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497 Clinical Science (1984) 66,497-508 EDITORL4L REVIEW Calmodulin and cell function S . TOMLINSON’, S . MACNEIL’, S . W. WALKER’, C. A. OLLIS’, J. E. MERRITT3 AND B. L. BROWN’ ’ ‘Department of Medicine, University Clinical Sciences Centre, Northern General Hospital, Sheffield, Department of Chemical Pathology, Northern General Hospital, Sheffield and ’Department of Human Metabolism and Clinical Biochemistry, The Medical School, Sheffield, U.K. Introduction The importance of calcium in the regulation of cell function has become increasingly recognized in the past 20 years.. It is now known that changes in intracellular calcium concentration, like changes in adenosine 3’:5’-monophosphate (cyclic AMP), are of crucial importance in stimulus-response coup ling. Thus, the second messenger theory originally proposed by Sutherland et al. [l], in which a hormone or nerve impulse is fust messenger and cyclic AMP the second intracellular messenger, has been expanded to include calcium ions as well as cyclic nucleotides. Moreover, there is now evidence Correspondence: Dr S. Tomlinson, Department of Medicine, University Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, U.K. in many cellular systems that calcium ions and cyclic nucleotides act as dual interrelated messengers [2], and, therefore, some cellular processes are regulated by calcium as well as by cyclic nucleotides, as shown in Table 1. Evidence has accumulated that it is changes in free intracellular calcium concentration that are, in some way, responsible for activation of the enzymes involved in the processes shown in t h i s Table. The resting concentration of free intracellular calcium is between lo-’ and lO-’mol/l (0.01-0.1 pnolll) and an increase in this concentration by ten- to a hundred-fold effects the appropriate response [3]. Changes in the intracellular free calcium can be derived from either extracellular or intracellular sources. Calcium channels allow calcium to enter the cell from the extracellular TABLE1. Cellular processes regulated by calcium, calmodulin and cyclic nucleotides Cellular process Insulin secretion Thyroid secretion pituitary secretion Adrenal secretion Neurohormone secretion Intestinal secretion Cell proliferation Cell architecture Lysosome release Smooth muscle contraction Lymphocyte mediated cytotoxicity Prostaglandin synthesis Disassembly of microtubules Histamine release Ciliary motility Fast axonal transport Neurohormone synthesis Neurohormone super sensitivity Phagocytosis Initiation of DNA synthesis Cast dependent Role for calmodulin cyclic nucleotides + + + + + + + + + + i i + + + i + + + i i + + i + + i + + i + + + + + + + i + i + + + + i + + i i + + + + 498 S. Tomlinson et al. fluid [4]; these calcium channels can be voltage dependent (for example in glucose stimulated insulin release and in secretion of some pituitary hormones, such as prolactin) or agonist dependent (for example in adrenergic and cholinergic effects in the salivary glands). There is increasing evidence that hydrolysis of the membrane phospholipid, phosphoinositol, is connected with mechanisms for opening agonist dependent calcium channels PI. Although some cells can respond to stimuli even in the absence of extracellular calcium, it has been established that increases in intracellular calcium do still occur. The sources of increased cytosolic free calcium are probably the mitochondria, endoplasmic reticulum, plasma membrane and possibly the nucleus of the cell. If an increase in intracellular calcium activates a specific calcium dependent process, the activation must be terminated by a corresponding reduction in the intracellular free calcium concentration. This is achieved by a number of mechanisms: first, there are so-called energy requiring calcium pumps located in the cell membranes which transport calcium from the intracellular to the extracellular compartment; second, translocation of the calcium from cytosol to cellular organelles can occur and, third, calcium binding molecules not involved in the activation process may also modulate intracellular free calcium concentration. The question arises: how does calcium act as a second messenger? It has become clear over the last 10 years that many calcium dependent events are mediated by its binding to and activation of a ubiquitous intracellular calcium binding protein called calmodulin. The calcium-calmodulin complex is then able to activate a wide variety of enzymes, including those which affect cyclic nucleotide metabolism. TABLE2 . Chronological order of findings about calmodulin Year Discovery 1970 Protein activator of phosphodiesterase discovered Protein found to actjvate Ca2+,Mg2'dependent phosphodiesterase 