Effective ferroptotic small-cell lung cancer cell death from SLC7A11 inhibition by sulforaphane.

Small-cell lung cancer (SCLC) is a highly aggressive cancer with poor prognosis, due to a lack of therapeutic targets. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables and has shown anticancer effects against numerous types of cancer. However, its anticancer effect against SCLC remains unclear. The present study aimed to demonstrate the anticancer effects of SFN in SCLC cells by investigating cell death (ferroptosis, necroptosis and caspase inhibition). The human SCLC cell lines NCI-H69, NCI-H69AR (H69AR) and NCI-H82 and the normal bronchial epithelial cell line, 16HBE14o- were used to determine cell growth and cytotoxicity, evaluate the levels of iron and glutathione, and quantify lipid peroxidation following treatment with SFN. mRNA expression levels of cystine/glutamate antiporter xCT (SLC7A11), a key component of the cysteine/glutamate antiporter, were measured using reverse transcription-quantitative PCR, while the levels of SLC7A11 protein were measured using western blot analysis. Following the addition of SFN to the cell culture, cell growth was significantly inhibited, and cell death was shown in SCLC and multidrug-resistant H69AR cells. The ferroptotic effects of SFN were confirmed following culture with the ferroptosis inhibitor, ferrostatin-1, and deferoxamine; iron levels were elevated, which resulted in the accumulation of lipid reactive oxygen species. The mRNA and protein expression levels of SLC7A11 were significantly lower in SFN-treated cells compared with that in the control cells (P<0.0001 and P=0.0006, respectively). These results indicated that the anticancer effects of SFN may be caused by ferroptosis in the SCLC cells, which was hypothesized to be triggered from the inhibition of mRNA and protein expression levels of SLC7A11. In conclusion, the present study demonstrated that SFN-induced cell death was mediated via ferroptosis and inhibition of the mRNA and protein expression levels of SLC7A11 in SCLC cells. The anticancer effects of SFN may provide novel options for SCLC treatment.

Oral administration of porcine liver decomposition product for 4 weeks enhances visual memory and delayed recall in healthy adults over 40 years of age: A randomized, double-blind, placebo-controlled study.

BACKGROUND AND OBJECTIVES: Porcine liver decomposition product (PLDP) contains neurofunctional phospholipids. We previously reported that PLDP enhances cognitive function in healthy adult humans, based on clinical evaluations using Hasegawa's Dementia Scale-Revised. In this study, we evaluated the effect of PLDP on memory indicators of the Wechsler Memory Scale-Revised (WMS-R), an internationally recognized battery for memory assessment. METHODS: We conducted a double-blind parallel-group placebo-controlled trial to evaluate the effect of PLDP on memory. Fifty-eight participants competed the trial: 28 participants were in the PLDP group and 30 participants were in the placebo group. Each group was administered PLDP (4 capsules) or a placebo (4 capsules) for 4 continuous weeks. WMS-R was administered before and 4 weeks after PLDP or placebo intake. The data were also subdivided by age for participants under 40 years (N = 15 in PLDP; N = 15 in placebo) and over 40 years (N = 13 in PLDP, N = 15 in placebo). Changes in Verbal Memory, Visual Memory, Attention/Concentration, and Delayed Recall were analyzed. RESULTS: No significant differences were found in any memory indicators between the PLDP group and the placebo group in pooled participants and in participants under 40 years of age. However, for participants over 40 years of age, PLDP administration resulted in a significant enhancement than placebo administration in Delayed Recall (14.1 ± 7.1 points vs. 7.1 ± 6.8 points) (P < 0.05), Visual Recall I (20.1 ± 23.1 percentile vs 1.9 ± 22.8 percentile) (P < 0.05), and Visual Recall II (24.2 ± 25.8 percentile vs 6.7 ± 19.0 percentile) (P < 0.05), respectively. The composition ratio of men to women in each group was imbalanced but no significant difference existed between the two groups. LIMITATIONS: A modest sample size, single-center design, and a fairly short follow-up period. CONCLUSION: PLDP enhanced Visual Memory and Delayed Recall in healthy adults over 40 years of age but not in healthy adults under 40 years of age. Therefore, PLDP may represent a promising nutraceutical that could improve cognitive function in healthy adults over 40 years of age. Further studies are required to evaluate if long term PLDP administration can prevent or delay cognitive dysfunction in healthy adults over 40 years of age.

