Download Intracellular calcium: friend or foe?

ClinicalScience (1987)72,l-10
1
EDITORIAL REVIEW
Intracellular calcium: friend or foe?
ANTHONY K. CAMPBELL
Department of Medical Biochemistry, Universityof Wales College ofMedicine, Cardifi U.K.
Introduction
“Oh, you’re interested in calcium? Isn’t that something to do with bones and teeth?” This is a typical
layman’s response on learning that one is interested
in the biological role of calcium. Until relatively
recently a similar reaction might have been
expected from many clinical colleagues, though
they would, of course, be aware of the importance
of dystrophic or metastatic tissue calcification in
several pathological conditions. The realization that
calcium outside the cell plays a key role in maintaining the skeletal structures of metazoans can be
traced back to the nineteenth century. The idea that
calcium within the cells of soft tissues alters their
behaviour, either to activate or to injure them, is
also nearly a century old [l]. The investigation and
direct measurement of intracellular Ca2+ is now
playing a vital part in the experimental investigation
of disease processes [l-41. These include diseases
where tissues have been attacked by components of
the immune system such as diabetes, rheumatoid
arthritis, multiple sclerosis and thyroid disease.
They include conditions such as coronary artery
disease where intracellular Ca2+plays a key role in
the physiology of healthy smooth and cardiac
muscle. Several genetic abnormalities also involve
disturbances of intracellular processes capable of
regulation in healthy cells by Ca2+, for example
secretion in cystic fibrosis or muscle contraction in
muscular dystrophy, whereas abnormalities of cell
division and tissue growth, for example in embryogenesis and in cancer, may interfere with more longterm regulatory mechanisms dependent on
intracellular Ca2+:
In 1883 Ringer [5] demonstrated that external
calcium was necessary for the normal contraction of
frog heart. Other distinguished physiologists such as
Correspondence: Dr A. K. Campbell, Department of
Medical Biochemistry, University of Wales College of
Medicine, Heath Part, Cardiff CF4 4XN, U.K.
Straub, Mines, Loeb, Locke, Herbst and Overton
working in the early years of this century showed
similar requirements for extracellular calcium in egg
development, in cell adhesion, in the transmission
of impulses from nerve to muscle, and in the actions
of adrenaline and cardiac glycosides (see [l]for
references). Some of these experiments were even
interpreted as evidence of a role for intracellular
calcium in the activation of the cell concerned.
Several of these workers also realized that, under
certain conditions, calcium was necessary for cell
necrosis. As early as 1928 Pollack [6] tried to
measure calcium inside a living amoeba by microinjecting into it the red dye alizarin sulphonate,
claiming that pseudopod formation was provoked
by a rise in internal calcium close to the site of the
membrane event. Furthermore, some 50 years ago
the American physiologist Lewis Heilbrunn [7]
documented much convincing evidence for intracellular calcium as a ‘universal’cell regulator.
Why then has it taken so long, particularly in
medicine, for the importance of intracellular
calcium as a mediator of cell activation and cell
injury to be recognized? Four possible reasons can
be identified for scepticism about the ‘calcium
hypothesis’, a scepticism which was to be found in
many biologists until the early 1960s. Firstly, the
cell and molecular biology of many phenomena
involving cell activation were often poorly defined.
Even when the proteins responsible had been purified, for example actin and myosin from muscle,
they failed to respond physiologically to calcium.
Secondly, how could a cation, Ca2+,acting at low
concentrations provide enough energy for cell activation? Thirdly, the extremely low concentration of
free CaZ+in the cell cytoplasm compared with that
outside the cell was not generally realized. Fourthly,
the experimental approach necessary for definitive
identification of intracellular Ca2+ as the mediator
of a cellular event had not been developed.
The rightful place of intracellular Ca2+ in cell
physiology and pathology is now fully recognized as
2
A. K . Campbell
a result of the identification and characterization of
the initiators of cell activation and cell injury,
together with the discovery of Ca2+ binding proteins responsible for transforming a change in the
concentration of intracellular Ca2+ into a cellular
event. The energy for these events comes not from
Ca2+ itself but from cell metabolism. Furthermore,
indicators for intracellular free Ca2+ have been
developed, together with other methods, enabling
chemical events to be quantified and localized
whilst the cell remains intact. The resolution of
these conceptual and experimental problems now
provides the basis for seeing whether changes or
disturbances in intracellular Ca2+ are involved in
the molecular mechanisms underlying disease processes.
