scholarly journals Stimulation of astrocyte Na+/H+ exchange activity in response to in vitro ischemia depends in part on activation of ERK1/2

2005 ◽  
Vol 289 (4) ◽  
pp. C934-C945 ◽  
Author(s):  
Douglas B. Kintner ◽  
Andy Look ◽  
Gary E. Shull ◽  
Dandan Sun
Keyword(s):  
Glucose Deprivation ◽  
Oxygen And Glucose Deprivation ◽  
In Vitro Ischemia ◽  
Reverse Mode ◽  
Intracellular Ca ◽  
Nhe1 Activity ◽  
Underlying Mechanisms ◽  
Stimulation Of

We recently reported that Na+/H+ exchanger isoform 1 (NHE1) activity in astrocytes is stimulated and leads to intracellular Na+ loading after oxygen and glucose deprivation (OGD). However, the underlying mechanisms for this stimulation of NHE1 activity and its impact on astrocyte function are unknown. In the present study, we investigated the role of the ERK1/2 pathway in NHE1 activation. NHE1 activity was elevated by ∼75% in NHE1+/+ astrocytes after 2-h OGD and 1-h reoxygenation (REOX). The OGD/REOX-mediated stimulation of NHE1 was partially blocked by 30 μM PD-98059. Increased expression of phosphorylated ERK1/2 was detected in NHE1+/+ astrocytes after OGD/REOX. Moreover, stimulation of NHE1 activity disrupted not only Na+ but also Ca2+ homeostasis via reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX led to a 103% increase in intracellular Ca2+ concentration ([Ca2+]i) in NHE1+/+ astrocytes in the presence of thapsigargin. Inhibition of NHE1 activity with the NHE1 inhibitor HOE-642 decreased OGD/REOX-induced elevation of [Ca2+]i by 73%. To further investigate changes of Ca2+ signaling, bradykinin-mediated Ca2+ release was evaluated. Bradykinin-mediated intracellular Ca2+ transient in NHE1+/+ astrocytes was increased by ∼84% after OGD/REOX. However, in NHE1−/− astrocytes or NHE1+/+ astrocytes treated with HOE-642, the bradykinin-induced Ca2+ release was increased by only ∼34%. Inhibition of the reverse mode of Na+/Ca2+ exchange abolished OGD/REOX-mediated Ca2+ rise. Together, our data suggest that ERK1/2 is involved in activation of NHE1 in astrocytes after in vitro ischemia. NHE1-mediated Na+ accumulation subsequently alters Ca2+ homeostasis via Na+/Ca2+ exchange.

Role of Na+-K+-Cl− cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

2007 ◽  
Vol 292 (3) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Douglas B. Kintner ◽  
Jing Luo ◽  
Josiah Gerdts ◽  
Andy J. Ballard ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Glucose Deprivation ◽  
Cytochrome C Release ◽  
Genetic Ablation ◽  
In Vitro Ischemia ◽  
Reverse Mode ◽  
Intracellular Ca ◽  
Acting In Concert

Na+-K+-Cl− cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na+/Ca2+ exchanger (NCX) contribute to intracellular Na+ and Ca2+ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca2+ (Cam2+) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca2+ ( P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca2+ was reduced by ∼42% ( P < 0.05). Genetic ablation of NKCC1 also reduced Cam2+ accumulation. Moreover, OGD/REOX in NKCC1+/+ astrocytes caused dissipation of the mitochondrial membrane potential (Ψm) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of Ψm was attenuated significantly (66 ± 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca2+ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Cam2+ was accompanied by loss of Ψm and cytochrome c release in NKCC1+/+ astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Cam2+ homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia.

scholarly journals ERK1/2-p90RSK-mediated Phosphorylation of Na+/H+ Exchanger Isoform 1

2007 ◽  
Vol 282 (38) ◽  
pp. 28274-28284 ◽  
Author(s):  
Jing Luo ◽  
Douglas B. Kintner ◽  
Gary E. Shull ◽  
Dandan Sun
Keyword(s):  
Nuclear Translocation ◽  
Neuronal Damage ◽  
Glucose Deprivation ◽  
Mek Inhibitor ◽  
Oxygen And Glucose Deprivation ◽  
Genetic Ablation ◽  
Extracellular Signal ◽  
Nhe1 Activity ◽  
Ribosomal S6 Kinase

The function and regulation of Na+/H+ exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the Vmax for NHE1 but also shifted the Km toward decreased [H+]i. These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90RSK) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90RSK, a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90RSK. Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90RSK pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.