1973-1974 The protein's mechanism of action elucidated The protein activates brain adenylate cyclase 1975 Trifluoperazine found to inactivate the 1976 protein 1977 The protein activates Ca2+-ATPase The protein is the 8 subunit of phosphorylase 1978 kinase The protein activates myosin lightchain kinase The protein activates NAD kinase Galmodulin is given as a proper name Calmodulin activates Caz+dependentprotein kinase Calmodulin enhances microtubule disassemblv 1979-1980 Calmoduh regulates synthesis and release of neurotransmitters Amino acid sequence of calmodulin elucidated Calmodulin activates glycogen synthase kinase Calmodulii important in DNA synthesis and cell division Naphthalene sulphonamides (W compounds) increasingly recognized as more specific inhibitors of calmodulin Calmodulin involved in prostaglandin synthesis Immunoassays for calmodulin developed ? Calmodulin activation of adenylate cyclase possibly a general phenomenon 1981-1983 Calmodulin activates guanylate cyclase Calmodulin increasingly recognized as important in stimulus-secretion coupling in endocrine cells DNA sequence of calmoduli elucidated Alterations in calmodulin in disease: increased activity in cystic fibrosis; increased concentration in psoriasis and in experimental diabetes Calmodulin A brief summary of the chronological order in which calmodulin's role has been elucidated is shown in Table 2. The protein was first discovered as a heat-stable, dissociable activator of brain cyclic nucleotide phosphodiesterase independently by Cheung and Kachiuchi et al. in 1970 [6,7]. In 1973 Teo & Wang [8] elucidated the protein's mechanism of action and showed that the two proteins described by the original investigators were, in fact, identical. Subsequently, Brostrom et al. [9] found that the protein activated brain adenylate cyclase and Levin & Weiss in 1976 [lo] described the inactivation of the protein by trifluoperazine, which provided a useful tool for the study of the role of calmodulin in cellular systems. In 1978, the now accepted name calmodulin was coined by Cheung et al. [l 11. It is remarkable that calmodulin is very widely distributed, being found in both the plant and animal kingdoms and probably in all eukaryotic cells. Brain and testicular tissue are particularly rich sources of the protein but possibly the richest source is the electroplax of the electric eel, where calmodulin accounts for 5-10% of the total protein [ 121. Properties of calmodulin Calmodulin is a heat-stable, acidic polypeptide of 148 amino acids. It contains four calcium binding Calmodulin and cell function TABLE 3. Properties of calmodulin Straight chain polypeptide; 148 amino acids Mol. wt. 16 500 Isoelectric point. pH approximately 4 .O Calcium binding, four sites; affinity approximately mol Internal amino acid homology at calcium binding sites Binds to phenothiazines: Caz* dependent Resistant to denaturation sites with affinities for calcium in the range which corresponds closely to the free intracellular calcium concentration in the stimulated state. The primary structure of all calmodulins studied so far is very similar and biological studies show that it is neither tissue nor species specific. Some of the important properties of calmodulin are summarized in Table 3. It is of interest that the four calcium binding domains in calmodulin show nearly identical amino acid sequences and it seems likely that the protein has arisen by gene duplication. Each domain has the basic ‘EF hand’ structure as determined by X-ray crystallography [ 131. The protein has a similar amino acid sequence, particularly in the calcium binding regions, to a number of other calcium binding proteins known as the troponin C super-family of calcium binding proteins. Not only is the basic structure of calmodulin similar throughout the plant and animal kingdoms, but recent evidence suggests that base triplets coding for amino acids are also conserved since DNA coding for electric eel calmodulin hybridizes to DNA from several other species [14,15]. Mechanism of action of calmodulin Current evidence suggests that under normal conditions calmodulin activity is not altered by changes in its concentration within the cell but Inactive mlrnodulin Active oalrnodulin 499 mainly by changes in the concentration of free intracellular calcium [161. Under resting conditions, the concentration of intracellular free calcium is too low to allow for any significant binding of calcium to calmodulin. When a cell is stimulated, there is an increase in intracellular free calcium concentration due to the movement of calcium through channels in the plasma membrane or its release from intracellular membranes or organelles. Calcium then binds to calmodulin, which