Expression of the high capacity calcium-binding domain of calreticulin increases bioavailable calcium stores in plants

Modulation of cytosolic calcium levels in both plants and animals is achieved by a system of Ca²⁺-transport and storage pathways that include Ca²⁺ buffering proteins in the lumen of intracellular compartments. To date, most research has focused on the role of transporters in regulating cytosolic calcium. We used a reverse genetics approach to modulate calcium stores in the lumen of the endoplasmic reticulum. Our goals were two-fold: to use the low affinity, high capacity Ca²⁺ binding characteristics of the C-domain of calreticulin to selectively increase Ca²⁺ storage in the endoplasmic reticulum, and to determine if those alterations affected plant physiological responses to stress. The C-domain of calreticulin is a highly acidic region that binds 20–50 moles of Ca²⁺ per mole of protein and has been shown to be the major site of Ca²⁺ storage within the endoplasmic reticulum of plant cells. A 377-bp fragment encoding the C-domain and ER retention signal from the maize calreticulin gene was fused to a gene for the green fluorescent protein and expressed in Arabidopsis under the control of a heat shock promoter. Following induction on normal medium, the C-domain transformants showed delayed loss of chlorophyll after transfer to calcium depleted medium when compared to seedlings transformed with green fluorescent protein alone. Total calcium measurements showed a 9–35% increase for induced C-domain transformants compared to controls. The data suggest that ectopic expression of the calreticulin C-domain increases Ca²⁺ stores, and that this Ca²⁺ reserve can be used by the plant in times of stress.     

Calcium Rapidly Down-Regulates Human Renal Epithelial Sodium Channels Via a W-7-Sensitive Mechanism

Increases in intracellular calcium (Ca²⁺) inhibit renal sodium (Na⁺) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca²⁺ (using 10 µmol/L A23187, a Ca²⁺ ionophore) on wild-type and Liddle-mutated human epithelial Na⁺ channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na⁺ current by 55 % in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 μmol/L W-7 (to inhibit the Ca²⁺/calmodulin complex). By contrast, co-exposure to 50 μmol/L calphostin (to inhibit protein kinase C) or 5 μmol/L KN-62 (to inhibit Ca²⁺/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na⁺ current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na⁺ current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na⁺ channels in situ was unchanged by the rise in intracellular Ca²⁺. These data suggest that the A23187-induced rise in intracellular Ca²⁺ inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na⁺ channels from the cell membrane by endocytosis. We, therefore, propose that Na⁺ absorption in cortical collecting duct cells is inhibited by Ca²⁺, possibly when complexed with calmodulin.     

Calcium-induced protein phosphorylation and changes in levels of calmodulin and calreticulin in maize sperm cells

 To examine possible calcium (Ca²⁺)-mediated prefertilization events in male gametes of higher plants, we studied protein phosphorylation and the Ca²⁺-binding proteins, calmodulin and calreticulin, in sperm cells isolated from maize (Zea mays L.) pollen in the presence and absence of Ca²⁺. Using immunoblotting, we detected calmodulin and calreticulin and Ca²⁺-induced variations. Exposure of sperm cells to 1 mM Ca²⁺ for 1 h increased calmodulin content by 136% compared with the control. Ca²⁺ had little effect on calreticulin at 1 h, but induced a 34% increase after 3 h. Phosphorylation of proteins was low in 1 h-control and Ca²⁺-treated cells. However, a 13-fold increase in phosphorylation of a 18-kDa protein was found at 12 h in the presence of Ca²⁺. Ca²⁺-induced changes in calmodulin, calreticulin and protein phosphorylation observed in maize sperm cells may reflect prefertilization changes in vivo that facilitate sperm cell fusion with egg and central cells. Received: 26 July 1996 / Revision accepted: 7 February 1997     

The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors

Tetrastigma hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production. The effects of calcium ions (Ca²⁺) on induction were also studied. It was found that the most effective elicitors were 50 μM of the heavy metal ion copper (Cu²⁺) and 100 μM of the rare earth element cerium (Ce³⁺). The maximal biomass levels under respective treatments over a 16-d culture period increased by 1.3- and 1.6-fold, and the total flavonoid content was 1.8- and 1.6-fold greater than the control, respectively. Reducing the exogenous Ca²⁺ concentration or adding Ca²⁺ antagonists (1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N-tetraacetc acid (EGTA) or 1 mM verapamil) strengthened inductive effects of metal elicitors and enhanced flavonoid production. However, 0.5 μM of the calcium ionophore A23187 showed contrary results. The increase in exogenous Ca²⁺ concentration in the presence of A23187 suppressed H₂O₂ bursts and peroxidase activity caused by metal elicitors. The results suggest that Ca²⁺ plays an inhibitory role in the plant cell response to metal elicitors. This suppression could have been caused by Ca²⁺ preventing the cells from absorbing metal ions and then easing the induction, or because the decrease of Ca²⁺ concentration worked as an induction signal. Therefore, reducing the Ca²⁺ concentration in culture medium, or adding Ca²⁺ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T. hemsleyanum suspension cells.     

