Perturbation of Akt Signaling, Mitochondrial Potential, and ADP/ATP Ratio in Acidosis-Challenged Rat Cortical Astrocytes.

Cells switch to anaerobic glycolysis when there is a lack of oxygen during brain ischemia. Extracellular pH thus drops and such acidosis causes neuronal cell death. The fate of astrocytes, mechanical, and functional partners of neurons, in acidosis is less studied. In this report, we investigated the signaling in acidosis-challenged rat cortical astrocytes and whether these signals were related to mitochondrial dysfunction and cell death. Exposure to acidic pH (6.8, 6.0) caused Ca2+ release and influx, p38 MAPK activation, and Akt inhibition. Mitochondrial membrane potential was hyperpolarized after astrocytes were exposed to acidic pH as soon as 1 h and lasted for 24 h. Such mitochondrial hyperpolarization was prevented by SC79 (an Akt activator) but not by SB203580 (a p38 inhibitor) nor by cytosolic Ca2+ chelation by BAPTA, suggesting that only the perturbation in Akt signaling was causally related to mitochondrial hyperpolarization. SC79, SB203580, and BAPTA did not prevent acidic pH-induced cell death. Acidic pH suppressed ROS production, thus ruling out the role of ROS in cytotoxicity. Interestingly, pH 6.8 caused an increase in ADP/ATP ratio and apoptosis; pH 6.0 caused a further increase in ADP/ATP ratio and necrosis. Therefore, astrocyte cell death in acidosis did not result from mitochondrial potential collapse; in case of acidosis at pH 6.0, necrosis might partly result from mitochondrial hyperpolarization and subsequent suppressed ATP production. J. Cell. Biochem. 118: 1108-1117, 2017. © 2016 Wiley Periodicals, Inc.

Parthenolide-Induced Cytotoxicity in H9c2 Cardiomyoblasts Involves Oxidative Stress.

BACKGROUND: Cardiac cellular injury as a consequence of ischemia and reperfusion involves nuclear factor-κB (NF-κ B), amongst other factors, and NF-κ B inhibitors could substantially reduce myocardial infarct size. Parthenolide, a sesquiterpene lactone compound which could inhibit NF-κ B, has been shown to ameliorate myocardial reperfusion injury but may also produce toxic effects in cardiomyocytes at high concentrations. The aim of this study was to examine the cytotoxic effects of this drug on H9c2 cardiomyoblasts, which are precursor cells of cardiomyocytes. METHODS: Cell viability and apoptosis were examined by MTT and TUNEL assay, respectively, and protein expression was analyzed by western blot. Reactive oxygen species (ROS) production was measured using DCFH-DA as dye. Cytosolic Ca(2+) concentration and mitochondrial membrane potential were measured microfluorimetrically using, respectively, fura 2 and rhodamine 123 as dyes. RESULTS: Parthenolide caused apoptosis at 30 µ M, as judged by TUNEL assay and Bax and cytochrome c translocation. It also caused collapse of mitochondrial membrane potential and endoplasmic reticulum stress. Parthenolide triggered ROS formation, and vitamin C (antioxidant) partially alleviated parthenolide-induced cell death. CONCLUSIONS: The results suggested that parthenolide at high concentrations caused cytotoxicity in cardiomyoblasts in part by inducing oxidative stress, and demonstrated the imperative for cautious and appropriate use of this agent in cardioprotection. KEY WORDS: Cardiomyoblast; Endoplasmic reticulum stress; Oxidative stress; Parthenolide; Reperfusion injury.

Protein carbonylation in THP-1 cells induced by cigarette smoke extract via a copper-catalyzed pathway.