Yet there is still a problem: Ca2+is not the only
intracellular mediator of cellular events. What
about cyclic AMP and cyclic GMP, and their ability
to activate protein kinases within the cell [8, 9]?
What is the significance of the activation of other
protein kinases, such as protein kinase C by diacylglycerol [lo-121, and the concomitant release of
inositol phosphates from membrane phospholipids
which occurs in many cells within seconds of their
activation [13,14]? There have been many attempts
over the past 20 years to rationalize the relative significance of these intracellular regulators [ l ,
15-17]. Much emphasis has been placed on their
biochemistry, the investigation of their mode of formation, action and interactions with organelles
being based mainly on analyses from broken cell
preparations. Important as these conventional
‘grind and find’ biochemical approaches have been,
and will be, it is my view [ l ] that the full comprehension of the role of intracellular Ca2+,together with
that of the other intracellular messengers involved
in cell physiology and pathology, will only be
achieved by a re-examination of the cell biology of
the phenomena concerned. In particular, the
quanta1 or stochastic nature of individual cell
responses, together with differences in the location
and function of events within them, must be taken
into account.
Cell biology
Eukaryotic organisms depend for their survival and
reproduction on the ability of their cells to change
their behaviour in response to physical or chemical
stimuli. Cellular events are provoked by primary
stimuli and modified by secondary regulators
(Table 1).Thus action potentials in the cell membrane activate muscle contraction and neurosecretion, hormones and neurotransmitters activate
endocrine or exocrine secretion as well as intermediary metabolism, growth factors and components of the immune system can provoke cell transformation and cell division, and contact of the egg by
sperm results in the activation of many intracellular
processes required for fertilization. The efficacy of
the primary stimulus can be modified, in terms of its
time of onset, duration or magnitude, by secondary
regulators. A further complicating factor is the
ability of some primary stimuli to activate more
than one event within a cell, or for a cell to do different things when responding to different stimuli.
For example, thrombin provokes both platelet
aggregation and secretion of ADP. On the other
hand, bacteria coated with complement fragment
C3b activate phagocytosis in neutrophils, whereas
fragment C5a provokes chemotaxis and activates
secretion.
A significant characteristic of many phenomena
involving cell activation, often ignored by those
interested in molecular mechanisms, is the fact that
they can involve ‘thresholds’within individual cells.
Thus a heart cell beats or it does not; the cell body
TABLE1. Some examples of cell activation provoked by a rise in intracellular free Ca
Phenomenon
Cell movement
Secretion from vesicles
Cell aggregation
Cell transformation
Intermediary metabolism
Example
Muscle: skeletal
smooth
cardiac
Amoeboid: neutrophil chemotaxis
Ciliate: Paramecium reversal
Nerve terminal
Endocrine: mast cell
Exocrine: pancreas
Platelet
Lymphocyte
Oocyte maturation: starfish
Egg fertilization
Glycogenolysis in liver
+
Primary stimulus
Secondary regulators
Acetylcholine
Noradrenaline
Action potential
C5a
Touch
Eicosanoids ( + , - )
Adrenaline ( + )
Eicosanoids ( +, - )
-
Action potential
Antigen of IgE
Cholecystokinin
Thrombin
Antigen
I-Methyladenine
Sperm
Adrenaline (a)
Adenosine ( - )
Adrenaline ( - )
?
Eicosanoids ( + , - )
T-cells/macrophages
?
?
