Calmodulin Concentration Research Articles

Molecular cloning of the cDNA encoding beta-cell calcium/calmodulin-dependent protein kinase II.

Current hypotheses postulate that the metabolism of glucose within islets results in a transient increase in the ATP:ADP ratio, leading to depolarization and opening of voltage-dependent Ca2+ channels, promoting influx of Ca2+ into the β-cell. The increase in intracellular calcium may activate Ca2+-dependent protein kinases, which in turn phosphorylate key components in the secretory machinery. The nature and identity of these components are at present unclear. In addition, the β-cell contains a high (15–50 μM) concentration of calmodulin (CaM) and it has been shown that inhibitors of CaM block insulin secretion. Possible candidates for Ca2+/calmodulin-dependent protein kinases acting as regulatory enzymes in the exocytotic release process are phosphorylase kinase, myosin light chain kinase and CaM kinases.KeywordsNorthern AnalysisMyosin Light Chain KinasePhosphorylase KinaseAutographa Californica Nuclear Polyhedrosis VirusEncode Protein KinaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The Regulatory Ser262 of Microtubule-associated Protein Tau Is Phosphorylated by Phosphorylase Kinase

Abnormally phosphorylated tau is the major component of paired helical filaments found in the brains of patients suffering from Alzheimer's disease. Therefore, the identification of kinases that phosphorylate tau is of considerable interest. A DEAE-Sepharose column resolved porcine brain extract into five tau kinase activity peaks. Among these peaks, two were completely inhibited by EGTA, indicating that these two activity peaks contained Ca2+-dependent tau kinases. One of the above two Ca2+-dependent tau kinase activity peaks also contained phosphorylase kinase activity. The tau kinase and phosphorylase kinase activities associated with this peak could not be separated from each other by Superose 12 gel filtration, hydroxylapatite, and calmodulin-agarose affinity chromatographies. Phosphorylase kinase, purified from rabbit skeletal muscle, phosphorylated tau to a stoichiometry of 2.1 mol of phosphate/mol of tau and converted tau to a species with a retarded mobility on SDS-polyacrylamide gel electrophoresis. The apparent Km and kcat values for tau phosphorylation by muscle phosphorylase kinase were 6.9 microM and 47.4 min-1, respectively. As a substrate of muscle phosphorylase kinase, phosphorylase was eight times better than tau. Sequence analyses of tryptic and thermolytic phosphopeptides derived from tau phosphorylated by muscle phosphorylase kinase revealed five phosphorylation sites, Ser237, Ser262, Ser285, Ser305, and Ser352. Among these sites, Ser262 was previously shown to be phosphorylated in human tau from fetal, adult, and Alzheimer's diseased brains (Seubert, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V. W., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J. Q., and Lee, V. M. Y. (1995) J. Biol. Chem. 270, 18917-18922); and its phosphorylation abolished tau's binding to microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Slot-blot analysis using a monoclonal antibody against muscle phosphorylase kinase and an activity assay using phosphorylase revealed that phosphorylase kinase was present in microtubules extensively purified by repeated cycles of polymerization and depolymerization. Taken together, these results suggest that in neurons, phosphorylase kinase may be one of the kinases that participate in the phosphorylation of tau.

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol.

The effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl2 (0.5 mM) plus calmodulin (10 microg/ml) in the enzyme reaction mixture . This increase was completely prevented by the addition of trifluoperazine (25 microM), an antagonist of calmodulin. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 microM. The cytosolic Ca2+/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca2+ (100-1000 microM). This increase was markedly blocked by the presence of regucalcin (0.1 microM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 microg/ml). These results demonstrate that regucalcin can regulate Ca2+/calmodulin-dependent protein kinase activity in renal cortex cells.

Inhibition of G Protein-coupled Receptor Kinase Subtypes by Ca2+/Calmodulin

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous betaARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gbetagamma-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 approximately 2 microM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.

Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle.

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca(2+)-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca(2+)-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 microM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.

Inhibition of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes by deprenyl.

Intracellular concentrations of cyclic nucleotides is regulated by cyclic nucleotide phosphodiesterases and calmodulin-dependent cyclic nucleotide phosphodiesterases (CaMPDE), one of the most intensively studied and best characterized phosphodiesterases. In the present study, the effect of an antiparkinsonian agent, deprenyl (selegeline hydrochloride) which is believed to be a selective inhibitor of monoamine oxidase-B, on bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) isozymes have been investigated. The findings indicated that deprenyl inhibited brain 60 kDa isozyme, however the inhibition for brain 63 kDa CaMPDE was observed to a lesser extent. The inhibition of brain 60 kDa CaMPDE was overcome by increasing the concentration of calmodulin suggesting that deprenyl may be calmodulin antagonist or act specifically and reversibly on the action of calmodulin. The 60 kDa CaMPDE isozyme is predominantly expressed in brain and its inhibition can result in increased intracellular levels of cAMP. The increased intracellular levels of cAMP have a protective role for dopaminergic neurons. Therefore, deprenyl may be a valuable tool to investigate the physiological roles of brain CaMPDE isozymes in progression of Parkinson's disease and gives a new insight into the action of this drug.

