Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa.

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H2 O2 ) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H2 O2 , and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.

Effect of immunosuppressive drugs on cytokine production in canine whole blood stimulated with lipopolysaccharide or a combination of ionomycin and phorbol 12-myristate 13-acetate.

A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12-myristate 13-1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7-day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin-10 (IL-10), interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα) were significantly activated by PMA/I stimulation and IL-6, IL-10 and TNFα by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL-10, IFNγ and TNFα production was significantly reduced after stimulation with PMA/I compared to pre-treatment. After prednisone treatment, TNFα production was significantly reduced after stimulation with PMA/I or LPS compared to pre-treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

Apoptotic death in erythrocytes of lamprey Lampetra fluviatilis induced by ionomycin and tert-butyl hydroperoxide.

The work examined the effects of Ca2+ overload and oxidative damage on erythrocytes of river lamprey Lampetra fluvialtilis. The cells were incubated for 3h with 0.1-5µM Ca2+ ionophore ionomycin in combination with 2.5mM Ca2+ and 10-100µM pro-oxidant agent tert-butyl hydroperoxide (tBHP). The sensitivity of lamprey RBCs to studied compounds was evaluated by the kinetics of their death. Both toxicants induced dose- and time dependent phosphatidylserine (PS) externalization (annexin V-FITC labeling) and loss of membrane integrity (propidium iodide uptake). Highest doses of ionomycin (1-2µM) increased the number of PS-exposed erythrocytes to 7-9% within 3h, while 100µM tBHP produced up to 50% of annexin V-FITC-positive cells. Caspase inhibitor Boc-D-FMK (50µM), calpain inhibitor PD150606 (10µM) and broad protease inhibitor leupeptin (200µM) did not prevent ionomycin-induced PS externalization, whereas tBHP-triggered apoptosis was blunted by Boc-D-FMK. tBHP-dependent death of lamprey erythrocytes was accompanied by the decrease in relative cell size, loss of cell viability, activation of caspases 9 and 3/7, and loss of mitochondrial membrane potential, but all these processes were partially attenuated by Boc-D-FMK. None of examined death-associated events were observed in ionomycin-treated erythrocytes except activation of caspase-9. Incubation with ionomycin did not alter intracellular K+ and Na+ content, while exposure to tBHP resulted in 80% loss of K+ and 2.8-fold accumulation of Na+. Thus, lamprey erythrocytes appear to be more susceptible to oxidative damage. Ca2+ overload does not activate the cytosolic death pathways in these cells.

Lycopene inhibits reactive oxygen species production in SK-Hep-1 cells and attenuates acetaminophen-induced liver injury in C57BL/6 mice.

Our aim was to investigate the antioxidant potential of lycopene in different experimental liver models: in vitro, to evaluate the influence of lycopene on reactive oxygen species (ROS) production mediated by the PKC pathway and in vivo, to evaluate the protective effects of lycopene in an experimental model of hepatotoxicity. The in vitro study assessed the lycopene antioxidant potential by the quantification of ROS production in SK-Hep-1 cells unstimulated or stimulated by an activator of the PKC pathway. The role of NADPH oxidase was evaluated by measuring its inhibition potential using an inhibitor of this enzyme. In the in vivo study, male C57BL/6 mice received lycopene (10 or 100 mg/kg by oral gavage) and 1 h later, acetaminophen (APAP) (500 mg/kg) was administrated. Lycopene decreased ROS production in SK-Hep-1 cells through inhibition of NADPH oxidase, brought about in the PKC pathway. Lycopene improved hepatotoxicity acting as an antioxidant, reduced GSSG and regulated tGSH and CAT levels, reduced oxidative damage primarily by decreasing protein carbonylation, promoted the downregulation of MMP-2 and reduced areas of necrosis improving the general appearance of the lesion in C57BL/6 mice. Lycopene is a natural compound that was able to inhibit the production of ROS in vitro and mitigate the damage caused by APAP overdose in vivo.

Morphine Suppresses T helper Lymphocyte Differentiation to Th1 Type Through PI3K/AKT Pathway.