Fatty Acid Composition and Applications of Eriobotrya japonica Seed Oil.

The loquat (Eriobotrya japonica) is commonly found in Japan. Its fruits are consumed raw or used in processed foods, and its leaves are used as a traditional medicine and in the manufacturing of cosmetics. Additionally, its seeds have several industrial applications. Therefore, this study aimed to estimate the fatty acid composition of loquat seed oil, and to evaluate its potential application as a deodorant. Palmitic acid, linoleic acid, behenic acid, and lignoceric acid were found to be the primary fatty acids present in the seeds, among which linoleic acid was involved in the deodorization of allyl methyl sulfide. Based on these results, loquat seed oil has potential for use in deodorant production.

Characteristics of scratching behavior in ADJM mice (atopic dermatitis from Japanese mice).

In order to elucidate the characteristics of scratching behavior in atopic dermatitis from Japanese mice (ADJM) mice, the effects of some antagonists of pruritogens on this behavior were studied. Both male and female ADJM mice showed frequent scratching behavior around the face, abdomen and back. The number of scratching behavior around the face was greater than on the abdomen and back, and scratching behavior in female mice was significantly more frequent than in male mice. Histamine H1 antagonist, chlorpheniramine, p.o., inhibited this behavior potently and dose-dependently. Histamine H1 antagonist with serotonin 5-TH(5-hydroxytryptamine)2 antagonist, cyproheptadine, also inhibited this behavior. However, NK1 antagonist, aprepitant, p.o., had no significant inhibitory effect even at a dose of 100 mg/kg, p.o., Mu antagonist, naloxone, and kappa agonist, nalfurafine, significantly inhibited this behavior at doses of 0.3 mg/kg, s.c., and 0.01 mg/kg, p.o., respectively. Histamine contents in the skin of ADJM mice were significantly higher than in BALB/c mice. These results strongly indicate that scratching behavior in ADJM mice is related with histamine H1, opioid mu and opioid kappa receptors.

Structure-activity relationship of a series of 17 parabens and related compounds for histamine release in rat peritoneal mast cells and skin allergic reaction in guinea pigs.

Parabens, which are a homologous series of esters of p-hydroxybenzoic acid, have been used as preservatives in cosmetics, medicines and foods because of their antimicrobial activity. However, parabens in cosmetics have been suspected to cause allergic contact dermatitis. In this study, we examined paraben-induced histamine release from rat peritoneal mast cells and skin reaction in guinea pigs using a series of 17 parabens with different alcohol side chains, ranging from methylparaben to dodecylparaben. Octylparaben showed the greatest histamine release-inducing activity from mast cells, and the activity was decreased in shorter- and longer-side-chain parabens. Octyl benzoate, octyl o-hydroxybenzoate and phenyloctane caused no significant degranulation of mast cells, whereas octyl m-hydroxybenzoate, octyl p-hydroxybenzoate and octyl phenol induced concentration-related degranulation. Metabolites of these parabens (p-hydroxybenzoic acid and alcohols) did not show histamine release-inducing activity. In the guinea pig skin reaction test, heptylparaben induced a typical strong skin reaction, while butylparaben induced a typical weak skin reaction, and methylparaben and dodecylparaben were inactive. Metabolites of parabens (p-hydroxybenzoic acid and alcohols) were also inactive. These results indicate that interaction of parabens with rat mast cells requires a minimum length and adequate lipophilicity of the alkyl side chain. Since metabolites of parabens were inactive, parabens appear to be direct-acting allergens.

Mitochondrial superoxide mediates mitochondrial and endoplasmic reticulum dysfunctions in TRAIL-induced apoptosis in Jurkat cells.

