W7 Treatment Research Articles

Calcineurin inhibitors block sodium-chloride cotransporter dephosphorylation in response to high potassium intake

Dietary potassium intake is inversely related to blood pressure and mortality. Moreover, the sodium-chloride cotransporter (NCC) plays an important role in blood pressure regulation and urinary potassium excretion in response to potassium intake. Previously, it was shown that NCC is activated by the WNK4-SPAK cascade and dephosphorylated by protein phosphatase. However, the mechanism of NCC regulation with acute potassium intake is still unclear. To identify the molecular mechanism of NCC regulation in response to potassium intake, we used adult C57BL/6 mice fed a 1.7% potassium solution by oral gavage. We confirmed that acute potassium load rapidly dephosphorylated NCC, which was not dependent on the accompanying anions. Mice were treated with tacrolimus (calcineurin inhibitor) and W7 (calmodulin inhibitor) before the oral potassium loads. Dephosphorylation of NCC induced by potassium was significantly inhibited by both tacrolimus and W7 treatment. There was no significant difference in WNK4, OSR1, and SPAK expression after high potassium intake, even after tacrolimus and W7 treatment. Another phosphatase, protein phosphatase 1, and its endogenous inhibitor I-1 did not show a significant change after potassium intake. Hyperkaliuria, induced by high potassium intake, was significantly suppressed by tacrolimus treatment. Thus, calcineurin is activated by an acute potassium load, which rapidly dephosphorylates NCC, leading to increased urinary potassium excretion.

In vitro effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide on differentiation from human adipose-derived mesenchymal stem cells to endothelial cells

To explore the effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide(W7) on the differentiation from human adipose-derived mesenchymal stem cells (hADSCs) to endothelial cells. hADSCs were cultured with serum-free differential medium containing 40 ng/ml vascular endothelial growth factor (VEGF) and 10ng/ml basic fibroblast growth factor (bFGF). Cells were divided into control group (differential medium without W7), high-dose group (containing 30 μmol/L W7), medium-dose group (containing 20 μmol/L W7), and low-dose group ( containing 10 μmol/L W7). The hADSCs were cultured for 8 days, and then the changes in the phenotypes of von Willebrand factor (vWF) and vessel-selective cadherin (VE-Cadherin) were detected by flow cytometry (FCM). The intracellular Ca(2+) labeled with Fluo-3 was detected by laser confocal microscopy. After hADSCs planting on Matrigel, their angiogenic potentials were observed under the inverted phase contrast microscope, and the expression of extracellular regulated kinase (ERK) and phosphorylated extracellular regulated kinase (p-ERK) were evaluated by Western blot. After the hADSCs were cultured for 8 days, compared with the control group, the expressions of vWF and VE-Cadherin significantly increased along with the decrease of W7 level and the intracellular Ca(2+) also significantly increased (Pü0.01). Lumina-like vascular structure was formed in W7 treatment groups, but not in the blank control group. Compared with the blank control group, the expression of ERK showed no significant in W7 treatment groups (high-, medium-, and low-dose groups)(P>0.05); however, along with the decrease of W7 levels, the expression of p-ERK significantly increased(P<0.05). W7 in proper levels can effectively induce the differentiation from hADSCs to endothelium by increasing the intracellular Ca(2+) level and thus activating the ERK/MAPK pathway.