To investigate the effect of morphine on T helper lymphocyte differentiation and PI3K/AKT pathway mechanism, CD4+ lymphocytes were treated by phorbol-myristate-acetate (25 ng/ml) (PMA) plus ionomycin (1 µg/ml) in the presence of various concentrations of morphine (25, 50, 100, 200 ng/ml) for 4 h. Th1 and Th2 subsets, supernatant cytokines, and PI3K, AKT, and protein kinase C-theta (PKC-θ) levels were detected. The Th1 cell percentage, Th1-derived cytokines, and ratio of Th1/Th2 decreased in the presence of morphine in a concentration-dependent manner. However, Th2 cell percentage kept stable after morphine treatment. The phosphorylation of PI3K and AKT decreased, but the phosphorylation of PKC-θ did not change in the presence of morphine. The decreased percentage of Th1 cells and ratio of Th1/Th2 was recovered by naloxone concentration-dependently. Morphine can inhibit the differentiation of Th1 lymphocytes and decrease the ratio of Th1/Th2 via the pathway of PI3K/AKT. The effect can be inhibited by naloxone.

Environmental mold and mycotoxin exposures elicit specific cytokine and chemokine responses.

BACKGROUND: Molds can cause respiratory symptoms and asthma. We sought to use isolated peripheral blood mononuclear cells (PBMCs) to understand changes in cytokine and chemokine levels in response to mold and mycotoxin exposures and to link these levels with respiratory symptoms in humans. We did this by utilizing an ex vivo assay approach to differentiate mold-exposed patients and unexposed controls. While circulating plasma chemokine and cytokine levels from these two groups might be similar, we hypothesized that by challenging their isolated white blood cells with mold or mold extracts, we would see a differential chemokine and cytokine release. METHODS AND FINDINGS: Peripheral blood mononuclear cells (PBMCs) were isolated from blood from 33 patients with a history of mold exposures and from 17 controls. Cultured PBMCs were incubated with the most prominent Stachybotrys chartarum mycotoxin, satratoxin G, or with aqueous mold extract, ionomycin, or media, each with or without PMA. Additional PBMCs were exposed to spores of Aspergillus niger, Cladosporium herbarum and Penicillium chrysogenum. After 18 hours, cytokines and chemokines released into the culture medium were measured by multiplex assay. Clinical histories, physical examinations and pulmonary function tests were also conducted. After ex vivo PBMC exposures to molds or mycotoxins, the chemokine and cytokine profiles from patients with a history of mold exposure were significantly different from those of unexposed controls. In contrast, biomarker profiles from cells exposed to media alone showed no difference between the patients and controls. CONCLUSIONS: These findings demonstrate that chronic mold exposures induced changes in inflammatory and immune system responses to specific mold and mycotoxin challenges. These responses can differentiate mold-exposed patients from unexposed controls. This strategy may be a powerful approach to document immune system responsiveness to molds and other inflammation-inducing environmental agents.

New methymycin derivatives of Streptomyces venezuelae ATCC 15439 and their inhibitory effects on human T cell proliferation mediated by PMA/ionomycin.

Two new methymycin derivatives, 3'-demethylmethymycin (1) and 3'-demethyldeoxymethymycin (2), together with seven known ones (3-9), were obtained from the strain Streptomyces venezuelae ATCC 15439. Their structures were determined on the basis of IR, MS, 1D and 2D NMR data. In addition, the inhibitory effects of all the compounds on human T cell proliferation mediated by PMA/ionomycin were evaluated. The data suggested for the first time that methymycin derivatives have potential anti-inflammatory activity.

Involvement of calpain 2 in ionomycin-induced cell death in cultured mouse lens epithelial cells.

PURPOSE: Calpains are calcium-activated, intracellular, non-lysosomal, cysteine proteases that hydrolyze lens crystallins and cytoskeletal proteins. Elevated calcium is a frequent finding in both rodent and human cataracts, and calpain 2 is present in lenses of both species. Lens epithelium forms a critical barrier to influx of calcium, but the role of calpain 2 in lens epithelium is poorly characterized. Thus, the purpose of the present experiment was to determine the role of calpain 2 in lens epithelial cell death. METHODS: Mouse lens epithelial cells (α-TN4) were cultured with the calcium ionophore ionomycin to promote calcium influx. Release of LDH into the culture medium was measured as a general marker of cell death, while necrosis and apoptosis were detected by staining with ethidium homodimer III (EtD-III) or FITC-annexin V. Calpain activity was determined by zymography and immunoblotting for activation-associated, fragments of calpain. Breakdown products of calpain substrate α-spectrin were also detected by immunoblotting as additional markers of calpain activation. RESULTS: Calpain 2 was found to be the major calpain isozyme in α-TN4 cells. Ionomycin caused leakage of LDH into the medium, activation of calpain 2, proteolysis of α-spectrin, and changes in α-TN4 cell morphology and staining characteristic of necrotic cell death. Calpain inhibitor SNJ-1945 significantly inhibited these changes. CONCLUSIONS: The ability of mouse lens epithelium to maintain lens transparency would be compromised by activation of calpain 2 and associated necrotic cell death. Since calpain 2 is ubiquitously present in all animal lenses so far observed, the current results may predict the pathological consequences of calpain 2 activation in animal lenses including those of man.

