Calmodulin Levels Research Articles

Platelet calmodulin correlates with platelet turnover

We measured the calmodulin content in platelets in 13 normal persons and in 62 patients with hematological diseases. The level of platelet calmodulin was higher in patients with idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus, myeloproliferative disorders, acute leukemia in a recovery phase, aplastic anemia, thrombosis and hypersplenism as compared to the controls. Among the patients with ITP, calmodulin was lower in responders than in nonresponders and those at the initial diagnosis. We also measured the volume, life-span and aggregation of the platelets and demonstrated a significant relationship between the calmodulin level and the platelet volume, and a negative relationship between the calmodulin level and platelet life-span, there was no correlation between the calmodulin level and platelet aggregation. We thus conclude that platelet calmodulin is inversely correlated with platelet turnover.

Calcium and calmodulin levels in late embryonic and early hatched japanese quail brain

Characterizing the last phases of embryonic avian brain development are increased brain activity and increased absorption of shell calcium. The calcium-binding protein, calmodulin, regulates many activities of calcium. In neural tissue, calmodulin modulates neural transmission, and is required for the phosphorylation of synaptosomal proteins. Therefore, the objective was to compare levels of brain calcium and calmodulin in the Japanese quail. Brain extracts from embryonic days 11, 15, hatch, and 5 days post-hatch (n = 7/group) were analysed for calcium, protein and calmodulin. Despite increases in protein between embryonic (X̄ = 0.126 and 0.145 mg/mg wet wt) and hatched groups (X̄ = 0.183 and 0.221 mg), no significant increases in calmodulin were observed (237–279 ng/mg protein). Calcium levels in the brain were U-shaped with low levels at embryonic day 1 (341 μg/mg wet wt) and post-hatch day 5 (315 μg/mg wet wt) with higher levels on embryonic day 15 (425 μg/mg wet wt) and at hatch (433 μg/mg wet wt). Calmodulin levels do not show a developmental pattern similar to calcium and protein levels or with reports of brain activity.

Elevated calmodulin levels and reduced calmodulin‐stimulated calcium‐ATPase in Duchenne progressive muscular dystrophy

We determined the calmodulin concentration and Ca2+-ATPase activity in subcellular fractions recovered from samples of vastus lateralis muscle obtained from 18 patients with Duchenne muscular dystrophy, 10 patients with other primary myopathies, 5 with spinal muscular atrophy, and 16 age-matched controls. Calmodulin levels were increased in the cytosol, plasmalemma, and heavy sarcoplasmic reticulum fractions from Duchenne dystrophy patients; the greatest increases occurred at early stages of disease or in mildly progressive cases. The total Ca2+-ATPase activities were decreased in the Duchenne dystrophy muscles; calmodulin caused a minimal stimulation of the activity in calmodulin-depleted membranes from Duchenne dystrophy compared with control membranes. The changes in calmodulin concentration and Ca2+-ATPase activity complement previous observations of reduced calsequestrin and dystrophin concentrations in Duchenne dystrophy muscles and suggest that these muscles lose calcium regulatory functions at early stages of the disease process.

Impairement of vascular reactivity and changes in intracellular calcium and calmodulin levels of smooth muscle cells in canine basilar arteries after subarachnoid hemorrhage.