A Role in Enzyme Activation for the N-terminal Leader Sequence in Calmodulin

We have found that deletion of residues 2-8 from the N-terminal leader sequence: Ala1-Asp2-Gln-Leu4-Thr-Glu6-Glu-Gln8, in calmodulin abolishes calmodulin-dependent activation of skeletal muscle myosin light chain kinase activity and reduces calmodulin-dependent activation of smooth muscle myosin light chain kinase activity to approximately 50% of the maximum level measured at a saturating calmodulin concentration. Calmodulin-dependent activation of cerebellar nitric oxide synthase activity is not affected by this deletion. Overlapping tripeptide deletions from the leader sequence indicate that the acidic cluster, Glu6-Glu-Gln8, contains the determinants necessary for activation of myosin light chain kinase activity. Deletion of Asp2-Gln-Leu4 has no effect on activation of enzyme activity. Based on enzyme kinetic analyses, deletions in the leader sequence have little or no effect on the apparent affinities of calmodulin for the synthase or the two kinases. Since the N-terminal leader does not appear to play a significant structural role in the complexes between calmodulin and peptides representing the calmodulin-binding domains in the two kinases, our results indicate that it participates in secondary interactions with these enzymes that are important to activation, but not to recognition or binding of calmodulin.

Calmodulin inhibits calcium influx current in vascular endothelium

Utilizing the whole-cell configuration of the patch-clamp technique the effect of calmodulin (CaM) on thapsigargin-induced Ca 2+ current has been studied. Addition of several concentrations of CaM to the patch pipette induced concentration-dependent inhibition of thapsigargin-induced Ca 2+ current in bovine aortic endothelial cells. The effect of CaM was Ca 2+ dependent and was not observed when the intracellular Ca 2+ was buffered to 1 nM with EGTA. CaM produced two major effects on the thapsigargin-induced Ca 2+ current. First CaM slow down activation of the current by thapsigargin from a control value of 16 ± 5 to 31 ± 6 s with 1 μM CaM in the pipette solution. The second effect of CaM was to reduce the current amplitude in a concentration-dependent manner. The inhibition of Ca 2+ current was observed at the peak of the current and at the sustained current level. The reduction of current at the sustained level was observed 15–20 s after onset of the thapsigargin response. The half inhibitory concentration determined from these experiments was 0.1 μM. These results indicate that CaM can modulate thapsigargin-induced Ca 2+ current in this endothelium, suggesting a possible role for CaM in the regulation of store-operated Ca 2+ influx.

Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1.

Smooth muscle myosin light chain kinase (MLCK) features several consensus sites of phosphorylation by proline-directed protein serine/threonine kinases. The phosphorylation of MLCK by two proline-directed kinases isolated from sea star oocytes, i.e., p44mpk (Mpk, a mitogen-activated protein kinase homologue) and cyclin-dependent kinase-1 (CDK1, also known as p34cdc2), was investigated. Chicken gizzard MLCK was phosphorylated on seryl and threonyl residues by both Mpk and CDK1. Phosphorylation of MLCK to 0.6 mol Pi/mol by Mpk increased the Vmax of phosphotransferase activity towards a synthetic peptide corresponding to residues 11-23 of the 20-kDa light chain of myosin by 1.6-fold. Phosphorylation of MLCK to 1.0 mol Pi/mol by CDK1 increased the Vmax by 2.3-fold. Phosphorylation by either kinase had no significant effect on the concentration of calmodulin required for half-maximal activation of MLCK. Analysis of the phosphorylation of synthetic peptides containing consensus phosphorylation sites for Mpk and CDK1 indicated that the major site of phosphorylation in MLCK by Mpk was Ser-834, and by CDK1 was Thr-283. Both of these sites are located outside the calmodulin-binding site (residues 796-815), consistent with the observation that phosphorylation by Mpk or CDK1 was unaffected by the presence of bound Ca2+/calmodulin. These results indicate that MLCK activity may be regulated by phosphorylation catalyzed by proline-directed kinases, possibly directed at Thr-40 and Thr-43 at the amino terminus of MLCK.

Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5′[γ-thio]triphosphate

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calmodulin in fibroblast proliferation.

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 +/- 0.04 microgram/mmol creatinine) (P < 0.001), when compared to polycythaemia vera (PV) (0.14 +/- 0.02), essential thrombocythaemia (ET) (0.13 +/- 0.04), chronic myeloid leukaemia (CML) (0.16 +/- 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 +/- 0.02) and age-matched controls (0.1 +/- 0.02) (P < 0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age-matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS-induced proliferation of low-density fibroblasts but had little or no inhibitory effect on high-density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely, IFG-1, EGF, bDGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG-beta and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.

P-71 - A novel Ca2+/calmodulin-dependent protein kinase lacking autophosphorylation activity in the rabbit heart

Abstract We report the discovery, semi-purification and characterization of a novel Ca2+/calmodulin-dependent protein kinase (peak I kinase) using syntide 2 as a substrate from the rabbit heart. In the study of dependence of peak I kinase on the concentration of calmodulin, half-maximal activation was obtained at approx. 2.0 × 10−7 M calmodulin. Peak I kinase did not undergo autophosphorylation. This kinase phosphorylates the synthetic peptides such as syntide 2, autocamtide-2, site 3 in a Ca2+/CaM-dependent manner, but not myosin light chain-peptide, γ-peptide, and cAMP Response Element Binding Protein (CREB) peptide. Elongation Factor-2, α-casein and histone-IIIs were not phosphorylated. These data indicate that this CaM kinase is different from other identified Ca2+/calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.

Distribution and role of calmodulin in tip growing hyphae of Saprolegnia ferax

Since direct staining of growing hyphae with trifluoperazine (TFP) could result in the migration of protoplasm in hyphae of water mold Saprolegnia ferax, an improved staining method for fixation of growing hyphae was developed. When hyphae of Saprolegnia ferax were pre-fixed with paraformaldehyde, the TFP fluorescence showed a clear tip-high Ca2+-activated calmodulin (CaM) gradient. By indirect immunohistochemical staining, we proved that total CaM also mainly concentrated in the apex of hyphae. It is noticeble that when the hyphal tip growth ceased, the high intensity of TFP fluorescence usually disappeared in non-growing tips and became evident in the newly emerged growing tips, which implied that the concentration of CaM shifted in concord with the transfer of growing point in hyphae. 2 × 10−5 mol /L CaM inhibitor TFP could reduce the tip growth rate and the percentage of growing hyphal tips to about 50%, and induce multibranching of hyphal tips. These findings strongly suggest that the high concentration of calmodulin in the hyphal tip may be involved in regulation of hyphal tip growth.

Enhancing effect of calmodulin on Ca(2+)‐induced Ca2+ release in the sarcoplasmic reticulum of rabbit skeletal muscle fibres.

1. We analysed the effect of calmodulin on Ca(2+)-induced Ca2+ release (CICR) in the sarcoplasmic reticulum (SR) using chemically skinned fibres of rabbit psoas muscle. Ca2+ release was measured using fura-2 microfluorometry. 2. In saponin-skinned fibres, calmodulin potentiated Ca2+ release at low Ca2+ concentrations (< 3 microM), while it showed an inhibitory effect at high Ca2+ concentrations (3-30 microM). 3. Co-application of ryanodine and calmodulin at 0.3 microM Ca2+, but not ryanodine alone, induced a decline in the Ca2+ uptake capacity of the SR, an effect expected from the open-lock of active CICR channels by ryanodine. Thus, potentiation of Ca2+ release by calmodulin at low Ca2+ concentrations can be regarded as a result of the activation of the ryanodine receptor. 4. Greater concentrations of calmodulin were required for potentiation of CICR at low Ca2+ concentrations (1 microM) than for inhibition at high Ca2+ concentrations (10 microM). 5. In beta-escin-permeabilized fibres in which intrinsic calmodulin was retained, the rates of CICR were similar to those measured in the presence of 1 microM calmodulin in saponin-permeabilized fibres. 6. These results suggest that calmodulin plays an important role in the regulation of CICR channels in intact skeletal muscle fibres.

Changes in calmodulin concentration and cyclic 3′,5′-nucleotide phosphodiesterase activity in skeletal muscle of hyper- and hypothyroid rats

Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.

