Oleanolic acid protects against mast cell-mediated allergic responses by suppressing Akt/NF-κB and STAT1 activation.

BACKGROUND: Oleanolic acid (OA) is an active compound found in a variety of medicinal herbs and plants. Though OA has been widely attributed with a variety of biological activities, studies focused on its anti-allergic inflammation properties are insufficient. PURPOSE: Given the rapid increase in allergic diseases and the lack of fundamental treatment options, this study aimed to find a safe and effective therapy for allergic disorders. METHODS: We evaluated the inhibitory effect of OA on allergic inflammatory response and the possible mechanisms underlying the effect using phorbol-12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-stimulated human mast cell (HMC)-1, and a mouse model of compound 48/80-induced anaphylactic shock. RESULTS: OA suppressed pro-inflammatory cytokine expressions in PMACI-induced HMC-1 cells by inhibiting activation of the Akt, p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription (STAT) 1 signaling pathways. Moreover, OA showed a protective effect against compound 48/80-induced anaphylactic shock through inhibition of histamine release and immunoglobulin E level via regulation of NF-κB and STAT1 activation. CONCLUSION: The results showed that OA suppressed mast cell-mediated allergic response by transcriptional regulation. We suggest that OA has potential effect against allergic inflammatory disorders, including anaphylaxis, and might be a useful therapeutic agent for allergic disease.

Novel Peptidomic Approach for Identification of Low and High Molecular Weight Tauopathy Peptides Following Calpain Digestion, and Primary Culture Neurotoxic Challenges.

Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products (Tau-BDP; 45K, 35K, and 17K). Tau proteolysis might also generate low molecular weight (LMW ≤10K) proteolytic peptides after neurodegenerative damage. In this study, we have subjected purified tau protein (phospho and non-phospho) and mouse brain lysate to calpain-1 digestion to characterize the LMW generated by nano-liquid chromatography coupled to electrospray ionization to tandem mass spectrometry (nano-LC-ESI-MS/MS). We have also challenged differentiated primary cerebrocortical neuronal cultures (CTX) with neurotoxic agents (calcium ionophore calcimycin (A23187), staurosporine (STS), N-methyl-D-aspartate (NMDA), and Maitotoxin (MTX)) that mimic neurodegeneration to investigate the peptidome released into the conditioned cell media. We used a simple workflow in which we fractionate LMW calpain-mediated tau peptides by ultrafiltration (molecular weight cut-off value (MWCO) of 10K) and subject filtrate fractions to nano-LC-MS/MS analysis. The high molecular weight (HMW) peptides and intact proteins retained on the filter were analyzed separately by western blotting using total and phospho-specific tau antibodies. We have identified several novel proteolytic tau peptides (phosphorylated and non-phosphorylated) that are only present in samples treated with calpain or cell-based calpain activation model (particularly N- and C-terminal peptides). Our findings can help in developing future research strategies emphasizing on the suppression of tau proteolysis as a target.

The epithelial barrier-protecting properties of a soy hydrolysate.

Enhancing the epithelial barrier function could be a possible strategy to prevent food allergy or reduce its symptoms. Soy hydrolysates containing bioactive peptides could be instrumental in this. In this study, the protective effects of pretreatment with 6 soy hydrolysates on calcium ionophore A23187-induced TEER reduction were studied in T84 cells. The effects of the most potent soy hydrolysate on tight junction gene expression were studied. In order to identify the underlying pathways involved, the barrier disruptor specificity of the effect was studied by comparing the protective effects on TEER and Lucifer Yellow flux after the exposure to barrier disruptors that work via different intracellular pathways, i.e. the disruptors A23187, mellitin, and deoxynivalenol (DON). Preincubation with one of the six hydrolysates protected the epithelial cells from a decrease in TEER induced by A23187 (restored to 105% of the starting point, while A23187 alone decreased to 53% of the starting value) and mellitin (restored to 11% of the starting point, while mellitin alone decreased to 3.8% of the starting value). This soy hydrolysate was found to increase claudin-1 and decrease claudin-2 expression. The protective effect of the hydrolysate on TEER was specific for the barrier disruptors A23187 and mellitin, but was not observed for DON. This observation suggests that the soy hydrolysate may act via PKC isoforms, which are known to lead to changes in the expression of claudin-1 and 2. Our data suggest that specific soy hydrolysates may be designed to strengthen the epithelial barrier which might be instrumental in the management of the barrier function in individuals at risk of developing food allergy.