Cigarette smoke is a mixture of chemicals that cause direct or indirect oxidative stress in different cell lines. We investigated the effect of nonfractionated cigarette smoke extract (CSE) on protein carbonylation in human THP-1 cells. Cells were exposed to various concentrations (2.5-20%) of CSE for 30 min, and protein carbonylation was assessed by use of the sensitive 2,4-dinitrophenylhydrazine immuno-dot blot assay. CSE-induced protein carbonylation exhibited a dose-response relation with CSE concentrations. However, with prolonged exposure to CSE, significant decrements were observed when compared with the 30 min exposure. Cotreatment of THP-1 cells with antioxidants (N-acetyl-cysteine, S-allyl-cysteine, and alpha-tocopherol) and copper(II) ion chelators (d-penicillamine) during CSE exposure significantly reduced protein carbonylation, whereas cotreatment with antioxidants (vitamin C and trolox) and a metal chelator (EDTA), iron chelator (1,10-phenanthroline), or copper(I) chelator (neocuprin) did not decrease CSE-induced protein carbonylation in THP-1 cells. These results suggest that protein carbonylation is induced by CSE in THP-1 cells via a copper(II)-catalyzed reaction and not an iron-catalyzed reaction. Furthermore, the copper(II) ions involved in this CSE-induced protein carbonylation are derived from the intracellular pool, not via uptake from the extracellular medium. We speculate that natural copper(II) chelators may prevent some of the health problems caused by cigarette smoking, including lung disease, renal failure, and diabetes.

Lysophosphatidylcholine-induced cytotoxicity and protection by heparin in mouse brain bEND.3 endothelial cells.

A pathological feature in atherosclerosis is the dysfunction and death of vascular endothelial cells (EC). Oxidized low-density lipoprotein (LDL), known to accumulate in the atherosclerotic arterial walls, impairs endothelium-dependent relaxation and causes EC apoptosis. A major bioactive ingredient of the oxidized LDL is lysophosphatidylcholine (LPC), which at higher concentrations causes apoptosis and necrosis in various EC. There is hitherto no report on LPC-induced cytotoxicity in brain EC. In this work, we found that LPC caused cytosolic Ca2+ overload, mitochondrial membrane potential decrease, p38 activation, caspase 3 activation and eventually apoptotic death in mouse cerebral bEND.3 EC. In contrast to reported reactive oxygen species (ROS) generation by LPC in other EC, LPC did not trigger ROS formation in bEND.3 cells. Pharmacological inhibition of p38 alleviated LPC-inflicted cell death. We examined whether heparin could be cytoprotective: although it could not suppress LPC-triggered Ca2+ signal, p38 activation and mitochondrial membrane potential drop, it did suppress LPC-induced caspase 3 activation and alleviate LPC-inflicted cytotoxicity. Our data suggest LPC apoptotic death mechanisms in bEND.3 might involve mitochondrial membrane potential decrease and p38 activation. Heparin is protective against LPC cytotoxicity and might intervene steps between mitochondrial membrane potential drop/p38 activation and caspase 3 activation.

Repressed Ca(2+) clearance in parthenolide-treated murine brain bEND.3 endothelial cells.

Parthenolide is a sesquiterpene lactone compound isolated from the leaves and flowerheads of the plant feverfew (Tanacetum parthenium). The anticancer effects of parthenolide have been well studied and this lactone compound is currently under clinical trials. Parthenolide is also a protective agent in cardiac reperfusion injury via its inhibition of nuclear factor-κB (NF-κB). Not much is known if this compound affects signal transduction in non-tumor cells. We investigated whether parthenolide affected Ca(2+) signaling in endothelial cells, key components in regulating the vascular tone. In this work using mouse cortical microvascular bEND.3 endothelial cells, we found that a 15-h treatment with parthenolide resulted in amplified ATP-triggered Ca(2+) signal; the latter had a very slow decay rate suggesting suppression of Ca(2+) clearance. Evidence suggests parthenolide suppressed Ca(2+) clearance by inhibiting the plasmalemmal Ca(2+) pump; such suppression did not result from decreased expression of the plasmalemmal Ca(2+) pump protein. Rather, such suppression was possibly a consequence of endoplasmic reticulum (ER) stress, since salubrinal (an ER stress protector) was able to alleviate parthenolide-induced Ca(2+) clearance suppression. Given the current deployment of parthenolide as an anti-cancer drug in clinical trials and the potential usage of this lactone as a cardioprotectant, it is important to examine in details the perturbing effects of parthenolide on Ca(2+) homeostasis in endothelial cells and neighboring vascular smooth muscle cells, activities of which exert profound effects on hemodynamics.