Bcl-x(L) blocks a mitochondrial inner membrane channel and prevents Ca2+ overload-mediated cell death.

Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca(2+) is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-x(L) on Ca(2+) overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-x(L) neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca(2+)-mediated mitochondrial multiple conductance channel opening. Bcl-x(L) neuroprotective actions were antagonized by the Bcl-x(L) inhibitor ABT-737 and potentiated by the Ca(2+) chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death.

A Na+/Ca2+ exchanger isoform, NCX1, is involved in retinal cell death after N-methyl-D-aspartate injection and ischemia-reperfusion.

We investigated the expression of Na(+)/Ca(2+) exchanger (NCX) and the functional role of NCX in retinal damage by using NCX1-heterozygous deficient mice (NCX1(+/-)) and SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy] phenoxy]-5-ethoxyaniline), a selective NCX inhibitor in vivo. We also examined the role of NCX in oxygen-glucose deprivation (OGD) stress with a retinal ganglion cell line (RGC-5) cell culture in vitro. The expression of NCX1 was confirmed and entirely localized in retina by immunoblotting and immunohistochemistry, respectively. NCX1(+/-) mice possessed significant protection against retinal damage induced by intravitreal injection of N-methyl-D-aspartate (NMDA). SEA0400 at 3 and 10 mg/kg significantly reduced NMDA- or high intraocular pressure-induced retinal cell damage in mice. Furthermore, SEA0400 reduced the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells and the expression of phosphorylated mitogen-activated protein kinases (ERK1/2, JNK, p38) induced by NMDA injection. In RGC-5, SEA0400 at 0.3 and 1 microM significantly inhibited OGD-induced cell damage. OGD-induced cell damage was aggravated by ouabain (a Na(+),K(+)-ATPase inhibitor) at 100 microM, and this increased damage was significantly reduced by SEA0400 at 1 microM. In conclusion, these results suggest that NCX1 may play a role in retinal cell death induced by NMDA and ischemia-reperfusion.

Quantitative nitric oxide production by rat, bovine and porcine macrophages.

The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-gamma) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-gamma together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.

Enhancement of allergic responses in vivo and in vitro by butylated hydroxytoluene.

The effect of butylated hydroxytoluene (BHT), which is used widely as an antioxidant, on IgE-dependent allergic responses in vivo and in vitro was investigated. For in vivo study, passive cutaneous anaphylaxis (PCA) was elicited in rats by i.d. injection of anti-DNP IgE and 48 h later by i.v. injection of DNP-HSA. BHT was i.p. given immediately after anti-DNP IgE injection. For in vitro studies, the rat mast cell line RBL2H3 sensitized with monoclonal anti-dinitrophenol (DNP) IgE was challenged with the multivalent antigen DNP-human serum albumin (DNP-HSA) in the presence or absence of BHT. beta-Hexosaminidase and histamine released from RBL2H3 cells, as indicators of degranulation of the cells, the concentration of intracellular Ca2+, the level of phosphorylated-Akt, and global tyrosine phosphorylation as indicators of mast cell activation, were measured. The results showed that BHT given to anti-DNP IgE-sensitized rats augmented DNP-specific PCA in a dose-dependent manner. In the presence of BHT, IgE-induced releases of beta-hexosaminidase and histamine from RBL2H3 cells were increased. BHT also further elevated IgE-mediated increased concentrations of intracellular Ca2+ and the levels of phosphorylated-Akt, but did not affect global tyrosine phosphorylation, in RBL2H3 cells. Moreover, the PI3K inhibitor LY294002 inhibited IgE-dependent degranulation and its enhancement by BHT. These findings indicate that BHT may upregulate PCA by enhancing mast cell degranulation associated with enhancements of intracellular Ca2+ concentration and PI3K activation, suggesting that BHT might affect allergic diseases such as allergic rhinitis and asthma.

Mutation of the circadian clock gene Per2 alters vascular endothelial function.

