Calcium channel blocking agents protect against acetaminophen-induced cytotoxicity in rat hepatocytes.

The effect on acetaminophen-induced cytotoxicity of three calcium channel blocking agents--diltiazem, verapamil and gallopamil--was studied in primary cultures of rat hepatocytes and compared with the chelating agent EGTA. Using the measurement of cytosolic lactate dehydrogenase (LDH) as an index of cytotoxicity, it was demonstrated that a 1-hr pretreatment with calcium channel blocking agents protected cells against acetaminophen cytotoxicity, but were less effective than EGTA. These data suggest that influx of extracellular Ca2+ into the cells could have a role in the genesis of hepatocyte injury by acetaminophen.     

Efficiency of histidine-associating compounds for blocking the alzheimer's Abeta channel activity and cytotoxicity

The opening of the Alzheimer's Aβ channel permits the flux of calcium into the cell, thus critically disturbing intracellular ion homeostasis. Peptide segments that include the characteristic histidine (His) diad, His¹³ and His¹⁴, efficiently block the Aβ channel activity, blocking Aβ cytotoxicity. We hypothesize that the vicinal His-His peptides coordinate with the rings of His in the mouth of the pore, thus blocking the flow of calcium ions through the channel, with consequent blocking of Aβ cytotoxicity. To test this hypothesis, we studied Aβ ion channel activity and cytotoxicity after the addition of compounds that are known to have His association capacity, such as Ni²⁺, imidazole, His, and a series of His-related compounds. All compounds were effective at blocking both Aβ channel and preventing Aβ cytotoxicity. The efficiency of protection of His-related compounds was correlated with the number of imidazole side chains in the blocker compounds. These data reinforce the premise that His residues within the Aβ channel sequence are in the pathway of ion flow. Additionally, the data confirm the contribution of the Aβ channel to the cytotoxicity of exogenous Aβ.     

The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl₂ with EGTA. Calcium channel blockers, CoCl₂ and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.     

Ca-independent effects of BAPTA and EGTA on single-channel Cl currents in brown adipocytes

The Cl⁻ channels of brown adipocytes electrophysiologically resemble outwardly rectifying Cl⁻ channels (ORCC). To study tentative Ca²⁺ regulation of these channels, we attempted to control Ca²⁺ levels at the cytoplasmic side of the inside-out membrane patches with Ca²⁺-chelating agents. However, we found that the commonly used Ca²⁺-chelators EGTA and BAPTA by themselves influenced the Cl⁻ channel currents, unrelated to their calcium chelating effects. Consequently, in this report we delineate effects of Ca²⁺-chelators (acting from the cytoplasmic side) on the single Cl⁻ channel currents in patch-clamp experiments. Using fixed (1-2 mM) concentrations of chelators, two types of Cl⁻ channels were identified, as discriminated by their reaction to the Ca²⁺-chelators and by their conductance: true-blockage channels (31 pS) and quasi-blockage channels (52 pS). In true-blockage channels, EGTA and BAPTA inhibited channel activity in a classical flickery type manner. In quasi-blockage channels, chelators significantly shortened the duration of individual openings, as in a flickering block, but the overall channel activity tended to increase. This dual effect of mean open time decrease accompanied by a tendency of open probability to increase we termed a quasi-blockage. Despite the complications due to the chelators as such, we could detect a moderate inhibitory effect of Ca²⁺. The anionic classical Cl⁻ channel blockers DIDS and SITS could mimic the true/quasi blockage of EGTA and BAPTA. It was concluded that at least in this experimental system, standard techniques for Ca²⁺ level control in themselves could fundamentally affect the behaviour of Cl⁻ channels.     

In vivo evoked specific cell mediated cytotoxicity in carp (Cyprinus carpio L.) uses mainly a perforin/granzyme-like pathway

A specific cytotoxicity assay has been developed for carp using the Epithelioma Papulosum Cyprini (EPC) cell line as target cells and the release of lactate dehydrogenase (LDH) as read out system. Non-specific cytotoxicity against EPC was not observed, but animals immunized with EPC clearly showed specific killing by effector cells present in kidney, blood and spleen. This killing was strongly calcium dependent, suggesting the utilization of a perforin/granzyme-like pathway. However, blocking with EGTA was not complete indicating the existence of other additional killing routes.     

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid,an intracellular calcium chelator

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca²⁺), mPB1^- (modified PBS without Ca²⁺), mPB1⁺/EGTA (mPB1⁺ containing EGTA), mPB1^-/EGTA (mPB1^- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.     

