Intracellular Calcium Accumulation Research Articles

Lipid peroxidation end product 4-hydroxy-trans-2-nonenal triggers unfolded protein response and heme oxygenase-1 expression in PC12 cells: Roles of ROS and MAPK pathways

This study investigates the roles of ROS overproduction and MAPK signaling pathways in the induction of unfolded protein response (UPR) and the expression of Phase II enzymes in response to 4-hydroxy-trans-2-nonenal (4-HNE) in a neuronal-like catecholaminergic PC12 cells. Our results showed that 4-HNE triggered three canonical pathways of UPR, namely IRE1-XBP1, PERK-eIF2α-ATF4 and ATF6, and induced the expression of UPR-targeted genes, GRP78, CHOP, TRB3, PUMA, and GADD34, as well as Phase II enzymes, HO-1 and GCLC. 4-HNE also induced apoptosis, intracellular calcium accumulation, caspase-3 activation, and G0/G1 cell cycle arrest, which was correlated with the increased expression of GADD45α. The addition of tiron, a cellular permeable superoxide scavenger, scavenged 4-HNE-mediated ROS formation, but did not alleviate cytotoxicity, or the expression of UPR-targeted genes or Phase II enzymes, indicating that ROS overproduction per se did not play a major role in 4-HNE-caused deleterious effects. HO-1 expression was attenuated by Nrf2 siRNA and chemical chaperone 4-phenylbutyrate (4-PBA), suggesting HO-1 expression was regulated by Nrf2-ARE, which may work downstream of ER stress. 4-HNE treatment promptly induced ERK, JNK and p38 MAPK activation. Addition of p38 MAPK specific inhibitor SB203580 attenuated HO-1 upregulation, but enhanced expression of CHOP, PUMA and TRB3, and cytotoxicity. These results indicate that 4-HNE-induced transient p38 MAPK activation may serve as an upstream negative regulator of ER stress and confer adaptive cytoprotection against 4-HNE-mediated cell injury.

Transglutaminase‐2 mediates calcium‐regulated crosslinking of the Y‐Box 1 (YB‐1) translation‐regulatory protein in TGFβ1‐activated myofibroblasts

Myofibroblast differentiation is required for wound healing and accompanied by activation of smooth muscle α-actin (SMαA) gene expression. The stress-response protein, Y-box binding protein-1 (YB-1) binds SMαA mRNA and regulates its translational activity. Activation of SMαA gene expression in human pulmonary myofibroblasts by TGFβ1 was associated with formation of denaturation-resistant YB-1 oligomers with selective affinity for a known translation-silencer sequence in SMαA mRNA. We have determined that YB-1 is a substrate for the protein-crosslinking enzyme transglutaminase 2 (TG2) that catalyzes calcium-dependent formation of covalent γ-glutamyl-isopeptide linkages in response to reactive oxygen signaling. TG2 transamidation reactions using intact cells, cell lysates, and recombinant YB-1 revealed covalent crosslinking of the 50 kDa YB-1 polypeptide into protein oligomers that were distributed during SDS-PAGE over a 75-250 kDa size range. In vitro YB-1 transamidation required nanomolar levels of calcium and was enhanced by the presence of SMαA mRNA. In human pulmonary fibroblasts, YB-1 crosslinking was inhibited by (a) anti-oxidant cystamine, (b) the reactive-oxygen antagonist, diphenyleneiodonium, (c) competitive inhibition of TG2 transamidation using the aminyl-surrogate substrate, monodansylcadaverine, and (d) transfection with small-interfering RNA specific for human TG2 mRNA. YB-1 crosslinking was partially reversible as a function of oligomer-substrate availability and TG2 enzyme concentration. Intracellular calcium accumulation and peroxidative stress in injury-activated myofibroblasts may govern SMαA mRNA translational activity during wound healing via TG2-mediated crosslinking of the YB-1 mRNA-binding protein.