Possible Involvement of the Inhibition of NF-κB Factor in Anti-Inflammatory Actions That Melatonin Exerts on Mast Cells.

Melatonin is a molecule endogenously produced in a wide variety of immune cells, including mast cells (RBL-2H3). It exhibits immunomodulatory, anti-inflammatory and anti-apoptotic properties. The physiologic mechanisms underlying these activities of melatonin have not been clarified in mast cells. This work is designed to determine the anti-inflammatory effect and mechanism of action of melatonin on activated mast cells. RBL-2H3 were pre-treated with exogenous melatonin (MELx) at physiological (100nM) and pharmacological (1 mM) doses for 30 min, washed and activated with PMACI (phorbol 12-myristate 13-acetate plus calcium ionophore A23187) for 2 h and 12 h. The data shows that pre-treatment of MELx in stimulated mast cells, significantly reduced the levels of endogenous melatonin production (MELn), TNF-α and IL-6. These effects are directly related with the MELx concentration used. MELx also inhibited IKK/NF-κB signal transduction pathway in stimulated mast cells. These results indicate a molecular basis for the ability of melatonin to prevent inflammation and for the treatment of allergic inflammatory diseases through the down-regulation of mast cell activation. J. Cell. Biochem. 117: 1926-1933, 2016. © 2016 Wiley Periodicals, Inc.

Melatonin protects mast cells against cytotoxicity mediated by chemical stimuli PMACI: possible clinical use.

Melatonin has documented cytoprotective effects on a wide variety of immune cells. The mechanism of action on mast cells (RBL-2H3) still remains in the dark. We found that melatonin significantly attenuated phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI)-induced cytotoxicity in a concentration and time-dependent manner. It appears that the effect of melatonin on mast cells is two-fold: dependent (MT1 and MT2) and independent membrane receptors. In conclusion, melatonin treatment reduced the cytotoxicity, mediated by PMACI, and could provide a useful therapeutic option in processes where an excessive activation of mast cells occurs.

Age-dependent rat retinal ganglion cell susceptibility to apoptotic stimuli: implications for glaucoma.

BACKGROUND: This paper seeks to investigate differences between the neonatal and adult retinal ganglion cell populations to apoptotic death stimuli. DESIGN AND SAMPLES: In vitro and ex vivo paradigms involving P6 and P60 Sprague-Dawley rat retinal explants and retinal ganglion cells were employed. METHODS: Postnatal day 6 (P6) and 60 (P60) Sprague-Dawley retinal ganglion cells and retinal explants were either serum starved or subjected to excitotoxicity using calcium ionophore A23187. MAIN OUTCOME MEASURES: Apoptosis was detected in both models using terminal dUTP nick end labelling. Expression of Apaf-1, active caspases-3 and 9 in P6 and P60 retinas, and in the ganglion cell layer was examined using Western blotting. RESULTS: In both the dissociated retinal ganglion cell and retinal explant models, P60 retinal ganglion cells were significantly less susceptible to excitoxicity and serum starvation than their P6 counterparts. Western blotting indicated that active caspase-3 and Apaf-1 are downregulated in the Sprague-Dawley rat retina at P60 compared with P6. CONCLUSIONS: We demonstrate that neonatal Sprague-Dawley retinal ganglion cells are more susceptible to glaucoma-related death stimuli than their adult counterparts in dissociated retinal ganglion cells and axotomized retinal explant models. It is apparent that these different retinal ganglion cell populations are inherently designed to react differently to death stimuli. Thus caution should be exercised when noting the high susceptibility of neonatal retinal ganglion cells to glaucomatous death stimuli.

Cytoprotective action of the potassium channel opener NS1619 under conditions of disrupted calcium homeostasis.

Cytoprotective properties of potassium channel openers (KCOs) have been demonstrated in several models of cell injury, mainly in ischemia-reperfusion-induced damage of cardiac muscle. The mechanism responsible for the observed cytoprotection and the relative contribution of plasma membrane or inner mitochondrial membrane potassium channels regarding the beneficial effects exerted by KCOs remain unclear. Our work demonstrates the cytoprotective properties of NS1619, an opener of large-conductance calcium-activated potassium channels (BKCa channels), using C2C12 myoblasts injured by calcium ionophore A23187 treatment. Application of two BKCa channel inhibitors, paxilline and iberiotoxin, abolished this cytoprotective effect. At concentrations of 10-100 µM, NS1619 increased the respiration rate and decreased mitochondrial membrane potential (Δψ) in C2C12 cells in a dose-dependent manner. At a concentration of 0.2 µM, paxilline, which effectively abolished the protective effect of NS1619, failed to counteract the opener-induced mitochondrial depolarization and increase in cellular respiration. This result indicates that the NS1619-mediated increase in the survival rate of A23187-treated C2C12 cells occurs in a manner distinct from its effect on mitochondrial functioning and suggests that activation of BKCa channels in the plasma membrane is the mechanism responsible for cytoprotection by NS1619.