Influence of calcium on blue-light-induced chloroplast movement in Lemna trisulca L.

The presence of calcium is essential for chloroplast movement induced by blue light in Lemna trisulca L. The regulatory role of calcium was confirmed by the inhibition of chloroplast movement by cytochalasin B and trifluoperazine. The calcium concentration in tissues was modified by ethylene glycol-bis(2-aminoethylether)-N,N,N, N-tetraacetic acid (EGTA), the calcium ionophore A23187 and La³⁺. Only a long period of incubation (12h) in EGTA or La³⁺ caused distrubances in chloroplast movement. This indicates that calcium influx is not essential for chloroplast movement. Those conditions that dramatically changed the internal calcium concentration, either applications of calcium ionophore A23187 and EGTA, or ionophore and La³⁺, markedly decreased the amplitude of response to blue-light pulses. This demonstrates that disturbances of chloroplast movement are observable only when internal stores of calcium are affected by Ca²⁺-antagonists. We suggest that the calcium involved in blue-light-induced chloroplast movement is derived from intracellular stores. The addition of Mg²⁺ to EGTA buffer counteracted its effect, indicating that Mg²⁺, as well as Ca²⁺, might possibly be involved in chloroplast movement.Abbreviations EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes 4(2-hydroxyethyl-1-piperazine) ethanesulfonic acid - A23187 calcium ionophore We express our gratitude to Professor W. Korohoda for valuable critical comments on this paper and stimulating discussion. We also thank Mr. P. Malec for help in preparing the experiment with trifluoperazine and Mr. A. Waloszek for taking the photographs. We are indebted to Mr. Tim Kline (International House, Krakow, Poland) for improving the English style. This research was supported by grant No. 1042/P2/92/03 from the State Committe for Scientific Research.     

Changes of cytosolic Ca2+ fluorescence intensity and plasma membrane calcium channels of maize root tip cells under osmotic stress

The changes of cytosolic Ca²⁺ fluorescence intensity and the activities of calcium channel of primary maize root tip cells induced by PEG6000 or abscisic acid(ABA) were studied by both confocal techniques and the whole-cell patch clamping in this study. The Ca²⁺ fluorescence intensity increased while treated with PEG or ABA within 10 min, illuminating that Ca²⁺ participated in the process of ABA signal transduction. For further proving the mechanism and origin of cytosolic Ca²⁺ increase induced by PEG treatments, N,N,N′,N′-tetraacetic acid (EGTA), Verapamil (VP) and Trifluoperazine (TFP) were added to the PEG solution in the experiments separately. The results showed that Ca²⁺ fluorescence intensity induced by PEG was suppressed by both EGTA and VP obviously in the root tip cells. The Ca²⁺ fluorescence intensity of plants changed after the addition of CaM inhibitor TFP while subjected to osmotic stress, which seemed to show that CaM participated in the process of signal transduction of osmotic stress too. The mechanism about it is unknown today. Further, a hyperpolarization-activated calcium permeable channel was recorded in plasma membrane of maize root tip cells. The Ca²⁺ current (I_Ca) intensity increased remarkably after PEG treatment, and the open voltage of the calcium conductance increased. Similar changes could be observed after ABA treatment, but the channel opened earlier and the current intensity was stronger than that of PEG treatment. The activation of calcium channel initiated by PEG strongly was inhibited by EGTA, VP or TFP respectively. The results revealed that Ca²⁺ participated in the signals transduction process of osmotic stress, and the cytosolic free Ca²⁺ increase by osmotic stress mainly came from the extracellular, and some came from the release of cytoplasmic calcium pool.     

Calcium channels mediate phase shifts of the Bulla circadian pacemaker

1. Light-induced phase advances of the activity rhythm of the Bulla ocular circadian pacemaker are blocked when the extracellular calcium concentration is reduced with EGTA to 0.13 microM. Phase advances are also blocked in low calcium solutions without EGTA [( Ca] less than 50 microM). 2. The dependence of light-induced phase delays on extracellular calcium concentration in EGTA-free seawater was determined. Phase delays are blocked at calcium concentrations below 400 microM, and reduced at concentrations of 1 mM and 3.5 mM (relative to shifts in normal ASW, [Ca] = 10 mM). Phase delays are also reduced and blocked at calcium concentrations higher than normal (60 mM and 110 mM, respectively). 3. Low calcium EGTA also blocked both phase delays and phase advances induced by pulses of depolarizing high K+ seawater. Low calcium EGTA pulses presented alone at the same times did not generate significant phase shifts. 4. The organic calcium channel antagonists verapamil, diltiazem and nitrendipine as well as the inorganic calcium channel antagonists La3+, Co2+, Cd2+, and Mn2+ were applied along with light pulses, however, the treated eyes were either phase shifted by these substances, or these substances were found to be toxic. 5. The inorganic calcium channel antagonist Ni2+ blocked both light-induced phase delays and advances at a concentration of 5 mM. Ni2+ applied alone did not generate significant phase shifts. Phase delays induced by high K+ seawater were blocked in the presence of 50 mM Ni2+ but not in 5 mM Ni2+. The light-induced CAP activity of the putative pacemaker cells was not inhibited by Ni2+, suggesting that its blocking action was probably via its known role as a calcium channel antagonist.     