We examined vascular reactivity to various vasoconstrictors and dilators, and the changes in calcium-calmodulin levels in canine basilar arteries after subarachnoid hemorrhage (SAH). Contractile responses to noradrenaline, serotonin, and potassium chloride were markedly attenuated at 48 hours (P less than 0.05), and further attenuated at 7 and 14 days after SAH (P less than 0.01). Dilation responses to calcium antagonist were maintained at 48 hours after SAH, but were markedly reduced at 7 and 14 days after SAH (P less than 0.05). Transmission electron micrographs of the basilar artery showed contraction of the media between 48 hours and 7 days and degeneration of smooth muscle cells over the 7 days after SAH. Electron microscopic cytochemical examination for calcium showed that intracellular deposits of calcium pyroantimonate increased in smooth muscle cells of basilar arteries at 1 hour after the first intracisternal injection of blood (early spasm), but decreased in smooth muscle cells at 48 hours after SAH (at the beginning of delayed vasospasm). They decreased further in the vessels 7 days after SAH. The calmodulin contents in the basilar arteries were decreased slightly at 6 hours, and significantly (P less than 0.05) at 48 hours after SAH, as determined by radioimmunoassay and phosphodiesterase assay. Therefore, it is considered that delayed vasospasm is not simply an active contraction of the vessels, but a functional or structural derangement of contractile elements of smooth muscle cells after 48 hours after SAH.

Vertebrate and yeast calmodulin, despite significant sequence divergence, are functionally interchangeable.

Yeast strains relying solely on vertebrate (Xenopus laevis) calmodulin, expressed under control of a yeast (GAL1) promoter, grew at the same rate as yeast cells containing their endogenous calmodulin. Therefore, the ability to perform essential functions has been conserved between yeast and vertebrate calmodulins, suggesting that calmodulin performs the same (or overlapping) roles in yeast as it does in higher eukaryotes. Successful substitution of vertebrate for yeast calmodulin also suggests that the two proteins can adopt similar conformations in vivo, despite the large number of amino acid differences between them (60 out of 148 residues). Strains overproducing either vertebrate or yeast calmodulin about 100-fold and a strain producing a normal level of yeast calmodulin were essentially indistinguishable in many characteristics, including microtubule distribution, rate of secretion, response to mating pheromone, sporulation, and adaptation to nutrient limitation. Calmodulin overproduction did not confer elevated resistance to a phenothiazine drug, trifluoperazine, thought to be a calmodulin-specific inhibitor. These results have important implications for understanding the role of calmodulin in intracellular calcium signaling.

Phytochrome Mediated Photoregulation of NAD Kinase in Terminal Buds of Pea Seedlings

Summary Exposure of etiolated pea ( Pisum sativum L.) seedlings for 5 min to red light increased NAD kinase activity in the terminal buds after a lag of about 12 h. Maximal increase occurred at 48 h and the activity returned to control level at 96 h of dark incubation. The red-light-mediated increase was reversed by immediate exposure to far-red light demonstrating the involvement of phytochrome in the regulation of NAD kinase by light. The escape period for far-red reversibility was 3 min. Repeated red light exposures given every 24 h caused a much higher increase, which was also reversed by immediate far-red light treatment. Calmodulin levels remain unaltered in seedlings treated with different lights. The phytochromemediated increase in NAD kinase was due to increased synthesis of the enzyme since the effect was abolished when the seedlings were sprayed with actinomycin D and cycloheximide immediately after red light treatment.

Very high levels of calmodulin (CAL) in outer hair cells (OHC) of organ of Corti (OC)

Using immunohistochemical techniques, Slepecky et al. [Hear. Res. 321, 11–22 (1988)] demonstrated a selective accumulation of CAL, an ubiquitous calcium binding protein, in the inner hair cells (IHC) and OHC of the OC. By quantitative polyacrylamide gel electrophoresis, it was found that in chinchilla OC, CAL is by far the highest in OHC, where levels of 6%–8% of total protein are reached. For comparison, the highest known accumulation of CAL (in the heads of spermatozoa) amounts to 12%. CAL is considerably lower in IHC (about 1%), and is not detectable with usual protein loads in other cell types of the OC. CAL is also very low in stria vascularis and Reissner's membrane. The relative distribution of CAL in the OC is similar to that of glycogen, where the exceedingly high level of 600-mmol/kg dry weight is reached in OHC [Thalmann et al., Laryngoscope 80, 1619–1645 (1970)]. This circumstantial evidence suggests that activation of phosphorylase kinase may be an important function of CAL in OHC, whereby ultimately energy for contraction is mobilized from glycogen. Most likely CAL-induced activation of myosin light chain kinase also takes place as part of the contractile process. [Work supported by NIH and DRF.]