Involvement of Ca2+ Channel Signalling in Sclerotial Formation of Polyporus umbellatus

Growth and morphogenesis transformation in Polyporus umbellatus were examined in the presence of various pharmacological compounds, to investigate signal transduction pathways that influence the development of sclerotia. Both the calcium channel blocker nifedipine and the calcium ionophor A23187 reduced sclerotial production in P. umbellatus; four classes of Ca²⁺ signal agent—including calcium chelators, calcium channel blockers, calcium ionophors and calmodulin inhibitors—were further studied. Among them, EGTA and BAPTA, as calcium chelators, exhibited a complete inhibitory effect on sclerotial formation, among the levels tested. Calcium channel blockers and calcium ionophors at the concentrations used in this study could not eliminate sclerotia formation completely, but did greatly reduce sclerotial production. Notoginsenoside in dosages >250 μg/ml produced a significant negative effect on mycelial growth, and it prevented sclerotial formation entirely at a dosage of 500 μg/ml; no other drug influenced vegetative growth at all. The calcium ionophor A23187 did not decrease sclerotial mean weight at low doses (20 nM); at higher doses (200 nM), however, sclerotial development was significantly reduced, albeit not completely halted. The CaM inhibitors (W-7 and chlorpromazine) could each completely stop sclerotial formation. Using Fluo-3/AM as the indicator of cytosolic free calcium, the Ca²⁺ content in the cytoplasm was found to have decreased significantly when hyphae were treated with different drugs, and there was no active Ca²⁺ signal in the sclerotial mycelium. In general, the results suggest that Ca²⁺ signal transduction may play an important role in sclerotial formation in P. umbellatus.     

The effect of calcium ionophore A23187 on the ATP level of human erythrocytes

Incubation of red cells at 37° with the ionophore A23187 results in a loss of ATP that is dependent on the concentrations of A23187 and Ca²⁺ in the medium. ATP hydrolysis is greatest at micromolar concentrations of Ca²⁺ and decreases as Ca²⁺ in the medium is raised to millimolar levels. The ATP depletion is due to stimulation of calcium ATPase by A23187-mediated Ca²⁺ influx into the cell. The biphasic nature of Ca²⁺-stimulated ATP depletion in whole cells reflects the activity of Ca²⁺-ATPase in membrane preparations at varying Ca²⁺ concentrations. The ionophore can be removed by washing the cells with plasma or bovine serum albumin-containing medium and the ATP levels restored to normal by reincubating with 5 mM adenosine for 1 hr.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.     

PPARδ activation inhibits angiotensin II induced cardiomyocyte hypertrophy by suppressing intracellular Ca2+ signaling pathway

Peroxisome proliferator‐activated receptors δ (PPARδ) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARδ in cardiac cells except that PPARδ ligand treatment protects cardiac hypertrophy by inhibiting NF‐κB activation. Thus, in the present study, we examined the effect of selective PPARδ ligand L‐165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L‐165041 (10 µM) inhibited AngII‐induced [³H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L‐165041 pretreatment significantly inhibited AngII‐induced intracellular Ca²⁺ increase and subsequent phosphorylation of FAK. Further experiment using Ca²⁺ ionophore A23187 confirmed that Ca²⁺ induced FAK phosphorylation, and this was also blocked by L‐165041 pretreatment. In addition, overexpression of PPARδ using adenovirus significantly inhibited AngII‐induced intracellular Ca²⁺ increase and FAK expression, while PPARδ siRNA treatment abolished the effect of L‐165041. These data indicate that PPARδ ligand L‐165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca²⁺/FAK/ERK signaling pathway in a PPARδ dependent mechanism. J. Cell. Biochem. 106: 823–834, 2009. © 2009 Wiley‐Liss, Inc.     

