Low parotid saliva calmodulin in patients with taste and smell dysfunction

Parotid saliva calmodulin was found both in 32 normal volunteers and in 60 patients with taste and smell dysfunction; salivary calmodulin concentration was significantly lower in the patients than in the volunteers. There were no differences in salivary calmodulin concentration with respect to age, sex, or salivary flow rate in either normal volunteers or patients. When patients were categorized by diagnosis, calmodulin concentration was found to be decreased in all patient groups. The concentration of calmodulin in saliva was about 10 times that found in serum, suggesting that the parotid gland is a major source of this protein.     

A simple procedure for the purification of calmodulin bound to membranes; calmodulin bound to the particulate fraction of AH-66 hepatoma ascites cells

We have demonstrated the identity of calmodulin tightly bound to the particulate fractions of AH-66 hepatoma cells and normal liver with authentic soluble calmodulin and have compared the particulate calmodulin content of AH-66 cells with that of normal liver. Calmodulin bound to the particulate fractions of the hepatoma and normal liver cells was purified to electrophoretic homogeneity by a simple procedure which involves solubilization of the particulate fractions with LIS, extraction of the solubilized solution with 37.5% phenol, gel filtration, and affinity chromatography on Fluphenazine-Sepharose. There were no detectable differences between the particulate calmodulin thus purified and authentic soluble calmodulin of rat brain. The particulate calmodulin in the hepatoma and normal liver cells was assayed based on its ability to activate calmodulin-deficient phosphodiesterase after partial purification of calmodulin from the particulate fractions by solubilization with LIS and extraction with phenol as described above. In addition, the particulate calmodulin content in the hepatoma and normal liver cells was also measured after solubilization of the particulate fractions with Lubrol PX. The results obtained by these two different procedures indicate that calmodulin content in the particulate fraction as well as in the soluble fraction of the hepatoma is significantly higher than that in the corresponding fractions of normal liver.     

The abundance of calmodulin mRNAs is regulated in phosphorylase kinase-deficient skeletal muscle

In the I/Lyn mouse strain a mutation on the X chromosome results in a deficiency of the major calmodulin-regulated enzyme in skeletal muscle, phosphorylase kinase. Calmodulin has been identified as the delta-subunit of phosphorylase kinase, and it is estimated that approximately 40% of the total calmodulin in rabbit skeletal muscle is associated with the phosphorylase kinase hexadecamer (alpha, beta, gamma, delta)4. The absence of phosphorylase kinase in I/Lyn skeletal muscle results in a reduction in the total amount of calmodulin. The mechanisms affecting this reduction were investigated by comparing the abundance and heterogeneities in calmodulin mRNAs between normal and phosphorylase kinase-deficient skeletal muscles. The results demonstrate that in normal tissue there are four species of calmodulin mRNA distinguished by their molecular weight. All four of these species are present in the deficient tissue, and none of them are preferentially reduced. However, there is a 54% reduction in all four mRNAs as well as in calmodulin in the deficient skeletal muscle relative to normal skeletal muscle. These results indicate that the expression of calmodulin mRNAs is coordinated with the expression of its major enzyme target in skeletal muscle.     

Elevation of calmodulin in avian muscular dystrophy

In an attempt to understand the mechanism of calcium accumulation in myopathies, changes in the major calcium-binding protein, calmodulin, was studied in genetically dystrophic chickens. Measurements by radioimmunoassay revealed an increase in the calmodulin concentration of dystrophic chicken muscles. Poly A-containing RNA(s) of fast and slow muscles from the normal and dystrophic chicks were hybridized with [32P]-labeled calmodulin cDNA probe by the dot-hybridization technique. Densitometric scan of the autoradiogram showed that the calmodulin mRNA levels of dystrophic fast muscles (pectoralis and posterior latissimus dorsi) were approximately two-fold higher than those of the corresponding normal muscles. No significant change in calmodulin and calmodulin messenger RNA of slow muscle (ALD) was found in dystrophic chickens. Our results suggest that increased calcium flux within the dystrophic muscle may be modulated by calmodulin.     