Central dopamine-synthesis regulation by the calcium-calmodulin-dependent system

The effects of the intraventricular (IVT) administration of calcium on the amount of dopamine (DA) in various regions of the mouse brain were analyzed immunohistochemically by using a microphotometry system. The DA levels in the nucleus accumbens and the lateral part of the neostriatum were increased by approximately 45% ( p < 0.01) and 25–35% ( p<0.01), respectively, by the IVT administration of CaCl 2 (10 μmol/kg). It was also found that this effect was abolished by the calmodulin antagonist, W-7 (4.2 μg/mouse, IVT). The brain regions in which the amount of DA was increased by calcium were areas where high levels of calmodulin and tyrosine hydroxylase are distributed. These findings suggest that the synthesis of central DA is regulated by calcium through a calmodulin-dependent system.

The presence of parvalbumin in a nonmuscle cell line attenuates progression through mitosis.

Based on studies that have examined the effect of calcium chelators on cells, it has been proposed that this cation plays a role in regulating cell proliferation. In this study a novel approach was used to indirectly examine the role of calcium in cell cycle progression. A cDNA for the Ca2+-binding protein parvalbumin has been expressed in mouse C127 cells, using a bovine papilloma virus-based expression vector. The normal role of parvalbumin is that of a calcium buffer in vertebrate fast twitch muscle, and the C127 cells do not normally express this protein. The presence of parvalbumin had several effects on the growth of C127 cells. The most striking phenotype was an increase in cell cycle duration which analysis showed was the result of an increase the length of G1 and mitosis (predominantly at prophase). Since changes in cell cycle duration typically occur as a result of changes in G1 duration, the observed increase in the length of mitosis is most unusual. The present results indicate that the previously observed increase in the rate of cell proliferation in cells with elevated calmodulin levels is not the result of a general increase in the level of cytoplasmic calcium-binding protein, but is specific to calmodulin. In addition, the results suggest that calcium regulates progression through mitosis by both calmodulin-dependent (metaphase transition) and -independent (prophase) mechanisms.

Abscisic acid and temperature modify the levels of calmodulin in embryonic axes of Cicer arietinum

Changes in calmodulin (CaM) levels in embryonic axes of Cicer arietinum L. cv. Castellana germinated under three different conditions were measured. Abscisic acid (ABA) and a temperature of 30°C, which delay chick‐pea germination, respectively decreased and increased the concentration of CaM compared to the values obtained under normal germinating conditions (H2O‐25°C). The CaM concentration was higher in those zones of the axes undergoing an active cell division.The compartmentalization of CaM in 36‐h‐old embryonic axes grown under these three conditions was also measured. Cytosolic and mitochondrial CaM was higher in axes where a delayed germination occurred as well as in the cell walls of normally grown ones. On the other hand, CaM was higher in nuclear and microsomal fractions extracted from H2‐O‐25°C‐treated axes. From these data we postulate that delayed germination could be an effect of altered CaM distribution in chick‐pea embryonic axes.

A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division.

The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed. The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12-15 h. The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12 h, no intracellular calmodulin protein was detectable. Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle. Thus, the defect was mainly in nuclear division. Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud. Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.

Calmodulin is required for cell-cycle progression during G1 and mitosis.

In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine-papilloma-virus (BPV)-based expression vectors capable of inducibly synthesizing CaM sense (BPV-MCM) or anti-sense (BPV-CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV-MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV-CaMAS vector transiently produce CaM anti-sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti-sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell-cycle progression under normal conditions of growth.

