Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway

The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca²⁺]_i ), and modulation of [Ca²⁺]_i via treatment with IP ₃R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca²⁺]_i, followed by Ca²⁺-ERK and Ca²⁺-mitochondria-caspase signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 05 July 2008; received after revision 25 August 2008; accepted 17 September 2008     

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca²⁺ and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca²⁺], derived from intracellular stores, followed, only in myotubes, by a large Ca²⁺ influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca²⁺ permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca²⁺ mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death. Received 21 January 2009; received after revision 05 March 2009; accepted 11 March 2009     

HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca²⁺ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca²⁺ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721C and T7721N cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721C cells, but not affected in T7721N cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca²⁺ mobilization although each domain may play different roles.Received 1 April 2004; received after revision 15 June 2004; accepted 22 June 2004     

In vitro dopaminergic neuroprotective and in vivo antiparkinsonian-like effects of Δ3,2-hydroxybakuchiol isolated from Psoralea corylifolia (L.)

Cocktail recipes containing Psoralea corylifolia seeds (PCS) are used to empirically treat Parkinson disease. A PCS isolate Δ³,2-hydroxybakuchiol (BU) can inhibit dopamine uptake in dopamine transporter (DAT) transfected Chinese hamster ovary (CHO) cells, and dopamine reuptake blockade may provide an alternative approach for ameliorating parkinsonism. Here, we assessed the potential dopaminergic neuroprotective, and antiparkinsonian-like activity of BU. BU sample size was increased by using a scale-up extraction paradigm. Pharmacologically, BU significantly protected SK-N-SH cells from 1-methyl-4-phenylpyridinium (MPP⁺) insult, produced striking inhibitory actions on dopamine/norepinephrine uptake and WIN35,428 binding in synaptosomes on in vivo administration, and significantly preventing poor performance on rotarod and dopaminergic loss in substantia nigra in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice. BU acts by protecting dopaminergic neurons from MPP⁺ injury and preventing against MPTP-induced behavioral and histological lesions in the Parkinson’s disease (PD) model, possibly by inhibiting monoamine transporters. These findings suggest that BU could be meaningful in PD treatment. Received 14 January 2009; received after revision 22 February 2009; accepted 10 March 2009     

Digoxin and ouabain increase the synthesis of cholesterol in human liver cells

Digoxin and ouabain are steroid drugs that inhibit the Na⁺/K⁺-ATPase, and are widely used in the treatment of heart diseases. They may also have additional effects, such as on metabolism of steroid hormones, although until now no evidence has been provided about the effects of these cardioactive glycosides on the synthesis of cholesterol. Here we report that digoxin and ouabain increased the synthesis of cholesterol in human liver HepG2 cells, enhancing the activity and the expression of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of the cholesterol synthesis. This effect was mediated by the binding of the sterol regulatory element binding protein-2 (SREBP-2) to the HMGCR promoter, and was lost in cells silenced for SREBP-2 or loaded with increasing amounts of cholesterol. Digoxin and ouabain competed with cholesterol for binding to the SREBP-cleavage-activating protein, and are critical regulators of cholesterol synthesis in human liver cells. Received 10 January 2009; received after revision 11 February 2009; accepted 6 March 2009     

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotinconjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 15 September 2008; received after revision 17 December 2008; accepted 06 January 2009     

Influence of ethanol on calcium,inositol phospholipids and intracellular signalling mechanisms

Summary Studies have implicated Ca⁺⁺ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca⁺⁺ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca⁺⁺/Mg⁺⁺-ATPase, the high affinity membrane Ca⁺⁺ pump.Current research suggests that a subtype of the voltage-dependent Ca⁺⁺ channel, the dihydropyridine-sensitive Ca⁺⁺ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases⁴⁵Ca⁺⁺-influx and [³H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca⁺⁺/Mg⁺⁺-ATPase activity in neuronal membranes. Changes in Ca⁺⁺ channel and Ca⁺⁺/Mg⁺⁺-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca⁺⁺ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca⁺⁺/Mg⁺⁺-ATPase activity.The increased sensitivity of three Ca⁺⁺-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

Role of calcium in the histamine release induced by D-galactosamine from rat mast cells

