Medication errors during simulated paediatric resuscitations: a prospective, observational human reliability analysis.

INTRODUCTION: Medication errors during paediatric resuscitation are thought to be common. However, there is little evidence about the individual process steps that contribute to such medication errors in this context. OBJECTIVES: To describe the incidence, nature and severity of medication errors in simulated paediatric resuscitations, and to employ human reliability analysis to understand the contribution of discrepancies in individual process steps to the occurrence of these errors. METHODS: We conducted a prospective observational study of simulated resuscitations subjected to video microanalysis, identification of medication errors, severity assessment and human reliability analysis in a large English teaching hospital. Fifteen resuscitation teams of two doctors and two nurses each conducted one of two simulated paediatric resuscitation scenarios. RESULTS: At least one medication error was observed in every simulated case, and a large magnitude (>25% discrepant) or clinically significant error in 11 of 15 cases. Medication errors were observed in 29% of 180 simulated medication administrations, 40% of which considered to be moderate or severe. These errors were the result of 884 observed discrepancies at a number of steps in the drug ordering, preparation and administration stages of medication use, 8% of which made a major contribution to a resultant medication error. Most errors were introduced by discrepancies during drug preparation and administration. CONCLUSIONS: Medication errors were common with a considerable proportion likely to result in patient harm. There is an urgent need to optimise existing systems and to commission research into new approaches to increase the reliability of human interactions during administration of medication in the paediatric emergency setting.

Far red/near infrared light-induced protection against cardiac ischemia and reperfusion injury remains intact under diabetic conditions and is independent of nitric oxide synthase.

Far red/near-infrared light (NIR) promotes a wide range of biological effects including tissue protection but whether and how NIR is capable of acutely protecting myocardium against ischemia and reperfusion injury in vivo is not fully elucidated. Our previous work indicates that NIR exposure immediately before and during early reperfusion protects the myocardium against infarction through mechanisms that are nitric oxide (NO)-dependent. Here we tested the hypothesis that NIR elicits protection in a diabetic mouse model where other cardioprotective interventions such as pre- and postconditioning fail, and that the protection is independent of nitric oxide synthase (NOS). NIR reduced infarct size dose dependently. Importantly, NIR-induced protection was preserved in a diabetic mouse model (db/db) and during acute hyperglycemia, as well as in endothelial NOS(-/-) mice and in wild type mice treated with NOS inhibitor L-NAME. In in vitro experiments NIR light liberates NO from nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) in a wavelength-(660-830 nm) and dose-dependent manner. Irradiation at 660 nm yields the highest release of NO, while at longer wavelengths a dramatic decrease of NO release can be observed. Similar wavelength dependence was observed for the protection of mice against cardiac ischemia and reperfusion injury in vivo. NIR-induced NO release from deoxymyoglobin in the presence of nitrite mildly inhibits respiration of isolated mitochondria after hypoxia. In summary, NIR applied during reperfusion protects the myocardium against infarction in an NO-dependent, but NOS-independent mechanisms, whereby mitochondria may be a target of NO released by NIR, leading to reduced reactive oxygen species generation during reperfusion. This unique mechanism preserves protection even during diabetes where other protective strategies fail.

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.

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.

Exercise affects both ovarian follicular dynamics and hormone concentrations in mares.