The cytoprotective action of the potassium channel opener BMS-191095 in C2C12 myoblasts is related to the modulation of calcium homeostasis.

UNLABELLED: BMS-191095 is an opener of the mitochondrial ATP-regulated potassium channel, which has been shown to provide cytoprotection in models of ischemia-reperfusion induced injury in various tissues. This study aimed at checking the protective action of BMS-191095 under the conditions of oxidative stress or disruption of intracellular calcium homeostasis. METHODS: The cytoprotective potential of BMS-191095 was tested in C2C12 myoblasts injured by treatment with H(2)O(2) or calcium ionophore A23187. The influence of the opener on intracellular calcium levels, calpain activity and respiration rates were determined. RESULTS: BMS-191095 protected myoblasts from calcium ionophore A23187-induced injury, but not from H H(2)O(2)-induced injury. A23187-mediated cell damage was also prevented by calpain inhibitor PD 150606. A23187 administration led to a transient increase in cytosolic calcium levels, concomitant activation of calpains and a decrease in state 3 respiration rates, indicating mitochondrial dysfunction. Co-administration of BMS-191095 diminished calpain activation in A23187-treated cells but did not prevent mitochondrial damage. In the presence of BMS-191095, restoration of cytosolic calcium concentrations to basal levels after A23187 treatment was considerably faster which may underly the reduced activation of calpains. CONCLUSION: The BMS-191095-mediated cytoprotection observed in C2C12 myoblasts results probably from modulation of intracellular calcium transients leading to prevention of calpain activation.

Genipin suppresses A23187-induced cytotoxicity in neuro2a cells.

Genipin is an iridoid compound and an aglucon of geniposide isolated from Gardenia fructus. We have previously reported that genipin induces neurite outgrowth in PC12h and Neuro2a cells and protects against cytotoxicity induced by several conditions such as beta-amyloid peptide, serum deprivation, and oxidative stress in rat primary hippocampal neurons and Neuro2a cells. In this paper, we examined the protective effect of genipin on A23187 (a calcium ionophore)-induced cytotoxicity in Neuro2a cells. A23187 induced cytotoxicity in concentration- and time-dependent manners as assayed by measurements of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide (MTT) reduction activity and lactate dehydrogenase (LDH) release. The cytotoxicity was significantly suppressed by genipin in a concentration-dependent manner. A23187 also significantly activated caspase3/7, which is known to be the critical mediator of apoptosis, after 1 h, and the cytotoxicity was clearly blocked by an inhibitor of caspase 3/7. Furthermore, A23187 induced the expression of immunoglobulin-binding protein/glucose-regulated protein of 78 kDa (BiP/GRP78) protein, which is an endoplasmic reticulum (ER) stress marker protein, and the expression was suppressed by genipin. These results suggest that genipin protects Neuro2a cells from A23187-induced cytotoxicity mediated by caspase 3/7 and ER stress. Therefore, genipin may be effective in preventing neurodegeneration observed in Alzheimer's disease and Parkinson's disease involving ER stress.

Cytoprotective roles of senescence marker protein 30 against intracellular calcium elevation and oxidative stress.

Senescence marker protein 30 (SMP30) is identified as an important aging marker molecule and known to play multifunctional roles as an intracellular calcium regulatory protein in the signaling process. To elucidate the functional significance of SMP30, we established the stably transfected P19 cell line with SMP30 expression vector. Overexpression of SMP30 slightly suppressed the proliferation of P19 cells. However, SMP30 overexpression was cytoprotective against calcium-mediated stress such as calcium ionophore (A23187), and thapsigargin. We found that SMP30 overexpression reduced the elevated intracellular calcium levels induced by A23187, but not by thapsigargin. In addition, SMP30 transfected P19 cells were more protective to tert-butylhydroperoxide induced cytotoxicity, indicating the antioxidative properties of SMP30. Taken together, our results suggest that external calcium regulation and antioxidant properties are involved in the cytoprotective mechanism of SMP30.