A fast Ca 2+-induced Ca 2+-release mechanism in Dictyostelium discoideum

In vertebrate cells calcium- induced calcium release (CICR) is thought to be responsible for rapid cytosolic Ca 2+ elevations despite the occurrence of strong Ca 2+ buffering within the cytosol. In Dictyostelium, a CICR mechanism has not been reported. While analyzing Ca 2+ regulation in a vesicular fraction of Dictyostelium rich in Ca 2+-flux activity, containing contractile vacuoles (CV) as the main component of acidic Ca 2+ stores and ER, we detected a rapid Ca 2+ change upon addition of Ca 2+ (CIC). CIC was three times larger in active stores accumulating Ca 2+ than before Ca 2+ uptake and in inactivated stores. Ca 2+ release was demonstrated with the calmodulin antagonist W7 that inhibits the V-type H +ATPase activity and Ca 2+ uptake of acidic Ca 2+ stores. W7 caused a rapid and large increase of extravesicular Ca 2+ ([Ca 2+] e), much faster and larger than thapsigargin (Tg), a Ca 2+-uptake inhibitor of the ER. W7 treatment blocked CIC indicating that a large part of CIC is due to Ca 2+ release. The height of CIC depended on the filling state of the Ca 2+ stores. CIC was virtually unchanged in the iplA − strain that lacks a putative IP 3 or ryanodine receptor thought to be located at the endoplasmic reticulum. By contrast, CIC was reduced in two mutants, HGR8 and lvsA −, that are impaired in acidic Ca 2+-store function. Purified Ca 2+ stores enriched in CV still displayed CIC, indicating that CV are a source of Ca 2+-induced Ca 2+ release. CIC-defective mutants were altered in their oscillatory properties. The irregularity of the HGR8 oscillation suggests that the principal oscillator is affected in this mutant.

Differential calreticulin expression affects focal contacts via the calmodulin/CaMK II pathway

Calreticulin is an ER calcium-storage protein, which influences gene expression and cell adhesion. In this study, we analysed the differences in adhesive properties of calreticulin under- and overexpressing fibroblasts in relation to the calmodulin- and calcium/calmodulin-dependent kinase II (CaMK II)-dependent signalling pathways. Cells stably underexpressing calreticulin had elevated expression of calmodulin, activated CaMK II, activated ERK and activated c-src. Inhibition of calmodulin by W7, and CaMK II by KN-62, caused the otherwise weekly adhesive calreticulin underexpressing cells to behave like the overexpressing cells, via induction of increased cell spreading. Increased vinculin, activated paxillin, activated focal adhesion kinase and fibronectin levels were observed upon inhibition of either the calmodulin or the CaMK II signalling pathways, which was accompanied by an increase in cell spreading and focal contact formation. Both KN-62 and W7 treatment increased cell motility in underexpressing cells, but W7 treatment led to loss of directionality. Thus, both the calmodulin and CaMK II signalling pathways influence cellular spreading and motility, but subtle differences exist in their distal effects on motility effectors.

Calmodulin regulates the post-anaphase reposition of centrioles during cytokinesis

A transient postanaphase repositioning of the centriole is found to control the completion of cytokinesis. Using a green fluorescent protein-calmodulin fusion protein as a living cell probe, we have previously found that calmodulin is associated with the initiation and progression of cytokinesis. In this study, we further studied the effect of calmodulin on the repositioning of the centriole and subsequent cell cycle progression. When activity of calmodulin is inhibited, the regression of the centriole from the intercellular bridge to the cell center is blocked, and thus the completion of cell division is repressed and two daughter cells are linked by longer cell bridge in perturbed cells. W7 treatment during cytokinesis also results in unfinished cytokinesis and stopped G1 phase. These results suggest that calmodulin activity is required for centriole repositioning and can affect the completion of cytokinesis and cell cycle progression.

The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase–anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 μM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.

The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein.

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM-agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase–anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 μM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.

Calmodulin-dependent regulation of hypotonicity-induced translocation of ENaC in renal epithelial A6 cells