The objectives were to evaluate the effects of exercise on ovarian folliculogenesis and related hormones in mares. Mares (n = 11) were randomly assigned into a control (non-exercised) or treatment (exercised) group. Treatment mares (n = 5) were moderately exercised for 30 min, 6 d/wk. All mares underwent daily transrectal ultrasonographic examinations and ovarian follicles > 6 mm were measured. Blood samples were collected during the first (Cycle 1) and last (Cycle 4) cycle, and serum concentrations of cortisol, LH, and FSH were determined. Mean cortisol concentrations were elevated (P < 0.05) in exercised mares, 6.29 ± 0.22 compared with 5.62 ± 0.16 ng/dL (mean ± SEM), 30 min post exercise. There were no significant differences between groups in mean FSH concentrations; however, exercised mares had lower (17.3 ± 6.4 vs 41.1 ± 5.5 ng/mL; P < 0.05) peak LH concentrations. Furthermore, exercised mares experienced a longer (24.7 ± 0.8 vs 22.2 ± 0.8 d; P < 0.05) mean interovulatory interval for all cycles combined, fewer (P < 0.05) follicles 6 to 20 mm in diameter, and an increased (P < 0.05) number of follicles >20 mm following deviation. The dominant and largest subordinate follicle in exercised mares had a greater (P < 0.05) mean diameter on the day of deviation, suggesting delayed deviation. Exercised mares also tended (P = 0.06) to have an increased number of cycles with at least two dominant follicles compared to control (62 vs 36%, respectively), indicating a decreased ability of the largest follicle to assert dominance. Under the conditions of this study, moderately exercising mares induced higher cortisol concentrations, lowered peak LH concentrations, and altered ovarian follicular dynamics.

Cardiac-specific overexpression of GTP cyclohydrolase 1 restores ischaemic preconditioning during hyperglycaemia.

AIMS: Hyperglycaemia (HG) decreases intracellular tetrahydrobiopterin (BH(4)) concentrations, and this action may contribute to injury during myocardial ischaemia and reperfusion. We investigated whether increased BH(4) by cardiomyocyte-specific overexpression of the GTP cyclohydrolase (GTPCH) 1 gene rescues myocardial and mitochondrial protection by ischaemic preconditioning (IPC) during HG through a nitric oxide (NO)-dependent pathway. METHODS AND RESULTS: Mice underwent 30 min of myocardial ischaemia followed by 2 h of reperfusion with or without IPC elicited with four cycles of 5 min ischaemia/5 min of reperfusion in the presence or absence of HG produced by d-glucose. In C57BL/6 wild-type mice, IPC increased myocardial BH(4) and NO concentrations and decreased myocardial infarct size (30 ± 3% of risk area) compared with control (56 ± 5%) experiments. This protective effect was inhibited by HG (48 ± 3%) but not hyperosmolarity. GTPCH-1 overexpression increased myocardial BH(4) and NO concentrations and restored cardioprotection by IPC during HG (32 ± 4%). In contrast, a non-selective NO synthase inhibitor N(G)-nitro-l-arginine methyl ester attenuated the favourable effects of GTPCH-1 overexpression (52 ± 3%) during HG. Mitochondria isolated from myocardium subjected to IPC required significantly higher in vitro Ca(2+) concentrations (184 ± 14 µmol mg(-1) protein) to open the mitochondrial permeability transition pore when compared with mitochondria isolated from control experiments (142 ± 10 µmol mg(-1) protein). This beneficial effect of IPC was reversed by HG and rescued by GTPCH-1 overexpression. CONCLUSION: Increased BH(4) by cardiomyocyte-specific overexpression of GTPCH-1 preserves the ability of IPC to elicit myocardial and mitochondrial protection that is impaired by HG, and this action appears to be dependent on NO.

Cigarette smoking impairs Na+-K+-ATPase activity in the human coronary microcirculation.