Neferine, an alkaloid from lotus seed embryo, inhibits human lung cancer cell growth by MAPK activation and cell cycle arrest

Neferine is the major bisbenzylisoquinoline alkaloid isolated from the seed embryo of a traditional medicinal plant Nelumbo nucifera (Lotus). Epidemiological studies have revealed the therapeutic potential of lotus seed embryo. Although several mechanisms have been proposed, a clear anticancer action mechanism of neferine on lung cancer cells is still not known. Lung cancer is the most common cause of cancer death in the world, and the patients with advanced stage of nonsmall lung cancer require adjunct chemotherapy after surgical resection for the eradication of cancer cells. In this study, the effects of neferine were evaluated and characterized in A549 cells. Neferine induced apoptosis in a dose-dependent manner with the hypergeneration of reactive oxygen species, activation of MAPKs, lipid peroxidation, depletion of cellular antioxidant pool, loss of mitochondrial membrane potential, and intracellular calcium accumulation. Furthermore, neferine treatment leads to the inhibition of nuclear factor kappaB and Bcl2, upregulation of Bax and Bad, release of cytochrome C, activation of caspase cascade, and DNA fragmentation. In addition, neferine could induce p53 and its effector protein p21 and downregulation of cell cycle regulatory protein cyclin D1 thereby inducing G1 cell cycle arrest. These results suggest a novel function of neferine as an apoptosis inducer in lung cancer cells.

Cannabinoids: well-suited candidates for the treatment of perinatal brain injury.

Perinatal brain injury can be induced by a number of different damaging events occurring during or shortly after birth, including neonatal asphyxia, neonatal hypoxia-ischemia and stroke-induced focal ischemia. Typical manifestations of these conditions are the presence of glutamate excitoxicity, neuroinflammation and oxidative stress, the combination of which can potentially result in apoptotic-necrotic cell death, generation of brain lesions and long-lasting functional impairment. In spite of the high incidence of perinatal brain injury, the number of clinical interventions available for the treatment of the affected newborn babies is extremely limited. Hence, there is a dramatic need to develop new effective therapies aimed to prevent acute brain damage and enhance the endogenous mechanisms of long-term brain repair. The endocannabinoid system is an endogenous neuromodulatory system involved in the control of multiple central and peripheral functions. An early responder to neuronal injury, the endocannabinoid system has been described as an endogenous neuroprotective system that once activated can prevent glutamate excitotoxicity, intracellular calcium accumulation, activation of cell death pathways, microglia activation, neurovascular reactivity and infiltration of circulating leukocytes across the blood-brain barrier. The modulation of the endocannabinoid system has proven to be an effective neuroprotective strategy to prevent and reduce neonatal brain injury in different animal models and species. Also, the beneficial role of the endocannabinoid system on the control of the endogenous repairing responses (neurogenesis and white matter restoration) to neonatal brain injury has been described in independent studies. This review addresses the particular effects of several drugs that modulate the activity of the endocannabinoid system on the progression of different manifestations of perinatal brain injury during both the acute and chronic recovery phases using rodent and non-rodent animal models, and will provide a complete description of the known mechanisms that mediate such effects.

Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro

BackgroundOsteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).MethodsOsteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.ResultsBCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.ConclusionsTogether these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Surfactin-Induced Apoptosis Through ROS–ERS–Ca2+-ERK Pathways in HepG2 Cells

Although surfactin is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism responsible for this process remain elusive. In this study, the signaling network underlying the apoptosis of human hepatoma (HepG2) cells induced by surfactin was investigated. It is found that the reaction oxygen species (ROS) production and intracellular calcium ([Ca2+]i) accumulation are both induced HepG2 cells apoptosis. The [Ca2+]i exaltation was partly depended on the Ca2+ release from inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry) receptors channels, which both triggered endoplasmic reticulum stress (ERS). The results showed that surfactin induced the ROS production and ROS production led to ERS. The occurrence of ERS increased the [Ca2+]i level and the processes associated with blocking extracellular signal-regulated kinase (ERK) pathway. According to a comprehensive review of all the evidence, it is concluded that surfactin induces apoptosis of HepG2 cells through a ROS–ERS–Ca2+ mediated ERK pathway.

The Phospholipase C Inhibitor U73122 Attenuates trans-10, cis-12 Conjugated Linoleic Acid-Mediated Inflammatory Signaling and Insulin Resistance in Human Adipocytes