Hypotonicity stimulates translocation of epithelial Na + channel (ENaC) to the apical membrane from the intracellular store site of ENaC by activating protein tyrosine kinase (PTK) in renal epithelial A6 cells. Based upon the fact that calmodulin shows its action on other enzymes through PTK caused phosphorylation of tyrosine residues of calmodulin itself, we studied whether a calmodulin-dependent pathway is involved in the action of hypotonicity on ENaC. W7, an antagonist of calmodulin, diminished the stimulatory action of hypotonicity on ENaC, irrespective of W7 treatment before or after application of hypotonicity. Calmodulin is known to regulate three pathways: (1) protein phosphatase 2B (PP2B), (2) Ca 2+/calmodulin-dependent protein kinase II (CaMK II), and (3) myosin light chain kinase (MLCK). Pretreatment with cyclosporin A, an inhibitor of PP2B, did not influence the hypotonicity action on ENaC. The hypotonicity action on ENaC was partially inhibited by pretreatment with KN93, an inhibitor of CaMK II, but not by addition of KN93 after hypotonic stimulation had been applied. ML-7, an inhibitor of MLCK, showed the action similar to KN93. These observations indicate that: (1) the hypotonicity-induced translocation of ENaC depends on CaMK II and MLCK and (2) ENaC translocated to the apical membrane by hypotonicity is maintained in its activity and/or stability at the apical membrane through a calmodulin-dependent pathway.

Intracellular distribution of a cytoplasmic progesterone receptor mutant and of immunophilins cyclophilin 40 and FKBP59: effects of cyclosporin A, of various metabolic inhibitors and of several culture conditions

The effect of cyclosporin A (CsA) on the intracellular distribution of a mutated NLS minus rabbit progesterone receptor (PRm) and the receptor-associated immunophilins, cyclophilin 40 (Cyp40) and FKBP59, was tested in Lcl3 cells by indirect immunofluorescent staining. PRm, which is cytoplasmic in absence of progesterone, is shifted to the nucleus by the hormone as well as by CsA, but not by FK506 or Rapamycin [I. Jung-Testas, M.-C. Lebeau, E.E. Baulieu. C.R. Acad. Sci. Paris 318 (1995) 873-878]. However the time course of nuclear import due to CsA and its sensitivity to N-ethyl maleimide (NEM) and to a calmodulin inhibitor (W7) was different from those observed for the hormonal effect. Cyp40 in Lcl3 cells is localized mainly in the nucleoli. CsA treatment increased nucleolar staining, while NEM and W7 caused it to decrease; after actinomycin D (1 μM) nucleolar staining of Cyp40 disappeared. FKBP59 is mainly cytoplasmic and concentrated in the perinuclear region, never in the nucleoli. CsA, actino D and W7 treatment did not influence FKBP59 localization. In serum-deprived medium FKBP59 was cytoplasmic, but when the culture medium was enriched (20% serum, insulin and EGF) FKBP59 became perinuclear and hsp 86 was partly shifted to the nucleus, but PRm remained cytoplasmic. CsA has an effect on PRm distribution, while it does not influence Cyp40 and FKBP59 localization. In presence of actino D the labelling of Cyp40 disappears from the nucleoli, while the distribution of PRm and FKBP59 is unaffected. Growth factors influence FKBP59 but not PRm or Cyp40. These results suggest that these proteins shuttle independently and that their association is transient.

Effect of calcium and calmodulin antagonists on ethylene biosynthesis in tomato fruits

The aim of this work was to elucidate the mode of action of calcium in calcium-inhibited ethylene biosynthesis. In one experiment, mature green tomatoes were treated with 1.5% (w/v) CaCl 2 + 60 μM N-6(aminohexyl)-1-naphthalene-sulphonamide (W5). 1.5% (w/v) CaCl 2 + 60 μM N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), 1.5% (w/v) CaCl 2 alone or distilled water (control). The control and the W7 treatment had much higher ethylene production than either the CaCl 2 treatment alone or the CaCl 2 + W5 treatment. In another experiment, similar tomatoes were treated with 1.5% (w/v) CaCl 2 + 15 μM trifluoperazine (TFP), 1.5% (w/v) CaCl 2 only, or distilled water (control). The treatment with 1.5% CaCl 2 + 15 μM TFP gave higher ethylene production than 1.5% (w/v) CaCl 2 without TFP, and nearly the same level as the control towards the end of the experimental period. For enzyme and intermediate metabolite assays, mature green tomatoes were treated with 1.5% (w/v) CaCl 2 plus either W5 or W7. W5 treatment had a lower ethylene forming enzyme (EFE) activity, higher 1-aminocyclopropane-l-carboxylic acid (ACC) concentration, similar levels of ACC synthase activity and 1-(malonylamino)-cyclopropane-1-carboxylic acid concentration, compared to the control and W7 treatment. As W7 is a more potent calmodulin antagonist than W5, these results suggest a calcium-calmodulin inactivation of EFE activity.