Reactive oxygen species (ROS), such as superoxide (O2(•-)) and hydrogen peroxide (H2O2), have been reported to be important mediators of the apoptosis induced by death ligands, including Fas, tumor necrosis factor-α, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Conversely, there is evidence that H2O2 and prooxidative conditions are protective. Therefore, the roles of ROS in death ligand-induced apoptosis are a matter of debate. In this study, we attempted to define the oxidant species mediating TRAIL-induced apoptosis in human tumor cells. The generation of intracellular O2(•-), but not H2O2, was correlated with apoptosis in the cells. TRAIL treatment resulted in increased mitochondrial O2(•-) generation and the oxidation of cardiolipin. The O2(•-)-selective scavenger MnTBaP [Mn(III) tetrakis (4-benzoic acid) porphyrin chloride] specifically blocked TRAIL-induced apoptosis and proapoptotic events including mitochondrial membrane collapse and caspase-3/7 activation. TRAIL also induced endoplasmic reticulum (ER) stress responses including caspase-12 activation, while inhibition of caspase-12 prevented the apoptosis. In addition, increased mitochondrial O2(•-) generation by uncoupling of oxidative phosphorylation or inhibition of the electron transport chain amplified the TRAIL-induced apoptosis and proapoptotic events. This amplification was also significantly abolished by MnTBaP treatment. Our data indicate that mitochondrial O2(•-) mediates mitochondrial and ER dysfunctions during TRAIL-induced apoptosis in Jurkat cells. The present findings suggest that pharmacological agents increasing mitochondrial O2(•-) may serve as clinical drugs that amplify TRAIL effectiveness toward cancer cells.

Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation.

Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O2()) are thought to mediate apoptosis induced by death receptor ligands, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the role of H(2)O(2) is controversial, since some evidence suggests that H(2)O(2) acts as an anti-apoptotic factor. Here, we show that exogenously applied H(2)O(2) (30-100 µM) induces cell death in TRAIL-resistant human melanoma cells via intracellular superoxide (O(2)-) generation. H(2)O(2) induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied; low concentrations of H(2)O(2) preferentially activated the caspase-dependent apoptotic pathway, while high concentrations of H(2)O(2) induced apoptotic and necrotic cell death in a caspase-independent manner. The H(2)O(2)-induced cell death was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation and ER stress responses including caspase-12 and X-box-binding protein-1 (XBP-1) activation. H(2)O(2) induced intracellular O2- generation even within the mitochondria, while TRAIL did not. The superoxide dismutase mimetic antioxidant MnTBaP [Mn (III) tetrakis (4-benzonic acid) porphyrin chloride] inhibited the H(2)O(2)-induced O(2)- generation, apoptosis and XBP-1 and caspase-12 activation at comparable concentrations. Importantly, H(2)O(2) treatment caused minimal O(2)- generation and apoptosis in normal primary melanocytes. These data show that H(2)O(2) induces endoplasmic reticulum-associated cell death via intracellular O(2)- generation and that malignant melanoma cells are more susceptible than normal cells to this oxidative cell death. The findings suggest that H(2)O(2) has therapeutic potential in the treatment of TRAIL-resistant melanoma.

Diallyl trisulfide sensitizes human melanoma cells to TRAIL-induced cell death by promoting endoplasmic reticulum-mediated apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment because of its selective cytotoxicity toward tumor cells. However, some cancer cell types including malignant melanoma cells are resistant to TRAIL cytotoxicity. Here, we show that diallyl trisulfide (DATS), a garlic organosulfur compound, sensitizes melanoma cells to TRAIL-induced apoptosis while sparing normal cells. DATS also potentiates apoptosis induced by agonistic antibodies against death receptors (DR) 4 and DR5. The amplification of DR-mediated apoptosis was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation. However, these events were not sufficient for full sensitization. TRAIL also induced endoplasmic reticulum (ER) stress, as indicated by the activation of X-box-binding protein 1 and caspase-12 and DATS poten-tiated both events. Moreover, inhibition of caspase-12, but not caspase-4, abolished the amplification of apoptosis, indicating that ER stress plays a crucial role. On the other hand, DATS and/or TRAIL induced minimal apoptosis and caspase-12 activation in melanocytes despite their substantial expression of DR4 and DR5 on the cell surface. Our data suggest that DATS amplifies death ligand-induced melanoma cell death by disrupting their adaptation to ER-mediated death pathway. The present findings raise the possibility that DATS may be combined with death ligands to treat TRAIL-resistance melanoma cells without impairing its tumor selectivity.

Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: role for ATP-sensitive K+ channels and endoplasmic reticulum stress.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment owing to its selective cytotoxicity against malignant cells. However, some cancer cell types, including malignant melanoma cells, are resistant to TRAIL-induced apoptosis. Therefore, drugs that can amplify TRAIL cytotoxicity are urgently required. Depolarization of the plasma membrane potential is associated with apoptosis induced by a variety of death-inducing agents but its role in apoptosis remains a matter of debate. We found that TRAIL treatment resulted in robust depolarization in human melanoma cells with a considerable lag (2-4 h). Moreover, membrane-depolarizing agents, including K+ and ATP-sensitive K+ (KATP) channel inhibitors glibenclamide and U37883A enhanced TRAIL-induced apoptosis. On the contrary, inhibitors of calcium- and voltage-dependent K+ channels and mitochondrial KATP channels had no such effects. Melanocytes were insensitive to TRAIL-induced depolarization and apoptosis as well as to the sensitization by membrane-depolarizing agents despite their substantial surface expression of death receptors. TRAIL induced robust activation of X-box-binding protein-1 and caspase-12, both of which were enhanced by the K+ and KATP channel inhibitors, but not by other K+ channel inhibitors. Finally, caspase-12-selective inhibitor completely abolished the amplification of apoptosis. These findings suggest that depolarization promotes endoplasmic reticulum stress-mediated death pathway, thereby amplifying TRAIL cytotoxicity. Thus, membrane-depolarizing agents such as KATP channel inhibitors may have therapeutic potential in the treatment of TRAIL-resistant cancer cells without impairing tumor-selectivity.

Calcium signaling in mast cells: focusing on L-type calcium channels.

Mast cells play central roles in adaptive and innate immunity. IgE-dependent stimulation of the high-affinity IgE receptor (FcεRI) results in rapid secretion of various proinflammatory chemical mediators and cytokines. All of the outputs depend to certain degrees on an increase in the intracellular Ca(2+) concentration, and influx of Ca(2+) from the extracellular space is often required for their full activation. There is strong evidence that FcεRI stimulation induces two different modes of Ca(2+) influx, store-operated Ca(2+) entry (SOCE) and non-SOCE, which are activated in response to endoplasmic reticulum Ca(2+) store depletion and independently of Ca(2+) store depletion, respectively, in mast cells. Although Ca(2+) release-activated Ca(2+) channels are the major route of SOCE, recent evidence indicates that they are not the only Ca(2+) channels activated by Ca(2+) store depletion. The recent data suggest that L-type Ca(2+) channels, which were thought to be a characteristic feature of excitable cells, exist in mast cells to mediate non-SOCE, which is critical for protecting mast cells against activation-induced mitochondrial cell death. In this chapter, we provide an overview of recent advances in our understanding of Ca(2+) signaling in mast cells with a special attention to the emerging role for the L-type Ca(2+) channels as a regulator of mast cell survival.

Autoimmunity-inducing metals (Hg, Au and Ag) modulate mast cell signaling, function and survival.

The three heavy metals, mercury, gold and silver commonly and specifically induce aberrant immunological responses leading to autoimmune disorders in genetically susceptible animals and humans. The disorders are characterized by autoantibody production, increases in serum IgG and IgE, polyclonal activation of B and T lymphocytes and renal immune complex deposition and glomerulonephritis. Mast cells play key roles in allergic and inflammatory reactions. A growing body of evidence suggests that mast cells are key players in innate and adaptive immunity and involved in autoimmune diseases. Mast cells are also direct targets for autoimmunity-inducing metals both in vitro and in vivo and play a role in the development of metal-induced autoimmune disorders. The three metals specifically modulate mast cell function, including degranulation and secretion of arachidonic acid metabolites and cytokines such as interleukin-4. Divergent signaling components, including mitogen-activated protein kinase activation, reactive oxygen and nitric oxide generation and Ca2+ influx are modulated by the metals. Furthermore, the metals have considerable impacts on mast cell survival, which also species seems to be involved in the development of metal-induced autoimmune disorders. In this review, we provide an overview of recent advances in our understanding of the impacts of the three metals on mast cell signaling, function and survival and their possible roles in the pathologies of metal-induced autoimmunity.