Summary Rat peritoneal mast cells were isolated and purified by differential centrifugation in Ficoll. Cells pooled from three to four rats were suspended at approximately 10⁶ cells/ml in a buffered salt solution and incubated for 1 h at 37°C in 300 l volumes in the absence or presence (9×10^–4 M) of calcium chloride. Addition of D-galactosamine hydrochloride (DGM; 2.8×10^–4 M) caused (in addition to basal release) a mean ±SEM percent histamine release of 15.7±5.2 in the presence of Ca⁺⁺ and 19±4.9 in the absence of Ca⁺⁺ (p>0.05). It is suggested that D-galactosamine does not require extracellular Ca⁺⁺ for the release of histamine from the rat mast cell.A preliminary analysis of these results was presented at the International Symposium on calcium entry blockers and tissue protection, Rome, 15–16 March 1984.     

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Activation of δ-opioid receptors (DOR) attenuates anoxic K⁺ leakage and protects cortical neurons from anoxic insults by inhibiting Na⁺ influx. It is unknown, however, which pathway(s) that mediates the Na⁺ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na⁺ influxes mediated by voltage-gated Na⁺ channels; (2) DOR activation inhibited Na⁺ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K⁺ derangement; and (3) DOR activation had little effect on Na⁺/Ca²⁺ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K⁺ derangement by restricting Na⁺ influx mediated by Na⁺ channels and NMDA receptors, and that non-NMDA receptors and Na⁺/Ca²⁺ exchangers, although involved in anoxic K⁺ derangement in certain degrees, are less likely the targets of DOR signal. Received 26 November 2008; received after revision 26 December 2008; accepted 13 January 2009     

Overexpression of HAb18G/CD147 promotes invasion and metastasis via α3β1 integrin mediated FAK-paxillin and FAK-PI3K-Ca2+ pathways

Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin α3β1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin α3β1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca²⁺ mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin α3β1-mediated FAK-paxillin and FAKPI3K-Ca²⁺ signal pathways. Received 5 June 2008; received after revision 16 July 2008; accepted 23 July 2008     

Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid which regulates multiple biological parameters in a number of cell types, including stem cells. Here we report, for the first time, that S1P dose-dependently stimulates differentiation of adipose tissue-derived mesenchymal stem cells (ASMC) towards smooth muscle cells. Indeed, S1P not only induced the expression of smooth muscle cell-specific proteins such as α-smooth muscle actin (αSMA) and transgelin, but also profoundly affected ASMC morphology by enhancing cytoskeletal F-actin assembly, which incorporated αSMA. More importantly, S1P challenge was responsible for the functional appearance of Ca²⁺ currents, characteristic of differentiated excitable cells such as smooth muscle cells. By employing various agonists and antagonists to inhibit S1P receptor subtypes, S1P₂ turned out to be critical for the pro-differentiating effect of S1P, while S1P₃ appeared to play a secondary role. This study individuates an important role of S1P in AMSC which can be exploited to favour vascular regeneration. Received 06 March 2009; accepted 17 March 2009     

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action

A large variety of snake toxins evolved from PLA₂ digestive enzymes through a process of ‘accelerated evolution’. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA₂s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca²⁺ concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells. Received 05 March 2008; received after revision 08 April 2008; accepted 29 April 2008     

Impaired apoptosis in lymphoblasts from Alzheimer’s disease patients: Cross-talk of Ca<Superscript>2+</Superscript>/calmodulin and ERK1/2 signaling pathways

We have analyzed the intracellular signals that allow lymphoblasts from Alzheimer’s disease (AD) patients to escape from serum deprivation-induced apoptosis. The following observations suggested that modulation of ERK1/2 activity by Ca²⁺/calmodulin (CaM) is involved in preventing apoptosis: (i) ERK1/2 activity seems to support lethality in control cells, as PD98059, the inhibitor of the activating MEK prevented cell death; (ii) control cells show a persistent and higher stimulation of ERK1/2 than that of AD cells in the absence of serum; (iii) CaM antagonists have no effects on control cells, but sensitize AD cells to death induced by serum withdrawal and increased ERK1/2 phosphorylation, and (iv) no apoptotic effects of CaM antagonists were observed in AD cells treated with PD98059. These results suggest the existence of an activation threshold of the ERK1/2 pathway setting by Ca²⁺/CaM-dependent mechanisms, which appears to be the critical factor controlling cell survival or death decision under trophic factor withdrawal. F. Bartolomé, N. de las Cuevas: These authors contributed equally to this work. Received 14 February 2007; received after revision 16 April 2007; accepted 23 April 2007     