undergoes a conformational change, allowing interaction with inactive enzyme to form an active complex. A reduction in intracellular free calcium concentration by extrusion from the cell (brought about by calmodulin-dependent Ca2’ATPase) or by translocation within the cell results in dissociation of the active calmodulin-enzyme complex, thus decreasing enzyme activity. This simplified scheme is illustrated in Fig. 1. However, it must be emphasized that there may be other factors which regulate calmodulin’s activity, in addition to alterations in intracellular free calcium concentration. As well as enzymes that bind to calmodulin and are thus activated, there are other proteins to which calmodulin will bind and these proteins themselves may regulate the activity of calmodulin [171. One example of such an inhibitory protein is calcineurin, which has been found in brain. Calcineurin is composed of two subunits: a large molecular weight subunit which binds to calmodulin, and a small molecular weight subunit which itself is a calcium binding protein. This complex can bind to and inactivate calmodulin, thus preventing, for example, activation of cyclic nucleotide phosphodiesterase or adenylate cyclase. However, recent work has shown that calcineurin itself has an enzymatic function since it appears indistinguishable from protein phosphatase 2b [18]. A third mechanism for the regulation of lnactiw receptor protein Active receptor protein FIG. 1. Mechanism by which calmodulin mediates the biological action of calcium ions is depicted in this highly schematic diagram. Neither calcium alone nor calmodulin alone is active. The binding of 4 Ca2+ to calmodulin changes the shape of the protein. As a result, calmodulin is able to interact with an enzyme (‘receptor protein’), which is thereby activated. 500 S. Tomlinson et al. calmodulin activity may be by the localization of the protein within the cell. This has been described both during cell division [19] and in the capping of cell surface receptors for concanavalin A in a human lymphoblastoid cell line [20]. It is possible that this relocation of calmodulin is in some way influenced by cyclic AMP. Conversely, calmodulin may promote relocation of its own binding proteins within the cell, as has been described in exocytosis [21]. Finally, chemical modification of the calmodulin molecule itself may influence its activity; for example, carboxylmethylation of calmodulin occurs in intact cells and there is evidence that carboxylmethylated calmodulin shows reduced stimulation of cyclic nucleotide phosphodiesterase [22]. The regulation of calmodulin’s activity is clearly an area of great importance and one might speculate (see below) that it could be abnormalities in the regulation of this activity which might be important in some disease processes. Inhibitors of calmodulin activity One of the advantages of using these drugs is that they cross cell membranes and, therefore, can be used in studies of whole cell systems. Specific inhibitors of calmodulin, such as monoclonal antibodies against the protein, can only be used provided that calmodulin in the test system is available to the antibody. A further disadvantage of antibodies is that they tend to have lower affinity for calmodulin than calmodulin has for its receptor proteins or enzymes and, therefore, a large amount of antiserum may be required to exert its inhibitory effect. The calmodulin binding protein, calcineurin, may be more useful in such studies since it is specific and binds with high affinity to the protein. However, even using this specific inhibitor, one has to bear in mind that it also has intrinsic activity of its own which may affect the system under investigation. Calmodulin and cyclic nucleotide synthesis and degradation [29] Calmodulin was first described as a protein activaA major observation for subsequent studies of the tor of cyclic nucleotide phosphodiesterase [6, 71. function of calmodulin was that phenothiazines Subsequently, it was found that as well as influencbind to and inactivate calmodulin in the presence ing cyclic nucleotide degradation, calmodulin of calcium [lo]. After this initial observation, it could also influence cyclic nucleotide synthesis by was found that many other drugs, including the activation of adenylate cyclase in brain [9, 111; butyrophenones, tricyclic antidepressants, some more recently, it has been reported that calmodulin dopamine antagonists, some opiate derivatives, is involved in the activation of adenylate cyclase 0-receptor blockers and local anaesthetics, could in guinea-pig sperm [30], pancreatic islets [3 1, 321, inhibit, with varying potencies, the activity of the bovine adrenal medulla [33], a vasopressin sensitive protein [23-251. The mechanism of this inhibition adenylate cyclase in a pig kidney cell line [34] and has been partially elucidated; when calcium binds in rat testicular germ cells [35]. In addition, our to calmodulin it exposes a hydrophobic domain own recent studies suggest the involvement of which appears to be essential for interaction with, calmodulin in mouse B16 melanoma adenylate and subsequent activation of, its receptor proteins cyclase activity [36] and human thyroid adenylate or enzymes [26]. Phenothiazines appear to bind to cyclase activity [37]. Interestingly, although prothis exposed domain and interfere with the bind- karyotes do not appear to have calmodulin, activaing of calmodulin to its receptor proteins. tion of prokaryote adenylate cyclase by calmodulin Unfortunately, none of these drugs is specific for has recently been reported in Bordetella pertussis calmodulin; this has two consequences. The first [381. is that the therapeutic effect of the drugs is unExperimentally, we have found it easier to likely to be due to their ability to inhibit calmodu- demonstrate calmodulin stimulated phospholin; indeed, using a variety of chlorpromazine diesterase activity in a cell, using calmodulin analogues, no correlation between tranquillizer depleted cytosol, than to demonstrate calmodulin activity and calmodulin inhibition was found [27]. activation of membrane adenylate cyclase, because Secondly, although the drugs can be used to indi- of the difficulty of removing calmodulin from cell cate a possible role for calmodulin in cellular membranes. Reports of attempts to remove calmoprocesses, investigators must be aware that the dulin from particulate preparations vary considerdrugs have a variety of other effects related to ably, with up to 76% being removable from brain their hydrophobicity rather than their specific membranes [39], but none was removable from effect on calmodulin. There is evidence that a guinea-pig sperm membranes [30]. In our own new agent N-(6-aminohexyl)-5 -chloro-1-naphtha- experience, addition of exogenous calmodulin lene sulphonamide (W7) which we have used in to membranes containing calmodulin produces some of our own studies is more specific for no further activation of adenylate cyclase. This calmodulin than previously described drugs [28]. makes it easier to understand why the earliest Calmodulin and cell firnction demonstrations of calmodulin activation of adenylate cyclase used solubilized enzyme from which calmodulin was removed by chromatography [9],and why the number of tissues in which calmodulin has been demonstrated to activate adenylate cyclase is still relatively few. With respect to guanosine 3': 5'-monophosphate (cyclic GgP) synthesis and degradation, guanylate cyclase activity in Tetrahymena pynformis and Paramecium has now been shown to be activated by calmodulin [40,40a] and cyclic GMP is known to be hydrolysed by calmodulin dependent phosphodiesterase activity in brain. It is therefore clear that calmodulin is involved in cyclic AMP metabolism and, indeed, cyclic AMP itself may modulate the calcium signalling system [4]. Cyclic AMP may not only be important in influencing the opening of calcium channels but may also play a role in releasing calcium from - h .5 intracellular stores and regulating intracellular translocation of the calcium-calmodulin complex. In attempting to understand the relationship between the calcium and cyclic AMP signalling systems, one is presented with the apparent paradox that the enzymes for synthesis and degradation of cyclic AMP can both be activated by calmodulin in the same cell. For example, we have shown that there is not only calmodulin dependent adenylate cyclase in the mouse B16 melanoma, but also a calmodulin dependent phosphodiesterase (see Fig. 2). This is also true of other cell systems. A partial and simplified explanation for this may lie in the sequential activation concept proposed by Cheung [41]. This is dependent upon the fact that calmodulin dependent adenylate cyclase will be located in the cell membrane, whereas the calmodulin activated phosphodiesterase will be in cytosol. Stimulus promoted entry of calcium ions h .5 ( a ) Adenylate cyclase 501 (6)Phosphodiesterase B * b) ea 2a T Q c NaF - +C+W? No treatment - - l+C+WJ CDR depletion ~ FIG. 2. ( a ) Inhibition of basal activity and m S H ( molt and NaF (10 mmol/l)stimulated adenylate cyclase activity in mouse B16 melanoma cultured cell lysates by the calmodulin antagonist prochlorperazine (PCP) (100 pmol/l). Results shown are means f SEM of triplicate determinations of a typical experiment in which the inhibition produced by PCP is most marked for hormone stimulated activity ( 8 5 % ) compared with that produced for basal (73%)or