The extracellular K(+) concentration ([K(+)](o)) has been proposed to link cardiac metabolism with coronary perfusion and arrhythmogenesis, particularly during ischemia. Several animal studies have also supported K(+) as an EDHF that activates Na(+)-K(+)-ATPase and/or inwardly rectifying K(+) (K(ir)) channels. Therefore, we examined the vascular reactivity of human coronary arterioles (HCAs) to small elevations in [K(+)](o), the influence of risk factors for coronary disease, and the role of K(+) as an EDHF. Changes in the internal diameter of HCAs were recorded with videomicroscopy. Most vessels dilated to increases in [K(+)](o) with a maximal dilation of 55 ± 6% primarily at 12.5-20.0 mM KCl (n = 38, average: 16 ± 1 mM). Ouabain, a Na(+)-K(+)-ATPase inhibitor, alone reduced the dilation, and the addition of Ba(2+), a K(ir) channel blocker, abolished the remaining dilation, whereas neither endothelial denudation nor Ba(2+) alone reduced the dilation. Multivariate analysis revealed that cigarette smoking was the only risk factor associated with impaired dilation to K(+). Ouabain significantly reduced the vasodilation in HCAs from subjects without cigarette smoking but not in those with smoking. Cigarette smoking downregulated the expression of the Na(+)-K(+)-ATPase catalytic α(1)-subunit but not Kir2.1 in the vessels. Ouabain abolished the dilation in endothelium-denuded vessels to a same extent to that with the combination of ouabain and Ba(2+) in endothelium-intact vessels, whereas neither ouabain nor ouabain plus Ba(2+) reduced EDHF-mediated dilations to bradykinin and ADP. A rise in [K(+)](o) dilates HCAs primarily via the activation of Na(+)-K(+)-ATPase in vascular smooth muscle cells with a considerable contribution of K(ir) channels in the endothelium, indicating that [K(+)](o) may modify coronary microvascular resistance in humans. Na(+)-K(+)-ATPase activity is impaired in subjects who smoke, possibly contributing to dysregulation of the coronary microcirculation, excess ischemia, and arrhythmogenesis in those subjects. K(+) does not likely serve as an EDHF in the human coronary arteriolar dilation to bradykinin and ADP.

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 microm). 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 inhibitor 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 microm, 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.

Isoflurane postconditioning protects against reperfusion injury by preventing mitochondrial permeability transition by an endothelial nitric oxide synthase-dependent mechanism.

BACKGROUND: The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. The authors addressed this issue using eNOS mice. METHODS: In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before and 3 min after reperfusion. Ca+-induced mitochondrial permeability transition (MPT) pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function. RESULTS: Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 +/- 10% (n = 10) in control to 48 +/- 10%, 41 +/- 8% (n = 8, P < 0.05), and 38 +/- 10% (n = 8, P < 0.05), respectively, and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by N-nitro-L-arginine methyl ester (a nonselective nitric oxide synthase inhibitor) administered either before ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca+ loading than did controls (78 +/- 29 microm vs. 40 +/- 25 microm CaCl2 per milligram of protein, n = 10, P < 0.05) to open the MPT pore. Hearts from eNOS mice displayed no marked differences in infarct size, cardiac function, and sensitivity of MPT pore to Ca+, compared with wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function, or the amount of Ca+ necessary to open the MPT pore in mitochondria isolated from the eNOS hearts compared with control hearts. CONCLUSIONS: IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.

Enhancement of nitric oxide release from nitrosyl hemoglobin and nitrosyl myoglobin by red/near infrared radiation: potential role in cardioprotection.

Nitric oxide is an important messenger in numerous biological processes, such as angiogenesis, hypoxic vasodilation, and cardioprotection. Although nitric oxide synthases (NOS) produce the bulk of NO, there is increasing interest in NOS independent generation of NO in vivo, particularly during hypoxia or anoxia, where low oxygen tensions limit NOS activity. Interventions that can increase NO bioavailability have significant therapeutic potential. The use of far red and near infrared light (R/NIR) can reduce infarct size, protect neurons from methanol toxicity, and stimulate angiogenesis. How R/NIR modulates these processes in vivo and in vitro is unknown, but it has been suggested that increases in NO levels are involved. In this study we examined if R/NIR light could facilitate the release of NO from nitrosyl heme proteins. In addition, we examined if R/NIR light could enhance the protective effects of nitrite on ischemia and reperfusion injury in the rabbit heart. We show both in purified systems and in myocardium that R/NIR light can decay nitrosyl hemes and release NO, and that this released NO may enhance the cardioprotective effects of nitrite. Thus, the photodissociation to NO and its synergistic effect with sodium nitrite may represent a noninvasive and site-specific means for increasing NO bioavailability.

The mechanism of helium-induced preconditioning: a direct role for nitric oxide in rabbits.