動脈硬化性疾患の急性虚血による血小板凝集能の変動機序に関する研究

Platelets and ischemia play an important role in the progression of atherosclerosis. In order to clarify the pathogenetic mechanism triggered by ischemia in atherosclerotic disease, the relationship between ischemia and platelet function was studied in 25 patients with atherosclerotic disease (A group) and in 25 healthy subjects (H group).Early in the morning, at a fasting state, blood was drawn from an antecubital vein. The upper arm was compressed at a pressure of 20mmHg above systolic arterial pressure. Five minutes after compression, and immediately after releasing it, blood was obtained again. In each blood sample, we measured platelet counts, platelet aggregability, cytoplasmic free calcium ion concentration ([Ca++]i) in platelets, calmodulin concentration in platelets, plasma β-thromboglobulin (β-TG), plasma platelet factor 4 (PF4), plasma c-AMP, plasma 6-keto PGF1α, plasma thromboxane B2, pH, P02, PCO2 and serum lactic acid concentration.Platelet counts decreased significantly 5 minutes after compression. Platelet aggregability in the H group was suppressed during and after compression. In the A group, however, aggregability increased slightly or showed no change.A significant increase in [Ca++]i was noted in both groups 5 minutes after compression. The calmodulin concentration in group A was markedly lower than in group H. Calmodulin levels in group A increased significantly during and after compression, but decreased significantly in group H. Plasma β-TG and PF4 levels increased significantly during and after compression in both groups. The plasma c-AMP level increased significantly during and after compression in group A, while it tended to increase in group H during compression.Plasma 6-keto PGF1α concentrations showed no changes caused by ischemia in either group. A significant increase was noted during and after compression in both groups. PH and P02 concentrations decreased after 5 minutes of ischemia in both groups. PCO2 and lactic acid concentrations increased after 5 minutes of ischemia in both groups. The magnitude of pH, P02, PCO2 and lactate changes was greater in group A than in group H.The consumption of easy aggregative platelets may induce decreased platelet counts after 5 minutes of ischemia. Ischemia brought about a significant increase of [Ca++]i and activated platelets. On the other hand, the suppression of platelet aggregability in group H was related to the decreased calmodulin in the platelets. Platelet aggregability was not suppressed during ischemia in group A because calmodulin levels in the platelets increased. The platelets were activated by ischemia and released β-TG, PF4 and thromboxane A2. C-AMP decreases [Ca++]i and suppresses platelet function. C-AMP was increased by ischemia in group A to maintain homeostasis in vivo. The changes of pH, P02, PCO2 and lactate reflected a certain ischemia in our methods.In conclusion, vascular accidents frequently occur in atherosclerotic patients. Therefore, controlling calmodulin levels might be important in reducing vascular accidents following ischemia.

Quantitation of calmodulin in the mammary gland of the lactating sow

It has been suggested that calmodulin, a calcium-binding protein, has a functional role during milk secretion. High levels of calmodulin are present during lactation in rat mammary glands and a substantial increase has been observed in the bovine mammary gland prior to parturition. In the sow, regressed glands involute while suckled glands remain highly active even though they are under the same hormonal influence. In this study, tissue samples were taken from suckled and regressed glands of the same sow at both peak and late lactation. Calmodulin and total protein were measured in tissue homogenate supernatants. Residual milk was apparent in regressed glands during mid lactation but not in the same glands by late lactation. Calmodulin levels in tissue were the same for both suckled and regressed glands. There was a slight but non-significant increase in the tissue calmodulin level from peak to late lactation. Protein levels declined significantly from mid to the late stage of lactation. There was no change in protein level between the suckled and regressed glands. Calmodulin may be responsible for casein phosphorylation and/or the mediation of prolactin action on the gland. The precise regulatory mechanisms relating hormonal control to calmodulin levels during lactation need further investigation.

