Penehyclidine hydrochloride protects against anoxia/reoxygenation injury in cardiomyocytes through ATP‐sensitive potassium channels,and the Akt/GSK‐3β and Akt/mTOR signaling pathways

Penehyclidine hydrochloride (PHC) can protect against myocardial ischemia/reperfusion (I/R) injury. However, the possible mechanisms of PHC in anoxia/reoxygenation (A/R)‐induced injury in H9c2 cells remain unclear. In the present study, H9c2 cells were pretreated with PI3K/Akt inhibitor LY294002, ATP‐sensitive K⁺ (KATP) channel blocker 5‐hydroxydecanoate (5‐HD), PHC, or KATP channel opener diazoxide (DZ) before subjecting to A/R injury. Cell viability and cell apoptosis were determined by cell counting kit‐8 assay and annexin V/PI assay, respectively. Myocardial injury was evaluated by measuring creatine kinase (CK) and lactate dehydrogenase (LDH) activities. Intracellular Ca²⁺ levels, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨ_m), and mitochondrial permeability transition pore (mPTP) were measured. The levels of cytoplasmic/mitochondrial cytochrome c (Cyt‐C), Bax, Bcl‐2, cleaved caspase‐3, KATP channel subunits (Kir6.2 and SUR2A), and the members of the Akt/GSK‐3β and Akt/mTOR signaling pathways were determined by western blotting. We found that PHC preconditioning alleviated A/R‐induced cell injury by increasing cell viability, reducing CK and LDH activities, and inhibiting cell apoptosis. In addition, PHC preconditioning ameliorated intracellular Ca²⁺ overload and ROS production, accompanied by inhibition of both mPTP opening and Cyt‐C release into cytoplasm, and maintenance of ΔΨ_m. Moreover, PHC preconditioning activated mitochondrial KATP channels, and modulated the Akt/GSK‐3β and Akt/mTOR signaling pathways. Similar effects were observed upon treatment with DZ. Pretreatment with LY294002 or 5‐HD blocked the beneficial effects of PHC. These results suggest that the protective effects of PHC preconditioning on A/R injury may be related to mitochondrial KATP channels, as well as the Akt/GSK‐3β and Akt/mTOR signaling pathways.     

Inhibition of Rac1 reduces store overload‐induced calcium release and protects against ventricular arrhythmia

Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload‐induced Ca²⁺ release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1^f/f and cardiac‐specific Rac1 knockdown (Rac1^ckd) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1^f/f mice. Remarkably, I/R‐induced ventricular arrhythmia was significantly decreased in Rac1^ckd compared to Rac1^f/f mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R‐induced ventricular arrhythmia. Ca²⁺ imaging analysis showed that in response to a 6 mM external Ca²⁺ concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca²⁺ waves in Rac1^f/f cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R‐induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1^ckd mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease.     

Plant-originated glycoprotein (24 kDa) has an inhibitory effect on proliferation of BNL CL.2 cells in response to di(2-ethylhexyl)phthalate

Di(2‐ethylhexyl)phthalate (DEHP) is one of the many environmental chemicals that are widely used in polyvinyl chloride products, vinyl flooring, food packaging and infant toys. They cause cell proliferation or dysfunction of human liver. The purpose of this study is to investigate the inhibitory effect of a glycoprotein (24 kDa) isolated from Zanthoxylum piperitum DC (ZPDC) on proliferation of liver cell in the DEHP‐induced BNL CL. 2 cells. [³H]‐thymidine incorporation, intracellular reactive oxygen species (ROS), intracellular Ca²⁺ mobilization and activity of protein kinase C (PKC) were measured using radioactivity and fluorescence method respectively. The expression of mitogen‐activated protein kinases [extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK)], activator protein (AP)‐1 (c‐Jun and c‐Fos), proliferating cell nuclear antigen (PCNA) and cell cycle‐related factors (cyclin D1/cyclin‐dependent kinase [CDK] 4) were evaluated using Western blotting or electrophoretic mobility shift assay. The results in this study showed that the levels of [³H]‐thymidine incorporation, intracellular ROS, intracellular Ca²⁺ mobilization and activity of PKCα were inhibited by ZPDC glycoprotein (100 µg/ml) in the DEHP‐induced BNL CL. 2 cells. Also, activities of ERK, JNK and AP‐1 were reduced by ZPDC glycoprotein (100 µg/ml). With regard to cell proliferation, activities of PCNA and cyclin D1/CDK4 were significantly suppressed at treatment with ZPDC glycoprotein (100 µg/ml) in the presence of DEHP. Taken together, these findings suggest that ZPDC glycoprotein significantly normalized activities of PCNA and cyclin D1/CDK4, which relate to cell proliferation factors. Thus, ZPDC glycoprotein appears to be one of the compounds derived from natural products that are able to inhibit cell proliferation in the phthalate‐induced BNL CL. 2 cells. Copyright © 2011 John Wiley & Sons, Ltd.     

Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

     

Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

Acidic microenvironment is commonly observed in ischaemic tissue. In the kidney, extracellular pH dropped from 7.4 to 6.5 within 10 minutes initiation of ischaemia. Acid‐sensing ion channels (ASICs) can be activated by pH drops from 7.4 to 7.0 or lower and permeates to Ca²⁺entrance. Thus, activation of ASIC1a can mediate the intracellular Ca²⁺ accumulation and play crucial roles in apoptosis of cells. However, the role of ASICs in renal ischaemic injury is unclear. The aim of the present study was to test the hypothesis that ischaemia increases renal epithelia cell apoptosis through ASIC1a‐mediated calcium entry. The results show that ASIC1a distributed in the proximal tubule with higher level in the renal tubule ischaemic injury both in vivo and in vitro. In vivo, Injection of ASIC1a inhibitor PcTx‐1 previous to ischaemia/reperfusion (I/R) operation attenuated renal ischaemic injury. In vitro, HK‐2 cells were pre‐treated with PcTx‐1 before hypoxia, the intracellular concentration of Ca²⁺, mitochondrial transmembrane potential (?ψm) and apoptosis was measured. Blocking ASIC1a attenuated I/R induced Ca²⁺ overflow, loss of ?ψm and apoptosis in HK‐2 cells. The results revealed that ASIC1a localized in the proximal tubular and contributed to I/R induced kidney injury. Consequently, targeting the ASIC1a may prove to be a novel strategy for AKI patients.     

A tip-high,Ca<Superscript>2+</Superscript>-interdependent,reactive oxygen species gradient is associated with polarized growth in <Emphasis Type="Italic">Fucus serratus</Emphasis> zygotes

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2′,7′-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca²⁺]_cyt gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca²⁺]_cyt gradient. Such modulation of intracellular [Ca²⁺]_cyt signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.     

Mechanisms of Cigarette Smoke Effects on Human Airway Smooth Muscle

Cigarette smoke contributes to or exacerbates airway diseases such as asthma and COPD, where airway hyperresponsiveness and airway smooth muscle (ASM) proliferation are key features. While factors such as inflammation contribute to asthma in part by enhancing agonist-induced intracellular Ca²⁺ ([Ca²⁺]i) responses of ASM, the mechanisms by which cigarette smoke affect ASM are still under investigation. In the present study, we tested the hypothesis that cigarette smoke enhances the expression and function of Ca²⁺ regulatory proteins leading to increased store operated Ca²⁺ entry (SOCE) and cell proliferation. Using isolated human ASM (hASM) cells, incubated in the presence and absence cigarette smoke extract (CSE) we determined ([Ca²⁺]i) responses and expression of relevant proteins as well as ASM proliferation, reactive oxidant species (ROS) and cytokine generation. CSE enhanced [Ca²⁺]i responses to agonist and SOCE: effects mediated by increased expression of TRPC3, CD38, STIM1, and/or Orai1, evident by attenuation of CSE effects when siRNAs against these proteins were used, particularly Orai1. CSE also increased hASM ROS generation and cytokine secretion. In addition, we found in the airways of patients with long-term smoking history, TRPC3 and CD38 expression were significantly increased compared to life-long never-smokers, supporting the role of these proteins in smoking effects. Finally, CSE enhanced hASM proliferation, an effect confirmed by upregulation of PCNA and Cyclin E. These results support a critical role for Ca²⁺ regulatory proteins and enhanced SOCE to alter airway structure and function in smoking-related airway disease.     

