Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca²⁺ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca²⁺ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl₂ (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca²⁺ influx through the addition of extracellular CaCl₂ suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl₂ in the medium, the addition of Ca²⁺ chelator EGTA (1, 2.5, and 5 mM) or Ca²⁺ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca²⁺ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca²⁺ concentration and a low extracellular Ca²⁺ concentration (3 mM) enhanced MJ-induced ajmalicine production.     

Ca<Superscript>2+</Superscript>-Dependence and Inhibition of Transformation by Trifluoperazine and Chlorpromazine in <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

Ca²⁺ enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl₂ upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01–0.04 mM along with optimal CaCl₂ concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca²⁺-stimulated transformation. The results of present investigation suggest the involvement of a Ca²⁺-dependent protein activator in the development of Ca²⁺-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete. Received: 21 May 2002 / Accepted: 21 June 2002     

Role of Ca2+ in induction and secretion of dengue virus-induced cytokines

The present study was undertaken to investigate the role of calcium ions (Ca₂₊) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca₂₊ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Epigallocatechin‐3‐gallate induces mesothelioma cell death via H2O2−dependent T‐type Ca2+ channel opening

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin‐3‐gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H₂O₂ release in cell cultures, and exogenous catalase (CAT) abrogated EGCG‐induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3‐loaded, EGCG‐exposed MMe cells showed significant [Ca²⁺]_i rise, prevented by CAT, dithiothreitol or the T‐type Ca²⁺ channel blockers mibefradil and NiCl₂. Cell loading with dihydrorhodamine 123 revealed EGCG‐induced ROS production, prevented by CAT, mibefradil or the Ca²⁺ chelator BAPTA‐AM. Direct exposure of cells to H₂O₂ produced similar effects on Ca²⁺ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Ca_v3.2 T‐type Ca²⁺ channels in these cells, compared to normal mesothelium. Also, Ca_v3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Ca_v3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T‐type Ca²⁺ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Effects on N6, O2′-dibutyryladenosine-3′,5′ cyclic monophosphate on calcium accumulation by mouse mastocytoma cells

The accumulation of ⁴⁵Ca²⁺ by intact mouse mastocytoma cells was examined before and after treatment of the cells with N⁶,O^2′-dibutyryladenosine 3′,5′, cyclic monophosphate and theophylline to inhibit growth. In the presence of phosphate either glycolysis, respiration or ATP supported ⁴⁵Ca²⁺ uptake by the cells and in each case the accumulated ⁴⁵Ca²⁺ appeared to be retained by mitochondria. Inhibition of growth by drug treatment for 20h increased subsequent ⁴⁵Ca²⁺ accumulation when cells were incubated with ⁴⁵CaCl₂, succinate and phosphate. Since prior drug treatment did not increase ⁴⁵Ca²⁺ accumulation with glucose, ATP or malate the drugs appeared to increase ⁴⁵Ca²⁺ accumulation by affecting succinate metabolism.     

The Role of Calcium in the Artificially Induced Decidual Cell Reaction in Pseudopregnant Mice

The present study was designed to test the hypothesis that Ca²⁺is required for the successful induction of the decidual cell reaction (DCR) in mice following stimulation with concanavalin A (Con A). Con A (125 μg) administered intraluminally on Day 4 of pseudopregnancy increased uterine vascular permeability, increased uterine weight, and induced morphological and histological transformations that were clearly indicative of decidualization. Radioactive⁴⁵CaCl₂(1 mmol liter⁻¹, 600 mCi mmol⁻¹) introduced into the uterine lumen with either Con A or saline was subsequently incorporated into the uterine tissue and detected only in the luminal epithelium by microautoradiography techniques. The intraluminal administration of CaCl₂in combination with Con A increased the magnitude of the lectin-induced DCR. In contrast, the administration of other cationic chloride solutions, at various concentrations and tonicity, either had no effect (viz. Na⁺, Mg²⁺, and Ba²⁺) or reduced (viz. K⁺, Zn²⁺, Cd²⁺, and La³⁺) this uterine response. While ionophore A23187 was also deciduogenic, it suppressed the DCR when administered before Con A and enhanced the DCR when administered after Con A. The Ca²⁺channel blockers, nifedipine, verapamil, nicardipine, and diltiazem, the Ca²⁺-calmodulin inhibitor, W7, and the Ca²⁺-ATPase inhibitor, thapsigargin, also effectively reduced the uterine response to Con A when administered intraluminally. However, the Con A-induced DCR was not influenced by the Ca²⁺chelators, EGTA, EDTA, BAPTA, and BAPTA-AM. The results confirm that Con A is deciduogenic in pseudopregnant mice and suggest that luminal Ca²⁺plays an important role in facilitating the induction of the lectin-induced DCR by influencing the metabolism of the luminal epithelium.     