AbstractWe have demonstrated that trans-10, cis-12 conjugated linoleic acid (18:2t10,c12)-mediated delipidation of human adipocytes was dependent on increased intracellular calcium and activation of inflammatory signaling in human primary adipocytes. These data are consistent with the actions of diacylglycerol and inositol triphosphate derived from phospholipase C (PLC)-dependent cell signaling. To test the hypothesis that PLC was an upstream activator of these cellular responses to 18:2t10,c12, primary cultures of human adipocytes were pretreated with 1-[6-((17β-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), a universal PLC inhibitor, followed by 18:2t10,c12 treatment. U73122 attenuated 18:2t10,c12-mediated insulin resistance within 48 h and suppression of the mRNA levels of peroxisome proliferator-activated receptor (PPAR)γ, insulin-stimulated glucose transporter-4, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase-1, and the protein levels of PPARγ within 18–24 h. U73122 inhibited 18:2t10,c12-mediated induction of the inflammatory-related genes calcium/calmodulin-dependent protein kinase-β, cyclooxygenase-2, monocyte chemoattractant protein-1, interleukin (IL)-6, and IL-8, secretion of IL-6 and IL-8, and the activation of extracellular signal-related kinase, c-Jun N-terminal kinase, and c-Jun within 18–24 h. Moreover, 18:2t10,c12 increased the mRNA levels of heat shock proteins within 6–24 h and intracellular calcium concentrations within 3 min, which were inhibited by U73122. Lastly, 18:2t10,c12 increased the abundance of PLCγ1 in the plasma membrane within 3 min. Taken together, these data suggest that PLC plays an important role in 18:2t10,c12-mediated activation of intracellular calcium accumulation, inflammatory signaling, delipidation, and insulin resistance in human primary adipocytes.

Ketamine Inhibits Calcium Elevation and Hydroxyl Radical and Nitric Oxide Production in Lipopolysaccharide-Stimulated NR8383 Alveolar Macrophages

Macrophages play a critical role in mediating inflammatory processes; activated macrophages respond to endotoxin by releasing pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. Ketamine, a widely used anesthetic agent, has unequivocally anti-inflammatory effects in vivo and in vitro. However, the detailed mechanisms for the anti-inflammatory effects of ketamine in microglia have not been elucidated yet. This study aimed to evaluate the effects of ketamine on lipopolysaccharide (LPS)-induced nitric oxide (NO), hydroxyl radical (·OH) production, and intracellular calcium accumulation in macrophages. Macrophages were pretreated with ketamine at the concentrations of 10, 100, and 1,000 μM 1 h before LPS stimulation. The production of NO and ·OH in the culture supernatant of macrophages was assayed by Griess Reagent Kit. LPS enhanced NO and ·OH production and provoked a significant intracellular calcium elevation. With the concentrations higher than 100 μM, ketamine inhibited LPS-induced NO and ·OH accumulation and intracellular calcium elevation. However, a low concentration of ketamine (10 μM) did not exert anti-inflammatory effects. These results suggest that intracellular calcium elevation is, at least, partially involved in the inhibitory effect of ketamine.

Effects of paeonol on intracellular calcium concentration and expression of RUNX3 in LoVo human colon cancer cells

Paeonol, a major phenolic component of the root bark of Paeonia moutan, is known to exhibit antitumor effects. However, the underlying mechanisms remain unknown. In the present study, the effects of paeonol on cell viability, intracellular calcium concentration and the expression of runt‑related transcription factor3 (RUNX3) were analyzed in LoVo human colon cancer cells. Results revealed that paeonol markedly reduced LoVo cell viability in a time‑ and dose‑dependent manner. Flow cytometry assays demonstrated that paeonol blocked the cell cycle at the G1 to S transition and significantly induced apoptosis in LoVo cells. Intracellular calcium accumulation occurred following a 48h treatment with paeonol. Furthermore, RUNX3 gene expression was increased in paeonol‑treated cells. These observations indicate that paeonol possesses antiproliferative properties and apoptosis‑inducing activity. One of the antitumor mechanisms of paeonol may be its apoptosis‑inducing activity through an increased intracellular calcium concentration and the upregulation of RUNX3 expression. Paeonol may be a promising antitumor agent for colon carcinoma treatment.

Thalamic involvement in patients with neurologic impairment due to Shiga toxin 2

The outbreak of hemolytic-uremic syndrome and diarrhea caused by Shiga toxin-producing Escherichia coli O104:H4 in Germany during May to July 2011 involved severe and characteristic neurologic manifestations with a strong female preponderance. Owing to these observations, we designed a series of experimental studies to evaluate the underlying mechanism of action of this clinical picture. A magnetic resonance imaging and electroencephalographic study of patients was performed to evaluate the clinical picture in detail. Thereafter, combinations of different experimental settings, including electrophysiological and histological analyses, as well as calcium imaging in brain slices of rats, were conducted. We report on 7 female patients with neurologic symptoms and signs including bilateral thalamic lesions and encephalopathic changes indicative of a predominant involvement of the thalamus. Experimental studies in rats revealed an enhanced expression of the Shiga toxin receptor globotriaosylceramide on thalamic neurons in female rats as compared to other brain regions in the same rats and to male animals. Incubation of brain slices with Shiga toxin 2 evoked a strong membrane depolarization and intracellular calcium accumulation in neurons, associated with neuronal apoptosis, predominantly in the thalamic area. These findings suggest that the direct cytotoxic effect of Shiga toxin 2 in the thalamus might contribute to the pathophysiology of neuronal complications in hemolytic-uremic syndrome.