Chemical Hypoxia-Induced Cell Death in Human Glioma Cells: Role of Reactive Oxygen Species,ATP Depletion,Mitochondrial Damage and Ca2+

This study was undertaken to evaluate whether chemical hypoxia-induced cell injury is a result of reactive oxygen species (ROS) generation, ATP depletion, mitochondrial permeability transition, and an increase in intracellular Ca²⁺, in A172 cells, a human glioma cell line. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport, in a glucose-free medium. Exposure of cells to chemical hypoxia resulted in cell death, ROS generation, ATP depletion, and mitochondrial permeability transition. The H₂O₂ scavenger pyruvate prevented cell death, ROS generation, and mitochondrial permeability transition induced by chemical hypoxia. In contrast, changes mediated by chemical hypoxia were not affected by hydroxyl radical scavengers. Antioxidants did not affect cell death and ATP depletion induced by chemical hypoxia, although they prevented ROS production and mitochondrial permeability transition induced by chemical hypoxia. Chemical hypoxia did not increase lipid peroxidation even when antimycin A was increased to 50 M, whereas the oxidant t-butylhydroperoxide caused a significant increase in lipid peroxidation, at a concentration that is less effective than chemical hypoxia in inducing cell death. Fructose protected against cell death and mitochondrial permeability transition induced by chemical hypoxia. However, ROS generation and ATP depletion were not prevented by fructose. Chemical hypoxia caused the early increase in intracellular Ca²⁺. The cell death and ROS generation induced by chemical hypoxia were altered by modulation of intracellular Ca²⁺ concentration with ruthenium red, TMB-8, and BAPTA/AM. However, mitochondrial permeability transition was not affected by these compounds. These results indicate that chemical hypoxia causes cell death, which may be, in part, mediated by H₂O₂ generation via a lipid peroxidation-independent mechanism and elevated intracellular Ca²⁺. In addition, these data suggest that chemical hypoxia-induced cell death is not associated directly with ATP depletion and mitochondrial permeability transition.     

ACE2‐EPC‐EXs protect ageing ECs against hypoxia/reoxygenation‐induced injury through the miR‐18a/Nox2/ROS pathway

Oxidative stress is one of the mechanisms of ageing‐associated vascular dysfunction. Angiotensin‐converting enzyme 2 (ACE2) and microRNA (miR)‐18a have shown to be down‐regulated in ageing cells. Our previous study has shown that ACE2‐primed endothelial progenitor cells (ACE2‐EPCs) have protective effects on endothelial cells (ECs), which might be due to their released exosomes (EXs). Here, we aimed to investigate whether ACE2‐EPC‐EXs could attenuate hypoxia/reoxygenation (H/R)‐induced injury in ageing ECs through their carried miR‐18a. Young and angiotensin II‐induced ageing ECs were subjected to H/R and co‐cultured with vehicle (medium), EPC‐EXs, ACE2‐EPCs‐EXs, ACE2‐EPCs‐EXs + DX600 or ACE2‐EPCs‐EXs with miR‐18a deficiency (ACE2‐EPCs‐EXs^{anti‐miR‐18a}). Results showed (1) ageing ECs displayed increased senescence, apoptosis and ROS production, but decreased ACE2 and miR‐18a expressions and tube formation ability; (2) under H/R condition, ageing ECs showed higher rate of apoptosis, ROS overproduction and nitric oxide reduction, up‐regulation of Nox2, down‐regulation of ACE2, miR‐18a and eNOS, and compromised tube formation ability; (3) compared with EPC‐EXs, ACE2‐EPC‐EXs had better efficiencies on protecting ECs from H/R‐induced changes; (4) The protective effects were less seen in ACE2‐EPCs‐EXs + DX600 and ACE2‐EPCs‐EXs^{anti‐miR‐18a} groups. These data suggest that ACE‐EPCs‐EXs have better protective effects on H/R injury in ageing ECs which could be through their carried miR‐18a and subsequently down‐regulating the Nox2/ROS pathway.     

α1‐adrenergic stimulation mediates Ca2+‐dependent inositol phosphate formation through the α1B‐like adrenoceptor subtype in adult rat cardiac myocytes