BACKGROUND: The circadian clock regulates biological processes including cardiovascular function and metabolism. In the present study, we investigated the role of the circadian clock gene Period2 (Per2) in endothelial function in a mouse model. METHODS AND RESULTS: Compared with the wild-type littermates, mice with Per2 mutation exhibited impaired endothelium-dependent relaxations to acetylcholine in aortic rings suspended in organ chambers. During transition from the inactive to active phase, this response was further increased in the wild-type mice but further decreased in the Per2 mutants. The endothelial dysfunction in the Per2 mutants was also observed with ionomycin, which was improved by the cyclooxygenase inhibitor indomethacin. No changes in the expression of endothelial acetylcholine-M3 receptor or endothelial nitric oxide synthase protein but increased cyclooxygenase-1 (not cyclooxygenase-2) protein levels were observed in the aortas of the Per2 mutants. Compared with Per2 mutants, a greater endothelium-dependent relaxation to ATP was observed in the wild-type mice, which was reduced by indomethacin. In quiescent aortic rings, ATP caused greater endothelium-dependent contractions in the Per2 mutants than in the wild-type mice, contractions that were abolished by indomethacin. The endothelial dysfunction in the Per2 mutant mice is not associated with hypertension or dyslipidemia. CONCLUSIONS: Mutation in the Per2 gene in mice is associated with aortic endothelial dysfunction involving decreased production of NO and vasodilatory prostaglandin(s) and increased release of cyclooxygenase-1-derived vasoconstrictor(s). The results suggest an important role of the Per2 gene in maintenance of normal cardiovascular functions.

Neuroprotective effects of galanthamine and tacrine against glutamate neurotoxicity.

We examined the mechanisms of the neuroprotective effects of two central-type acetylcholinesterase inhibitors, galanthamine and tacrine, on nitric oxide-mediated glutamate neurotoxicity using primary cultures from the cerebral cortex of fetal rats. Galanthamine and tacrine showed prominent protective effects against glutamate neurotoxicity. Mecamylamine, a nicotinic acetylcholine receptor antagonist, but not scopolamine, a muscarinic acetylcholine receptor antagonist, inhibited the protective effects of these inhibitors on glutamate neurotoxicity. Furthermore, dihydro-beta-erythroidine, an alpha4-nicotinic receptor antagonist, and methyllycaconitine, an alpha7-nicotinic receptor antagonist, inhibited the neuroprotective effects of galanthamine but not tacrine. Next, we investigated the site of action where galanthamine and tacrine prevent glutamate neurotoxicity. Both these acetylcholinesterase inhibitors prevented glutamate- and ionomycin-induced neurotoxicity, but only tacrine prevented S-nitrosocysteine-induced neurotoxicity. These results suggest that galanthamine and tacrine protect cortical neurons from glutamate neurotoxicity via different mechanisms.

Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx.

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.

Oxidative stress inhibits ionomycin-mediated cell death in cortical neurons.

Thiol-proteases play important roles in many cellular processes, including maintenance of protein homeostasis and execution of cell death. Therefore, determining how this family of enzymes is regulated is critical for our understanding of both physiological and pathological conditions. Because these proteases require a reduced cysteine residue for activity, the cellular redox state plays a crucial role in regulating the activity of thiol proteases. Importantly, increased oxidative stress can result in the direct modification of the active site cysteine, leading to enzyme inactivation. This would suggest that oxidative stress that occurs during pathological insults could prolong cell survival by preventing the execution of thiol-protease-dependent cell death pathways. To test this hypothesis, cultured rat cortical neurons were treated with the oxidizing agent diamide or doxorubicin in the presence or absence of the calcium ionophore ionomycin. Under normoxic conditions, ionomycin treatment resulted in approximately 70% cell death, which was prevented by addition of the calpain-selective inhibitor benyzloxycarbonyl-Leu-Leu-Tyr fluoromethylketone. Similarly, pretreatment of neurons with either oxidant was also protective. Protection resulting from oxidative stress was not due to new protein synthesis, insofar as cycloheximide did not affect oxidant-mediated protection. Interestingly, treatment with the antioxidant Trolox to reverse or prevent oxidative stress blocked the protective effects of both oxidants against ionomycin-induced cell death. We interpret these findings to suggest that, in diseases or conditions in which oxidative stress is increased, the ability of thiol-proteases to execute cell death pathways fully is decreased and may prolong cell survival.