Cordycepin induced eryptosis in mouse erythrocytes through a Ca2+-dependent pathway without caspase-3 activation.

Cordyceps sinensis is a prized traditional Chinese medicine and its major component cordycepin is found to have anti-leukemia activities. However, its cytotoxicity in erythrocytes was unclear. To examine the effect of cordycepin on the induction of eryptosis (an apoptosis-like process in enucleated erythrocytes), flow cytometric assays based on membrane integrity and asymmetry were employed. For comparison, analyses were performed in parallel with two other anti-leukemia agents, indirubin 3'-monoxime (IDM) and As2O3. We found that at the IC50 against leukemia HL-60, cordycepin elicited eryptosis while IDM and As2O3 showed no erythrotoxicity in mouse erythrocytes. Mechanistically, cordycepin increased the [Ca2+]i and activated mu-calpain protease in a dose-dependent manner. Yet, no caspase-3 activation was observed in the cordycepin-treated erythrocytes. When extracellular Ca2+ was depleted, both the cordycepin-induced eryptosis and mu-calpain cleavage were suppressed. Our study therefore demonstrated for the first time that cordycepin induces eryptosis through a calcium-dependent pathway in the absence of mitochondria and caspase-3 activation.

Reciprocal effects of glucose on the process of cell death induced by calcium ionophore or H2O2 in rat lymphocytes.

We have examined the effects of glucose at high concentrations on the process of cell death induced by excessive increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) or oxidative stress in rat lymphocytes. The cell death elicited by the excessive increase in [Ca(2+)](i) seemed to be induced by an activation of Ca(2+)-dependent K(+) channels because the inhibitors for Ca(2+)-dependent K(+) channels attenuated the decrease in cell viability. Glucose at 30-50mM augmented the decrease in cell viability by the excessive increase in [Ca(2+)](i). It was not specific for glucose because it was the case for sucrose or NaCl, suggesting an involvement of increased osmolarity in adverse action of glucose. On the contrary, glucose protected the cells suffering from oxidative stress induced by H(2)O(2), one of reactive oxygen species. It was also the case for fructose or sucrose, but not for NaCl. The process of cell death induced by H(2)O(2) started, being independent from the presence of glucose. Glucose delayed the process of cell death induced by H(2)O(2). Sucrose and fructose also protected the cells against oxidative stress. The reactivity of sucrose to reactive oxygen species is lower than those of glucose and fructose. The order in the reactivity cannot explain the protective action of glucose. Glucose at high concentrations exerts reciprocal actions on the process of cell death induced by the oxidative stress and excessive increase in [Ca(2+)](i).

The effects of organic pesticides on inner membrane permeability in Escherichia coli ML35.

We have tested whether some pesticides might cause inner membrane leakage in ML35 Escherichia coli cells, which express beta-galactosidase (lacZ; EC 3.2.1.23) constitutively but lack the permease (lacY) required for substrate entry. The activity of beta-galactosidase (indicative of substrate leakage through the inner membrane) was increased by various concentrations of pesticides, including the organometallic fungicides maneb and mancozeb, the insecticide Thiodan, and the herbicide Ally, as well as by antibiotics such as ampicillin, gramicidin D, and the calcium ionophore A23187. The enzyme activity was increased by up to approximately 30% when the E. coli ML35 strain was exposed to various concentrations (between 50 and 250 ppm) of both fungicides. Thiodan had only a slight effect on beta-galactosidase activity (increase of 12.8%), whereas, among the antibiotics, the calcium ionophore at 20 microg/ml caused a significant increase in enzyme activity by up to 61.8%. This effect is similar to that of sodium dodecyl sulfate, used as positive control ( approximately 70% increase). Accumulation of maneb and mancozeb by bacterial cells was also studied taking advantage of their metal content and using atomic absorption spectrophotometry. In parallel with the increase in enzyme activity, both fungicides accumulated in the cells as a function of their concentration. Time course experiments (3, 6, and 9 h) of fungicide accumulation and of bacterial growth at various pesticide concentrations were also carried out. Maneb seems to inhibit the bacterial growth better than mancozeb. In addition, maneb uptake increases with time up to 9 h at all tested concentrations, whereas the accumulation of mancozeb is similar at all the exposure times tested. This indicates a different uptake and/or metabolizing strategy by E. coli cells for the two fungicides.