Insulin
Intracellular ca1ciurn:friend or foe?
of a neuron in the brain will only generate an action
potential when the necessary excitatory transmitters
are able to combat the inhibitory ones; a platelet
aggregates or it does not; a cell in a luminous animal
flashes or is invisible. The relationship between the
dose of stimulus to the time course and magnitude
of response in a cell population therefore depends
on the time of onset and duration of the
phenomenon in each cell, together with the number
of cells capable of being ‘switched on’ under a particular balance of primary and secondary regulators. Thus adrenaline speeds up the response of an
individual myocyte to the primary stimulus, the
action potential, and increases the magnitude of the
contraction itself. In contrast, during platelet activation secretion of ADP provoked by thrombin acts
to provoke a cascade or chain reaction of aggregation in other platelets, thereby accelerating blood
clot formation. A similar conceptual framework
based on thresholds in individual cells can help to
explain how cells respond to drugs and pathogens.
For example, the killing of cells by viruses, bacterial
toxins, T-cells and the terminal part of the complement pathway require a critical threshold point
beyond which irreversible cell damage occurs, but
before which the cell may be able to recover if it can
remove the potentially lethal attacking agent in time
[ l , 3,18-201.
A rise in cytoplasmic free Ca2+ is ideally suited
to provoke these threshold events. In resting cells
the intracellular free Ca2+ is about 0.1 pmol/l,
whereas that outside is around 1 mmol/l. There is
thus a 10000-fold concentration gradient of Ca2+
across the cell membrane with a negative membrane potential of some 50-100 mV also pulling
Ca2+ into the cell. This large electrochemical Ca2+
gradient is maintained by a Ca2+ pump in the cell
membrane. The energy utilized by this pump,
through MgATP hydrolysis, is considerably less
than that used by the sodium pump since the passive permeability of the membrane to CaZ+is some
10-100 times lower than that to monovalent
cations. Since the total cell calcium is some 2-15
mmol of calcium/l of cell water [ l ] this means that
>99.9% of the cell’s calcium is bound within the
cell: in the endoplasmic reticulum, the mitochondria, specialized vesicles, with some probably in the
nucleus. Estimates for the calcium bound to the
outer surface of the cell vary from 5 to 50%.
A small increase in the permeability of the cell
membrane to Ca2+,or a small fractional release of
CaZ+from an internal store, induced by a physiological stimulus or evoked by a toxin or pathogen,
will lead to a large fractional rise in cytoplasmic free
Ca2+. The resulting cytoplasmic free Ca2+ is
usually some 10-100 times that in the resting cell.
Furthermore, Ca2 buffering systems, such as mito+
3
chondria within the cell, are able to localize any
change in internal free Ca2+ to a particular region
of the cell. Activation of an individual cell will only
occur if the rise in intracellular free Ca2+ is sufficient, and in the necessary place, to activate the
Ca2+dependent mechanism within the cell. Secondary regulators can then act by altering the magnitude and localization of the Ca2+ transient, or the
mode of action of Ca2+,or by a mechanism independent of Ca2+,thereby increasing or decreasing
the number of cells switched on at any given time
together with the extent of their response.
A small disturbance in the cell membrane caused
by a pathogen will cause Ca2+to flood into the cell
[ l , 3, 7, 201. This leads not only to the activation of
Ca2+dependentpathways but also to disturbances
in cell structure and function, and even death. The
normal physiology of cells therefore depends critically on careful control of the electrochemical
gradient of Ca2+across both the cell membrane and
the membranes of intracellular organelles. Physiological and pathological effects on the cell membrane will inevitably lead to alterations in this
gradient. The crucial question is whether these
changes in intracellular Ca2+ are a cause, as
opposed to a consequence, of cellular changes
induced by the stimulus or pathogen. The only way
to answer directly this question is to measure the
concentration of free Ca2+ in the living cell and to
correlate any changes with the time course and
magnitude of the cellular event. In particular, it is
necessary to show that any rise in intracellular free
Ca2+occurs before the onset of this event, and that
prevention of the Ca2+ rise prevents activation or
injury to the cell.
Evidence for intracellular Ca2+as a cell activator
Since Ringer’s pioneering experiments 100 years
ago there have been many attempts to provide the
necessary unequivocal evidence that a change in
free Ca2+within the cell is responsible for mediating its response to a primary stimulus. These
include effects of manipulation of external CaZ+
and Ca2+ ionophores, effects of so-called Ca2+
antagonists, measurement of Ca2+ fluxes, and
effects of Caz+ on isolated intracellular systems [ 11.