Calcium-Dependent Activation of Skeletal-Muscle Ca 2+ Release Channel (Ryanodine Receptor) by Calmodulin

In this study terminal cisternae vesicles from rabbit skeletal muscle were fused into planar bilayers and the effect of calmodulin on single Ca 2+ release channel currents was investigated. In the presence of 10 −7 and 10 −9 M free [Ca 2+], nanomolar concentrations of calmodulin activated the channel by increasing the open probability of single-channel events in a dose dependent manner. The activatory effect of calmodulin was reversed by 10 μM ruthenium red. At 10 −5 M free [Ca 2+], calmodulin (0.1-1 μM) inhibited channel activity. Calmodulin overlays were carried out using concentrations of Ca 2+ similar to those used for the planar lipid bilayer assays. In the presence of 10 −7 M [Ca 2+], calmodulin bound to the ryanodine receptor, to a region defined by residues 2937-3225 and 3546-3655. These results suggest that calmodulin may activate the Ca 2+-release channel (ryanodine-receptor) by interacting with binding sites localized in the central portion of the RYR protomer.

A Novel Ca 2+/Calmodulin-Dependent Protein Kinase Lacking Autophosphorylation Activity in the Rabbit Heart

We report the discovery, semi-purification and characterization of a novel Ca 2+/calmodulin-dependent protein kinase (peak I kinase) using syntide 2 as a substrate from the rabbit heart. In the study of dependence of peak I kinase on the concentration of calmodulin, half-maximal activation was obtained at approx. 2.0 × 10 −7 M calmodulin. Peak I kinase did not undergo autophosphorylation. This kinase phosphorylates the synthetic peptides such as syntide 2, autocamtide-2, site 3 in a Ca 2+/CaM-dependent manner, but not myosin light chain-peptide, γ-peptide, and cAMP Response Element Binding Protein (CREB) peptide. Elongation Factor-2, α-casein and histone-IIIs were not phosphorylated. These data indicate that this CaM kinase is different from other identified Ca 2+/calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.

Sensitization of Calcium-induced Calcium Release by Cyclic ADP-ribose and Calmodulin

Cyclic ADP-ribose (cADPR) is emerging as an endogenous regulator of Ca2+-induced Ca2+ release (CICR), and we have recently demonstrated that its action is mediated by calmodulin (CaM) (Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E., and Walseth, T. F. (1994) Nature 370, 307-309). In this study we show by immunoblot analyses that the protein factor in sea urchin eggs responsible for conferring cADPR sensitivity to egg microsomes was CaM. This was further supported by the fact that bovine CaM was equally effective as the egg factor. In contrast, plant CaM was only partially active even at 10-20-fold higher concentrations. This exquisite specificity was also shown by binding studies using 125I-labeled bovine CaM. The effectiveness of various CaMs (bovine > spinach > wheat germ) in competing for the binding sites was identical to their potency in conferring cADPR sensitivity to the microsomes. A comparison between bovine and wheat germ CaM in competing for the sites suggests only 10-14% of the total binding was crucial for the activity. Depending on the CaM concentration, the sensitivity of the microsomes to cADPR could be changed by several orders of magnitude. The requirement for CaM could be alleviated by raising the divalent cation concentration with Sr2+. Results showed that CaM, cADPR, and caffeine all act synergistically to increase the divalent cation sensitivity of the CICR mechanism. The combined action of any of the three agonists was sufficient to sensitize the mechanism so much that even the nanomolar concentration of ambient Ca2+ was enough to activate the release. Unlike the CICR mechanism, the microsomal inositol 1,4,5-trisphosphate-sensitive Ca2+ release showed no dependence on CaM. Using an antagonist of CaM, W7, it was demonstrated that the cADPR-but not the inositol 1,4,5-trisphosphate-dependent release mechanism could be blocked in live sea urchin eggs. These results indicate cADPR can function as a physiological modulator of CICR and, together with CaM, can alter the sensitivity of the release mechanism to divalent cation by several orders of magnitude.

Interaction of calponin with actin and its functional implications

Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.

Inhibition of constitutive endothelial nosynthase activity by tannin and quercetin

The effect of natural polyphenols on three isoforms of NO-synthase was investigated. Among the compounds tested, tannin was the most potent, inhibiting endothelial constitutive NO synthase (eNOS) with an ic 50 of 2.2 μM. Other NOS isoforms (i.e. neuronal constitutive NOS and smooth muscle inducible NOS) were also inhibited but at much higher concentrations (selectivity ratio of approx. 20–30). Quercetin was also an effective but less potent inhibitor of eNOS ( C 50 = 220 μM). The kinetics of tannin inhibition were investigated to gather information on the mechanism of action. Tannin did not interfere with the interaction of the enzyme with the co-substrates l-arginine and NADPH nor with the cofactor tetrahydrobiopterin. The inhibition level was also independent of free Ca 2+ concentration as well as of the presence of high exogenous calmodulin concentrations.