The role of calcium in depigmentation of iris epithelial cells during cell-type conversion

During the conversion of newt iris epithelial cells into lens cells, melanosomes disappear from the cytoplasm. In this “depigmentation,” exocytosis of melanosomes is involved. The role of Ca²⁺ in this process has been the subject of this work. The intracellular Ca²⁺ concentration of cultured iris epithelial cells was increased by three methods: microinjection of 10^?3, M CaCl₂ into the cytoplasm, fusion of phospholipid vesicles containing 10^?3, M CaCl₂ with the cell membrane, and exposure to the calcium ionophore A23187. Each of these treatments caused an increase in the release of melanosomes. Further experiments suggest that cAMP stimulates exocytosis probably by liberating Ca²⁺ from intracellular stores.     

Ketoconazole-Evoked [Ca2+]i Rises and Non-Ca2+-Triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effect of ketoconazole on cytosolic free Ca^{2 +} concentrations ([Ca^{2 +}]_i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca^{2 +} levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca^{2 +}]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 μ M and above increased [Ca^{2 +}]_i in a concentration-dependent manner. The Ca^{2 +} signal was reduced partly by removing extracellular Ca^{2 +}. The ketoconazole-induced Ca^{2 +} influx was insensitive to L-type Ca^{2 +} channel blockers and protein kinase C modulators. In Ca^{2 +}-free medium, after pretreatment with 50 μ M ketoconazole, thapsigargin-(1 μ M)-induced [Ca^{2 +}]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca^{2 +}]_i rises. Inhibition of phospholipase C with 2 μ M U73122 did not change ketoconazole-induced [Ca^{2 +}]_i rises. At concentrations between 5 and 100 μ M, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 μ M ketoconazole was not reversed by prechelating cytosolic Ca^{2 +} with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca^{2 +}]_i rises by causing Ca^{2 +} release from the endoplasmic reticulum and Ca^{2 +} influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca^{2 +}]_i rise.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

Apoptosis-inducing factor of a cytotoxic T cell line: involvement of a secretory phospholipase A2

Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A₂ (sPLA₂) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37°C in the presence of 1 mM Ca²⁺ enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca²⁺-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA₂). The involvement of an sPLA₂ was confirmed by blocking the pro-apoptotic activity of the Ca²⁺-treated cell extract with an anti-sPLA₂ polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA₂ was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA₂ is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Programa de Núcleos de Excelência (PRONEX–CNPq).     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Influence of calcium on fungal growth, hyphal morphology and citric acid production inaspergillus niger

Addition of 0.5 g/L CaCl₂ to the fermentation medium lowered the final biomass dry mass by 35% and increased the uptake of phosphate and sucrose, and the production of citric acid by 15, 35 and 50%, respectively. In a medium deprived of Ca²⁺ the microorganism displayed both a pelleted and a filamentous form of growth, the hyphae being scarcely branched, without bulbous cells. An addition of Ca²⁺ induced a pelleted form of growth, highly branched hyphae and numerous bulbous cells. Bulbous cells growing in the presence of Ca²⁺ exhibited cell walls composed of laminated layers, and featured vesicles associated with the wall and/or the cell membrane, containing numerous inclusions. The cytotoxic effect of high concentrations of citric acid in the medium as well as an increase of the activity of N-acetyl-β-d-glucosaminidase, a lytic enzyme, might be involved in these morphological changes.     

Extra-matrix Mg2+ limits Ca2+ uptake and modulates Ca2+ uptake–independent respiration and redox state in cardiac isolated mitochondria

Cardiac mitochondrial matrix (m) free Ca²⁺ ([Ca²⁺]_m) increases primarily by Ca²⁺ uptake through the Ca²⁺ uniporter (CU). Ca²⁺ uptake via the CU is attenuated by extra-matrix (e) Mg²⁺ ([Mg²⁺]_e). How [Ca²⁺]_m is dynamically modulated by interacting physiological levels of [Ca²⁺]_e and [Mg²⁺]_e and how this interaction alters bioenergetics are not well understood. We postulated that as [Mg²⁺]_e modulates Ca²⁺ uptake via the CU, it also alters bioenergetics in a matrix Ca²⁺–induced and matrix Ca²⁺–independent manner. To test this, we measured changes in [Ca²⁺]_e, [Ca²⁺]_m, [Mg²⁺]_e and [Mg²⁺]_m spectrofluorometrically in guinea pig cardiac mitochondria in response to added CaCl₂ (0–0.6 mM; 1 mM EGTA buffer) with/without added MgCl₂ (0–2 mM). In parallel, we assessed effects of added CaCl₂ and MgCl₂ on NADH, membrane potential (ΔΨ_m), and respiration. We found that >0.125 mM MgCl₂ significantly attenuated CU-mediated Ca²⁺ uptake and [Ca²⁺]_m. Incremental [Mg²⁺]_e did not reduce initial Ca²⁺uptake but attenuated the subsequent slower Ca²⁺ uptake, so that [Ca²⁺]_m remained unaltered over time. Adding CaCl₂ without MgCl₂ to attain a [Ca²⁺]_m from 46 to 221 nM enhanced state 3 NADH oxidation and increased respiration by 15 %; up to 868 nM [Ca²⁺]_m did not additionally enhance NADH oxidation or respiration. Adding MgCl₂ did not increase [Mg²⁺]_m but it altered bioenergetics by its direct effect to decrease Ca²⁺ uptake. However, at a given [Ca²⁺]_m, state 3 respiration was incrementally attenuated, and state 4 respiration enhanced, by higher [Mg²⁺]_e. Thus, [Mg²⁺]_e without a change in [Mg²⁺]_m can modulate bioenergetics independently of CU-mediated Ca²⁺ transport.     