Calreticulin is an upstream regulator of calcineurin

Ca²⁺ is a signalling molecule involved in virtually every aspect of cell function. The endoplasmic reticulum (ER) is an important and dynamic organelle responsible for storage of the majority of intracellular Ca²⁺. Within the ER lumen are proteins that function as Ca²⁺ buffers and/or molecular chaperones including calreticulin, a multifunctional Ca²⁺-binding protein. Calreticulin-deficiency is lethal in utero due to impaired cardiac development. In the absence of calreticulin Ca²⁺ storage capacity in the ER and InsP₃ receptor mediated Ca²⁺ release from ER are compromised. Remarkably, over-expression of constitutively active calcineurin in the hearts of calreticulin deficient mice rescues them from embryonic lethality and produces live calreticulin deficient animals. These observations provide first evidence that calreticulin is a key upstream regulator of calcineurin in the Ca²⁺-signalling cascade and they highlight the importance of ER during early stages of cellular commitment and tissue development during organogenesis.     

Expression regulation and function of Pref-1 during adipogenesis of human mesenchymal stem cells (MSCs)

Preadipocyte Factor 1 (Pref-1), also known as Delta-like Protein 1 (DLK-1) is an epidermal growth factor-like domain-containing trans-membrane protein that is involved in adipogenesis and cell fate decision. Its function in adipogenesis is reported inconsistently based on different cellular model systems. Here, by using human mesenchymal stem cells (MSCs), we show that Pref-1 is modulated by both dexamethasone and 3-isobutyl-1methylxanthine (IBMX), two components of the adipogenic induction mixture during the adipogenesis in vitro. IBMX induces the expression of Pref-1 in a time- and dose-dependent manner through cyclic AMP and cyclic GMP independent pathway and attenuates adipocyte differentiation by down-regulating PPARγ (peroxisome proliferator activated receptor gamma) expression. Dexamethasone, on the other hand, is capable of subduing the inhibitory effect of IBMX-induced Pref-1 and initiating the adipogenesis by up-regulating PPARγ expression. Moreover, the treatment of IBMX or dexamethasone alone fails to develop MSCs into mature adipocytes, however, treating cells with both IBMX and dexamethasone leads to a complete adipocyte differentiation as evaluated by lipid-droplet formation. Taken together, our study demonstrates that IBMX accelerates accumulation of lipid in MSCs only under the circumstance that the negative effect of Pref-1 induced by IBMX on the adipogenesis is overcome by dexamethasone.     

PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca²⁺ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca²⁺ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis.     

The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca²⁺]i, in bovine adrenal chromaffin cells was determined. At 10^–4M carbamylcholine maximal release occurred with an accompanying increase i n [Ca²⁺]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca²⁺]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca²⁺]i.     

Hysteretic activation of the Ca pump revealed by calcium transients in human red cells

The enzymatic basis for the Ca²⁺ pump in human red cells is an ATPase with hysteretic properties. The Ca²⁺-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca²⁺ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133–143). In order to study whether the Ca²⁺ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Ca_i), induced by the divalent cation ionophore A23187. The time-dependent changes of Ca_i were measured by use of radioactive calcium (⁴⁵Ca²⁺) and analysed with the aid of a mathematical model, based partly on the Ca²⁺-dependent parameters obtained from Ca²⁺-ATPase experiments, partly on the A23187-induced Ca²⁺ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca²⁺ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca²⁺ pump in human red cells responds hysteretically. It is suggested that Ca²⁺ pumps in other types of cell also have hysteretic properties.     

Participation of the microtubular-microfilamentous system on intracellular Ca transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[¹⁴C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca²⁺. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca²⁺, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca²⁺ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca²⁺. In addition, an increase in cytosolic Ca²⁺ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca²⁺ increase induced by A23187 in a Ca²⁺-free medium. On the other hand, in a Ca²⁺-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca²⁺ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca²⁺ uptake. These observations indicate that intracellular Ca²⁺ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca²⁺ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca²⁺-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca²⁺-activated protease similar to myofibrillar Ca²⁺-activated protease has been described in platelets. To explore the role of Ca²⁺-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca²⁺-activated protease-like activity. The hydrolysis of actin-binding protein by Ca²⁺-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca²⁺-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca²⁺-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca²⁺-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E₁, prostaglandin I₂, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca²⁺-activated protease in platelets and indicate that the source of calcium important in Ca²⁺-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca²⁺-activated protease-like activity as measured by the present analytical technique.     

Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with EGTA and A23187. Ca²⁺ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca²⁺ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca²⁺ indicator, demonstrated that verapamil inhibited Ca²⁺ release from them induced by valinomycin in the presence of K⁺ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca²⁺ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.