NaF-stimulated enzyme activity (43%).Hatched histograms, controls; open histograms, in the presence of PCP. (b) Effects of calmodulin and the calmodulin antagonist W7 on phosphodiesterase activity. The left-hand set of histograms show inhibition of untreated soluble cyclic AMP phosphodiesterase activity of mouse B16 melanoma by W7 (5 x 10-5mol/l) (4- W7).Addition of 1pg of calmodulin/ ml (1.5 x lO-'mol/l) (+ C) had no significant effect on enzyme activity. The righthand set of histograms shows the same soluble phosphodiesterase activity after passage over fluophenazine Sepharose 6B gel, which removes most of the endogenous calmodulin. It can be seen that when endogenous calmodulin was removed, addition of exogenous calmodulin (1.5 x lO-'mol/l) (+ C) now activated phosphodiesterase activity and addition of W7 (5 x 10-5mol/l) (4- W7) had no effect on basal enzyme activity. CDR, Calcium dependent regulator, i.e. calmodulin. 502 S. Tomlinson et al. into the cell membrane would be the primary signal resulting in activation of calmodulin and then stimulation of adenylate cyclase. Subsequent increase in intracellular calcium concentration would activate calmodulin in the cytosol, leading to stimulation of calmodulin dependent phosphodiesterase which would then hydrolyse cyclic AMP, terminating the cyclic AMP signal. In addition, there is some evidence that the two enzymes have different requirements for calcium; calmodulin dependent adenylate cyclase activity appears to require low concentrations of calcium and higher concentrations (10-5mol/l and higher) are, in fact, inhibitory to the enzyme [42]. Thus it is now clear that the two signalling systems are interrelated and play a fundamental role in cellular metabolism. Some of the relationships between the two systems are considered in subsequent sections on the role of calmodulin in stimulus-response coupling, neurotransmitter release and neuronal function. Calmodulin and secretion It is now well established that calcium ions play a major role in hormone secretion. There is good evidence that calmodulin mediates calcium regulated secretion of insulin. Release of insulin from the /3 cell requires the presence of extracellular calcium ions [43,44]. Calmodulin is present in the /3 cell of the islets of Langerhans and phenothiazines can inhibit insulin secretion [45]. Both calmodulin activated adenylate cyclase and cyclic AMP phosphodiesterase have been found in islet cells [45]. This is rather indirect evidence that calmodulin has a role in insulin secretion; however, more direct evidence comes from the fact that calmodulin can stimulate specific protein phosphorylations in islet cells, independent of those stimulated by cyclic AMP [46]. The secretion of anterior pituitary hormones appears to be dependent on calcium ions [47-501. A calcium dependent activator of phosphodiesterase has been found in anterior pituitary tissue and calmodulin has been purified from anterior pituitaries of freshly killed pigs [51]. A cyclic nucleotide phosphodiesterase activated by calmodulin has been identified in pituitary tissue [52]. In addition, inhibitors of calmodulin, such as the phenothiazines and the naphthalene sulphonamide W7, have been shown to inhibit thyrotropin (TSH) secretion, prolactin secretion [53-551 and luteinizing hormone secretion from pituitary cells [56]. In some of these studies the drugs had the same order of potency on inhibition of hormone secretion as reported for their effect on inhibition of calmodulin activated phosphodiesterase [25]. As in the islets of Langerhans, there also appear to be calmodulin specific protein phosphorylations in anterior pituitary cells. We have shown that one of the major proteins phosphorylated in the presence of calmodulin has an approximate molecular weight of 53 000 daltons. The phosphorylation of this protein was also enhanced by cyclic AMP in the absence of calcium, suggesting that it might be a substrate for two independently regulated protein kinases. Interestingly, similar results have been obtained with pancreatic islets [57] and so there is the possibility that the same (or similar) proteins exist in both tissues, and are subject to similar regulation. The thyroid gland is of particular interest because, unlike the /3 cells of the islets or anterior pituitary cells, TSH-stimulated thyroid hormone release is not dependent on the presence of extracellular calcium. Nevertheless, extracellular calcium is required for a number of other TSH-stimulated intracellular processes in the thyroid (for example, glucose oxidation and iodide binding to protein) and these processes are also affected by manipulations which alter intracellular calcium levels. It appears, therefore, that calcium is involved