The neuronal calcium sensor protein VILIP-1 is associated with amyloid plaques and extracellular tangles in Alzheimer's disease and promotes cell death and tau phosphorylation in vitro: a link between calcium sensors and Alzheimer's disease?

To investigate whether the observed association of intracellular neuronal calcium sensor (NCS) proteins with amyloid plaques and neurofibrillar tangles in Alzheimer brains is linked to a possible neuroprotective or neurotoxic activity of the protein, we performed cytotoxicity tests in PC12 cells transfected with the calcium sensor protein VILIP-1 (visinin-like protein) and the calcium buffer protein calbindin-D28K. Whereas VILIP-1 expression enhanced the neurotoxic effect of ionomycin already at low ionophore concentrations, calbindin-D28K protected against ionomycin-induced cytotoxicity only at high ionomycin and therefore calcium concentrations. However, in double-transfected cells calbindin-D28K rescued VILIP-1-mediated cytotoxicity at low ionomycin concentrations. Since VILIP-1 was found to be associated with fibrillar tangles in Alzheimer brains, we tested whether VILIP-1 has an influence on tau hyperphosphorylation. VILIP-1 expression enhanced hyperphosphorylation of tau protein compared to nontransfected or calbindin-D28K-transfected cells. These results raise the possibility that the observed reduction in VILIP-1-expressing cells may indicate a selective vulnerability of these neurons and that the calcium sensor protein is involved in the pathophysiology of Alzheimer's disease. The calcium sensor protein may influence tau phosphorylation and have a role in calcium-mediated neurotoxicity opposed to the previously discovered protective effect of calcium buffer proteins.

Cutting edge: the class II transactivator prevents activation-induced cell death by inhibiting Fas ligand gene expression.

The Fas:Fas ligand pathway is critical in regulating immune homeostasis by eliminating activated T cells that proliferated in response to an infection. Here, we show that the MHC class II transactivator (CIITA) can suppress this pathway by inhibiting transcription of the Fas ligand gene. CIITA can effectively repress transcription from the Fas ligand promoter in both T cell lines as well as primary cells. The repression appears to be at least partly due to interference of NFAT-mediated induction of Fas ligand gene transcription. T cells that express CIITA constitutively do not up-regulate Fas ligand on the cell surface after activation via the TCR. Consequently, these cells lack the ability to undergo activation-induced cell death, and to kill Fas-bearing target cells.

Protective effect of bradykinin against glutamate neurotoxicity in cultured rat retinal neurons.

PURPOSE: To identify the localization and expression of bradykinin (BK)-B2 receptors in rat retina and examine the effects of BK on glutamate-induced neurotoxicity using cultured rat retinal neurons. METHODS: An immunohistochemical study using a specific antibody against BK-B2 receptor was performed with rat retina. Primary cultures were obtained from the retina of fetal rats (gestation day 17-19). Expression of BK-B2 receptor mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA obtained from cultured retinal neurons. Cultured cells were exposed to glutamate (1 mM) for 10 minutes and followed by incubation in glutamate-free medium for 1 hour. The effects of BK were assessed by simultaneous application of BK with glutamate. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method. RESULTS: Immunohistochemical study demonstrated that BK-B2 receptors were expressed in the ganglion cell, inner nuclear layers, and outer nuclear layers. Furthermore, BK-B2 receptor mRNA expression was observed in cultured retinal neurons. Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by a 1-hour incubation in glutamate-free medium. Simultaneous application of BK at concentrations of 0.001 to 1 microM with glutamate demonstrated dose-dependent protection against glutamate neurotoxicity. The protective action of BK (1 microM) was inhibited by simultaneous application of BK-B2 receptor antagonist, Hoe140 (1 microM). Furthermore, 1 microM BK had protective effects on neurotoxicity induced by 1 microM ionomycin, a calcium ionophore, and sodium nitroprusside (SNP, 500 microM), a nitric oxide (NO)-generating agent. However, BK did not inhibit neurotoxicity induced by 3-morpholinosydnonimine (SIN-1, 10 microM), an NO and oxygen radical donor. CONCLUSIONS: These results suggest that BK-B2 receptors were distributed in rat retinas and cultured retinal neurons and that BK had a protective action against glutamate neurotoxicity through BK-B2 receptors in cultured retinal neurons. It is suggested that BK-induced protection against glutamate neurotoxicity took place downstream to NO generation and upstream to oxygen radical generation.