Anna K. Brzezinska

Peroxynitrite is a contractile agonist of cerebral artery smooth muscle cells

On reperfusion of ischemic tissue, a prolonged phase of vasoconstriction occurs, the mechanism of which is poorly understood. However, it is known that peroxynitrite (ONOO-) is formed during reperfusion. In this study the contractile properties of ONOO- were investigated in Wistar rat middle cerebral arteries. The effects of ONOO- on vessel diameter were dose dependent. Low-dose ONOO- (10 microM) caused vessels to constrict by 15%. At an intermediate concentration of 25 microM, the effect of ONOO- was variable, whereas at the highest concentration (100 microM), vessels underwent persistent dilation and became insensitive to the endogenous vasoconstrictor 5-hydroxytryptamine. At the single cell level, ONOO- caused cerebral artery smooth muscle cells to contract. Reduced, but not oxidized, glutathione completely inhibited the contractile action of ONOO- on single cells. Vehicle and decomposed ONOO- each had minimal effect on cell length. These data show that ONOO- is a contractile agonist of middle cerebral arteries, at the single cell and whole vessel levels, suggesting that formation of ONOO- may contribute mechanistically to ischemic brain injury during stroke. Moreover, relatively high concentrations of ONOO- result in vascular paralysis.

Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca(2+) overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca(2+), and mPTP opening. The magnitude of APC-induced decrease in DeltaPsi(m) was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in DeltaPsi(m). In cardiomyocytes, DeltaPsi(m), ROS, mPTP opening, and cytosolic and mitochondrial Ca(2+) were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca(2+) uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca(2+) uptake. However, in stressed cardiomyocytes, a similar decrease in DeltaPsi(m) attenuated both cytosolic and mitochondrial Ca(2+) accumulation. In conclusion, a partial decrease in DeltaPsi(m) underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in DeltaPsi(m) primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca(2+) uptake.

Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

Background:Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Methods:Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or NG-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or NG-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. Results:APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. NG-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. Conclusion:The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

Hyperglycemia Adversely Modulates Endothelial Nitric Oxide Synthase during Anesthetic Preconditioning through Tetrahydrobiopterin– and Heat Shock Protein 90–mediated Mechanisms

Background:Endothelial nitric oxide synthase activity is regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and heat shock protein 90. The authors tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways. Methods:Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mm) or high (20 mm) glucose conditions, with or without sepiapterin (10 or 100 &mgr;m). Results:APC decreased myocardial infarct size compared with control experiments (26 ± 6% vs. 46 ± 3%, respectively; P < 0.05), and this action was blocked by hyperglycemia (43 ± 4%). Sepiapterin alone had no effect on infarct size (46 ± 3%) but restored APC during hyperglycemia (21 ± 3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitior N (G)-nitro-l-arginine methyl ester (47 ± 2%) and the BH4 synthesis inhibitor N-acetylserotonin (46 ± 3%). Isoflurane increased nitric oxide production to 177 ± 13% of baseline, and this action was attenuated by high glucose concentrations (125 ± 6%). Isoflurane increased, whereas high glucose attenuated intracellular BH4/7,8-dihydrobiopterin (BH2) (high performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase colocalization (confocal microscopy) and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149 ± 12% and 175 ± 9%; 10 and 100 &mgr;m, respectively). Conclusion:The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central role in cardioprotection that is favorably modulated by volatile anesthetics and dysregulated by hyperglycemia. Enhancing the production of BH4 may represent a potential therapeutic strategy.

