LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H₂DCF) but had no direct impact on the H₂O₂ production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H₂O₂ generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q_o site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na⁺ or K⁺ excluding a role for putative mitochondrial K_ATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes

Background

Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential.

Conclusions

Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function.

Methods

To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration.

Results

AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10–30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration.

General significance

These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.     

P2X7 receptor antagonists display agonist-like effects on cell signaling proteins

Background

The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca²⁺]_i. We report that some agents that can block P2X₇R receptors also promote diverse P2X₇R-independent effects on cell signaling.

Methods

We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X₇R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins.

Results

With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated ⁴⁵Ca²⁺ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X₇R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation.

Conclusions

Several agents used as P2X₇R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists.

General significance

Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.     

Role of estrogen receptors in pro-oxidative and anti-oxidative actions of estrogens: A perspective

Background

Estrogens are steroid hormones responsible for the primary and secondary sexual characteristics in females. While pre-menopausal women use estrogens as the main constituents of contraceptive pills, post-menopausal women use the same for Hormone Replacement Therapy. Estrogens produce reactive oxygen species by increasing mitochondrial activity and redox cycling of estrogen metabolites. The phenolic hydroxyl group present at the C3 position of the A ring of estrogens can get oxidized either by accepting an electron or by losing a proton. Thus, estrogens might act as pro-oxidant in some settings, resulting in complicated non-communicable diseases, namely, cancer and cardiovascular disorders. However, in some other settings the phenolic hydroxyl group of estrogens may be responsible for the anti-oxidative beneficial functions and thus protect against cardiovascular and neurodegenerative diseases.

Scope of review

To date, no single review article has mentioned the implication of estrogen receptors in both the pro-oxidative and anti-oxidative actions of estrogens.

Major conclusion

The controversial role of estrogens as pro-oxidant or anti-oxidant is largely dependent on cell types, ratio of different types of estrogen receptors present in a particular cell and context specificity of the estrogen hormone responses. Both pro-oxidant and anti-oxidant effects of estrogens might involve different estrogen receptors that can have either genomic or non-genomic action to manifest further hormonal response.

General significance

This review highlights the role of estrogen receptors in the pro-oxidative and anti-oxidative actions of estrogens with special emphasis on neuronal cells.     

Role of AMPK-mediated adaptive responses in human cells with mitochondrial dysfunction to oxidative stress

Background

Mitochondrial DNA (mtDNA) mutations are an important cause of mitochondrial diseases, for which there is no effective treatment due to complex pathophysiology. It has been suggested that mitochondrial dysfunction-elicited reactive oxygen species (ROS) plays a vital role in the pathogenesis of mitochondrial diseases, and the expression levels of several clusters of genes are altered in response to the elevated oxidative stress. Recently, we reported that glycolysis in affected cells with mitochondrial dysfunction is upregulated by AMP-activated protein kinase (AMPK), and such an adaptive response of metabolic reprogramming plays an important role in the pathophysiology of mitochondrial diseases.

Scope of review

We summarize recent findings regarding the role of AMPK-mediated signaling pathways that are involved in: (1) metabolic reprogramming, (2) alteration of cellular redox status and antioxidant enzyme expression, (3) mitochondrial biogenesis, and (4) autophagy, a master regulator of mitochondrial quality control in skin fibroblasts from patients with mitochondrial diseases.

Major conclusion

Induction of adaptive responses via AMPK–PFK2, AMPK–FOXO3a, AMPK–PGC-1α, and AMPK–mTOR signaling pathways, respectively is modulated for the survival of human cells under oxidative stress induced by mitochondrial dysfunction. We suggest that AMPK may be a potential target for the development of therapeutic agents for the treatment of mitochondrial diseases.

General significance

Elucidation of the adaptive mechanism involved in AMPK activation cascades would lead us to gain a deeper insight into the crosstalk between mitochondria and the nucleus in affected tissue cells from patients with mitochondrial diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.

Methods

NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.

Results

Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.

Conclusions

These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.

General Significance

Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.     

PEP-1-PEA-15 protects against toxin-induced neuronal damage in a mouse model of Parkinson's disease

Background

PEA-15 is abundantly expressed in both neurons and astrocytes throughout the brain. It is a multifunctional protein with the ability to increase cell survival via anti-apoptotic and anti-proliferative properties. However, the function of PEA-15 in neuronal diseases such as Parkinson's disease (PD) remains unclear. In this study, we investigated the protective effects of PEA-15 on neuronal damage induced by MPP⁺ in neuroblastoma SH-SY5Y and BV2 microglia cells and in a MPTP-induced PD mouse model using cell-permeable PEP-1-PEA-15.

Methods

PEP-1-PEA-15 was purified using affinity chromatography. Cell viability and DNA fragmentation were examined by MTT assay and TUNEL staining. Dopaminergic neuronal cell death in the animal model was examined by immunohistochemistry.

Results

PEP-1-PEA-15 transduced into the SH-SY5Y and BV2 cells in a time- and dose-dependent manner. Transduced PEP-1-PEA-15 protected against MPP⁺-induced toxicity by inhibiting intracellular ROS levels and DNA fragmentation. Further, it enhanced the expression levels of Bcl-2 and caspase-3 while reducing the expression levels of Bax and cleaved caspase-3. We found that PEP-1-PEA-15 transduced into the substantia nigra and prevented dopaminergic neuronal cell death in a MPTP-induced PD mouse. Also, we showed the neuroprotective effects in the model by demonstrating that treatment with PEP-1-PEA-15 ameliorated MPTP-induced behavioral dysfunctions and increased dopamine levels in the striatum.