The effect of trifluoperazine, a calmodulin antagonist, on the growth of normal and malignant epidermal keratinocytes in culture

Calmodulin, a cytoplasmic calcium binding protein, is present in concentrations two- to four-fold higher in malignant cells compared to normal cells. In an effort to learn the significance of these elevated levels, we examined the effect of calmodulin blockage on the growth of normal and malignant keratinocytes in vitro. The level of calmodulin in SCC 12.B 2, a line of keratinocytes derived from an epidermal squamous cell carcinoma (SCC), was about 3.5 times greater than in normal, human newborn foreskin keratinocytes. When exposed to trifluoperazine (TFP), an inhibitor of calmodulin, cell growth was reduced primarily in the cultures of normal keratinocytes. This growth inhibition resulted from two changes in the replicating population of cells, namely an increase in cell cycle length and an increase in rate of cell cycle withdrawal. Cell cycle withdrawal is the irreversible arrest of the cell cycle and is an early event in keratinocyte terminal differentiation. There was no measurable effect on the cell cycle time or withdrawal rate in SCC 12.B 2. The increased resistance to growth arrest in SCC cells may be a consequence of the elevated level of calmodulin in these cells.

Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation.

We have analyzed the levels, subcellular distribution, and target proteins of two calcium-modulated proteins, S100 and calmodulin, in differentiated and undifferentiated rat C6 glioma cells. Undifferentiated and differentiated C6 cells express primarily the S100 beta polypeptide, and the S100 beta levels are four-fold higher in differentiated compared to undifferentiated cells. Double fluorescent labeling studies of undifferentiated cells demonstrated that S100 beta staining localized to a small region of the perinuclear cytoplasm and colocalized with the microtubule organizing center and Golgi apparatus. Analysis of differentiated C6 cells demonstrated that S100 beta distribution and S100 beta-binding protein profile changed significantly upon differentiation. In addition, the brain-specific isozyme of one S100-binding protein, fructose-1,6-bisphosphate aldolase C, can be detected in differentiated but not undifferentiated C6 cells. While changes in the subcellular distribution of calmodulin were not observed during differentiation, calmodulin levels and calmodulin-binding protein profiles did change. Altogether these data suggest that S100 beta and calmodulin regulate different processes in glial cells and that the regulation of the expression, subcellular distribution, and target proteins of S100 beta and calmodulin during differentiation is a complex process which involves multiple mechanisms.

Calmodulin Inhibition by Anthralin

Increased epidermal calmodulin (CaM) levels have been reported in psoriatic lesions. It has been suggested that CaM inhibition might be of relevance in the treatment of psoriasis vulgaris. Therefore we investigated the possible CaM inhibition by the antipsoriatic drug anthralin in vitro and in vivo. For in vitro studies, anthralin (0.44 mM) effects on the CaM-dependent Ca++-ATPase of CaM-depleted erythrocyte ghosts were assessed. At 100 microM Ca++, no enzyme inhibition was measured either in the presence or absence of CaM. At 2 microM Ca++ anthralin inhibited the CaM stimulation of membrane-bound ATPase for 50% in presence of CaM. For in vivo studies, skin biopsies were taken from anthralin-treated psoriatic lesions without scaling but still palpable infiltration during the first 3 weeks of therapy. Lesions with anthralin-induced irritation were excluded. The epidermal CaM level was determined by measuring the ability of soluble epidermal protein to activate the Ca++-ATPase in CaM-depleted erythrocyte ghosts. Epidermal CaM was 7.07 +/- 4.57 (mean +/- SD) microgram CaM/mg soluble epidermal protein. This is a 6-fold increase compared to normal human epidermis and a 3-fold increase compared to nontreated lesional psoriatic epidermis (p less than 0.01 and 0.02, respectively). The results argue against CaM inhibition of psoriatic epidermal keratinocytes as the primary event in the antipsoriatic action of anthralin.