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

Involvement of double-stranded RNA-dependent protein kinase and phosphorylation of eukaryotic initiation factor-2alpha in neuronal degeneration.

Inhibition of protein translation plays an important role in apoptosis. While double-stranded RNA-dependent protein kinase (PKR) is named as it is activated by double-stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2alpha phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the beta-amyloid (Abeta) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2alpha and the number of apoptotic cells were enhanced in over-expressed wild-type PKR neuroblastoma cells exposed to Abeta peptide, while dominant-negative PKR reduced eIF2alpha phosphorylation and apoptosis induced by Abeta peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Abeta peptide toxicity. Activation of PKR and eIF2alpha pathway by Abeta peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA-AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2alpha phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.

Comparative effects of carboxylic ionophores on membrane potential and resistance of NG108-15 cells.

Comparative analyses were conducted to determine the effects of Na(+) (monensin, MON), K(+) (nigericin, NIG) and Ca(2+) (A23187) selective carboxylic ionophores on differentiated NG108-15 (neuroblastoma X glioma hybrid) cells. Alterations in membrane potential (V(m)), input resistance (Rin) and electrically induced action potential generation were measured using intracellular microelectrode techniques in cells treated with 0.1-30 microM MON and NIG and 0.1-10 microM A23187. Responses to the ionophores were similar in that membrane hyperpolarization and unchanged R(in) predominated with all three compounds. However, significant differences between the ionophores were also detected. MON- and A23187-induced hyperpolarization was generally maintained throughout the 24-min superfusion whereas that produced by NIG diminished with time or was replaced by depolarization. In addition, action potential generation was blocked by NIG, whereas MON had no effect and action potential alterations were evident only with the highest A23187 concentration (10 microM). This study represents the initial comprehensive analysis of the effects of carboxylic ionophores on membrane electrical characteristics of an intact cell system and forms the basis for subsequent work using NG108-15 cells as a model system to evaluate potential therapeutic treatments against the carboxylic ionophores.

Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic activity.

UDP-glucose:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that operates as a gatekeeper for quality control by preventing transport of improperly folded glycoproteins out of the ER. We report the isolation of two cDNAs encoding human UDP-glucose:glycoprotein glucosyltransferase homologues. HUGT1 encodes a 1555 amino acid polypeptide that, upon cleavage of an N-terminal signal peptide, is predicted to produce a soluble 173 kDa protein with the ER retrieval signal REEL. HUGT2 encodes a 1516 amino acid polypeptide that also contains a signal peptide and the ER retrieval signal HDEL. HUGT1 shares 55% identity with HUGT2 and 31-45% identity with Drosophila, Caenorhabditis elegans, and Schizosaccharomyces pombe homologues, with most extensive conservation of residues in the carboxy-terminal fifth of the protein, the proposed catalytic domain. HUGT1 is expressed as multiple mRNA species that are induced to similar extents upon disruption of protein folding in the ER. In contrast, HUGT2 is transcribed as a single mRNA species that is not induced under similar conditions. HUGT1 and HUGT2 mRNAs are broadly expressed in multiple tissues and differ slightly in their tissue distribution. The HUGT1 and HUGT2 cDNAs were expressed by transient transfection in COS-1 monkey cells to obtain similar levels of protein localized to the ER. Extracts from HUGT1-transfected cells displayed a 27-fold increase in the transfer of [(14)C]glucose from UDP-[(14)C]glucose to denatured substrates. Despite its high degree of sequence identity with HUGT1, the expressed recombinant HUGT2 protein was not functional under the conditions optimized for HUGT1. Site-directed alanine mutagenesis within a highly conserved region of HUGT1 identified four residues that are essential for catalytic function.

Oxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187-induced death of cultured striatal neurons.

Disruption of intracellular calcium homeostasis is thought to play a role in neurodegenerative disorders such as Huntington's disease (HD). To study different aspects of putative pathogenic mechanisms in HD, we aimed to establish an in vitro model of calcium-induced toxicity in striatal neurons. The calcium ionophore A23187 induced a concentration- and time-dependent cell death in cultures of embryonic striatal neurons, causing both apoptosis and necrosis. Cell death was significantly reduced by the cell-permeant antioxidant manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP). Cyclosporin A and its analogue N-MeVal-4-cyclosporin also reduced the incidence of cell death, suggesting the participation of mitochondrial permeability transition in this process. Furthermore, addition of either of two types of caspase inhibitors, Ac-YVAD-CHO (acetyl-Tyr-Val-Ala-Asp-aldehyde) and Ac-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde), to the striatal cells blocked A23187-induced striatal cell death in a concentration-dependent manner. These results suggest that oxidative stress, opening of the mitochondrial permeability transition pore and activation of caspases are important steps in A23187-induced cell death.

Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro.

PURPOSE: To compare effects of calpain inhibitors on in vitro light-scattering in rat lens soluble protein and calcium-ionophore (A23187)-induced cataract formation in cultured rat lenses. METHODS: Rat lens soluble protein was hydrolyzed for 24 hours by activation of endogenous lens calpain. Ten calpain inhibitors were tested in this model at 10 and 25 microM concentration. As an index of protein precipitation, light scattering was measured daily at 405 nm for 8 days. Lens proteins were analyzed by isoelectric-focussing. Subsequently, rat lenses were cultured for 5 days with 10 microM A23187. Calpain inhibitors (SJA6017, MDL28170, AK295 and PD150606), which inhibited light-scattering were tested at 100 microM concentration in this model. Cataract evaluation, isoelectric-focussing and calcium determinations were performed. RESULTS: At 25 microM concentration AK295, SJA6017, E-64, PD-150606 and MDL28170 produced greater than 25% inhibition of light-scattering. Isoelectric-focussing revealed that addition of Ca(2+) produced characteristic crystallin proteolysis and aggregation patterns. AK295, SJA6017, MDL28170 and E64c prevented these changes. Lenses cultured in A23187 exhibited nuclear cataract, elevated calcium and proteolysis and aggregation of crystallins. Co-culture with SJA6017, MDL28170 and E64c reduced A23187-induced nuclear opacities, proteolysis and aggregation of crystallins without affecting increased total calcium. CONCLUSIONS: Endogenous calpain-activation model and A23187-induced cataract model can be used sequentially to screen calpain inhibitors for potential anti-cataract activity. Proteolytic changes in lens cortex after exposure to A23187 are also due to calpain activation. AK295, SJA6017 and MDL28170 possess efficacy against calcium-induced models of rodent cataracts. Use of calpain inhibitors represents a promising approach to cataract therapy.

The role of phospholipase A2 in calcium-ionophore-mediated injury to rat gastric mucosal cells.

Although transient increases in intracellular Ca2+ ([Ca2+]i) underlie a number of important physiological processes, sustained elevations in [Ca2+]i mediate damage to a number of tissues and cell types including gastric mucosal cells. Increases in [Ca2+]i can activate phospholipid hydrolysis via increases in phospholipase A2 (PLA2) activity and subsequent cell injury. In the present study we have examined whether [Ca2+]i-induced gastric cellular injury is mediated by PLA2 activation. Gastric mucosal cells were harvested from rat stomachs after pronase digestion. Cell integrity was assessed using trypan blue dye exclusion and release of lysozomal enzymes. PLA2 activity was estimated colorimetrically by determination of thiol release from the substrate, arachidonyl thio-PC. In these studies calcium ionophore A23187 (3-25 microM) resulted in an increase in cell injury. The damage produced by A23187 (12.5 microM) was inhibited by preincubation of cells with the PLA2 inhibitor, quinacrine (1-100 microM). Quinacrine did not reduce ethanol (10% w/v) mediated-cell damage. Similarly Ca2+ ionophore A23187 treatment resulted in a concentration-dependent increase in PLA2 activity in gastric cells. The increase in PLA2 activity was attenuated if cells were incubated in Ca2+-depleted medium containing EGTA (4 mM). Furthermore lysophospholipids generated by PLA2 (lysophosphatidylethanolamine and lysophosphatidylcholine; 100 microM) also increased the degree of cell injury. Pretreatment of cells with the PAF antagonist WEB 2086 (10(-6) and 10(-5) M), the leukotriene synthase inhibitor 5,6-dehydroarachidonic acid (10 microM), or the thromboxane synthase inhibitor furegrelate (1 microM) decrease A23187-mediated cell injury. These data suggest that Ca2+ ionophore-mediated increases in [Ca2+]i result in gastric cell injury and this effect is mediated in part by PLA2 activation and subsequent release of free fatty acids and lysophosphatides.