scholarly journals The Application and Analytical Pathway of Dexmedetomidine in Ischemia/Reperfusion Injury

2019 ◽  
Vol 2019 ◽  
pp. 1-10
Author(s):  
Ying Tang ◽  
Changxin Jia ◽  
Jianshuai He ◽  
Yang Zhao ◽  
Huayong Chen ◽  
...  
Keyword(s):  
Cell Survival ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Potential Effect ◽  
Cerebral Injury ◽  
Oxygen And Glucose Deprivation ◽  
Calcium Uniporter

Ischemia/reperfusion cerebral injury can cause serious damage to nerve cells. The injured organelles are cleared by autophagy eventually, which is critical for cell survival. Dexmedetomidine is neuroprotective in various ischemia/reperfusion models. Mitochondrial calcium uniporter (MCU) is the most important channel of mitochondrial Ca2+ influx into mitochondria, where Ca2+ has a potential effect on mitochondrial autophagy. However, the role of MCU in the changes of mitophagy and autophagy caused by dexmedetomidine is unknown. In this study, we constructed an in vitro I/R model by subjecting the oxygen and glucose deprivation/reperfusion model to SH-SY5Y cells to mimic the cerebral I/R injury. We found that postconditioning with dexmedetomidine and 3-methyladenine (3MA, an autophagy inhibitor) increased the cell survival meanwhile reduced the production of autophagic vesicles and the expression of LC3 and Beclin 1. This process also increased the expression of BCL-2, P62, and TOM20. After applied with spermine (MCU-specific agonist), the expression of autophagy proteins by dexmedetomidine was reversed, and the same changes were also observed in immunofluorescence. The results of our study suggested that dexmedetomidine can inhibit MCU and reduce excessive mitophagy and autophagy for conferring protection against I/R injury.

Increased tolerance to oxygen and glucose deprivation in astrocytes from Na+/H+ exchanger isoform 1 null mice

2004 ◽  
Vol 287 (1) ◽  
pp. C12-C21 ◽  
Author(s):  
Douglas B. Kintner ◽  
Gui Su ◽  
Brett Lenart ◽  
Andy J. Ballard ◽  
Jamie W. Meyer ◽  
...  
Keyword(s):  
Regulatory Mechanism ◽  
Ischemic Injury ◽  
Glucose Deprivation ◽  
Cell Types ◽  
Oxygen And Glucose Deprivation ◽  
Genetic Ablation ◽  
Cortical Astrocyte ◽  
Nhe1 Activity ◽  
Acid Load

The ubiquitously expressed Na+/H+ exchanger isoform 1 (NHE1) functions as a major intracellular pH (pHi) regulatory mechanism in many cell types, and in some tissues its activity may contribute to ischemic injury. In the present study, cortical astrocyte cultures from wild-type (NHE1+/+) and NHE1-deficient (NHE1−/−) mice were used to investigate the role of NHE1 in pHi recovery and ischemic injury in astrocytes. In the absence of HCO3−, the mean resting pHi levels were 6.86 ± 0.03 in NHE1+/+ astrocytes and 6.53 ± 0.04 in NHE1−/− astrocytes. Removal of extracellular Na+ or blocking of NHE1 activity by the potent NHE1 inhibitor HOE-642 significantly reduced the resting level of pHi in NHE1+/+ astrocytes. NHE1+/+ astrocytes exhibited a rapid pHi recovery (0.33 ± 0.08 pH unit/min) after NH4Cl prepulse acid load. The pHi recovery in NHE1+/+ astrocytes was reversibly inhibited by HOE-642 or removal of extracellular Na+. In NHE1−/− astrocytes, the pHi recovery after acidification was impaired and not affected by either Na+-free conditions or HOE-642. Furthermore, 2 h of oxygen and glucose deprivation (OGD) led to an ∼80% increase in pHi recovery rate in NHE1+/+ astrocytes. OGD induced a 5-fold rise in intracellular [Na+] and 26% swelling in NHE1+/+ astrocytes. HOE-642 or genetic ablation of NHE1 significantly reduced the Na+ rise and swelling after OGD. These results suggest that NHE1 is the major pHi regulatory mechanism in cortical astrocytes and that ablation of NHE1 in astrocytes attenuates ischemia-induced disruption of ionic regulation and swelling.

scholarly journals Role of interleukins in the pathogenesis of pulmonary fibrosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yi Xin She ◽  
Qing Yang Yu ◽  
Xiao Xiao Tang
Keyword(s):  
Pulmonary Fibrosis ◽  
Therapeutic Strategy ◽  
Future Directions ◽  
Regulation Mechanisms ◽  
Underlying Mechanisms ◽  
Multiple Cells ◽  
The Relationship