Increased Response to High KCl-Induced Elevation in the Intracellular-Ca2+ Concentration in Differentiated NG108-15 Cell and the Inhibitory Effect of the L-Type Ca2+ Channel Blocker, Calciseptine

Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca²⁺]_i) by high-KCl application were investigated in the neuroblastoma×glioma hybrid NG108-15 cell line (NG108-15 cells). The present study confirmed that the increasing effect of [Ca²⁺]_i by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt₂cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1) The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine (CaS), an L-type Ca²⁺ channel blocker, but not by N-, P- and R-type Ca²⁺ channel blockers. The IC₅₀ values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was irreversible. (3) The increasing effect for [Ca²⁺]_i by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca²⁺]_i by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was exchanged for a high-KCl solution containing EGTA (without CaCl₂). Together, these results suggest that the enhancement of the response effect of [Ca²⁺]_i by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive calcium channels (VSCCs), which were irreversibly inhibited by CaS.     

Calcium is essential for fructan synthesis induction mediated by sucrose in wheat

The role of Ca²⁺ in the induction of enzymes involved in fructan synthesis (FSS) mediated by sucrose was studied in wheat (Triticum aestivum). Increase of FSS enzyme activity and induction of the expression of their coding genes by sucrose were inhibited in leaf blades treated with chelating agents (EDTA, EGTA and BAPTA). Ca²⁺ channel blockers (lanthanum chloride and ruthenium red) also inhibited the FSS response to sucrose, suggesting the participation of Ca²⁺ from both extra- and intra- cellular stores. Sucrose induced a rapid Ca²⁺ influx into the cytosol in wheat leaf and root tissues, shown with the Ca²⁺ sensitive fluorescent probe Fluo-3/AM ester. Our results support the hypothesis that calcium is a component of the sucrose signaling pathway that leads to the induction of fructan synthesis.     

The Cytotoxic and Proapoptotic Activities of Hypnophilin are Associated with Calcium Signaling in UACC‐62 Cells

Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC‐62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC‐62 cells, which was blocked in cells stimulated in Ca²⁺‐free media. HNP treatment with BAPTA‐AM, an intracellular Ca²⁺ chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC‐62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC‐62 cells, after treatment with HNP, is associated with Ca²⁺ influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:479‐485, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21507     

The calcium requirement for functional vesicle development and nitrogen fixation by Frankia strains EAN1pec and CpI1

A calcium requirement was shown for both vesicle development and nitrogenase activity by Frankia strains EAN1_pec and CpI1. Washing cells with EGTA or EDTA inhibited both vesicle development and nitrogenase activity. The inhibition of both was reversed by the addition of calcium. A variety of agents known to affect calcium-dependent biological processes, such as a Ca-ATPase inhibitor, Ca-channel blockers, Ca-ionophores, calmodulin antagonists and the local anaesthetics, tetracaine and dibucaine, inhibited nitrogenase activity. Respiratory studies showed that a CN-insensitive respiration process occurred only under nitrogen derepressing conditions. Respiration by NH₄Cl-grown cells was completely inhibited by KCN while N₂-grown cells were inhibited by only 70%. Removal of calcium ions by EGTA or by the addition of dibucaine or tetracaine blocked the CN-insensitive respiration. This CN-insensitive respiration may be involved in protecting nitrogenase inside the vesicles from oxygen.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-( amino-ethyl ether) N,N¹-tetraacetic acid - GI germination inhibitor - MOPS 3-[N-morpholino] propane sulfonic acid - PCMBS p-chloromercuribenzene sulphonate - TMB 8,8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate     

Extracellular calcium does not contribute to cryopreservation-induced cytotoxicity

