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PubMed2006

Targeting calmodulin in reversing multi drug resistance in cancer cells.

Calmodulin is a Ca2+ binding protein found in many eukaryotic cells. It is one of the most important intracellular mediators of Ca2+-dependant signaling in eukaryotic cells. It regulates diverse processes including mitosis, muscle contraction and nucleotide metabolism by modulating the activity of at least 30 different target enzymes in a calcium-dependant manner. Calmodulin plays an important role in the regulation of processes, such as the assembly and disassembly of microtubules by controlling protein kinase activities, by exerting an indirect influence upon a wide variety of cellular processes. It is observed that multi-drug resistant cells have a greater intracellular concentration of calcium than non-resistant cells which contributes to their increased sensitivity to calmodulin antagonism compared with that of non resistant cells. Calmodulin mediated processes can be effectively inhibited by a variety of pharmacological agents of different chemical structures, eg:The calcium channel blocker verapamil and antipsychotic drugs like the Phenothiazines. Many bioisosteres of phenothiazines like phenoxazines and acridones have been prepared and these have also shown very good calmodulin antagonism. These calmodulin antagonists have been shown to modulate multi-drug resistance (MDR) in cancer cells. This review highlights concepts of identification and optimization of new inhibitors of calmodulin in reversing MDR in cancer cells.

Animals↗

PubMed1984

The effects of aging and diabetes mellitus on human red and white cell calmodulin levels (chemotaxis/phagocytosis/calcium).

Both the calcium ion and calmodulin have been proposed to play a role in producing the intracellular effects of insulin. Abnormalities in calmodulin levels have previously been reported in tissues from diabetic animals. Thus, using a sensitive and specific radioimmunoassay, we measured calmodulin levels in red cells and polymorphonuclear leukocytes from humans with diabetes mellitus. Diabetic patients have significantly lower white cell calmodulin levels than age-matched controls. There were no significant differences in red cell calmodulin levels in diabetics compared with controls. Red cell calmodulin levels in normal subjects were decreased with advancing age. We conclude that the alteration in calmodulin levels in leukocytes from diabetics may be associated with the decreased neutrophil function that has been observed in diabetics.

Adolescent↗

PubMed1991

Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins.

The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules.

Adenosine Triphosphate↗

PubMed1988

Effect of calmodulin antagonists on calcium pump of ram spermatozoa plasma membrane.

Plasma membranes isolated from ram spermatozoa contain calmodulin, which represents approximately 0.03% of the total sperm calmodulin and 0.025% of the membrane protein. When membranes were isolated in the presence of ethylene glycol (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), the amount of calmodulin associated with the plasma membranes was reduced by only 20%. The ATP-dependent calcium transport activity of the isolated plasma membranes is not enhanced by adding calmodulin and not inhibited by the calmodulin antagonists trifluoperazinc (TFP), compound 48/80, or calmidazolium. In fact, there is an enhancement of calcium uptake by the calmodulin antagonists and this enhancement can be blocked by the Ca2+-channel blocker D-600. It is suggested that the ATP-dependent calcium transport activity in the plasma membrane of ram spermatozoa is not regulated by calmodulin.

Animals↗

PubMedDate not supplied

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin.

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.

Animals↗

PubMed1996

Interactions of amphiphilic peptides derived from alpha s2-casein with calmodulin.

Calmodulin-binding peptides, which had previously been isolated from a pepsin digest of alpha-CN, were synthesized and then examined for their inhibitory effects on the activation of cyclic nucleotide phosphodiesterase that was induced by calmodulin. The concentrations of the synthetic peptides corresponding to 164-179, LKKISQRYQKFALPQY; 183-206, VYQHQKAMKPWIQPKTKVIPYVRY; and 183-207, VYQHQKAMKPWIQPKTKVIPYVRYL, of alpha s2-CN that gave half-maximal inhibition were 65, 7.0, and 2.6 microM, respectively. These inhibitory effects were reversed by increasing the amount of calmodulin. Fragments and analogs were prepared to study the interactions of the peptides with calmodulin in more detail. The results indicated that modification of the carboxyl terminus enhanced the affinities of the three peptides for calmodulin, and a region involved in the inhibition by alpha s2-CN (f183-207) was located at the carboxyl terminus 191-207. Two predicted calmodulin-binding sequences, 164-179 and 191-207 of alpha s2-CN, despite rather divergent primary structures, shared the structural motif common to the calmodulin-binding domains of the target proteins in the previously proposed complex model.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

A strange calmodulin of yeast.

Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.

Animals↗

PubMed2000

Megalin-mediated transcytosis of thyroglobulin by thyroid cells is a calmodulin-dependent process.

Megalin, a multiligand receptor expressed on the apical surface of thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of thyroid hormone release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat thyroid cells (FRTL-5), an established, differentiated thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37 degrees C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To study the role of calmodulin in Tg transcytosis, cells were preincubated with one of two calmodulin antagonists, either trifluoperazine or W7. Both antagonists markedly reduced transcytosis of Tg by FRTL-5 cells. These inhibitory effects and those of a monoclonal antimegalin antibody were not additive, indicating that calmodulin acts on the megalin-mediated pathway. Furthermore, trifluoperazine increased the extent of triiodothyronine (T3) release from exogenously added Tg by FRTL-5 cells, indicating that Tg transported in the calmodulin-dependent, megalin-mediated pathway, bypasses the lysosomal pathway.