AbstractInterleukins, a group of cytokines participating in inflammation and immune response, are proved to be involved in the formation and development of pulmonary fibrosis. In this article, we reviewed the relationship between interleukins and pulmonary fibrosis from the clinical, animal, as well as cellular levels, and discussed the underlying mechanisms in vivo and in vitro. Despite the effects of interleukin-targeted treatment on experimental pulmonary fibrosis, clinical applications are lacking and unsatisfactory. We conclude that intervening in one type of interleukins with similar functions in IPF may not be enough to stop the development of fibrosis as it involves a complex network of regulation mechanisms. Intervening interleukins combined with other existing therapy or targeting interleukins affecting multiple cells/with different functions at the same time may be one of the future directions. Furthermore, the intervention time is critical as some interleukins play different roles at different stages. Further elucidation on these aspects would provide new perspectives on both the pathogenesis mechanism, as well as the therapeutic strategy and drug development.

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na+/Ca2+exchange

2002 ◽  
Vol 282 (5) ◽  
pp. C1000-C1008 ◽  
Author(s):  
Kara L. Kopper ◽  
Joseph S. Adorante
Keyword(s):  
Membrane Potential ◽  
Intracellular Calcium ◽  
Normal Cell ◽  
Buffer Capacity ◽  
Neuroblastoma Cells ◽  
Fold Increase ◽  
Scorpion Venom ◽  
Reverse Mode ◽  
Intracellular Ca

In fura 2-loaded N1E-115 cells, regulation of intracellular Ca2+ concentration ([Ca2+]i) following a Ca2+ load induced by 1 μM thapsigargin and 10 μM carbonylcyanide p-trifluoromethyoxyphenylhydrazone (FCCP) was Na+ dependent and inhibited by 5 mM Ni2+. In cells with normal intracellular Na+ concentration ([Na+]i), removal of bath Na+, which should result in reversal of Na+/Ca2+exchange, did not increase [Ca2+]i unless cell Ca2+ buffer capacity was reduced. When N1E-115 cells were Na+ loaded using 100 μM veratridine and 4 μg/ml scorpion venom, the rate of the reverse mode of the Na+/Ca2+ exchanger was apparently enhanced, since an ∼4- to 6-fold increase in [Ca2+]ioccurred despite normal cell Ca2+ buffering. In SBFI-loaded cells, we were able to demonstrate forward operation of the Na+/Ca2+ exchanger (net efflux of Ca2+) by observing increases (∼ 6 mM) in [Na+]i. These Ni2+ (5 mM)-inhibited increases in [Na+]i could only be observed when a continuous ionomycin-induced influx of Ca2+ occurred. The voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to measure changes in membrane potential. Ionomycin (1 μM) depolarized N1E-115 cells (∼25 mV). This depolarization was Na+dependent and blocked by 5 mM Ni2+ and 250–500 μM benzamil. These data provide evidence for the presence of an electrogenic Na+/Ca2+ exchanger that is capable of regulating [Ca2+]i after release of Ca2+ from cell stores.

Role of iPLA2 and store-operated channels in agonist-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries

2008 ◽  
Vol 294 (3) ◽  
pp. H1183-H1187 ◽  
Author(s):  
Kristen M. Park ◽  
Mario Trucillo ◽  
Nicolas Serban ◽  
Richard A. Cohen ◽  
Victoria M. Bolotina
Keyword(s):  
Carotid Arteries ◽  
Cerebral Arteries ◽  
Present Evidence ◽  
Voltage Gated ◽  
Functional Inhibition ◽  
Intracellular Ca ◽  
Dependent Pathway ◽  
Secondary Activation

Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (CaL2+) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a CaL2+ inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and CaL2+ channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2β totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by CaL2+ channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of CaL2+ channels and lead to Ca2+ entry and vasoconstriction.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

2007 ◽  
Vol 292 (4) ◽  
pp. L915-L923 ◽  
Author(s):  
Jaime Chávez ◽  
Patricia Segura ◽  
Mario H. Vargas ◽  
José Luis Arreola ◽  
Edgar Flores-Soto ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pigs ◽  
Smooth Muscle Contraction ◽  
In Vivo Experiments ◽  
Intracellular Ca ◽  
Neurokinin 1 ◽  
Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

scholarly journals Genotoxicity and inflammatory potential of stainless steel welding fume particles: an in vitro study on standard vs Cr(VI)-reduced flux-cored wires and the role of released metals