Epigallocatechin-3-gallate induces cytokine production in mast cells by stimulating an extracellular superoxide-mediated calcium influx.

The green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has various biological activities, including anti-inflammatory, anti-neoplastic, anti- and pro-apoptotic, and neuroprotective effects. Although these are often associated with increased intracellular reactive oxygen species (ROS) and Ca(2+) levels, their involvement in biological effects is poorly understood. Here we report that EGCG induces cytokine production in mast cells via Ca(2+) influx and ROS generation. EGCG at concentrations of ≥50 µM induced interleukin-13 and tumor necrosis factor-α production in RBL-2H3 and bone marrow-derived mast cells. The effects were dependent on extracellular Ca(2+), and EGCG induced Ca(2+) release from intracellular stores and Ca(2+) influx. Ca(2+) influx was suppressed by 2-aminoethoxydiphenyl borate, an inhibitor of store-operated Ca(2+) (SOC) channels, including Ca(2+) release-activated Ca(2+) channels and transient receptor potential canonical channels. EGCG failed to induce Ca(2+) influx through SOC channels. EGCG-activated Ca(2+) channels were genetically and pharmacologically distinct from Ca(v)1.2 L-type Ca(2+) channels, another route of Ca(2+) influx into mast cells. EGCG evoked release of superoxide (O(2)(·-)) into the extracellular space. Exogenous superoxide dismutase, but not catalase, inhibited EGCG-evoked Ca(2+) influx and cytokine production, indicating that extracellular O(2)(·-) regulates these events. EGCG can serve as a powerful tool for studying O(2)(·-)-regulated Ca(2+) channels, which may be selectively involved in the regulation of cytokine production but have yet to be elucidated.

Protease-mediated house dust mite allergen-induced reactive oxygen species production by neutrophils.

A growing body of evidence indicates that neutrophils may play an important role in the pathogenesis of asthma. However, the involvement of the house dust mite (HDM) in neutrophil activation associated with the pathogenesis of asthma is not fully understood yet. To address this situation, we harvested neutrophils isolated from 15 HDM-sensitized asthmatic subjects and 18 HDM-sensitized nonasthmatic subjects and measured the amounts of neutrophil reactive oxygen species (ROS) production in response to the major HDM allergens Der-f and Der-f1. Der-f and Der-f1 significantly increased ROS production in neutrophils isolated from asthmatic subjects versus nonasthmatic subjects. To assess the involvement of Der-f-specific IgE antibodies binding to their receptors in HDM allergen-induced ROS production, we examined whether neutrophils produce ROS by cross-linking of cell-bound IgE antibodies with anti-IgE. Treatment with anti-IgE antibodies did not induce ROS production by neutrophils isolated from 6 asthmatic subjects. On the other hand, pretreatment of Der-f with E-64, a cysteine protease inhibitor, eliminated Der-f-induced ROS production. These results suggest that HDM-allergen exposure may result in greater production of ROS in asthmatic patients and may be involved in the pathogenesis of asthma.

Prophylactic effects of the histamine H1 receptor antagonist epinastine and the dual thromboxane A2 receptor and chemoattractant receptor-homologous molecule expressed on Th2 cells antagonist ramatroban on allergic rhinitis model in mice.