Effects of cyclodiene compounds on calcium pump activity in rat brain and heart

The in vitro and in vivo effects of aldrin, dieldrin, and endrin on calmodulin regulated Ca 2+-pump activity in rat brain synaptosomes and heart sarcoplasmic reticulum were investigated. All the 3 cyclodiene compounds inhibited both brain synaptosomal and heart sarcoplasmic reticulum Ca 2+-pump activity in vitro in a concentration dependent manner. Calmodulin depleted Ca 2+-pump activity was insensitive to the action of toxic compounds. Oral administration of pesticides (0.5–10 mg/kg) to rats similarly decreased the Ca 2+-pump activity, in addition to decreasing the levels of calmodulin of both brain and heart thus indicating disruption in membrane Ca 2+ transport mechanisms. Exogenous addition of calmodulin (1–20 μg) could effectively reverse the pesticide induced inhibition. Ca 2+-pump activity is more sensitive to the 3 cyclodiene compounds in brain than in heart. The results of the present study indicate that the cyclodiene compounds may produce neurotoxic effects by altering calmodulin regulated calcium dependent events in neurons.

1,25 Dihydroxyvitamin D3 affects calmodulin distribution among subcellular fractions of skeletal muscle.

1,25 Dihydroxyvitamin D3 has been shown to stimulate calcium fluxes across skeletal muscle membranes. The involvement of calmodulin in the effects of the metabolite was investigated. Primary cultures of chick embryo skeletal muscle myoblasts and soleus muscles from vitamin D-deficient or 1,25 (OH)2D3-treated chicks were used. Culture of myoblasts and vitamin D-deficient soleus with 1,25 (OH)2D3 (0.05 ng/ml) for 24 and 1 hour, respectively, significantly increased 45Ca uptake by the preparations. In the presence of the calmodulin antagonists flufenazine or compound 48/80 in the uptake medium, no differences between control and treated cultures were observed. The calmodulin content of myoblasts and soleus homogenates and subcellular fractions derived therefrom was estimated by measuring their capacity to stimulate calmodulin-depleted cAMP phosphodiesterase. No changes in total calmodulin cellular content could be detected in response to 1,25(OH)2D3. However, the sterol produced an increase in calmodulin levels of microsomes, mitochondria, and crude myofibrillar fraction and a proportional decrease in cytosolic calmodulin concentration. The 1,25(OH)2D3-dependent changes in calmodulin distribution among subcellular fractions of soleus muscle were observed either in vivo or in vitro. The effects in vitro were already detectable after 5 minutes of treatment with the sterol and parallel 1,25(OH)2D3-dependent changes in tissue Ca uptake. The results suggest that changes in calmodulin intracellular distribution may underly part of the mechanism by which 1,25(OH)2D3 affects muscle calcium transport.

Changes in the Levels of Calmodulin and of a Calmodulin Inhibitor in the Early Phases of Radish (Raphanus sativus L.) Seed Germination

An inhibitor of Ca(2+)-calmodulin (Cam)-dependent brain phosphodiesterase was present in the soluble fraction of embryo axes from ungerminated radish (Raphanus sativus L.) seeds. This inhibitor is a Ca(2+)-dependent, Cam-binding protein; in fact: (a) its effect was strongly reduced by treatment with proteases; (b) the inhibition was counteracted by Cam but not by Ca(2+); (c) on gel filtration in the presence of Ca(2+), Cam co-chromatographed with the inhibitor. The inhibitor is heat stable and positively charged at pH 7.5. During early phases of germination, the fresh weight and the levels of DNA and RNA of embryo axes increased, the level of the inhibitor decreased, and the level of Cam increased. Abscisic acid (ABA) inhibited germination, the decrease of inhibitor, and the increase of Cam. Fusicoccin (FC) stimulated the increase in fresh weight but not the increase in the RNA and DNA levels; in this condition, the inhibitor level decreased and the increase in Cam level was higher than in the control. In the presence of both ABA and FC, there was an increase in fresh weight not accompanied by an increase in DNA and RNA levels; Cam increased and, on a fresh weight basis, reached the value of the control. These results indicate that the Ca(2+)-Cam system was activated in early germination of radish seeds by an increase in Cam and a decrease in the inhibitor levels, that FC, probably through the activation of membrane functions, increased Cam level, and that the ABA inhibition on germination was not mediated by the Ca(2+)-Cam system.