Evolutionary and functional epitopes of the Spätzle protein: New insights into activation of the Toll receptor

Sp?tzle, a dimeric ligand, binds to the Drosophila Toll receptor and activates the signal pathway functioning in both embryonic patterning and innate immunity. Here, we used the evolutionary trace approach based on phylogenetic information to predict the evolutionary epitope of Sp?tzle and found that it mainly clusters in several adjacent loops of Sp?tzle far from the cystine-knot structural domain. We designed six mutants of Sp?tzle based on the evolutionary epitope and transfected them into a stable cell line expressing the luciferase reporter gene under the control of the drosomycin promoter. Luciferase assays showed that these mutants cannot significantly activate the drosomycin promoter, suggesting the involvement of these sites in binding of Sp?tzle to the Toll receptor. These data highlight the importance of the Trp-loop of the mushroom-shaped Sp?tzle dimer in Toll receptor activation and demonstrate that evolution-guided site-specific mutagenesis represents a useful and promising strategy for understanding the ligand-receptor interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 14 January 2009; received after revision 14 February 2009; accepted 09 March 2009     

The effect of cyclic AMP on Na+ and K+ transport systems in mouse macrophages

Summary Exogenous cyclic AMP (cAMP) inhibits the Na⁺, K⁺-cotransport system and stimulates the Na⁺, K⁺-pump and Na⁺, Ca²⁺ exchange in mouse macrophages. These effects are enhanced by inhibition of phosphodiesterase with methylisobutylxanthine (MIX). MIX alone showed little or no effect. A similar response was observed after stimulation of endogenous production of cAMP by isoproterenol.     

The effect of a phorbol ester upon the cholinergic regulation of potassium permeability in the rat submandibular gland

Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca²⁺]_i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-0-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca²⁺]_o-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca²⁺]_o-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pig ovaries

The porcine antral follicles, 3–6 mm in diameter, were dissected from the ovaries of mature pigs, and then granulosa and cumulus cells were isolated from each follicle. In atretic follicles, high activity of neutral Ca²⁺/Mg²⁺-dependent endonuclease and DNA ladder formation, estimated by electrophoresis, were noted in granulosa cells but not in cumulus cells. Extremely low activity of the endonuclease and no DNA ladder formation were observed in both types of cells obtained from healthy follicles. Moreover, apoptotic cells were observed histochemically among granulosa cells only. A good correlation (r=0.987) between the endonuclease activity of granulosa cells and the progesterone/estradiol ratio of follicular fluid in each follicle was found. These results suggest that apoptosis occurs in granulosa cells but not cumulus cells in the atretic antral follicles in pigs.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.     

Zinc ions block the second but not the first phasic response to repetitive application of carbachol and histamine in guinea-pig taenia caeci

In the presence of Zn²⁺ (0.3 mM), carbachol (10^–6 M) or histamine (10^–5 M) induced the phasic response in guinea-pig taenia caeci while the tonic response was markedly inhibited. However, when the muscles were kept in Zn²⁺-containing medium following the first stimulation with either carbachol or histamine, neither application of carbachol nor of histamine elicited another phasic contraction. Caffeine (25 mM) did not induce contraction in the presence of Zn²⁺. After the washing out of caffeine in the presence of Zn²⁺, however, the muscle did then develop the phasic response on the application of carbachol or histamine. In conclusion, Zn²⁺ did not affect the carbachol or histamine-induced Ca²⁺ release from the storage sites. However, when Zn²⁺ was continuously present, Ca²⁺ was not supplied to the storage sites. Furthermore, carbachol and histamine mobilized a common cellular Ca²⁺ store, but they activated Ca²⁺ release channels different from the ones activated by caffeine in the Ca²⁺ storage sites.