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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. References Campbell, A.K. ( 1983) Intracellular Calcium: its Universal Role as Regulator John Wiley, Chichester. Dormer, R.L., Hallett, M.B.& Campbell, A.K. (1985) In: Measurement of Intracellular Free Ca2+in Control and Manipulation of Calcium Movement, pp. 1-27. Ed. Parratt, J.R. Raven Press, New York. Campbell, A.K. & Luzio, J.P. 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