None has proved definitive; several have led to
spurious conclusions [21-231. There are only two
direct experiments: to microinject Ca2+close to the
location of the process activated within the cell, or
to measure directly the concentration of free Ca2+
in the live cell and correlate this with the cell
response after exposure to the stimulus.The former
will show whether Ca2+ is sufficient, whereas the
latter will show whether a rise in intracellular Ca2+
is both necessary and sufficient for cell activation.
4
A. K . Campbell
The latter will also show whether secondary regulators modify the cell response by affecting the Ca2+
rise, the action of Ca2+,or by a mechanism independent of Ca2+. Once these direct experiments
have been carried out the source of the Ca2+ can
then be identified, together with its mode of action.
A rationalization of the role of Ca2+ in relation to
the other intracellular regulators, including Ca2+
independent mechanisms, completes the picture.
How then can we measure the concentration of
free Ca2+ in a live cell? An indicator is required
that is specific for Ca2+ over the intracellular
physiological and pathological range (about 20
nmol-100 ,umol/l), is quantitative, and is capable
ultimately not only of detecting and quantifying
changes in free Ca2+ in a particular cell but also
where in the cell the change occurs. Furthermore,
the indicator must be capable of incorporation into
the cell without serious disruption to cell structure
and function, and ideally without altering the Ca2+
balance of the cell.
Five methods for measuring intracellular free
Ca2+ have been developed over the past 20 years
(Fig. 1), using chemiluminescence, absorbance,
fluorescence, nuclear magnetic resonance or
microelectrodes [1,2, 24-37]. Between 1967 and
1981 most of the important new information about
intracellular free Ca2+came from using the chemiluminescent photoproteins aequorin and obelin [2,
24,25,33,34]. The ingenious invention by Tsien of
the fluorescent tetracarboxylate Ca2+ indicators
quin 2, indo-1 and fura-2 [27-29,35,36] has made
widely available a method for monitoring free Ca2+
in small cells. Nuclear magnetic resonance indicators [32] offer potential for future studies in whole
organs, though the accumulation time for a detectable nuclear magnetic resonance spectrum may be
long relative to that of the Ca2+ change. It will not
be easy to apply this method to individual cells, or
to the distribution of free Ca2+ within them.
Microelectrodes have been useful in the investigation of Ca2+release from internal stores by inositol
trisphosphate [38].The two most popular methods,
and the methods of choice, are the photoproteins
and the fluors. But how can one get the indicator
through the cell membrane into the cytoplasm?
The Ca2+activated photoproteins aequorin and
obelin have a molecular weight of about 20000 and
can be injected easily into giant cells such as
barnacle muscle or squid axons [2, 24, 25, 391. In
skillful hands they can be injected into small numbers of mammalian cells including oocytes, fibroblasts, hepatocytes and myocytes [40-421. Other
methods have had to be developed to get them into
large numbers of platelets [43], phagocytes [44,45]
and tissue culture cells [46,47]. We have developed
three methods over the past 10 years to achieve
this, based on reversible cell swelling, erythrocyte
ghost-cell fusion and release from micropinocytotic vesicles [2, 22, 33, 44, 451. Others have also
developed methods based on reversible cell swelling and special permeabilization media [46,47]. In
contrast, the fluorescent indicators invented by
Tsien (Fig. 1)are added to cells as their membrane
permeant ester. Once inside, esterases in the cytoplasm hydrolyse them back to the tetracarboxylate
which is now impermeant. From a few micromoles
outside, the cell is able to accumulate up to millimolar concentrations of the indicator inside.