Structural and Functional Divergence of Growth Hormone-Releasing Hormone Receptors in Early Sarcopterygians: Lungfish and Xenopus

The evolutionary trajectories of growth hormone-releasing hormone (GHRH) receptor remain enigmatic since the discovery of physiologically functional GHRH-GHRH receptor (GHRHR) in non-mammalian vertebrates in 2007. Interestingly, subsequent studies have described the identification of a GHRHR2 in chicken in addition to the GHRHR and the closely related paralogous receptor, PACAP-related peptide (PRP) receptor (PRPR). In this article, we provide information, for the first time, on the GHRHR in sarcopterygian fish and amphibians by the cloning and characterization of GHRHRs from lungfish (P. dolloi ) and X. laevis. Sequence alignment and phylogenetic analyses demonstrated structural resemblance of lungfish GHRHR to their mammalian orthologs, while the X. laevis GHRHR showed the highest homology to GHRHR2 in zebrafish and chicken. Functionally, lungfish GHRHR displayed high affinity towards GHRH in triggering intracellular cAMP and calcium accumulation, while X. laevis GHRHR2 was able to react with both endogenous GHRH and PRP. Tissue distribution analyses showed that both lungfish GHRHR and X. laevis GHRHR2 had the highest expression in brain, and interestingly, X. laevis GHRHR2 also had high abundance in the reproductive organs. These findings, together with previous reports, suggest that early in the Sarcopterygii lineage, GHRHR and PRPR have already established diverged and specific affinities towards their cognate ligands. GHRHR2, which has only been found in xenopus, zebrafish and chicken hitherto, accommodates both GHRH and PRP.

What Determines the Kinetics of the Slow Afterhyperpolarization (sAHP) in Neurons?

What Determines the Kinetics of the Slow Afterhyperpolarization (sAHP) in Neurons?

Nitric Oxide Donors as Neuroprotective Agents after an Ischemic Stroke-Related Inflammatory Reaction

Cerebral ischemia initiates a cascade of detrimental events including glutamate-associated excitotoxicity, intracellular calcium accumulation, formation of Reactive oxygen species (ROS), membrane lipid degradation, and DNA damage, which lead to the disruption of cellular homeostasis and structural damage of ischemic brain tissue. Cerebral ischemia also triggers acute inflammation, which exacerbates primary brain damage. Therefore, reducing oxidative stress (OS) and downregulating the inflammatory response are options that merit consideration as potential therapeutic targets for ischemic stroke. Consequently, agents capable of modulating both elements will constitute promising therapeutic solutions because clinically effective neuroprotectants have not yet been discovered and no specific therapy for stroke is available to date. Because of their ability to modulate both oxidative stress and the inflammatory response, much attention has been focused on the role of nitric oxide donors (NOD) as neuroprotective agents in the pathophysiology of cerebral ischemia-reperfusion injury. Given their short therapeutic window, NOD appears to be appropriate for use during neurosurgical procedures involving transient arterial occlusions, or in very early treatment of acute ischemic stroke, and also possibly as complementary treatment for neurodegenerative diseases such as Parkinson or Alzheimer, where oxidative stress is an important promoter of damage. In the present paper, we focus on the role of NOD as possible neuroprotective therapeutic agents for ischemia/reperfusion treatment.

Neuroprotective effects of SNX-185 in an In Vitro model of TBI with a second insult