Endothelial–cardiomyocyte crosstalk enhances pharmacological cardioprotection

Endothelial cells (EC) serve a paracrine function to enhance signaling in cardiomyocytes (CM), and conversely, CM secrete factors that impact EC function. Understanding how EC interact with CM may be critically important in the context of ischemia-reperfusion injury, where EC might promote CM survival. We used isoflurane as a pharmacological stimulus to enhance EC protection of CM against hypoxia and reoxygenation injury. Triggering of intracellular signal transduction pathways culminating in the enhanced production of nitric oxide (NO) appears to be a central component of pharmacologically induced cardioprotection. Although the endothelium is well recognized as a regulator for vascular tone, little attention has been given to its potential importance in mediating cardioprotection. In the current investigation, EC-CM in co-culture were used to test the hypothesis that EC contribute to isoflurane-enhanced protection of CM against hypoxia and reoxygenation injury and that this protection depends on hypoxia-inducible factor (HIF1α) and NO. CM were protected against cell injury [lactate dehydrogenase (LDH) release] to a greater extent in the presence vs. absence of isoflurane-stimulated EC (1.7 ± 0.2 vs. 4.58 ± 0.8 fold change LDH release), and this protection was NO-dependent. Isoflurane enhanced release of NO in EC (1103 ± 58 vs. 702 ± 92 pmol/mg protein) and EC-CM in co-culture sustained NO release during reoxygenation. In contrast, lentiviral mediated HIF1α knockdown in EC decreased basal and isoflurane stimulated NO release in an eNOS dependent manner (517 ± 32 vs. 493 ± 38 pmol/mg protein) and prevented the sustained increase in NO during reoxygenation when co-cultured. Opening of mitochondrial permeability transition pore (mPTP), an index of mitochondrial integrity, was delayed in the presence vs. absence of EC (141 ± 2 vs. 128 ± 2.5 arbitrary mPTP opening time). Isoflurane stimulated an increase in HIF1α in EC but not in CM under normal oxygen tension (3.5 ± 0.1 vs. 0.79 ± 0.15 fold change density) and this action was blocked by pretreatment with the Mitogen-activated Protein/Extracellular Signal-regulated Kinase inhibitor U0126. Expression and nuclear translocation of HIF1α were confirmed by Western blot and immunofluorescence. Taken together, these data support the concept that EC are stimulated by isoflurane to produce important cardioprotective factors that may contribute to protection of myocardium during ischemia and reperfusion injury.

Decreased tetrahydrobiopterin and disrupted association of Hsp90 with eNOS by hyperglycemia impair myocardial ischemic preconditioning

Cardioprotection by ischemic preconditioning (IPC) is impaired during hyperglycemia, but the mechanisms underlying this phenomenon are poorly understood. This study investigated the role of hyperglycemia to adversely modulate tetrahydrobiopterin (BH(4)) and heat shock protein 90 (Hsp90) during cardioprotection by IPC. Rabbits or mice underwent 30 min of coronary occlusion followed by reperfusion with or without IPC in the presence or absence of hyperglycemia. IPC significantly (P < 0.05) decreased myocardial infarct size (46 ± 1 to 19 ± 2% of the area at risk in control and IPC rabbits, respectively) and increased BH(4) concentrations (HPLC; 7.6 ± 0.2 to 10.2 ± 0.3 pmol/mg protein, respectively), Hsp90-endothelial nitric oxide synthase (eNOS) association (coimmunoprecipitation and Western blotting in mice; 4.0 ± 0.3 to 5.4 ± 0.1, respectively), and the ratio of phosphorylated eNOS/total eNOS. These beneficial actions of IPC on infarct size, BH(4), Hsp90/eNOS, and phosphorylated eNOS were eliminated by hyperglycemia. Pretreatment of animals with the Hsp90 inhibitor geldanamycin (0.6 mg/kg) or the BH(4) synthesis inhibitor diamino-6-hydroxypyrimidine (1.0 g/kg) also eliminated cardioprotection produced by IPC. In contrast, the BH(4) precursor sepiapterin (2 mg/kg iv) restored the beneficial effects of IPC on myocardial BH(4) concentrations, eNOS dimerization, and infarct size during hyperglycemia. A-23871 increased Hsp90-eNOS association (0.33 ± 0.06 to 0.59 ± 0.3) and nitric oxide production (184 ± 17%) in human coronary artery endothelial cells cultured in normal (5.5 mM) but not high (20 mM) glucose media. These data indicate that hyperglycemia eliminates protection by IPC via decreases in myocardial BH(4) concentration and disruption of the association of Hsp90 with eNOS. The results suggest that eNOS dysregulation may be a central mechanism of impaired cardioprotection during hyperglycemia.

4 – Redox Regulation of Ion Channels

This chapter presents electrophysiological concepts of ion channels and describes the role of redox regulation in channel function. Ion channels are protein macromolecules that form pores in the membranes of mammalian cells; they are an integral component of the cell membrane. Channels form one mechanism by which ions traverse the cell membrane. Channels are generally classified and named according to their functional characteristics. These characteristics include—ionic selectivity, the electrical conductance generated by the passage of ions, the relationship between current amplitude through the channel and the membrane potential, the molecular and biophysical factors that cause the channel to open as well as close, and the kinetics of activation and inactivation. In electrophysiology, the current-voltage relationship is shown as an x-y plot in which the amplitude and direction of current is plotted on the ordinate, the difference in potential across the membrane is plotted on the ordinate, and the difference in potential across the membrane is plotted on the abscissa. Rectification refers to that property of a channel wherein current flows preferentially in one direction.