Animals↗

PubMed2003

Calmodulin activity in aging rat heart.

Calcium has been known to play an important role in the aging process. Intracellular calcium is regulated by a number of calcium binding proteins including calmodulin. In cardiac tissue, calmodulin is known to regulate a variety of functions. Therefore, to identify possible relationships between calmodulin and the aging process, heart tissue was collected from rats aging 3 weeks to 1 year. Heart tissue was homogenized in a homogenizing buffer and processed for soluble and membrane fractions. These fractions were further subjected to heat treatment. The heat-treated extracts rich in heat stable proteins were used for different experiments. The calmodulin activity was determined as a measure of stimulation of activator deficient 3'-5' phosphodiesterase. The activity of calmodulin at different ages was compared, and no significant changes were observed between 3 weeks vs. 8 weeks, 3 weeks vs. 6 months, and 3 weeks vs. 1 year. The soluble fractions also followed a similar trend, but there was a significant difference between 3 weeks vs. 1 year. At this point we do not know whether the decrease in activity is due to lower expression of calmodulin or decreased ability to stimulate the target enzyme. However, these data suggest that calmodulin may have some association with the aging process.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1990

Calmodulin antagonistic action of KS-504a, a novel metabolite of the fungus Mollisia ventosa.

KS-504a inhibited bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) with an IC50 value of 122 microM. The inhibition was reversed by a high concentration of calmodulin. Calmodulin-independent activities of the enzyme were not affected by the compound at the same concentration ranges. Ca2(+)-dependent interaction of the compounds with calmodulin was shown using hydrophobic fluorescence probes. These data indicated that the compound exerted its effects on CaM-PDE by interacting with calmodulin. KS-504a also inhibited other calmodulin-dependent enzymes at different concentration ranges; myosin light chain kinase was inhibited at the lowest concentrations with an IC50 value of 6.3 microM. The inhibition mechanism was competitive with respect to calmodulin and non-competitive to ATP.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed2004

TRPC channel interactions with calmodulin and IP3 receptors.

Consistent with the conformational coupling mechanism, which suggests that store-operated channels are activated via physical interactions with intracellular calcium release channels, previous studies have demonstrated a functional coupling and a physical interaction between the transient receptor potential canonical type 3 (TRPC3) and inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R-TRPC binding domains were determined using in vitro binding assays. This work aimed to study the effect of IP3R-TRPC interaction on TRPC function. Pull-down experiments were used to study the binding of TRPC to IP3R and to calmodulin. Patch clamp recordings in whole-cell and inside-out configurations were used to examine the effect of a TRPC-binding IP3R fragment, Ca2+ and calmodulin on TRPC activity. We found that IP3R and calmodulin compete for a common binding site at the TRPC C-terminus. TRPC channels are activated either by a peptide representing the TRPC-binding domain of IP3R or by inactivation of calmodulin from the excised membrane patches. TRPC3 activity is inhibited by Ca2+ and calmodulin. Therefore, we have identified a critical IP3R-TRPC interaction that is involved in the activation of TRPC-formed channels. We propose that IP3Rs activate TRPC channels by displacing inhibitory calmodulin from a common calmodulin-IP3R binding site located at the C-terminus of TRPC.

Amino Acid Sequence↗

PubMed1990

A role for calmodulin in the growth of human hematopoietic progenitor cells.

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

Bone Marrow Cells↗

PubMed2006

[Expression of calmodulin in the articular process of vertebrae of adolescent idiopathic scoliosis patients].

OBJECTIVE: To investigate the expression of calmodulin in the articular process of vertebrae of adolescent idiopathic scoliosis (AIS) patients and congenital scoliosis (CS) patients and its possible effects on the pathogenesis of AIS. METHODS: Nine AIS patients aged 13 - 17, and 10 patients of CS, aged 11 - 18, underwent orthopedic surgery. Specimens of the inferior articular process from the apical vertebrae and upper end vertebrae discarded during operation were collected from the AIS patients and specimens of the. Inferior articular process from the upper end vertebrae wee collected from the CS patients. Immunohistochemistry was used to detect the protein expression of calmodulin, and in situ hybridization was adopted to examine the mRNA expression of calmodulin. RESULTS: Calmodulin were expressed in the bone cells. There was no difference between the expression of calmodulin at the concave side and convex side, or between the apical vertebrae and end vertebrae. The expression of calmodulin of the AIS group was significant lower than that of the CS group (P < 0.05). The result of immunohistochemistry and that of in situ hybridization corresponded with each other. CONCLUSION: The expression of calmodulin in the articular process of vertebrae of AIS patients is low, suggesting that it plays a role in the development and progression of AIS.

