morphological changes occur within apoptotic
cells, including widespread plasma and nuclear membrane blebbing, compacting of organelles, and chromatin condensation [60}.The most reliable and characteristic marker for this process is activation of a
Ca2+-dependentendonuclease, which results in cleavage of cell chromatin into oligonucleosome-length fragments. Endonuclease activation has been implicated in
the killing of target cells by cytotoxic T lymphocytes
and natural killer cells, as well as in thymocytes exposed to glucocorticoid hormones.
The results of several recent studies show that Ca”
Orrenius et aI: CaZ+ in Oxidative Injury 539
apoptosis, is appearance of a characteristic “ladder” of
discrete oligonucleosome-sized bands when the DNA
fragments are separated on an agarose gel. This fragmentation is believed to result from the enzyme’s preference for DNA cleavage at the internucleosomal
linker DNA sites. In contrast, fragmentation caused by
’OH might be expected to occur at random sites on
the sugar-phosphate backbone, resulting in generation
of DNA fragments of a continuous, rather than discrete, size range. However, much of the evidence suggesting that ’OH attacks DNA at random sites is from
radiation studies, in which the radical is generated at
relatively high concentrations in free, bulk solution via
radiolysis of water molecules. In contrast, results from
studies employing transition metal-catalyzed ‘OH formation suggest that DNA damage may occur specifically at metal binding sites [69, 701. Although many
metal ions, including iron, are known to form complexes with DNA, it appears that, at least quantitatively, copper is the most important transition metal
ion associated with DNA in cell nuclei 1711. Copper,
along with calcium, appears to have a key role in maintaining the higher order structure of chromatin, resulting in organization of DNA (along with its associated histones) into large loops anchored to matrix
proteins at specific sites E l l , 721. Clearly, this mechanism gives rise to the following questions: (1) Is this
pool of copper able to interact with, for example, the
superoxide and hydrogen peroxide generated during
the redox cycling of such reagents as DMNQ (or indeed, “leaks” from the mitochondrial respiratory chain)
and induce oxidative DNA damage; and (2) can transition metal-catalyzed oxidative damage to DNA be
considered truly random if it occurs only at the metal
binding sites? Findings from studies employing model
systems demonstrate that copper bound to DNA can
indeed interact with hydrogen peroxide and induce oxidative damage on the DNA [73}; because binding of
copper ions to DNA is known to occur preferentially
at guanine residues, damage is expected to be sitespecific.
In support of the site-specific model of damage,
Sagripanti and Kraemer [69] demonstrated that copper-mediated oxidative damage to plasmid DNA occurs preferentially at polyguanosine sequences (i.e.,
sites at which copper ions are expected to bind). In the
cell nucleus, any specificity in the sites of oxidative
damage would be expected to be determined also by
the binding of copper ions to protein (e.g., in the formation of DNA anchor points to the nuclear matrix)
17 1, 721. However, the biological significance of copper-mediated site-specific DNA damage has yet to be
fully evaluated, as well as the relative importance of
free radical versus enzymatic mechanisms of strand
breakage. Thus, further investigations are required before the relative significance of enzymatic and free radi-
cal mechanisms of DNA fragmentation during oxidative stress can be evaluated.
It appears safe to conclude that calcium ions have an
important role in development of oxidative cell injury.
Recent research reveals some of the biochemical mechanisms by which intracellular Ca2+ overload can cause
cytotoxicity; however, the relative importance of the
various Ca2+-dependent processes involved in cell
death needs to be clarified further. Finally, it should
be emphasized that different levels of oxidative stress
may exert different effects on the cell (i.e., proliferation at low levels and death by apoptosis or necrosis at
considerably higher levels).
Work reported from the authors’ laboratory was supported by grants
from the Swedish Medical Research Council (Proj. no. 03X-2471).
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