The prophylactic use of anti-allergic drugs has been proposed to be effective in the treatment of seasonal allergic rhinitis in humans. However, there is little information regarding the prophylactic effect of thromboxane A(2) (TXA(2)) receptor antagonist on allergic rhinitis. Recent studies revealed that a TXA(2) receptor antagonist ramatroban could block the prostaglandin D(2) (PGD(2)) receptor and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). In the present study, we investigated the prophylactic effects of the histamine H(1) receptor antagonist epinastine and the TXA(2) receptor antagonist ramatroban and seratrodast on mouse models of allergic rhinitis. Female BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin and alum on days 0, 5, 14 and 21. Seven days later, mice were sensitized by intranasal application of ovalbumin thrice a week. Drugs were administered once a day from day 22. The severity of allergic rhinitis was assessed by determining the extent of 2 nasal allergic symptoms (sneezing and nasal rubbing). Histamine sensitivity and eosinophil infiltration into the nasal mucosa were also determined. Epinastine and ramatroban significantly reduced nasal symptoms and the number of eosinophils in the nasal mucosa. Seratrodast showed no effect on nasal symptoms and eosinophil infiltration into the nasal mucosa. In addition, histamine sensitivity was reduced by epinastine and ramatroban. These results indicate that epinastine and ramatroban induce the prophylactic effect on allergic rhinitis.

Gold activates mast cells via calcium influx through multiple H2O2-sensitive pathways including L-type calcium channels.

Heavy metals, including gold, induce severe contact hypersensitivity and autoimmune disorders, which develop through an initial Th2-independent process followed by a Th2-dependent process. It has been shown that mast cell activation plays a role in the Th2-independent process and that gold stimulates histamine release in vitro. However, the mechanisms of the gold-induced mast cell activation remain largely unclear. Here we report that gold directly activates mast cells in a Ca2+-dependent manner. HAuCl4 [Au(III)] at nontoxic concentrations (≤50 µM) induced substantial degranulation and leukotriene C4 secretion in an extracellular Ca2+-dependent manner. Au(III) induced a robust Ca2+ influx but not Ca2+ mobilization from internal stores. Au(III) also stimulated intracellular production of reactive oxygen species, including H2O2, and blockade of the production abolished the mediator release and Ca2+ influx. Au(III) induced Ca2+ influx through multiple store-independent Ca2+ channels, including Cav1.2 L-type Ca2+ channels (LTCCs) and 2-aminoethoxydiphenyl borate (2-APB)-sensitive Ca2+ channels. The 2-APB-sensitive channel seemed to mediate Au(III)-induced degranulation. Our results indicate that gold stimulates Ca2+ influx and mediator release in mast cells through multiple H2O2-sensitive Ca2+ channels including LTCCs and 2-APB-sensitive Ca2+ channels. These findings provide insight into the roles of these Ca2+ channels in the Th2-independent process of gold-induced immunological disorders.

Deficiency of SHP1 leads to sustained and increased ERK activation in mast cells, thereby inhibiting IL-3-dependent proliferation and cell death.

SHP-1 plays an important role for the regulation of signaling from various hematopoietic cell receptors. In this study, we examined IL-3-induced cell proliferation and IL-3 depletion-induced apoptosis in bone marrow-derived mast cells (BMMC) established from motheaten (me) that lack SHP-1 expression, viable motheaten (me(v)) expressing phosphatase-deficient SHP-1, and wild-type (WT) mice. When BMMC were stimulated with IL-3, increased ERK activation was evident in resting state and sustained in me-BMMC relative to WT-BMMC. ERK is known to be involved in the regulation of cell proliferation and apoptosis in some cells. In accordance with sustained ERK activation, apoptosis was decreased in me- and me(v)-BMMC compared with WT-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me- and me(v)-BMMC than WT-BMMC. Stimulation with lower concentration of IL-3 or addition of PD98059, a MEK inhibitor, to the culture resulted in the suppression of decreased apoptosis and cell proliferation in me- and me(v)-BMMC. Collectively, these results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity through its phosphatase activity. Furthermore, our results indicate that ERK would act as a negative regulator for cell proliferation and induce apoptosis when its activity is highly increased.

Epigallocatechin-3-gallate inhibits mast cell degranulation, leukotriene C4 secretion, and calcium influx via mitochondrial calcium dysfunction.

