High Levels Of Calmodulin Research Articles

Mycorrhizal-induced calmodulin mediated changes in antioxidant enzymes and growth response of drought-stressed trifoliate orange.

Trifoliate orange [Poncirus trifoliata (L) Raf.] is considered highly arbuscular mycorrhizal (AM) dependent for growth responses through a series of signal transductions in form of various physiological responses. The proposed study was carried out to evaluate the effect of an AM fungus (Funneliformis mosseae) on growth, antioxidant enzyme (catalase, CAT; superoxide dismutase, SOD) activities, leaf relative water content (RWC), calmodulin (CaM), superoxide anion (), and hydrogen peroxide (H2O2) concentrations in leaves of the plants exposed to both well-watered (WW) and drought stress (DS) conditions. A 58-day of DS significantly decreased mycorrhizal colonization by 60% than WW. Compared to non-AM seedlings, AM seedlings displayed significantly higher shoot morphological properties (plant height, stem diameter, and leaf number), biomass production (shoot and root fresh weight) and leaf RWC, regardless of soil water status. AM inoculation significantly increased CaM and soluble protein concentrations and CAT activity, whereas significantly decreased and H2O2 concentration under both WW and DS conditions. The AM seedlings also exhibited significantly higher Cu/Zn-SOD and Mn-SOD activities than the non-AM seedlings under DS but not under WW, which are triggered by higher CaM levels in AM plants on the basis of correlation studies. Further, the negative correlation of Cu/Zn-SOD and Mn-SOD activities with and H2O2 concentration showed the DS-induced ROS scavenging ability of CaM mediated SODs under mycorrhization. Our results demonstrated that AM-inoculation elevated the synthesis of CaM in leaves and up-regulated activities of the antioxidant enzymes, thereby, repairing the possible oxidative damage to plants by lowering the ROS accumulation under DS condition.

The roles of calmodulin polar distribution during pollen hydration and germination

Polarity patterning of pollen germination is a vital process for angiosperm fertilization. In our study a new method was employed to investigate the real-time distribution of calmodulin (CaM) in living pollen grains and pollen tubes. The CaM–GFP fusion gene was constructed under the control of the pollen-specific promoter LAT52-7 and transformed into Nicotiana tabacum L. Through confocal laser scanning microscopy, high levels of CaM were observed to accumulate in the three germinal apertures, and a tip–base gradient of CaM was detected in elongating pollen tubes. During pollen-grain hydration and germination, one of the three germinal apertures aggregated a much higher level of CaM than the other two. In addition, CaM showed a directional migration from the cytoplasm to this germinal aperture, where the pollen tube would emerge. Interestingly, CaM was not detected in the reproductive nucleus of either pollen grains or pollen tubes. Our findings indicated that the directional migration of CaM existed during pollen hydration and germination, and this movement may play a crucial role in the normal polarity establishment of pollen germination.Key words: calmodulin, polarity, pollen grain, Nicotiana tabacum.

Quantitative imaging of tyrosine hydroxylase and calmodulin in the human brain

The distributions of tyrosine hydroxylase and calmodulin in adult normal postmortem human brain were analyzed quantitatively. Consecutive coronal sections were obtained from the anterior area of the right hemisphere and were stained immunohistochemically for tyrosine hydroxylase and calmodulin. Stained sections were divided into approximately 3 million microareas at 50 μm intervals, and the immunohistochemical fluorescence intensity in each area was measured by a human brain mapping analyzer, which is a microphotometry system for analysis of the distribution of neurochemicals in a large tissue slice. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all brain regions, but its intensity varied. Relatively high levels of calmodulin were observed in brain regions with high levels of tyrosine hydroxylase, though high levels of tyrosine hydroxylase were not always observed in brain regions where high levels of calmodulin were distributed. In particular, high levels of both of tyrosine hydroxylase and calmodulin were distributed in the caudate nucleus and putamen. Previously it was shown that tyrosine hydroxylase was activated and dopamine synthesis was enhanced in the neostriatum region in mice and rats by the intracerebroventricular administration of calcium through a calmodulin-dependent system. The present results combined with these previous findings suggest that the activity of tyrosine hydroxylase in the caudate nucleus and putamen of humans may also be regulated by a calcium/calmodulin-dependent system. J. Neurosci. Res. 63:369–376, 2001. © 2001 Wiley-Liss, Inc.

Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase.