Comment: Interpretation of Recycling IndicesResponse: An Alternative Interpretation of the Appropriateness and Correct Means for the Evaluation of CO2 Recycling Indices.

Pretreatment of maize (Zea mays L.) seeds withCaCl2 solution significantly raised calcium andcalmodulin levels and enhanced intrinsic heat tolerance in maize seedlings.This enhancement is specific to Ca2+ and cannot besubstituted by Mg2+. In contrast, treatments with theCa2+ chelator EGTA or plasma membraneCa2+ channel blockers La3+ orverapamil lowered the intrinsic heat tolerance. Concurrent treatment withCa2+ and Ca2+ -channel blockers(La3+, verapamil) also decreased theCa2+ -enhanced heat tolerance. In addition, calmodulinantagonists chlorpromazine or W7 treatment eliminated theCa2+ -enhanced heat tolerance, whilst W5, a less activeanalogue of W7, had little effect on the Ca2+ -enhancedheat tolerance. Measurement of antioxidant enzyme activities and lipidperoxidation showed that heat stress induced an oxidative stress in maizeseedlings. External Ca2+ treatment enabled the seedlingsto keep relatively higher activities of superoxide dismutase (SOD), catalase(CAT) and ascorbate peroxidase (APX) and lower levels of lipid peroxidationthan Ca2+ -deficient treatments; on the contrary, EGTAtreatment led to more rapid loss of SOD, CAT and APX activities and higherlevels of lipid peroxidation in the seedlings under heat stress. In addition,concurrent Ca2+ and W7 treatment weakened the effects ofCa2+ treatment on SOD, CAT and APX activities and causedmore severe lipid peroxidation. These results suggested that externalCa2+ can enhance the intrinsic heat tolerance of maizeseedlings. This enhancement requires the entry of externalCa2+ into cells across plasma membranes and themediation of intracellular calmodulin, and is associated with the increase ofantioxidant system activity.

Effect of Calcium and Calmodulin on Intrinsic Heat Tolerance in Relation to Antioxidant Systems in Maize Seedlings

Pretreatment of maize (Zea mays L.) seeds with CaCl2 solution significantly raised calcium and calmodulin levels and enhanced intrinsic heat tolerance in maize seedlings. This enhancement is specific to Ca2+ and cannot be substituted by Mg2+. In contrast, treatments with the Ca2+ chelator EGTA or plasma membrane Ca2+ channel blockers La3+ or verapamil lowered the intrinsic heat tolerance. Concurrent treatment with Ca2+ and Ca2+ -channel blockers (La3+, verapamil) also decreased the Ca2+ -enhanced heat tolerance. In addition, calmodulin antagonists chlorpromazine or W7 treatment eliminated the Ca2+ -enhanced heat tolerance, whilst W5, a less active analogue of W7, had little effect on the Ca2+ -enhanced heat tolerance. Measurement of antioxidant enzyme activities and lipid peroxidation showed that heat stress induced an oxidative stress in maize seedlings. External Ca2+ treatment enabled the seedlings to keep relatively higher activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) and lower levels of lipid peroxidation than Ca2+ -deficient treatments; on the contrary, EGTA treatment led to more rapid loss of SOD, CAT and APX activities and higher levels of lipid peroxidation in the seedlings under heat stress. In addition, concurrent Ca2+ and W7 treatment weakened the effects of Ca2+ treatment on SOD, CAT and APX activities and caused more severe lipid peroxidation. These results suggested that external Ca2+ can enhance the intrinsic heat tolerance of maize seedlings. This enhancement requires the entry of external Ca2+ into cells across plasma membranes and the mediation of intracellular calmodulin, and is associated with the increase of antioxidant system activity.