Though these fluors have now been used widely in
many cells types their precise intracellular location
has rarely been fully documented. Other complications can be ester hydrolysis, occurring extracellularly during loading in some cells, leakage out of
cells during cell activation or injury, binding to
transition metals such as Zn2+,and buffering of the
intracellular Ca2+,even when using fura-2, which is
more sensitively detectable than quin 2. Nevertheless, photoproteins have three particularly good
points. Firstly, because they have to bind three
Ca2+ to chemiluminesce the measured signal is
related approximately to the cube of the free Ca2+
concentration. There is thus an amplification factor
enabling smaller Ca2+changes to be detected than
is possible with the fluors, especially if these occur
only in one part of the cell. Secondly, they can be
used at concentrations which do not significantly
buffer the intracellular Ca2+. Thirdly, they are
much more suitable for studying free Ca2+ in
injured cells since their high molecular weight
means that they leak out of cells less readily than
the fluors [3,34].The two groups of indicators thus
complement each other. Both have enabled local
changes in intracellular Ca2+and Ca2+gradients to
be identified during egg fertilization [48, 491, cell
division [50] and in resting cells between the
nucleus and cytoplasm [5I], using image intensification to visualize the luminescent signals.
A number of discoveries illustrate how important
measurement of intracellular free Ca2 has been,
and will be in the future. Firstly, it has enabled four
broad ranges of free Ca2+ in cells to be identified:
(a) resting cells, 20-450 nmol/l, depending on the
cell type and indicator used [2]; (b)cells activated by
electrical o r chemical stimuli, 1-5 pmol/l; (c)reversibly injured cells, 5-30 ,umol/l; (d) irreversibly
injured cells on their way to cell death, > 50 pmol/l
Ca2+.These provide the range of affinity constants
necessary in the Ca2 binding proteins responsible
for mediating the effects of intracellular Ca2+.
Secondly, for the first time primary stimuli either
dependent or independent of a rise in intracellular
Ca2+ have been clearly identified [22, 35, 36, 52,
531, even though the latter may need extracellular
+
+
Intracellular ca1cium:friend orfoe?
5
5. Ca2+ microelectrode
1. Photoproteins
Arsenazo 111
o~--'~~~o
0
Antipyrylozo 111
0
Murexide (ammonium purpurate)
3. Fluorescent indicators
(Cod
Chlortetracycline
Quin 2
40-R
0
4. Nuclear magnetic resonance indicators
F-bis(o-aminophen0xy)ethanetetra-pcetate (FBAPTA)
FIG.1. Indicators of intracellular free Ca2+.
Ca2+.Phenomena requiring a rise in intracellular
Ca2+ include muscle contraction, secretion from
intracellular vesicles, chemotaxis, egg fertilization,
certain types of cell transformation, cell growth and
division, and some examples of activation of intermediary metabolism (Table 1).In contrast, the acute
response of vertebrate retinal rods and cones to
light does not appear to require an increase in intracellular Ca2+[54] as was once thought; cyclic GMP
is the mediator. In neutrophils activation of reactive
oxygen metabolite production by at least some
phagocytic stimuli also does not require a rise in
intracellular free Ca2+,whereas that activated by
chemotactic agents does [22, 23, 52, 531. Thirdly,
correlation of the magnitude of rises in intracellular
free Ca2+ in fertilized eggs, maturing oocytes [55]
and platelets [56, 571 has provided evidence for
Ca2+ provoking thresholds for cell activation.
Fourthly, secondary regulators have been identified
which act by altering the intracellular Ca2+
6
A . K . Campbell
transient provoked by the primary stimulus. For
example, adrenaline increases the magnitude, and
speeds up the time course, of the Ca2+ change in
heart muscle cells [58]. Adenosine on the other
hand acts as an inhibitor of the Ca2+ dependent,
but not the Ca2+ independent, stimuli in neutrophils and may alter the Ca2+ change [59]. Finally,
the few direct measurements of intracellular free
Ca2+ during cell injury have established whether a
free Ca2+ rise is a cause or a consequence of cell
injury. In the poisoned myocyte, the free CaZ+rise
appears to occur well after morphologically identifiable cell injury [60]. In contrast, in cells attacked by
the membrane attack complex of complement
(CSb-9,,) a rise in intracellular free Ca2+ precedes
both cell activation and cell lysis [3, 18, 19, 34,
61-63]. Furthermore, we have shown that
nucleated cells can protect themselves against complement attack by removing the potentially lethal
complex from the cell surface [ 19,621, a mechanism
which requires a rise in cytoplasmic Ca2+ (611
occurring at a concentration some ten times higher
than that in physiologically activated cells. Inhibition of the Ca2+pump by trifluoperazine increases
considerably the intracellular Ca2+rise [23].