Calcium/calmodulin and cAMP/protein kinase-A pathways regulate sperm motility in the stallion

In spite of the importance of sperm motility to fertility in the stallion, little is known about the signaling pathways that regulate motility in this species. In other mammals, calcium/calmodulin signaling and the cyclic AMP/protein kinase-A pathway are involved in sperm motility regulation. We hypothesized that these pathways also were involved in the regulation of sperm motility in the stallion. Using immunoblotting, calmodulin and the calmodulin-dependent protein kinase II β were shown to be present in stallion sperm and with indirect immunofluorescence calmodulin was localized to the acrosome and flagellar principal piece. Additionally, inhibition of either calmodulin or protein kinase-A significantly reduced sperm motility without affecting viability. Following inhibition of calmodulin, motility was not restored with agonists of the cyclic AMP/protein kinase-A pathway. These data suggest that calcium/calmodulin and cyclic AMP/protein kinase-A pathways are involved in the regulation of stallion sperm motility. The failure of cyclic AMP/protein kinase-A agonists to restore motility of calmodulin inhibited sperm suggests that both pathways may be required to support normal motility.     

Calmodulin level and cAMP-dependent protein kinase activity in rat spermatogenic cells and hormonal control of spermatogenesis

The changes in intracellular calmodulin levels and cAMP-dependent protein kinase activities have been studied in the testis of normally developing and hypophysectomized rats. It appears that the onset of spermatogenesis which occurs on the first days of the postnatal development, is associated with a major (over fivefold) increase in the calmodulin level and enhancement of the cAMP-dependent protein kinase activity. On the contrary, hypophysectomy of adult animals is associated with a progressive decline in the calmodulin level and a rapid and regular decrease in the cAMP-dependent protein kinase activity. Moreover, measurements of the intracellular calmodulin level and cAMP-dependent kinase activity of isolated testicular germ cells or epididymal spermatozoa have shown that testosterone, administered to hypophysectomized rats as subcutaneous implants, maintains the concentration of these regulatory proteins to normal values.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

The role of calmodulin in human renal cell carcinoma

Calmodulin is a ubiquitous intracellular calcium binding protein which has been shown to be associated with cell cycling. Previous studies using animal tumor models have suggested a positive correlation between tumor calmodulin content and rate of tumor growth. We studied the role of calmodulin in renal cell carcinoma (RCC) cell lines and compared this with short term normal fetal kidney cell lines. The effects of calmodulin inhibition was determined using the calmodulin inhibitor W13 (Naphthalene-sulfonamide) and its less active partner W12. Cell size, calmodulin content and inhibition studies using W13 did not reveal any simple correlations for the RCC cell lines, although the RCC lines did have a higher content than the fetal kidney cell lines. Calmodulin content determination of RCC and normal adult kidney tissue failed to show any difference. We conclude that, contrary to previous reports using animal models, there is no simple relationship between tumor growth rates and calmodulin content for human RCC.     

Abnormal expression of the calmodulin gene in muscle from the dystrophic chicken

Compared to that of genetically-related normal chickens, pectoralis muscle from the dystrophic chicken contained increased calmodulin measured by radioimmunoassay. Determined by the dot blot procedure, expression of the calmodulin gene was enhanced in muscle from affected animals. The bioactivity of the gene product was normal. Together with previous studies reporting increased cell Ca2+ content in dystrophic muscle, the current findings of increased sarcoplasmic calmodulin suggest the latter is a cellular response to defective Ca2+ transport at the level of cell efflux or intracellular organelle (sarcoplasmic reticulum) uptake.     