Summary The possible role of extracellular calcium ([Ca⁺²]e) in cryopreservation-induced cytotoxicity was tested using Madin-Darby canine kidney (MDCK) cells and a fluorescent multiple endpoint assay. MDCK cells maintained in 2 mM [Ca⁺²]e and treated with the calcium ionophore, ionomycin, increased their intracellular calcium ([Ca⁺²]i) as revealed by the calcium indicator dye, Fluo3 and the bottom-reading spectrofluorometer, CytoFluor 2300. The addition of 10 mM [ethylene bis (oxyethylenenitrilo)]-tetraacetic acid (EGTA) to the extracellular medium before treatment with ionomycin blocked this ionomycin-dependent increase in [Ca⁺²]i. A number of site and activity-specific fluorescent probes were surveyed to determine which indicator dye might best reveal the ionomycin-induced cytotoxic events during this increase in [Ca⁺²]i. Although most dyes changed their emission profiles in response to calcium, neutral red was found to best reflect the loss of [Ca⁺²]i homeostasis. The NR50 for a 15-min exposure to ionomycin in the presence of 2 mM [Ca⁺²]e was approximately 2μM ionomycin, but ionomycin had little apparent effect on neutral red retention when 10 mM EGTA was added to the extracellular medium. Thus it was clear that an increase in [Ca⁺²]i could be cytotoxic to MDCK cells and that neutral red could monitor this cytotoxic episode. To test if [Ca⁺²]e was similarly cytotoxic during cryopreservation, MDCK cells were subjected to cryopreservation in the presence of dimethylsulfoxide (DMSO). In contrast to previous studies, plasma membrane integrity, not lysosomal function, seemed to best correlate with cell survival subsequent to cryopreservation. In addition, decreasing [Ca⁺²]e had no discernable effect on the retention of plasma membrane indicator dyes, neutral red, or cell survival. It is concluded that a) plasma membrane indicator dyes, not neutral red, might be better indicators of cytotoxicity occurring during cryopreservation; b) DMSO might be toxic to lysosomes during cryopreservation of cultured cells; and c) although [Ca⁺²]e can contribute to cytotoxicity, the presence of [Ca⁺²]e might not influence cryopreservation-induced cytotoxicity.     

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.     

Calcium-dependent natural killer and calcium-independent natural cytotoxic activities in an IL-2-dependent killer cell line

Using a cloned murine cell line, NKB61A2, that concomitantly exhibits both NK and natural cytotoxic (NC) activities, we investigated the biochemical mechanisms involved in natural cell mediated cytotoxicity against NK-sensitive YAC-1 tumor cells and against the NC-sensitive WEHI-164 tumor cells. Recent reports have suggested that target cell lysis by cytotoxic lymphocytes occurs by either a calcium dependent and/or a calcium-independent mechanism(s). To determine the role of calcium in NK and NC activities of the NKB61A2 cell line, we evaluated the effect of: 1) extracellular Ca2+ depletion by the divalent cation chelator, EGTA, 2) Ca2+ influx blockade by the Ca2+ channel blocker verapamil, and 3) blocking of intracellular Ca2+ mobilization by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8). We found that EGTA, verapamil, and TMB-8 were all capable of inhibiting NK activity, but they had little effect on NC activity of the NKB61A2 cells. Using 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide which are inhibitors of protein kinase C and calmodulin respectively, we determined that protein kinase C and calmodulin do play a role in the NK activity of NKB61A2 cells. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and N-(6-aminohexyl)-5-chloro-1-naphthalanesulfonamide, similar to Verapamil and TMB-8, had no effect on NC activity. Thus, the data indicate that the NK activity of NKB61A2 cells is calcium dependent whereas NC activity is not. These results may explain the disparate reports seen in the literature of calcium-dependent and -independent lysis of tumor cells.     

Enhanced silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.     

Calcium and calcium-related proteins in endometrial cancer: opportunities for pharmacological intervention

Intracellular calcium ions are key second messengers and play an important role in malignant transformation and cancer progression. Estrogen can evoke intracellular calcium increases through membrane-initiated effects and activate subsequent kinase cascades within minutes in normal and cancerous epithelial cells. Ca²⁺-related proteins are expressed in normal epithelial cells or endometrial cancer cells, some of which are upregulated by estrogen. Both estrogen-induced transient calcium increases and long-term changes in protein expression levels may be involved in regulating cancer initiation, progression and metastasis. Calcium channel blockers are reported to regulate both the rapid estrogen-induced intracellular Ca²⁺ increase and cell proliferation, apoptosis and migration, thus having the potential for pharmacological modulators to be repurposed for the treatment of endometrial cancer.