Crucial as is measurement of intracellular free
Ca2+ it is only the beginning of the complete
characterization of the molecular basis of the role of
Ca2+ in cell activation and cell injury. Having
identified that a rise in intracellular Ca2+is necessary for the cellular event, three questions arise.
Where does the Ca2+ come from? How is it
released, and then removed during cell recovery?
How does the Ca2+act?
Sources of the rise in cytoplasmic free Ca2’
In principle, the Caz for cell activation could come
from outside the cell through an increase in membrane permeability, a release from internal stores,
or an inhibition of Ca2+ efflux. The latter appears
to be rare as the primary cause of a rise in intracellular CaZ+,whereas the other two often accompany
each other, even when the bulk of the Ca2+comes
from internal release. Although the possible existence of membrane Ca2+ ionophores has not been
ruled out, the two major mechanisms for increased
Ca2+entry into an activated cell are via opening of
voltage sensitive Ca2+channels or receptor operated Ca2+ channels [64]. The former are found at
nerve terminals, in cardiac and smooth muscle and
in several invertebrate excitable cells. Much has
been learnt about the ‘threshold’ opening of Single
channels by using patch clamping [64]. Receptor
mediated CaZ+channels are presumed to exist in
non-excitable cells, as well as in smooth muscle
+
opened by noradrenaline [65]. However, their
molecular basis has yet to be defied.
Although the sarcoplasmic reticulum in muscle
was the first regulated intracellular CaZt store to be
identified [l,661, much attention in the 1960s and
1970s focused on mitochondria as a potential
source of Ca2+for cell activation. The evidence was
based mainly on experiments using Ca2+ concentrations in vitro some 100-1000 times those in the
resting cell. Attention has recently refocused on the
endoplasmic reticulum, although several mitochondrial enzymes are regulated by Ca2+[67],and mitochondria probably do act to buffer and localize
intracellular Ca2+ changes during cell activation
and cell injury. The key to unravelling the mystery
of how intracellular Ca2+ stores could be released
into the cytoplasm was the discovery, originally
made by the Hokins in the 1950s [68], that activation of many small cells leads to a breakdown of
inositol phospholipids (for reviews see [13, 14,691).
The most important product of a phosphodiesterase, cleaving at the same site as phospholipase C, is
the release into cytoplasm of inositol 1,4,5-trisphosphate (IPJ. This then interacts with an internal
‘receptor’. It is possible that this is a ‘ G (GTP binding) protein [70,71] analogous to the family of proteins apparently involved at the cell membrane in
the activation and inhibition of adenylate cyclase by
hormones, ion channel activation, and possibly in
activation of the phospholipase itself. The result is a
rapid release of Ca2+from a vesicular store. Other
inositol trisphosphate isomers and polyphosphates
have been found, but have, as yet, unidentified
functions. The IP, mediated internal release of CaZ+
has been found in many eukaryotic cells including
exocrine pancreas, platelets, fly salivary gland,
neutrophils, smooth muscle and the hepatocyte.
There are thus three mechanisms by which Ca2+
can be released from the endoplasmic reticulum
depending on the cell type: electrical activation in
skeletal muscle, Caz+ induced release in cardiac
muscle, the IP, induced release in non-excitable
cells. The release of CaZ+ into the cytoplasm is
usually followed by an increase in CaZ+influx, if
this has not already occurred before internal
release, and an inhibition of Ca2+ efflux. These
enable the free Ca2+ rise to be sustained, minimizing loss of CaZ+from the cell. Three mechanisms of CaZ+ efflux have been identified in
eukaryotic cells: a Ca2+-MgATPase, a Na+-Ca2+
exchange (one CaZ+per two to three Na+) and a
Ca2+-H+ exchange. The first is mainly responsible
for maintaining the Ca2+ gradient in resting cells.
The second, only found in some mammalian cells,
comes into play when the elevation in cytoplasmic
free Ca2+ is prolonged in cell activation or cell
injury.
Intracellular ca1cium:friend or foe ?