BACKGROUND: Helium produces preconditioning against myocardial infarction by activating prosurvival signaling, but whether nitric oxide (NO) generated by endothelial NO synthase plays a role in this phenomenon is unknown. We tested the hypothesis that NO mediates helium-induced cardioprotection in vivo. METHODS: Rabbits (n = 62) instrumented for hemodynamic measurement were subjected to a 30-min left anterior descending coronary artery occlusion and 3 h reperfusion, and received 0.9% saline (control) or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture before left anterior descending coronary artery occlusion in the absence or presence of pretreatment with the nonselective NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg), the selective inducible NOS inhibitor aminoguanidine hydrochloride (AG; 300 mg/kg), or selective neuronal NOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg). In additional rabbits, the fluorescent probe 4,5-diaminofluroscein diacetate (DAF-2DA) and confocal laser microscopy were used to detect NO production in the absence or presence of helium with or without L-NAME pretreatment. RESULTS: Helium reduced (P < 0.05) infarct size (24% +/- 4% of the left ventricular area at risk; mean +/- sd) compared with control (46% +/- 3%). L-NAME, AG, and 7-NI did not alter myocardial infarct size when administered alone. L-NAME, but not 7-NI or AG, abolished helium-induced cardioprotection. Helium enhanced DAF-2DA fluorescence compared with control (26 +/- 8 vs 15 +/- 5 U, respectively). Pretreatment with L-NAME abolished these helium-induced increases in DAF-2DA fluorescence. CONCLUSIONS: The results indicate that cardioprotection by helium is mediated by NO that is probably generated by endothelial NOS in vivo.

Inhibition of glycogen synthase kinase or the apoptotic protein p53 lowers the threshold of helium cardioprotection in vivo: the role of mitochondrial permeability transition.

BACKGROUND: Prosurvival signaling kinases inhibit glycogen synthase kinase-3beta (GSK-3beta) activity and stimulate apoptotic protein p53 degradation. Helium produces cardioprotection by activating prosurvival kinases, but whether GSK and p53 inhibition mediate this process is unknown. We tested the hypothesis that inhibition of GSK or p53 lowers the threshold of helium cardioprotection via a mitochondrial permeability transition pore (mPTP)-dependent mechanism. METHODS: Rabbits (n = 85) instrumented for hemodynamic measurement and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), or 1, 3, or 5 cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture (fraction of inspired oxygen concentration = 0.30) before LAD occlusion. Other rabbits received the GSK inhibitor SB 216763 (SB21; 0.2 or 0.6 mg/kg), the p53 inhibitor pifithrin-alpha (PIF; 1.5 or 3.0 mg/kg), or SB21 (0.2 mg/kg) or PIF (1.5 mg/kg) plus helium (1 cycle) before LAD occlusion in the presence or absence of the mPTP opener atractyloside (5 mg/kg). RESULTS: Helium reduced (P < 0.05) myocardial infarct size (35 +/- 6 [n = 7], 25 +/- 4 [n = 7], and 20 +/- 3% [n = 6] of area at risk, 1, 3, and 5 cycles, respectively) compared with control (44 +/- 6% [n = 7]). SB21 (0.6 [n = 7] but not 0.2 mg/kg [n = 6]) and PIF (3.0 [n = 6] but not 1.5 mg/kg [n = 7]) also reduced necrosis. SB21 (0.2 mg/kg) or 1.5 mg/kg PIF (1.5 mg/kg) plus helium (1 cycle; n = 6 per group) decreased infarct size to an equivalent degree as three cycles of helium alone, and this cardioprotection was blocked by atractyloside (n = 7 per group). CONCLUSIONS: Inhibition of GSK or p53 lowers the threshold of helium-induced preconditioning via a mPTP-dependent mechanism in vivo.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans.