We studied the effects of increased Ca²⁺ influx on α₁‐adrenoceptor‐stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific α₁‐adrenoceptor subtype. [³H]InsP responses to adrenaline were dependent on extracellular Ca²⁺ concentration, from 0.1 μM to 2 mM, and were completely blocked by Ca²⁺ removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca²⁺ concentrations higher than 1 μM had no effect on adrenaline‐stimulated [³H]InsP formation. Taken together these results suggest that [³H]InsP formation induced by α₁‐adrenergic stimulation is in part mediated by increased Ca²⁺ influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [³H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the α_1B‐adrenoceptor alkylating agent, CEC, [³H]InsP formation remained unaffected by increased Ca²⁺ concentrations, a pattern similar to that observed when intracellular Ca²⁺ was chelated with BAPTA. In contrast, addition of the α_1A‐subtype antagonist, 5′‐methyl urapidil, did not affect the Ca²⁺ dependence of [³H]InsP formation. Neither nifedipine, a voltage‐dependent Ca²⁺ channel blocker nor the inorganic Ca²⁺ channel blockers, Ni²⁺ and Co²⁺, had any effect on adrenaline stimulated [³H]InsP, at concentrations that inhibit Ca²⁺ channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein‐mediated response, α₁‐adrenergic‐stimulated [³H]InsP formation is activated by increased Ca²⁺ influx mediated by the α_1B‐subtype. J. Cell. Biochem. 84: 201–210, 2002. © 2001 Wiley‐Liss, Inc.     

Role of LOX‐1 in monocyte adhesion‐triggered redox,Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) in monocyte adhesion‐induced redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in endothelial cells (ECs). LOX‐1 was blocked by an antibody‐neutralizing LOX‐1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47^phox, and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF‐κB phosphorylation within 1 h, resulting in redox‐sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX‐1 blunted the monocyte adhesion‐triggered redox‐sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca²⁺ mobilization and Ca²⁺ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX‐1 is involved in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of oxidized low‐density lipoprotein (ox‐LDL). Furthermore, blockade of Ca²⁺ inhibited monocyte adhesion‐triggered Rac1 and p47^phox activation and ROS generation in ECs, whereas Ca²⁺ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor‐α or ox‐LDL. We provide evidence that LOX‐1 plays a role in redox‐sensitive, Akt/eNOS and Ca²⁺ signaling pathways in monocyte adhesion to ECs independent of the ox‐LDL–LOX‐1 axis. J. Cell. Physiol. 220: 706–715, 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Erythropoietin Protects Cardiomyocytes from Cell Death during Hypoxia/Reperfusion Injury through Activation of Survival Signaling Pathways

Hypoxia/Reoxygenation (H/R) cardiac injury is of great importance in understanding Myocardial Infarctions, which affect a major part of the working population causing debilitating side effects and often-premature mortality. H/R injury primarily consists of apoptotic and necrotic death of cardiomyocytes due to a compromise in the integrity of the mitochondrial membrane. Major factors associated in the deregulation of the membrane include fluctuating reactive oxygen species (ROS), deregulation of mitochondrial permeability transport pore (MPTP), uncontrolled calcium (Ca²⁺) fluxes, and abnormal caspase-3 activity. Erythropoietin (EPO) is strongly inferred to be cardioprotective and acts by inhibiting the above-mentioned processes. Surprisingly, the underlying mechanism of EPO''s action and H/R injury is yet to be fully investigated and elucidated. This study examined whether EPO maintains Ca²⁺ homeostasis and the mitochondrial membrane potential (ΔΨ_m) in cardiomyocytes when subjected to H/R injury and further explored the underlying mechanisms involved. H9C2 cells were exposed to different concentrations of EPO post-H/R, and 20 U/ml EPO was found to significantly increase cell viability by inhibiting the intracellular production of ROS and caspase-3 activity. The protective effect of EPO was abolished when H/R-induced H9C2 cells were treated with Wortmannin, an inhibitor of Akt, suggesting the mechanism of action through the activation Akt, a major survival pathway.     

Native LDL‐induced oxidative stress in human proximal tubular cells: multiple players involved

Dyslipidemia is a well‐established condition proved to accelerate the progression of chronic kidney disease leading to tubulo‐interstitial injury. However, the molecular aspects of the dyslipidemia‐induced renal damage have not been fully clarified and in particular the role played by low‐density lipoproteins (LDLs). This study aimed to examine the effects of native non‐oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS‐induced ROS release mechanism. The LDL‐dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca²⁺ uptake and loss of cytochrome c. All the above LDL‐induced effects were completely abrogated by chelating extracellular Ca²⁺ as well as by inhibition of the Ca²⁺‐activated cytoplas‐mic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL‐induced intracellular redox unbalance is triggered by a Ca²⁺ inward flux‐dependent commencement of cPLA2 followed by activation of a lipid‐ and ROS‐based cross‐talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox‐ and Ca²⁺‐dependent dysfunctions leading to cell‐harming conditions. These findings may help to clarify the mechanism of dyslipidemia‐induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