How intracellular Ca2 acts
+
7
in intracellular Ca2 can occur in cells under pathological conditions well before any irreversible
damage occurs. Accumulation of intracellular Ca2
has been proposed to provoke not only cell death
induced by T-cells and biological and chemical
toxins, but also to be important in non-lethal
irreversible cell damage induced by infectious
agents, genetic abnormalities such as sickle cell
anaemia, cystic fibrosis and muscular dystrophy,
and attack by components of the immune system
(see [ l ] for references). Some pathogens such as
paramyxoviruses, bacterial toxins, perforins from
T-cells, and the membrane attack complex of complement appear to allow Ca2+into the cell before
formation of pores which let larger molecules leak
out of the cell [3, 18-20, 631. The rise in free Ca2+
becomes 'pathological' once above about 10 pmol/l.
The result is not only activation of physiological
Ca2+dependent mechanisms but also cellular processes not normally affected by Ca2+under physiological conditions. The latter include: membrane
events, such as cell shape changes, membrane
vesiculation and Ca2 pump activation; sealing of
gap junctions and ionic conductance changes; metabolic changes including activation of enzymes such
as proteases, nucleases, phospholipases and transglutaminases or inhibition of adenylate cyclase;
organelle disruption including mitochondrial overload and chromatin breakdown; precipitation of
Ca2+salts and protein denaturation. A dramatic fall
in ATP is also an inevitable consequence of Ca2+
pump activation and mitochondrial disruption.
However, not all of these changes are deleterious.
Removal of potentially lethal pathogens, such as
viruses, bacterial toxins, perforins, or the membrane attack complex of complement, from the cell
surface via vesiculation or endocytosis can enable
the cell to protect itself before the critical
'threshold' point for cell death occurs. We have pro+
The discovery of troponin C, by Ebashi [72] in the
early 1960s, as the Ca2+binding protein in skeletal
muscle responsible for unleasing the primed actomyosin contractile apparatus was the scenario for
the current dogma of how a rise in intracellular
Ca2+ triggers cell activation. It acts by binding to
special Ca2 binding proteins which then mediate
cell activation. In many cases, though not in skeletal
muscle contraction, the Ca2+binding protein then
activates protein kinases which phosphorylate
membrane bound and soluble proteins, for example
the M, 20000 myosin light chain. The ubiquitous
occurrence of calmodulin in eukaryotic cells, the
occurrence of calmodulin binding proteins, e.g. caldesmons, its highly conserved structure, and its
ability to activate kinases and other enzymes in vitro
has excited much interest in its role in cell activation
[73, 74) since it was first discovered in the late
1960s (see [ l ] for references). However, direct
evidence in live cells for its regulatory role in
mediating activation is still poor. Further, the calmodulin paradox, whereby opposing enzymes, e.g.
adenylate cyclase and cyclic AMP phosphodiesterase, can both be activated in vitro by Ca*+-calmodulin, needs to be resolved. Many other, higher
molecular weight, Ca2 binding proteins have now
been found (Table 2), particularly associated with
the cytoskeleton. These may be important in gel-sol
transitions, together with membrane events, during
cell activation. Some Ca2+proteins work by derepressing enzyme or protein systems in the cell (e.g.
in muscle contraction); others activate their system
(eg. phosphorylase kinase).
+
+
lntracellular Ca2 in cell injury - a foe?
+
It is well known that nectotic cells contain much
more Ca2 than normal healthy cells, yet increases
+
+
+
TABLE
2. Some calcium binding proteins associated with cell activation
For references see [l],[8], [72-741, [76].
Protein
Approx. mol. wt.
Cell source
Tropin C
Leiotonin C subunit
17 000
20 000
Skeletal and cardiac muscle
Smooth muscle
Calmodulin
Actin binding proteins (a-actinin,
actinogelin, fragmin, gelsolin,
villin, vinculin)
16 700
40000-1 50000
All eukaryotic cells
Many non-muscle cells, e.g. phagocytes, slime moulds,
fibroblasts, liver
Calcimedins
33000,35000,67000
20 000
Smooth muscle
CaZ' activated photoproteins
Luciferin binding proteins
Bacterial CaZ' binding proteins
18500
33 000,47 000,60000
Luminous anthozoans
Escherichia coli
Luminous coelenterates
Luminous radiolarians
A. K . Campbell
8
posed that the balance between such reversible and
irreversible cell damage holds the key to understanding the molecular basis of tissue injury in
immune based diseases such as diabetes, thyroid
disease and multiple sclerosis [3,4, 18, 19,211.