Abnormal response to calmodulin in vitro of dystrophic chicken muscle membrane Ca2+-ATPase activity

A skeletal muscle membrane fraction enriched in sarcoplasmic reticulum (SR) contained Ca2+-ATPase activity which was stimulated in vitro in normal chickens (line 412) by 6 nM purified bovine calmodulin (33% increase over control, P less than 0.001). In contrast, striated muscle from chickens (line 413) affected with an inherited form of muscular dystrophy, but otherwise genetically similar to line 412, contained SR-enriched Ca2+-ATPase activity which was resistant to stimulation in vitro by calmodulin. Basal levels of Ca2+-ATPase activity (no added calmodulin) were comparable in muscles of unaffected and affected animals, and the Ca2+ optima of the enzymes in normal and dystrophic muscle were identical. Purified SR vesicles, obtained by calcium phosphate loading and sucrose density gradient centrifugation, showed the same resistance of dystrophic Ca2+-ATPase to exogenous calmodulin as the SR-enriched muscle membrane fraction. Dystrophic muscle had increased Ca2+ content compared to that of normal animals (P less than 0.04) and has been previously shown to contain increased levels of immuno- and bioactive calmodulin and of calmodulin mRNA. The calmodulin resistance of the Ca2+-ATPase in dystrophic muscle reflects a defect in regulation of cell Ca2+ metabolism associated with elevated cellular Ca2+ and calmodulin concentrations.     

Expression of human calmodulin cDNA in Escherichia coli and characterization of the protein

A cDNA clone of human calmodulin, isolated from liver, was subcloned into the expression vector pKK233-2. The resulting expression plasmid, designated pCWCaM1, produced human calmodulin in Escherichia coli SG5. The cDNA was sequenced using novel primers designed for use in plasmid-sequencing protocols with pKK233-2 and pKK223-3. The expressed calmodulin was purified and subjected to NMR analysis which revealed a structure essentially the same as natural calmodulin isolated from human tissue. The activation of myosin light chain kinase by the genetically engineered human calmodulin and bovine brain calmodulin was studied and found to be comparable to a high degree. The expressed calmodulin appears to be comparable to normal calmodulin and can be used for site-directed mutagenesis and structure/function investigations.     

Protein product of a human intronless calmodulin-like gene shows tissue-specific expression and reduced abundance in transformed cells.

The recently identified NB-1 mRNA is transcribed from a single intronless gene, previously thought to be an unexpressed calmodulin pseudogene. Although expression levels of the three known human calmodulin genes fluctuate only slightly in all cell types and tissues examined, NB-1 expression is limited to certain cells of pseudostratified and stratified epithelial tissues. Like calmodulin, the protein encoded by NB-1 is heat stable and binds to phenyl-Sepharose in a calcium-dependent manner. Despite the shared identity of 85% of their 148 amino acids, however, calmodulin and NB-1 protein are easily distinguished electrophoretically and immunologically. Polyclonal antibodies prepared against recombinant NB-1 protein recognize a protein with an apparent molecular weight of 16,000 which is abundant in cultured normal human mammary epithelial cells, but which is absent or barely detectable in fibroblasts or tumor cell lines. The immunohistochemical distribution of NB-1 protein in histologically normal tissues suggests that expression of the gene is regulated during epithelial differentiation. The majority of a small number of malignant tissues examined had lowered or undetectable NB-1 protein expression relative to normal tissues. Given its restricted distribution, the NB-1 protein may be involved in the initiation or maintenance of certain differentiated functions. Its absence may be due to or necessary for the manifestation of the transformed phenotype in certain cell types.     

Mutational analysis of calmodulin in Saccharomyces cerevisiae.