Conclusions

PEP-1-PEA-15 can efficiently transduce into cells and protects against neurotoxin-induced neuronal cell death in vitro and in vivo.

General significance

These results demonstrate the potential for PEP-1-PEA-15 to provide a new strategy for protein therapy treatment of a variety of neurodegenerative diseases including PD.     

The role of mitochondria in the aetiology of insulin resistance and type 2 diabetes

Background

The prevalence of type 2 diabetes is rapidly increasing world-wide and insulin resistance is central to the aetiology of this disease. The biology underpinning the development of insulin resistance is not completely understood and the role of impaired mitochondrial function in the development of insulin resistance is controversial.

Scope of review

This review will provide an overview of the major processes regulated by mitochondria, before examining the evidence that has investigated the relationship between mitochondrial function and insulin action. Further considerations aimed at clarifying some controversies surrounding this issue will also be proposed.

Major conclusions

Controversy on this issue is fuelled by our lack of understanding of some of the basic biological interactions between mitochondria and insulin regulated processes in the context of insults thought to induce insulin resistance. Aspects that have not yet been considered are tissue/cell type specific responses, mitochondrial responses to site-specific impairments in mitochondrial function and as yet uncharacterised retrograde signalling from mitochondria.

General significance

Further investigation of the relationship between mitochondria and insulin action could reveal novel mechanisms contributing to insulin resistance in specific patient subsets. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways

Background

Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established.

Scope of Review

EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells.

Major Conclusions

Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways.

General significance

These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

Stimulation of cytosolic and mitochondrial calcium mobilization by indomethacin in Caco-2 cells: Modulation by the polyphenols quercetin,resveratrol and rutin

Background

The effect of indomethacin (INDO) on Ca^2 + mobilization, cytotoxicity, apoptosis and caspase activation and the potential protective effect of quercetin (QUE), resveratrol (RES) and rutin (RUT) were determined in Caco-2 cells.

Methods

Caco-2 cells were incubated with INDO in the presence or absence of QUE, RES or RUT. The concentrations of Ca^2 + in the cytosol (Fluo-3 AM) and mitochondria (Rhod-2 AM) were determined as well as the cytotoxicity (MTT reduction and LDH leakage), apoptosis (TUNEL) and caspase-3 and 9 activities.

Results

INDO promoted Ca^2 + efflux from the endoplasmic reticulum (ER), resulting in an early, but transient, increment of cytosolic Ca^2 + at 3.5 min, followed by a subsequent increment of intra-mitochondrial Ca^2 + at 24 min. INDO also induced cytotoxicity, apoptosis, and increased caspase activities and cytochrome c release. All these alterations were prevented by the inhibitors of the IP3R and RyR receptors, 2-Aminoethoxydiphenyl borate (2-APB) and dantrolene. QUE was the most efficient polyphenol in preventing Ca^2 + mobilization induced by INDO and all of its consequences including cytotoxicity and apoptosis.

Conclusions

In Caco-2 cells, INDO stimulates ER Ca^2 + mobilization, probably through the activation of IP3R and RyR receptors, and the subsequent entry of Ca^2 + into the mitochondria. Polyphenols protected the cells against the Ca^2 + mobilization induced by INDO and its consequences on cytotoxicity and apoptosis.

General significance

These results confirm the possibility of using polyphenols and particularly QUE for the protection of the gastroduodenal mucosa in subjects consuming NSAIDs.     

Stimulation of σ1-receptor restores abnormal mitochondrial Ca mobilization and ATP production following cardiac hypertrophy

Background

We previously reported that the σ₁-receptor (σ₁R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ₁R stimulation with the selective σ₁R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ₁R localized in the sarcoplasmic reticulum (SR).

Methods

We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ₁R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ₁R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).

Results

σ₁R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca^2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca^2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.

Conclusions

σ₁R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca^2 + mobilization and ATP production via σ₁R stimulation.

General significance

Our observations suggest that σ₁R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction.     

Changes in iron-regulatory gene expression occur in human cell culture models of Parkinson's disease

Background

Neuronal iron accumulation is thought to be relevant to the pathogenesis of Parkinson’s disease (PD), although the mechanism remains elusive. We hypothesized that neuronal iron uptake may be stimulated by functional mitochondrial iron deficiency.

Objective

To determine firstly whether the mitochondrial toxin, 1-methyl-4-phenylpyridinium iodide (MPP⁺), results in upregulation of iron-import proteins and transporters of iron into the mitochondria, and secondly whether similar changes in expression are induced by toxins with different mechanisms of action.

Methods

We used quantitative PCR and Western blotting to investigate expression of the iron importers, divalent metal transporter, transferrin receptor 1 and 2 (TfR1 and TfR2) and mitoferrin-2 and the iron exporter ferroportin in differentiated SH-SY5Y cells exposed to three different toxins relevant to PD, MPP⁺, paraquat (a free radical generator) and lactacystin (an inhibitor of the ubiquitin-proteasome system (UPS)).

Results

MPP⁺ resulted in increased mRNA and protein levels of genes involved in cellular iron import and transport into the mitochondria. Similar changes occurred following exposure to paraquat, another inducer of oxidative stress. Lactacystin also resulted in increased TfR1 mRNA levels, although the other changes were not found.

Conclusion

Our results support the hypothesis of a functional mitochondrial iron deficit driving neuronal iron uptake but also suggest that differences exist in neuronal iron handling induced by different toxins.     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.