It has been proposed that there are two types of
cell death, necrotic and apoptotic (see [75] for
references). The former is induced typically by
toxins, viruses, complement and anoxia, being
characterized by organelle and membrane disruption. The latter occurs in normal tissue turnover
and embryogenesis, and can be triggered by physiological regulators such as glucocorticoids, being
characterized by contraction of the cell without
immediate organelle disruption. The role of intracellular Ca2+in apoptosis has yet to be established.
Unlike irreversible cell injury leading to necrosis
apoptosis does not involve an initial increase in cell
membrane permeability to Ca2 . However, release
of Ca2+from internal stores may be required. Cell
death is a natural phenomenon in tissue development and turnover. Disturbances in intracellular
Ca2' involved in programmed cell death during
embryogenesis may also be involved in congenital
abnormalities such as cleft palate or spina bifida.
Intracellular Ca2+ is ideally suited to act as a
mechanism for the natural selection of cells
required to maintain a healthy tissue.
+
Perspectives
Changes in intracellular calcium thus play a vital
part in controlling the acute and long-term behaviour of healthy cells. The regulatory mechanisms dependent on intracellular Ca2+ can interact
with others involving different intracellular regulators, for example protein kinases activated by
cyclic AMP or cyclic GMP, or protein kinase C
activated by diacylglycerol or fatty acids whose
affinity for Ca2+may change as a result of cell activation, enabling it to be activated without a rise in
free Ca2+.There are three ways in which these
other intracellular cell regulatory mechanisms can
interact with Ca2+.Firstly, a change in intracellular
Ca2+ may alter the concentration of another
messenger. Secondly, a change in the intracellular
concentration of another messenger may alter the
concentration of intracellular Ca2+.Thirdly, some
enzymes can be co-regulated by more than one
messenger. For example, phosphorylase kinase is
activated by Ca2+ binding to its y-subunit, calmodulin, and by phosphorylation catalysed by
cyclic AMP dependent protein kinase. The occurrence of Ca2+binding proteins in the nucleus suggests that intracellular Ca2+ plays a key role in
long-term regulatory mechanisms, not only those
involving cell division but also those mediated by
oscillating changes in intracellular regulators. It has
long been known that denervation of muscle cells
leads to a redistribution of the receptors over the
cell surface and normally found at the endplate.
Circadian rhythms have been observed in populations of individual cells. In both of these situations
regular changes in intracellular Ca2 occur
throughout the daily cycle. The question now arises
as to whether, and if so how, these oscillating
changes control the long-term behaviour of the
cells.
Increases in intracellular Ca2 induced by pathogens can activate cells inappropriately. An understanding of the molecular basis of the role of
intracellular Ca2 in such inappropriate cell activation, or in controlling the balance between reversible and irreversible cell injury, provides exciting
prospects for developing novel, rational approaches
to therapeutic intervention. Increases in intracellular Ca2 provide a mechanism for taking individual
cells through the thresholds of activation or injury.
However, Ca2+ can also activate protection
mechanisms, thereby preventing a cell passing
through a lethal threshold. A full characterization of
intracellular Ca2+at the level of single cells is therefore essential if we are to intervene successfully in a
Ca2 dependent process. We need to know: is intracellular calcium a friend or a foe?
+
+
+
+
+
Acknowledgments
I have been fortunate enough to enjoy an active collaboration with several other enthusiasts. Particular
thanks go to Dr Paul Luzio, Dr Ken Siddle and Dr
Chris Ashley, and more recently to Dr Alastair
Compston and Dr Alan McGregor. I thank also my
research group for many years of hard work and the
Director and Staff of the Marine Biological Association, Plymouth. I am grateful to the MRC, the
SERC, the ARC and the DHSS for financial support. I thank Dr R. L. Dormer for helpful comments
on the manuscript.
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