Calmodulin is well characterized as an intracellular Ca2+ receptor in nonproliferating tissues such as muscle and brain. Several observations indicate that calmodulin is also required for cellular growth and division. Deletion of the calmodulin gene is a lethal mutation in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Expression of calmodulin antisense RNA in mouse C127 cells causes a transient arrest at G1 and metaphase. Although these results indicate calmodulin plays a critical function during proliferation, they do not reveal the function. S. cerevisiae offers an excellent system for identifying calmodulin functions. Because calmodulin mutants can be readily constructed by gene replacement the consequences of mutations in calmodulin can be directly examined in vivo without interference from wild-type calmodulin. The available wealth of information concerning all aspects of the yeast life cycle provides a large framework for interpretation of new results. The recent dissection of cell cycle regulation is just the latest example of the important insights provided by analyzing basic cellular processes in yeast. Whether studies of calmodulin in yeast will reveal a universal function is unknown. One encouraging result is that yeast cells relying on vertebrate calmodulin as their only source of calmodulin survive and grow well, even if the amount of vertebrate calmodulin is equivalent to the normal steady state levels of yeast calmodulin. This review discusses the varied techniques we are using to identify the functions of calmodulin in yeast. As part of the analysis, we are defining the essential elements of calmodulin structure.     

Regulation of hepatic eNOS by caveolin and calmodulin after bile duct ligation in rats

In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.     

Regulation of red cell membrane deformability and stability by skeletal protein network

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca2+ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca2+ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and physiologic concentrations of calmodulin. Our data show that Ca2+ concentrations in the range of 1 to 100 microM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca2+ concentrations higher than 100 microM, and calmodulin had no effect. Examination of the the effects of Ca2+ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca2+ and calmodulin on spectrin-protein 4.1-actin interaction.     

Inhibition of myogenesis by trifluoperazine and compound 48/80

When trifluoperazine (TFP), a calmodulin antagonist, was given to chick or rat myoblasts in cultures, formation of multinucleated myotubes was inhibited. The inhibition of cell fusion by TFP in rat cultures prevents the normal increase in the amount of acetylcholine receptors (AChR) and creatine kinase (CK), while the levels of these proteins in chick muscle cultures are hardly affected. Another calmodulin antagonist, compound 48/80, inhibits fusion at doses that correspond closely to its antagonistic effects on calmodulin. Thus, our results suggest a possible role for calmodulin in the regulation of myoblast fusion, but not on the appearance of muscle proteins.     

Calmodulin in lymphocyte mitogenic stimulation and in lymphoid cell line growth

Calmodulin levels are elevated three- to fourfold in the dividing cells, resulting from the lectin-induced stimulation of fresh human lymphocytes. This increase in calmodulin appears to be related mainly to progression into S phase and supports the hypothesis that calmodulin might be crucial in regulating the progression of lymphoblasts through their division cycle. Calmodulin levels are higher in a lymphoid cell line derived from human acute lymphoblastic leukemia blood cells than in a lymphoid cell line derived from normal human blood cells, suggesting that calmodulin could be an important mediator of the leukemogenetic process.     

Estrogen modulation of eNOS activity and its association with caveolin-3 and calmodulin in rat hearts

Previous studies have shown that estrogen modulation of endothelial nitric oxide (NO) synthase (eNOS) may confer protection against heart disease. Here, we demonstrate an association between reductions in baroreflex-mediated bradycardia and in cardiac NOS activity in ovariectomized (Ovx) rats compared with controls. The latter resulted, at least in part, from a reduction in cardiac eNOS protein. eNOS-derived NO and its biological effects are determined by the levels of eNOS protein and by eNOS catalytic activity; the latter is regulated partly through the dynamic interaction with an inhibitory protein (caveolin) and a stimulatory protein (calmodulin). The association of eNOS immunoprecipitated with caveolin-3 and calmodulin was examined. Caveolin-3 and calmodulin binding with eNOS was increased and decreased, respectively, in Ovx rats. 17 beta-Estradiol replacement restored, to within normal levels, the baroreflex-mediated bradycardic responses along with eNOS activity, eNOS expression, and the association of eNOS with caveolin-3 and calmodulin. Our findings may help to elucidate the molecular mechanism underlying the favorable effects of estrogen on cardiac responses to baroreflex activation.