in thyroid cell metabolism [58-601. However, there are few reports of the presence and possible role of calmodulin in thyroid cell metabolism. Calcium dependent activators of phosphodiesterase have been found in the thyroid but these were not, at that time, identified as calmodulin [61,62]. Our own studies have shown that calmodulin is present in the human thyroid and we have also shown that calmodulin is possibly involved in thyroid cell cyclic nucleotide metabolism [63] (Fig. 3). Recently, calmodulin dependent phosphodiesterase has been found in dog thyroid slices [64]. In addition, calmodulin binding proteins have been found in thyroid cell membranes [65]. There are very few data on the function of calmodulin in other endocrine tissues. Calmodulin has been detected in the adrenal cortex by an immunofluorescent technique [66] and calmodulin may be involved in the synthesis of corticosteroids [67]. The protein may also be important in the stimulatory action of potassium and angiotensin I1 on aldosterone secretion since these processes are completely blocked by trifluoperazine [68].In the adrenal medulla calmodulin is probably important in exocytosis of chromaffin granules [21]. In other secretory systems there is evidence that phenothiazines can inhibit intestinal ion secretion [69], histamine release from mast cells [70], serotonin release from platelets [71] and protein secretion from polymorphonuclear leucocytes [72]. The role (if any) of calmodulin in parathyroid hormone W7 (pmolll) FIG. 3. Effects of W7 on TSH (50 m-units/ml)-stimulated cyclic AMP production by cultured human thyroid cells. Cells were incubated for 60 min at 37'C. Each point is the mean f SEM of triplicate cultures. 0, Unstimulated cyclic AMP accumulation; 0, TSH-stimulated cyclic AMP accumulation. At low concentrations W7 appears to enhance cyclic AMP production, possibly as a result of inhibition of calmodulin dependent phosphodiesterase in the cells. At higher concentrations there is a dose dependent inhibition of cyclic AMP production, presumably related to inhibition of calmodulin dependent TSH-stimulated adenylate cyclase in the thyroid cell membranes. secretion and in calcitonin secretion has yet to be established [73,74]. Calmodulin, neutrotransmitter release and neuronal function Many studies have indicated that calmodulin may mediate the neuronal functions of calcium ions. Calcium in micromolar concentrations can stimulate the release of several neurotransmitter substances, including acetylcholine, noradrenaline and dopamine from preparations of synaptic vesicles [75]. These neurotransmitter effects have been shown to be mediated by calmodulin and inhibited by calmodulin inhibitors such as trifluoperazine and phenytoin. The rate limiting steps in the biosynthesis of serotonin and noradrenaline are catalysed by tryptophan-5-mono-oxygenaseand tyrosine-3-mono-oxygenaserespectively;both these enzymes are activated by calmodulin dependent protein kinase [76]. It has also been shown that calmodulin can regulate calcium stimulated phosphorylation of several proteins in synapse structures [75]. Calmodulin appears to play a part in dopaminergic activity in the brain and could be interacting with guanyl nucleotides at various sites on the dopamine sensitive adenylate cyclase [77]. The available evidence, therefore, clearly indicates that calmodulin is involved in neurosecretion and in neuronal function. Calmodulin and cellular proliferation An increase in cytosolic calcium is a control signal for the initiation of DNA synthesis [78] and it may be that calmodulin is involved in this event [79]. During cell division it has been shown by immunofiuorescence that calmodulin localizes on the cellular spindle, which itself is composed of microtubules, and there is evidence that calmodulin is important in the calcium dependent assembly and disassembly of microtubules [80]. Calmodulin is increase.d in transformed cells [81,82] and levels of calmodulin have been shown to be elevated in hepatomp and in regenerating liver; in addition, calmodulin can stimulate DNA synthesis in isolated liver cells [83-861. Furthermore, calmodulin antagonists, particularly when present at the GI-S transition phase, will inhibit cell cycling [82,87]. Our own studies, using a number of calmodulin antagonists, have indicated that calmodulin may be important in agonist-stimulated DNA synthesis in lymphocytes (see Fig. 4) [88]. One of the possible ways in which calciumcalmodulin could regulate DNA synthesis is by activation of a protein kinase. Iwasa et al. [89] have shown effects of calmodulin on histone phosphorylation and, hence, possibly on gene expression. Calcium-calmodulin itself also controls the activity of ornithine decarboxylase ; inhibition of this enzyme stops cell division through the blocking of polyamine synthesis [90]. One of the major $ 1 L ", I 0 \ Y ' 0 '10-6 10-5 \ 10-4 Drug concentration (mol/l) FIG. 4. Effect of W7 o n phytohaemagglutininstimulated [ 3H]thymidine incorporation into peripheral blood lymphocytes. Results are means k SEM of triplicate determinations from a single representative experiment. [3H]Thymidine uptake in the absence of W7 is expressed as 100%. W7 caused a dose-dependent inhibition of [3H]thymidine uptake in to stimulated lymphocytes . characteristics of neoplastic cells compared with normal cells is their ability to proliferate normally lin low calcium medium [91]. Some clues now exist as to how tumour cells may lose this calcium requirement for division. A tyrosine phosphokinase activity found at the cytoplasmic face of cell membranes is associated with the oncogene product of a number of classes of tumour viruses [89], and it has been reported that calmodulin itself mediates the ability of this tyrosine phosphokinase activity to promote cell proliferation. MacManus [92] has described a calcium binding protein of molecular weight 11 500 daltons, called oncomodulin, which appears to be specific for cancer cells. This protein has many of the properties of 'EF hand' proteins and is able t o activate otherwise calmodulin dependent enzymes such as cyclic nucleatide phosphodiesterase. Since this protein may activate at lower calcium concentration than calmodulin, it may not depend on raising the intracellular concentration of calcium for its activity. The role of calmodulin in DNA synthesis and cellular proliferation may be important in the neoplastic process and might, therefore, have therapeutic implications for cancer. non-muscle cells, calcium's effect is probably mediated by calmodulin. Contractile processes in smooth muscle and in non-muscle cells are believed to be myosin-linked and regulated by calcium dependent myosin light-chain kinase. The enzyme consists of two proteins, a large polypeptide and a small regulatory protein which binds in the presence of calcium to the larger protein and which is identical with calmodulin. The protein phosphorylation produced by the kinase ultimately catalyses the hydrolysis of ATP to release energy for muscle contraction. A similar mechanism presumably operates for non-muscle contractile proteins in the regulation of cellular motility [93]. Interestingly, calmodulin appears t o be involved not only in muscle contraction, but also in providing the energy necessary for this process. This is probably true of both skeletal and smooth muscle. It has been shown that calmodulin is the delta subunit of phosphorylase kinase and confers calcium sensitivity on the enzyme; hence, glycogen is broken down, providing glucose for energy metabolism. Complementary to this, calmodulin activates glycogen synthase kinase which phosphorylates and inactivates glycogen synthase [94]. In addition, levels of glucose 1,6-bisphosphate, which is a key regulator of several enzymes of carbohydrate metabolism, may be partially controlled by calmodulin; the phosphatase which breaks down glucose 1,6-bisphosphate appears to be stimulated by calmodulin [95]. Interestingly it has also been reported that one of the enzymes in the tricarboxylic acid (Krebs) cycle, succinate dehydrogenase, is regulated by calcium [96]. Thus the suppression of glycogen synthesis, the stimulation of glycogen breakdown and the intermediary metabolism of glucose are, in part, regulated in a co-ordinated fashion by calcium and calmodulin. These events, in turn, provide the energy for the contractile process, which is itself modulated by calmodulin. Calmodulin may also have a role in prostaglandin metabolism; it activates phospholipase A2, leading to the synthesis in platelets of PGHz and thromboxane. It is also possible that calmodulin may enhance the activity of cyclo-oxygenase. In addition, there is evidence that calmodulin may be involved in the synthesis of prostacyclin by the vascular endothelium. The influence of calmodulin on prostaglandin metabolism obviously has relevance to the role of platelets and the vascular endothelium in haemostasis [97,98]. Calmodulin, cellular contractile processes and iutracellular metabolism Calmodulin and disease Troponin C is the major calcium receptor in striated muscle but in smooth muscle and the filaments of The studies described in this Review clearly not only have implications for normal cell function Gdmodulin and cell function 505 but also potentially for abnormalities that occur Another condition which is the subject of in disease. The central role of calmodulin as a intensive investigation at the moment is essential mediator of the