Second insults following traumatic brain injury (TBI), such as ischemia and hypoxia, significantly worsen outcome in patients and in experimental models of TBI. Following TBI there is a pathological increase in intracellular calcium, triggering cellular mechanisms of dysfunction and death. N-type specific voltage gated calcium channel (VGCC) blockers reduce cell death in both in vitro mechanical strain injury (MSI) and in vivo models of TBI, but they have not been previously explored in a model of TBI followed by a second insult. In the following studies, cortical neurons and astrocytes experienced MSI followed by incubation in 20% CO2. Cultures were treated with the N-type VGCC blocker, ω-conopeptide SNX-185 (1 μM), 5-minutes post-injury and intracellular calcium accumulation was assessed at 3, 6 and 24 h. Neuronal viability was assessed 24 h after MSI. Increasing incubator CO2 to 20% significantly increased calcium accumulation and cell death regardless of MSI severity. Treatment with 1 μM of SNX-185 significantly reduced the accumulation of calcium 3 hours following injury and increased the number of viable neurons 24 h post-injury and incubation in 20% CO2. In vitro models provide a critical tool for identifying roles of cell specific mechanisms involved in neuronal dysfunction and death following injury. These data demonstrate the potential of N-type VGCC blockers in reducing the damaging effects of TBI and second insults.

The Protective Role of Resveratrol against Arsenic Trioxide-Induced Cardiotoxicity

Arsenic trioxide (As2O3) shows substantial anticancer activity in patients with acute promyelocytic leukemia (APL). Unfortunately, limiting the application of this effective agent to APL patients is severe cardiotoxicity. Resveratrol, the natural food-derived polyphenolic compound, is well known for its antioxidant properties and protects the cardiovascular system. But the potential role of resveratrol against As2O3 in heart via nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) is unclear. The present study evaluated the effects of pretreatment with resveratrol and As2O3 on oxidative stress and cardiac dysfunction in rat. In the present study, resveratrol decreased As2O3-induced reactive oxygen species generation, oxidative DNA damage, and pathological alterations. In addition, cardiac dysfunction parameters, intracellular calcium and arsenic accumulation, glutathione redox ratio, and cAMP deficiency levels were observed in As2O3-treated rats; these changes were attenuated by resveratrol. Furthermore, resveratrol significantly prohibited the downregulation of both Nrf2 and HO-1 gene expressions that were downregulated by As2O3, whereas resveratrol did not alter As2O3-induced nitric oxide formation. Thus, the protective role of resveratrol against As2O3-induced cardiotoxicity is implemented by the maintenance of redox homeostasis (Nrf2-HO-1 pathway) and facilitating arsenic efflux. Our findings suggest coadministration with resveratrol, and As2O3 might provide a novel therapeutic strategy for APL.

Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes.

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.

The cardioprotective effect of danshen and gegen decoction on rat hearts and cardiomyocytes with post-ischemia reperfusion injury

BackgroundDanshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been used for treating heart disease for several thousand years in China. It has been found that a Danshen and Gegen decoction (DG) exhibiting an anti-atherosclerosis effect, which improves the patients’ heart function recovery. Pre-treatment with DG was reported to have protective effects on myocardium against ischemia/reperfusion injury. In the present study, we aim to investigate the post-treatment effect of DG on ischemic-reperfusion injuries ex vivo or in vitro and the underlying mechanisms involved.MethodsThe rat heart function in an ischemia and reperfusion (I/R) model was explored by examining three parameters including contractile force, coronary flow rate and the release of heart specific enzymes within the heart perfusate. In vitro model of hypoxia and reoxygenation (H/R), the protective effect of DG on damaged cardiomyocytes was investigated by examining the cell structure integrity, the apoptosis and the functionality of mitochondria.ResultsOur results showed that DG significantly improved rat heart function after I/R challenge and suppressed the release of enzymes by damaged heart muscles in a dose-dependent manner. DG also significantly inhibited the death of cardiomyocytes, H9c2 cells, with a H/R challenge. It obviously decreased cell apoptosis, protected the mitochondrial function and cell membrane skeleton integrity on H9c2 cells. The cardio-protection was also found to be related to a decrease in intracellular calcium accumulation within H9c2 cells after I/R challenge.ConclusionThe potential application of DG in treating rat hearts with an I/R injury has been implied in this study. Our results suggested that DG decoction could act as an anti-apoptotic and anti-ion stunning agent to protect hearts against an I/R injury.