CD38 promotes angiotensin II‐induced cardiac hypertrophy

Cardiac hypertrophy is an early hallmark during the clinical course of heart failure and regulated by various signalling pathways. Recently, we observed that mouse embryonic fibroblasts from CD38 knockout mice were significantly resistant to oxidative stress such as H₂O₂‐induced injury and hypoxia/reoxygenation‐induced injury. In addition, we also found that CD38 knockout mice protected heart from ischaemia reperfusion injury through activating SIRT1/FOXOs‐mediated antioxidative stress pathway. However, the role of CD38 in cardiac hypertrophy is not explored. Here, we investigated the roles and mechanisms of CD38 in angiotensin II (Ang‐II)‐induced cardiac hypertrophy. Following 14 days of Ang‐II infusion with osmotic mini‐pumps, a comparable hypertension was generated in both of CD38 knockout and wild‐type mice. However, the cardiac hypertrophy and fibrosis were much more severe in wild‐type mice compared with CD38 knockout mice. Consistently, RNAi‐induced knockdown of CD38 decreased the gene expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and reactive oxygen species generation in Ang‐II‐stimulated H9c2 cells. In addition, the expression of SIRT3 was elevated in CD38 knockdown H9c2 cells, in which SIRT3 may further activate the FOXO3 antioxidant pathway. The intracellular Ca²⁺ release induced by Ang‐II markedly decreased in CD38 knockdown H9c2 cells, which might be associated with the decrease of nuclear translocation of NFATc4 and inhibition of ERK/AKT phosphorylation. We concluded that CD38 plays an essential role in cardiac hypertrophy probably via inhibition of SIRT3 expression and activation of Ca²⁺‐NFAT signalling pathway. Thus, CD38 may be a novel target for treating cardiac hypertrophy.     

Putative roles of Ca2+‐independent phospholipase A2 in respiratory chain‐associated ROS production in brain mitochondria: influence of docosahexaenoic acid and bromoenol lactone

Ca²⁺‐independent phospholipase A₂ (iPLA₂) is hypothesized to control mitochondrial reactive oxygen species (ROS) generation. Here, we modulated the influence of iPLA₂‐induced liberation of non‐esterified free fatty acids on ROS generation associated with the electron transport chain. We demonstrate enzymatic activity of membrane‐associated iPLA₂ in native, energized rat brain mitochondria (RBM). Theoretically, enhanced liberation of free fatty acids by iPLA₂ modulates mitochondrial ROS generation, either attenuating the reversed electron transport (RET) or deregulating the forward electron transport of electron transport chain. For mimicking such conditions, we probed the effect of docosahexaenoic acid (DHA), a major iPLA₂ product on ROS generation. We demonstrate that the adenine nucleotide translocase partly mediates DHA‐induced uncoupling, and that low micromolar DHA concentrations diminish RET‐dependent ROS generation. Uncoupling proteins have no effect, but the adenine nucleotide translocase inhibitor carboxyatractyloside attenuates DHA‐linked uncoupling effect on RET‐dependent ROS generation. Under physiological conditions of forward electron transport, low micromolar DHA stimulates ROS generation. Finally, exposure of RBM to the iPLA₂ inhibitor bromoenol lactone (BEL) enhanced ROS generation. BEL diminished RBM glutathione content. BEL‐treated RBM exhibits reduced Ca²⁺ retention capacity and partial depolarization. Thus, we rebut the view that iPLA₂ attenuates oxidative stress in brain mitochondria. However, the iPLA₂ inhibitor BEL has detrimental activities on energy‐dependent mitochondrial functions.

     

Cardiac‐specific overexpression of metallothionein attenuates L‐NAME‐induced myocardial contractile anomalies and apoptosis