intracellular calcium signal implies hypertension. There is increasing evidence that that major abnormalities in structure or expression membrane abnormalities occur in various cell of calmodulin are likely to be lethal. However, the types in hypertension. Decreased calcium binding number of receptor proteins and enzymes for to cell membranes and increased cellular calcium calmodulin and the number of calmodulin binding have been found in essential hypertension in man proteins of, as yet, unidentified function, make it as well as in experimental hypertension in animals. possible that abnormalities might exist amongst Whether these abnormalities in intracellular calcium these proteins in disease. If this is the case, then are a consequence of extracellular factors affecting secondary alterations in intracellular calcium or the sodium pump or intrinsic membrane abnorcalmodulin concentration may occur. malities has yet to be resolved [105]. Whether In this connection there are diseases in which calmodulin, through its activation of myosin lightabnormalities of intracellular calcium have been chain kinase and its effects on smooth muscle described. Thus calcium levels are reported to be contraction, is involved in the genesis of hyperelevated in the cells of patients with cystic fibrosis. tension is speculative. It would obviously be of Reduced activity of the plasma membrane CaZ+/ some interest to know whether intracellular MgZ'-ATPase in erythrocytes and fibroblasts of immunoassayable calmodulin and calmodulin patients with cystic fibrosis have been observed activity is altered in essential hypertension. and this enzyme is calmodulin activated. Gnegy The explosion of knowledge in the last decade et al. [99] have measured calmodulin in cultured concerning the role of calcium in regulating cell skin fibroblasts from patients with cystic fibrosis function and the recognition that many calcium and have reported elevated levels. Such observa- dependent processes are mediated by the calcium tions are clearly interesting in the light of the binding protein, calmodulin, seems likely to have involvement of calmodulin in the secretory process. important pharmacological, therapeutic and clinical One very important area of interest is the regu- implications. lation of cell proliferation by calcium and by cyclic AMP. As has been stated above, calmodulin levels are elevated in transformed cells and in re- Acknowledgments generating and neoplastic liver cells. Furthermore, calmodulin antagonists, when present at the criti- S.T. is a Wellcome Trust Senior Lecturer. We are cal G1-S transition phase, can prevent cell cycling. grateful to the Wellcome Trust, MRC, SERC and Yorkshire Cancer Research Campaign for financial The implications of these findings are, at present, support. Table 1 is reproduced with permission unclear but it is of considerable interest that in psoriasis, a proliferative disease of the skin, calmo- from a paper by Dr W. L. West in Federation dulin levels have been reported to be grossly Proceedings (1982), vol. 41. Table 2 is adapted elevated compared with those of uninvolved skin and reproduced with permission from a paper by and normal controls [loo]. Another condition in Dr W. Y. Cheung in Federation Proceedings (1982), which intracellular levels of calmodulin have been vol. 41. Fig. 1 is reproduced with permission from reported to be increased is in experimental dia- an article by Dr W. Y. Cheung in Scientific Ameribetes; Morley et al. [loll showed that immuno- can (June, 1982). Fig. 3 is reproduced with perassayable calmodulin was increased in several mission from a paper by Ollis et al. (1983) in the tissues in diabetic mice. The implications of these Journal of Endocrinology (In press). findings have yet to be established. In addition to the three conditions described References above, where there appear to be abnormalities in calmodulin concentration, there are other condiI . Sutherland, E.W., @ye, 1. & Butcher. R.W. (1965) The action of epinephrin and the role of thc adenyl cyclase system in hormone tions where abnormalities in intracellular calcium action. Recent Progress in Hormone Research, 2 1 . 6 2 3 4 4 6 . have been described but, as yet, there are no data 2. Rassmussen. H. & Goodman, D.B.P. (1977) Relationships between calcium and cyclic nucleotides in cell activation. Physiological on calmodulin itself. In muscular dystrophy, for Reviews, 57,421-509. 3. Borle. A.B. (1981) Control. modulation and regulation of cell example, intracellular calcium has been shown to calcium. Reviews oJ Physiolog.v. Biochemisrry and Pharmacology, be elevated and it has been suggested that it might 90, 13-153. 4. Berridge, M.J. 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