Suppression of Ca(2+) influx in endotoxin-treated mouse cerebral cortex endothelial bEND.3 cells.

Release of nitric oxide (NO) is triggered by a rise in endothelial cell (EC) cytosolic Ca(2+) concentration ([Ca(2+)]i) and is of prime importance in vascular tone regulation as NO relaxes vascular smooth muscle. Agonists could stimulate EC [Ca(2+)]i elevation by triggering Ca(2+) influx via plasma membrane ion channels, one of which is the store-operated Ca(2+) channel; the latter opens as a result of agonist-triggered internal Ca(2+) release. Endotoxin (lipopolysaccharide, LPS) could cause sepsis, which is often the fatal cause in critically ill patients. One of the LPS-induced damages is EC dysfunction, eventually leading to perturbations in hemodynamics. We obtained data showing that LPS-challenged mouse cerebral cortex endothelial bEND.3 cells did not suffer from apoptotic death, and in fact had intact agonist-triggered intracellular Ca(2+) release; however, they had reduced store-operated Ca(2+) entry (SOCE) after LPS treatment for 3h or more. Using real-time PCR, we did not find a decrease in gene expression of stromal interaction molecule 1 (STIM1) and Orai1 (two SOCE protein components) in bEND.3 cells treated with LPS for 15h. LPS inhibitory effects could be largely prevented by sodium salicylate (an inhibitor of nuclear factor-κB; NF-κB) or SB203580 (an inhibitor of p38 mitogen-activated protein kinases; p38 MAPK), suggesting that the p38 MAPK-NF-κB pathway is involved in SOCE inhibition.

Palmitic acid-induced lipotoxicity and protection by (+)-catechin in rat cortical astrocytes.

BACKGROUND: Astrocytes do not only maintain homeostasis of the extracellular milieu of the neurons, but also play an active role in modulating synaptic transmission. Palmitic acid (PA) is a saturated fatty acid which, when being excessive, is a significant risk factor for lipotoxicity. Activation of astrocytes by PA has been shown to cause neuronal inflammation and demyelination. However, direct damage by PA to astrocytes is relatively unexplored. The aim of this study was to identify the mechanism(s) of PA-induced cytotoxicity in rat cortical astrocytes and possible protection by (+)-catechin. METHODS: Cytotoxicity and endoplasmic reticulum (ER) markers were assessed by MTT assay and Western blotting, respectively. Cytosolic Ca(2+) and mitochondrial membrane potential (MMP) were measured microfluorimetrically using fura-2 and rhodamine 123, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: Exposure of astrocytes to 100µM PA for 24h resulted in apoptotic cell death. Whilst PA-induced cell death appeared to be unrelated to ER stress and perturbation in cytosolic Ca(2+) signaling, it was likely a result of ROS production and subsequent MMP collapse, since ascorbic acid (anti-oxidant, 100µM) prevented PA-induced MMP collapse and cell death. Co-treatment of astrocytes with (+)-catechin (300µM), an anti-oxidant found abundantly in green tea, significantly prevented PA-induced ROS production, MMP collapse and cell death. CONCLUSION: Our results suggest that PA-induced cytotoxicity in astrocytes may involve ROS generation and MMP collapse, which can be prevented by (+)-catechin.

Inhibition of voltage-gated K+ channels and Ca2+ channels by diphenidol.

BACKGROUND: Although diphenidol has long been deployed as an anti-emetic and anti-vertigo drug, its mechanism of action remains unclear. In particular, little is known as to how diphenidol affects neuronal ion channels. Recently, we showed that diphenidol blocked neuronal voltage-gated Na(+) channels, causing spinal blockade of motor function, proprioception and nociception in rats. In this work, we investigated whether diphenidol could also affect voltage-gated K(+) and Ca(2+) channels. METHODS: Electrophysiological experiments were performed to study ion channel activities in two neuronal cell lines, namely, neuroblastoma N2A cells and differentiated NG108-15 cells. RESULTS: Diphenidol inhibited voltage-gated K(+) channels and Ca(2+) channels, but did not affect store-operated Ca(2+) channels. CONCLUSION: Diphenidol is a non-specific inhibitor of voltage-gated ion channels in neuronal cells.