FK506 Induced Apoptosis Is Mediated by Endoplasmic Reticulum Derived Calcium Dependent Caspase-12 in Jurkat Cells

Purpose: The effect of FK506 on endoplasmic reticulum(ER) derived calcium and caspase-12 mediated apoptosis in Jurkat human T-lymphocyte was investigated. Method: Cell viability was measured by flow cytometry. Intracellular calcium generation was measured. Western blotting of procaspase-12 was performed. And the catalytic activity of caspase -3, -6, -8, and -9 proteases in Jurkat cells was also measured. Results: FK506 dose-dependently decreased the viability in Jurkat cells. Increased intracellular accumulation of calcium in FK506 treated Jurkat cells from 24hours. FK506 continuously increased calcium concentration from 24hours to 72hrs. There was no evidence of calcium ionorpore (A23187) increased intracellular calcium changes with or without FK506 but calcium ATPase inhibitor (Thapsigargin) increased intracellular calcium accumulation and FK506 more and more increased calcium ATPase inhibitor(Thapsigargin) derived intracellular calcium accumulation. Procaspase-12 protease analyzed from 48hours. Treatment of cells with FK506 increased activation of caspase-12 protease. FK506 increased the catalytic activity of caspase-3 but there was no evidence of increased catalytic activation of caspase-6, -8 and -9 proteases in Jurkat cells. Conclusion: These data indicate that understanding of ER derived calcium and caspase-12 mediated apoptosis in human Jurkat cell line.

A Novel Hydrogen Sulfide-releasing N-Methyl-d-Aspartate Receptor Antagonist Prevents Ischemic Neuronal Death

Physiological levels of H(2)S exert neuroprotective effects, whereas high concentrations of H(2)S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H(2)S and NMDAR antagonism, we synthesized a novel H(2)S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H(2)S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na(2)S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na(2)S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H(2)S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury.

P47 A novel hydrogen sulfide-releasing NMDA receptor antagonist prevents ischemic neuronal death

Background Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule. A number of studies examined therapeutic potential of H2S-donating compounds and H2S gas itself in a variety of animal models of human disease and injury. In the majority of experimental studies, sodium sulfide (Na2S) and sodium hydrosulfide (NaHS) have been used as H2S donor compounds. However, because the short half-lives of these sulfide salts in biological fluid, plasma sulfide levels rapidly increase after bolus administration of Na2S or NaHS and then return to baseline instantaneously. To sustain “physiological” levels of sulfide in circulation after bolus administration, many slow-releasing H2S donor compounds, including 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48), have been developed. While it has been reported that low and physiological levels of H2S protect neurons, H2S also exhibits neurotoxicity especially at high concentrations. Some investigators have suggested that H2S-induced neurotoxicity may be mediated via enhancement of N-methyl-D-aspartate receptor (NMDAR) activity, because toxicity of H2S was abolished by NMDAR antagonist in vitro and in vivo. Based on these observations, we hypothesized that a hybrid NMDAR antagonist that is capable of slowly releasing H2S in circulation is more effective in protecting neurons than H2S donor compounds alone. Method To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol- 4-yl)-benzamide (sulfur-memantine) by chemically combining a slow releasing H2S donor ACS48 with a NMDAR antagonist memantine. We examined protective effects of sulfur-memantine in cultured cells at baseline and after oxygen glucose deprivation (OGD) using a panel of cytotoxicity assays. Neuroprotective effects of sulfur-memantine were validated in a mice model of cerebral ischemia and reperfusion injury. Result While sulfur-memantine and ACS48 increased sulfide levels similarly in cell culture medium, sulfur-memantine augmented intracellular sulfide concentration in human neuroblastoma cells (SH-SY5Y) ten-fold higher than did ACS48 ( Fig. 1 ). Incubation with sulfur-memantine after reoxygenation following OGD protected SH-SY5Y and murine primary cortical neurons more markedly than did ACS48 or memantine. Although Na2S and ACS48 exhibited dose-dependent cytotoxicity to primary cortical neurons measured by lactate dehydrogenase release, memantine and sulfur-memantine showed no cytotoxicity up to 50 μM. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by Na2S or ACS48, but suppressed by memantine and sulfur-memantine. Sulfur-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y more markedly than did Na2S or ACS48. Sulfur-memantine prevented caspase-3 activation in SH-SY5Y subjected to OGD. The anti-apoptotic effects of sulfur-memantine were associated with maintenance of extracellular-signal regulated kinase 1/2 (ERK) phosphorylation but not with changes in Akt phosphorylation. Administration of sulfur-memantine 1 min after reperfusion following 45 min of bilateral carotid occlusion reduced cerebral infarct volume and improved survival and neurological function of mice compared to Na2S, ACS48, or memantine up to 60 days after the ischemic insult ( Fig. 2 ). Conclusion These results suggest that a novel H2S-releasing NMDAR antagonist derivative sulfur-memantine prevents cerebral ischemia–reperfusion injury. Hybrid H2S-releasing NMDAR antagonists may provide a novel therapeutic strategy for ischemic brain injury and other forms for neuredegeneration.