RGK protein-mediated impairment of slow depolarization- dependent Ca2+ entry into developing myotubes

Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (Ca_V1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca²⁺ channel, and (3) voltage-sensor for slow depolarization-dependent Ca²⁺ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of Ca_V1.1. However, it is not known whether the latter function that has been attributed to Ca_V1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca²⁺ entry that is dependent on the voltage-sensing capability of Ca_V1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca²⁺ entry. Our observations provide the first evidence of modulation of this enigmatic Ca²⁺ entry pathway peculiar to skeletal muscle.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

Glucose-induced mobilisation of intracellular Ca2+ in depolarised pancreatic islets

Perifused rat pancreatic islets, prelabelled with ⁴⁵Ca, were exposed for 90 min to a medium containing 30 mM K⁺, 0.25 mM diazoxide and 0.5 mM EGTA, but deprived of CaCl₂. Either verapamil (0.05 mM) or Cd²⁺ (0.05 mM) were also present in the perifusate. Under these conditions a rise in D-glucose concentrations from either 2.8 to 16.7 mM or zero to 8.3 mM increased both ⁴⁵Ca outflow and insulin release, after an initial and transient decrease in effluent radioactivity. These findings suggest that, in islets depolarised by exposure to a high extracellular concentration of K⁺, D-glucose provokes an intracellular redistribution of Ca²⁺ ions and subsequent stimulation of insulin release. The functional response to D-glucose is apparently not attributable to either the closing of ATP-sensitive K⁺ channels, which were actually activated by diazoxide, or stimulation of Ca²⁺ influx, which was prevented by the absence of extracellular Ca²⁺. The present experimental design thus reveals a novel component of the glucose-induced remodelling of Ca²⁺ fluxes in islet cells. Such an effect might also be operative under physiological conditions, when the hexose leads to depolarisation of the islet B-cells.     

Interactive effects of Ca2+ and NaCl salinity on the growth of two tomato genotypes differing in Ca2+ use efficiency

Two tomato (Lycopersicon esculentum Mill.) lines differing in Ca²⁺ use efficiency (Ca²⁺ use efficient line 113 and Ca²⁺ use inefficient line 67) were subjected to salinity treatments in two separate experiments to determine whether they differed in salt tolerance. In experiment I, three NaCl and two CaCl₂ treatments were imposed. The Na⁺ concentrations were 1.1, 100 and 150 mM and the Ca²⁺ concentrations were either 1.51 or 10 mM. In experiment II, one NaCl and three Ca²⁺ treatments (as CaCl₂ or CaSO₄) were imposed. The treatments consisted of 150 mM NaCl at either 1.51 mM CaCl₂, 10 mM CaCl₂, or 10 mM CaSO₄. Response to treatments was determined by analysis of growth parameters (shoot and root dry weights, plant height, and root length). Shoot and root dry weight, and root length were depressed as salinity increased in plants lacking additional Ca²⁺. No significant differences in salt tolerance were detected between the two tomato lines after 24 d of salinity treatment. An important finding of this study was that root growth and length appeared to be more sensitive to the effect of CaCI₂ treatment alone and to the effects of CaCl₂ × NaCl treatments. This suggests that over the long term, both root growth and root length may be more sensitive indicators of salinity effects than shoots. Supplemental CaCl₂ had no ameliorative effect on NaCl stress in shoot growth. The inability of Ca²⁺ to counter Cl⁻ entry or toxicity may account for the lack of amelioration. Additional Ca²⁺ as CaSO₄ improved shoot growth of plants exposed to 150 mM NaCl. In contrast, root growth and length were improved by 10 mM Ca²⁺ as either CaCl₂ or CaSO₄.