Adolescent↗

PubMed1991

The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump.

Dimerization (oligomerization) of the plasma membrane Ca2+ pump increases its activity (Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). Fluorescence titration on preparations of the purified eosin-labeled human erythrocyte ATPase has been used to monitor the oligomerization process. Calmodulin inhibits oligomerization, although it can bind to the oligomerized enzyme. Synthetic peptides corresponding to the calmodulin-binding domain of the pump stimulate its ATPase activity, indicating the formation of heterooligomers of the peptides with the pump. The oligomerization is prevented by the preincubation of the ATPase with calmodulin. Polyclonal antibodies against the synthetic calmodulin-binding domain inhibit its basal and its calmodulin-stimulated ATPase activity and prevent the formation of the oligomers. ATPase preparations truncated at the COOH terminus with calpain to a fragment of 124 kDa which does not contain the calmodulin-binding domain fail to oligomerize with the intact ATPase. The results show that the calmodulin-binding domain mediates the oligomerization of the Ca2+ pump.

Amino Acid Sequence↗

PubMed1990

High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli.

We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.

Amino Acid Sequence↗

PubMed1987

Metofenazate as a more selective calmodulin inhibitor than trifluoperazine.

The interaction of several phenothiazines, benzodiazepines, butyrophenones, polycyclic neuroleptics and tricyclic antidepressants with calmodulin and troponin C was investigated using the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide. In the presence of Ca2+, trifluoperazine (2-trifluoromethyl-10-[3-(1-methylpiperazinyl-4)propyl]-phenothiaz ine dihydrochloride, TFP), which is commonly used as a selective calmodulin inhibitor, half maximally increased the fluorescence of the complex formed of the fluorescent dye with calmodulin at a concentration of 4 mumol/l, and with troponin C at 24 mumol/l. TFP completely inhibited the calmodulin dependent stimulation of cyclic nucleotide phosphodiesterase with a Ki of 4 mumol/l and decreased the maximum Ca2+ dependent troponin C mediated activation of actomyosin ATPase by 35% at a concentration of 100 mumol/l. Metofenazate (3,4,5-trimethoxybenzoate-2-chlor-10-(3-[(beta-oxyethyl) piperazinyl-4]-propyl)phenothiazine diethanesulfonate, methophenazine, MP) produced half maximal fluorescence enhancement of the calmodulin dye complex at a concentration of 6 mumol/l and did not influence the fluorescence of the troponin C dye complex at concentrations of up to 1000 mumol/l. MP also completely inhibited the calmodulin dependent stimulation of phosphodiesterase with a Ki of 7 mumol/l but it had not effect on maximum Ca2+ stimulation of actomyosin ATPase. MP increased the Ca2+ sensitivity of skinned cardiac muscle with an about 10fold lower potency than TFP. In view of these results, we propose MP as a useful tool for distinction between processes mediated by either calmodulin or troponin C.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1989

Structure and expression of fungal calmodulin gene.

I report on the isolation, structural analysis, and in vivo expression patterns of a fungal calmodulin gene. The gene is intronless and encodes a protein of 148 amino acid residues that is 92% homologous with vertebrate calmodulins. Through S1 nuclease transcript mapping, it was determined that the cloned gene (a) is transcribed in vivo, (b) has a 5'-untranslated region of about 400 nucleotides, and (c) has a 3'-untranslated end of about 300 nucleotides. Southern blot hybridization analysis of the genomic DNA and the cloned gene provide evidence for the existence of only one type of calmodulin gene in the organism. The amino acid sequence deduced from the DNA sequence shows that Achlya klebsiana calmodulin has amino acid substitutions that are a mix of those seen in calmodulins from invertebrates such as Drosophila and trypanosome when compared to mammalian calmodulins. Not surprisingly, it has less resemblance to calmodulins from Saccharomyces and Dictyostelium.

Amino Acid Sequence↗

PubMed1985

Platelet membrane Ca(2+)-ATPase: blunted calmodulin-stimulation in essential hypertension.

To determine whether defective calcium-efflux pump activity may be responsible for calcium overload in platelets from patients with essential hypertension, the properties of a calmodulin-stimulated Ca(2+)-ATPase in membranes from 25 normotensive and 27 hypertensive subjects were compared. Calcium-ATPase did not differ in affinity for calmodulin or Ca2+ between groups. Stimulation of Ca(2+)-ATPase activity at saturating calmodulin concentrations is diminished in calmodulin-deficient membranes from established essential hypertensive patients (64%) compared with normotensive subjects (125%) (P < 0.01). The capacity for Ca(2+)-ATPase activity (basal and calmodulin-activated) is markedly greater (1.5 to 1.8-fold) in both native and calmodulin-deficient platelet membranes from hypertensive compared with normotensive subjects. The data suggest that calmodulin-stimulation of Ca(2+)-ATPase in platelets from patients with established essential hypertension does not effectively maintain Ca2+ homeostasis, and that the increased Ca(2+)-ATPase capacity may reflect an adaptation to increased intracellular calcium concentration.

Adult↗