2021 ◽  
Author(s):  
Sarah McCarrick ◽  
Valentin Romanovski ◽  
Zheng Wei ◽  
Elin M. Westin ◽  
Kjell-Arne Persson ◽  
...  
Keyword(s):  
Dna Damage ◽  
In Vitro Study ◽  
Human Monocyte ◽  
Welding Fumes ◽  
Welding Fume ◽  
Increased Risk ◽  
Underlying Mechanisms ◽  
Cored Wires

AbstractWelders are daily exposed to various levels of welding fumes containing several metals. This exposure can lead to an increased risk for different health effects which serves as a driving force to develop new methods that generate less toxic fumes. The aim of this study was to explore the role of released metals for welding particle-induced toxicity and to test the hypothesis that a reduction of Cr(VI) in welding fumes results in less toxicity by comparing the welding fume particles of optimized Cr(VI)-reduced flux-cored wires (FCWs) to standard FCWs. The welding particles were thoroughly characterized, and toxicity (cell viability, DNA damage and inflammation) was assessed following exposure to welding particles as well as their released metal fraction using cultured human bronchial epithelial cells (HBEC-3kt, 5–100 µg/mL) and human monocyte-derived macrophages (THP-1, 10–50 µg/mL). The results showed that all Cr was released as Cr(VI) for welding particles generated using standard FCWs whereas only minor levels (< 3% of total Cr) were released from the newly developed FCWs. Furthermore, the new FCWs were considerably less cytotoxic and did not cause any DNA damage in the doses tested. For the standard FCWs, the Cr(VI) released in cell media seemed to explain a large part of the cytotoxicity and DNA damage. In contrast, all particles caused rather similar inflammatory effects suggesting different underlying mechanisms. Taken together, this study suggests a potential benefit of substituting standard FCWs with Cr(VI)-reduced wires to achieve less toxic welding fumes and thus reduced risks for welders.

scholarly journals Synthesis, Neuroprotection, and Antioxidant Activity of 1,1′-Biphenylnitrones as α-Phenyl-N-tert-butylnitrone Analogues in In Vitro Ischemia Models

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1127 ◽  
Author(s):  
Beatriz Chamorro ◽  
David García-Vieira ◽  
Daniel Diez-Iriepa ◽  
Estíbaliz Garagarza ◽  
Mourad Chioua ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Antioxidant Activities ◽  
Antioxidant Properties ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
Human Neuroblastoma ◽  
In Vitro Ischemia ◽  
Lipoxygenase Inhibition ◽  
Antioxidant Agent

Herein, we report the neuroprotective and antioxidant activity of 1,1′-biphenyl nitrones (BPNs) 1–5 as α-phenyl-N-tert-butylnitrone analogues prepared from commercially available [1,1′-biphenyl]-4-carbaldehyde and [1,1′-biphenyl]-4,4′-dicarbaldehyde. The neuroprotection of BPNs1-5 has been measured against oligomycin A/rotenone and in an oxygen–glucose deprivation in vitro ischemia model in human neuroblastoma SH-SY5Y cells. Our results indicate that BPNs 1–5 have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN), and they are quite similar to N-acetyl-L-cysteine (NAC), which is a well-known antioxidant agent. Among the nitrones studied, homo-bis-nitrone BPHBN5, bearing two N-tert-Bu radicals at the nitrone motif, has the best neuroprotective capacity (EC50 = 13.16 ± 1.65 and 25.5 ± 3.93 μM, against the reduction in metabolic activity induced by respiratory chain blockers and oxygen–glucose deprivation in an in vitro ischemia model, respectively) as well as anti-necrotic, anti-apoptotic, and antioxidant activities (EC50 = 11.2 ± 3.94 μM), which were measured by its capacity to reduce superoxide production in human neuroblastoma SH-SY5Y cell cultures, followed by mononitrone BPMN3, with one N-Bn radical, and BPMN2, with only one N-tert-Bu substituent. The antioxidant activity of BPNs1-5 has also been analyzed for their capacity to scavenge hydroxyl free radicals (82% at 100 μM), lipoxygenase inhibition, and the inhibition of lipid peroxidation (68% at 100 μM). Results showed that although the number of nitrone groups improves the neuroprotection profile of these BPNs, the final effect is also dependent on the substitutent that is being incorporated. Thus, BPNs bearing N-tert-Bu and N-Bn groups show better neuroprotective and antioxidant properties than those substituted with Me. All these results led us to propose homo-bis-nitrone BPHBN5 as the most balanced and interesting nitrone based on its neuroprotective capacity in different neuronal models of oxidative stress and in vitro ischemia as well as its antioxidant activity.

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