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca2+-calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

Calmodulin-Dependent Autophosphorylation of Smooth Muscle Myosin Light Chain Kinase: Intermolecular Reaction Mechanism via Dimerization of the Kinase and Potentiation of the Catalytic Activity Following Activation

In the presence of Ca2+ and calmodulin (CM), purified smooth muscle myosin light chain kinase (MLCKase) was found to undergo autophosphorylation at a rate that was about 200-fold slower than its catalytic activity. Up to 1.7 mol of phosphate were incorporated per mole of kinase. Lower levels of incorporation could be correlated with the presence of an endogenous protein phosphatase which could be inhibited with okadaic acid or Microcystin-LR. The major autophosphorylation site was identified as Thr-863 or Thr-865 and was located on the 24-kDa C-terminal fragment of the kinase. In addition, there was a relatively low and variable contribution of a Ca/CM-independent autophosphorylation at Ser-814 or Ser-815. The initial autophosphorylation rates and maximal incorporation levels were highest at a molar ratio of 2 MLCKase to 1 CM and were inhibited at higher CM levels. This indicated that binding of one molecule of the kinase apoenzyme by a CM-kinase complex was necessary for the reaction to occur. Kinetic analysis of the autophosphorylation reaction was consistent with this interpretation and indicated a second-order intermolecular process that included MLCKase dimerization or oligomerization. In contrast, the low Ca/CM-independent contribution was of intramolecular type since it did not depend on the kinase concentration. The autophosphorylation appeared to be involved in a relatively slow modification of the oligomeric properties of the kinase leading to a 2-4-fold amplification of the kinase catalytic activity which followed its activation by CM. Oligomerization and dimerization of the kinase was independently demonstrated by light scattering measurements.

Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system

Immunohistochemical distributions of tyrosine hydroxylase and calmodulin in the rat forebrain were analyzed quantitatively to confirm our previous results that the activities of central catecholamine-synthesizing enzymes are regulated by a calcium-calmodulin-dependent system. The adjacent slices of adult rat brain were stained immunohistochemically for tyrosine hydroxylase and for calmodulin, and the distributions and amounts of these proteins were measured by a fluorescence microphotometry system that was developed in our laboratory. Immunohistochemical fluorescence intensity was measured stepwise at 40 μm intervals through a 6 μmφ (on the slice) pin hole. Each stained brain slice was divided into approximately 100,000 areas, and measured for fluorescence intensity and displayed two- and three-dimensionally. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all areas of the brain, but its intensity varied. The relatively high levels of calmodulin could be observed in brain regions with high levels of tyrosine hydroxylase distribution, though high levels of tyrosine hydroxylase could not always be observed in brain regions where high levels of calmodulin were distributed. In the present study, high levels of tyrosine hydroxylase and calmodulin were distributed in the nucleus accumbens septi and the lateral part of the neostriatum regions in which the amount of dopamine was increased by the intraventricular administration of calcium. These findings suggest that the synthesis of central catecholamines is regulated by a calcium-calmodulin-dependent system.

Effect of intraventricular administration of calcium on the lowering of brain dopamine level in epileptic mice

Dopamine (DA) levels in the various brain regions of epileptic mice (El mice) were compared immunohistochemically with those in ddY mice (the mother strain of El mice) using a fluorescence microphotometry system. The fluorescence intensities of DA in the neostriatum and nucleus accumbens septi in El mice were approximately 11–15% ( P < 0.01) and 13% ( P < 0.01) lower than in ddY mice. On the other hand, the lower DA amounts in these regions of El mice were improved by intraventricular administration of CaCl 2 (10 μmol/kg). 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. This finding reconfirmed our previous report that the biogenic amine level disorder in El mice was related to a calcium non level disorder through a central calcium-calmodulin-dependent biogenic amine-synthesizing mechanism, and this might increase their susceptibility to epileptic convulsions.

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.

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.

Vitamin D-3 induces the de novo synthesis of calmodulin in Phaseolus vulgaris root segments growing in vitro

Vitamin D-3 affects growth and Ca 2+ transport in Phaseolus vulgaris roots by a mechanism dependent on de novo protein synthesis. The objective of this work was to identify the protein(s) induced by the sterol. Phaseolus vulgaris root segments cultured in vitro were used. Protein extracts of control cultures and cultures treated with 10 −9 M vitamin D-3 for 2 h labelled with [ 14C]leucine and [ 3H]leucine, respectively, were mixed and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Examination of 3H: 14C ratios in slices of gels revealed that vitamin D-3 stimulates production of a protein which exhibited Ca 2+-dependent electrophoretic mobility. The apparent molecular weight was 14500 in the presence of 1 mM Ca 2+ and 18 000 in the presence of 5 mM EGTA. This protein was heat- and acid-stable, bound 45Ca 2+ and had an isoelectric pH of 3.8-4.1. These data are consistent with the properties of calmodulin. In agreement with these observations, higher levels of calmodulin were detected in roots treated with vitamin D-3 than in control roots by means of assays based on the activation of calmodulin-dependent enzymes. Moreover, estimation of calmodulin relative synthesis and degradation rates in roots cultures in the absence and presence of vitamin D-3 indicated that the sterol increases calmodulin synthesis without affecting its degradation. In addition, the results suggest that the synthesis of other low-molecular-weight Ca 2+-binding proteins may be affected by vitamin D-3.

Calmodulin N-methyltransferase. Partial purification and characterization.

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K 0.5 = 93 +/- 5 nM), glycogen synthase (K 0.5 = 127 +/- 10 nM), and κ-casein (K 0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.

Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts.

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.

Biosynthesis of Calmodulin in Normal and Virus-Transformed Chicken Embryo Fibroblasts

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.