The green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been shown to reduce allergic inflammatory responses in animal models, but the mechanisms involved are poorly understood. Despite the essential role for Ca(2+) influx in mediating proinflammatory mediator release from mast cells, little is known about the effects of EGCG on this response. In the present study we found that EGCG inhibited antigen-induced Ca(2+) influx and store-operated Ca(2+) entry (SOCE), the principal mode of Ca(2+) influx into mast cells. EGCG, but not (-)-epicatechin (EC), inhibited antigen-induced degranulation, leukotriene (LT) C(4) secretion, and Ca(2+) influx. EGCG also blocked SOCE without reducing Ca(2+) store emptying whereas EC did not, although it did reduce Ca(2+) store emptying. EGCG, but not EC, also evoked intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (Psi(m)) collapse, cardiolipin oxidation, and mitochondrial Ca(2+) ([Ca(2+)](m)) release. Furthermore, FCCP, a potent inducer of Psi(m) collapse, induced ROS production and [Ca(2+)](m) dysfunction and inhibited degranulation, LTC(4) secretion, Ca(2+) influx, and SOCE. These data suggest that ROS production and Psi(m) collapse are important mechanisms underlying the antiallergic effects of EGCG. These events may lead to [Ca(2+)](m) dysfunction and impair mitochondria-mediated facilitation of SOCE, thereby attenuating mast cell activation.

Endothelial nitric oxide synthase is essential for nitric oxide generation, L-type Ca2+ channel activation and survival in RBL-2H3 mast cells.

Recent pharmacological and molecular genetic approaches have revealed the existence of functional L-type Ca2+ channels (LTCCs) in a variety of hematopoietic cells. We previously reported that Ca(v)1.2 LTCCs are expressed on mast cell surfaces, activated by the high-affinity IgE receptor (FcvarepsilonRI) engagement and protect mast cells against activation-induced cell death (AICD). We also demonstrated that FcvarepsilonRI engagement evokes nitric oxide (NO) generation in a phosphatidylinositol-3-kinase- and NO synthase (NOS)-dependent manner, which is also required for mast cell survival. Here we demonstrate that this endogenous NO mediates Ca(v)1.2 LTCC activation. FcvarepsilonRI engagement but not thapsigargin, a potent Ca2+ release-activated Ca2+ (CRAC) channel agonist, induced Ca2+ influx via NOS-dependent NO generation. RT-PCR analyses revealed predominant expression of eNOS in mast cells. Subsequent experiments involving siRNA-mediated gene silencing of eNOS or Ca(v)1.2 LTCC revealed that eNOS was essential for NOS-dependent NO generation and Ca(v)1.2 LTCC activation but not CRAC channel activation. Similar to Ca(v)1.2 LTCCs, eNOS prevented the dissipation of the mitochondrial membrane potential and mitochondrial integrity collapse, thereby protecting mast cells against AICD. Taken together, the present findings demonstrate the key roles of the eNOS-NO-LTCC axis in mast cell survival after FcvarepsilonRI engagement.

L-type Ca2+ channels: a new player in the regulation of Ca2+ signaling, cell activation and cell survival in immune cells.

Ca(2+) is a highly versatile intracellular second messenger in many cell types, and regulates many complicated cellular processes, including cell activation, proliferation and apoptosis. Influx of Ca(2+) from the extracellular fluid is required for sustained elevation of the cytosolic Ca(2+) concentration and full activation of Ca(2+)-dependent processes. It is widely accepted that Ca(2+) release-activated Ca(2+) channels are the major routes of Ca(2+) influx in electrically non-excitable cells, including hematopoietic cells, whereas voltage-gated Ca(2+) channels such as L-type Ca(2+) channels (LTCCs) serve as the principal routes of Ca(2+) entry into electrically excitable cells such as neurons and myocytes. However, recent pharmacological and molecular genetic studies have revealed the existence of functional LTCCs and/or LTCC-like channels in a variety of immune cells including mast cells. In this article, we review recent advances in our understanding of Ca(2+) signaling in immune cells with a special interest in mast cells. We highlight roles for LTCCs in antigen receptor-mediated mast cell activation and survival.