Hypertension contributes to the high cardiac morbidity and mortality. Although oxidative stress plays an essential role in hypertensive heart diseases, the mechanism remains elusive. Transgenic mice with cardiac overexpression of metallothionein, a heavy metal‐binding scavenger, were challenged with N^G‐nitro‐L‐arginine methyl ester (L‐NAME) for 14 days prior to measurement of myocardial contractile and intracellular Ca²⁺ anomalies as well as cell signalling mechanisms using Western blot and immunofluorescence analysis. L‐NAME challenge elicited hypertension, macrophage infiltration, oxidative stress, inflammation and cardiac dysfunction manifested as increased proinflammatory macrophage marker F4/80, interleukin‐1β (IL‐1β), intracellular production, LV end systolic and diastolic diameters as well as depressed fractional shortening. L‐NAME treatment reduced mitochondrial membrane potential (MMP), impaired cardiomyocyte contractile and intracellular Ca²⁺ properties as evidenced by suppressed peak shortening, maximal velocity of shortening/relengthening, rise in intracellular Ca²⁺, along with elevated baseline and peak intracellular Ca²⁺. These unfavourable mechanical changes and decreased MMP (except blood pressure and macrophage infiltration) were alleviated by overexpression of metallothionein. Furthermore, the apoptosis markers including BAD, Bax, Caspase 9, Caspase 12 and cleaved Caspase 3 were up‐regulated while the anti‐apoptotic marker Bcl‐2 was decreased by L‐NAME treatment. Metallothionein transgene reversed L‐NAME‐induced changes in Bax, Bcl‐2, BAD phosphorylation, Caspase 9, Caspase 12 and cleaved Caspase 3. Our results suggest that metallothionein protects against L‐NAME‐induced myocardial contractile anomalies in part through inhibition of apoptosis.     

PPARδ activation inhibits angiotensin II induced cardiomyocyte hypertrophy by suppressing intracellular Ca2+ signaling pathway

Peroxisome proliferator‐activated receptors δ (PPARδ) is known to be expressed ubiquitously, and the predominant PPAR subtype of cardiac cells. However, relatively less is known regarding the role of PPARδ in cardiac cells except that PPARδ ligand treatment protects cardiac hypertrophy by inhibiting NF‐κB activation. Thus, in the present study, we examined the effect of selective PPARδ ligand L‐165041 on angiotensin II (AngII) induced cardiac hypertrophy and its underlying mechanism using cardiomyocyte. According to our data, L‐165041 (10 µM) inhibited AngII‐induced [³H] leucine incorporation, induction of the fetal gene atrial natriuretic factor (ANF) and increase of cardiomyocyte size. Previous studies have implicated the activation of focal adhesion kinase (FAK) in the progress of cardiomyocyte hypertrophy. L‐165041 pretreatment significantly inhibited AngII‐induced intracellular Ca²⁺ increase and subsequent phosphorylation of FAK. Further experiment using Ca²⁺ ionophore A23187 confirmed that Ca²⁺ induced FAK phosphorylation, and this was also blocked by L‐165041 pretreatment. In addition, overexpression of PPARδ using adenovirus significantly inhibited AngII‐induced intracellular Ca²⁺ increase and FAK expression, while PPARδ siRNA treatment abolished the effect of L‐165041. These data indicate that PPARδ ligand L‐165041 inhibits AngII induced cardiac hypertrophy by suppressing intracellular Ca²⁺/FAK/ERK signaling pathway in a PPARδ dependent mechanism. J. Cell. Biochem. 106: 823–834, 2009. © 2009 Wiley‐Liss, Inc.     

Tyrosine phosphorylation modulates store‐operated calcium entry in cultured rat epididymal basal cells

Store‐operated calcium entry (SOCE) is essential for many cellular processes. In this study, we investigated modulation of SOCE by tyrosine phosphorylation in rat epididymal basal cells. The intracellular Ca²⁺([Ca²⁺]i) measurement showed that SOCE occurred in rat epididymal basal cells by pretreating the cells with thapsigargin (Tg), the inhibitor of sarco‐endoplasmic reticulum Ca²⁺‐ATPase. To identify the role of Ca²⁺ channels in this response, we examined the effects of transient receptor potential canonical channel blockers 2‐aminoethoxydiphenyl borate (2‐APB), 1‐[β‐[3‐(4‐methoxyphenyl)pro‐poxy]‐4‐methoxyphenethyl]‐1H‐imidazole hydrochloride(SKF96365), Gd³⁺, and non‐selective cation channel blocker Ni²⁺ respectively on SOCE and found that these blockers could inhibit the Ca²⁺ influx to different extent. Furthermore, we studied the regulation of SOCE by tyrosine kinase pathway. The inhibitor of tyrosine kinase genistein remarkably suppressed the SOCE response, whereas sodium orthovanadate, the inhibitor of tyrosine phosphatase, greatly enhanced it. The results suggest that tyrosine kinase pathway plays a significant role in the initiation of SOCE and positively modulates SOCE in epididymal basal cells. J. Cell. Physiol. 226: 1069–1073, 2011. © 2010 Wiley‐Liss, Inc.