Atherosclerosis remains a major cause of death in the developed world despite the success of therapies that lower cholesterol and BP. The intermediate-conductance calcium-activated potassium channel KCa3.1 is expressed in multiple cell types implicated in atherogenesis, and pharmacological blockade of this channel inhibits VSMC and lymphocyte activation in rats and mice. We found that coronary vessels from patients with coronary artery disease expressed elevated levels of KCa3.1. In Apoe(-/-) mice, a genetic model of atherosclerosis, KCa3.1 expression was elevated in the VSMCs, macrophages, and T lymphocytes that infiltrated atherosclerotic lesions. Selective pharmacological blockade and gene silencing of KCa3.1 suppressed proliferation, migration, and oxidative stress of human VSMCs. Furthermore, VSMC proliferation and macrophage activation were reduced in KCa3.1(-/-) mice. In vivo therapy with 2 KCa3.1 blockers, TRAM-34 and clotrimazole, significantly reduced the development of atherosclerosis in aortas of Apoe(-/-) mice by suppressing VSMC proliferation and migration into plaques, decreasing infiltration of plaques by macrophages and T lymphocytes, and reducing oxidative stress. Therapeutic concentrations of TRAM-34 in mice caused no discernible toxicity after repeated dosing and did not compromise the immune response to influenza virus. These data suggest that KCa3.1 blockers represent a promising therapeutic strategy for atherosclerosis.

Reactive oxygen species and mitochondrial adenosine triphosphate-regulated potassium channels mediate helium-induced preconditioning against myocardial infarction in vivo.

OBJECTIVES: Helium produces preconditioning by activating prosurvival kinases, but the roles of reactive oxygen species (ROS) or mitochondrial adenosine triphosphate-regulated potassium (K(ATP)) channels in this process are unknown. The authors tested the hypothesis that ROS and mitochondrial K(ATP) channels mediate helium-induced preconditioning in vivo. DESIGN: A randomized, prospective study. SETTING: A university research laboratory. PARTICIPANTS: Male New Zealand white rabbits. INTERVENTIONS: Rabbits (n = 64) were instrumented for the measurement of systemic hemodynamics and subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. In separate experimental groups, rabbits (n = 7 or 8 per group) were randomly assigned to receive 0.9% saline (control) or 3 cycles of 70% helium-30% oxygen administered for 5 minutes interspersed with 5 minutes of an air-oxygen mixture before LAD occlusion with or without the ROS scavengers N-acetylcysteine (NAC; 150 mg/kg) or N-2 mercaptoproprionyl glycine (2-MPG; 75 mg/kg), or the mitochondrial K(ATP) antagonist 5-hydroxydecanoate (5-HD; 5 mg/kg). Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni's modification of a Student t test. MEASUREMENTS AND MAIN RESULTS: The myocardial infarct size was determined by using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium significantly (p < 0.05) reduced infarct size (23 +/- 4% of the area at risk; mean +/- standard deviation) compared with control (46 +/- 3%). NAC, 2-MPG, and 5-HD did not affect irreversible ischemic injury when administered alone (49 +/- 5%, 45 +/- 6%, and 45 +/- 3%), but these drugs blocked reductions in infarct size produced by helium (45 +/- 4%, 45 +/- 2%, and 44 +/- 3%). CONCLUSIONS: The results suggest that ROS and mitochondrial K(ATP) channels mediate helium-induced preconditioning in vivo.

Antinociception produced by 14,15-epoxyeicosatrienoic acid is mediated by the activation of beta-endorphin and met-enkephalin in the rat ventrolateral periaqueductal gray.

Cytochrome P450 genes catalyze formation of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The effects of 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET microinjected into the ventrolateral periaqueductal gray (vlPAG) on the thermally produced tail-flick response were studied in male Sprague-Dawley rats. 14,15-EET microinjected into vlPAG (3-156 pmol) dose-dependently inhibited the tail-flick response (ED50 = 32.5 pmol). In contrast, 5,6-EET, 8,9-EET, and 11,12-EET at a dose of 156 pmol were not active when injected into the vlPAG. 14,15-EET failed to displace the radiobinding of [3H][D-Ala2,NHPe4, Gly-ol5]enkephalin (mu-opioid receptor ligand) or [3H]naltrindole (delta-opioid receptor ligand) in crude membrane fractions of rat brain. Tail-flick inhibition produced by 14,15-EET from vlPAG was blocked by intra-vlPAG pretreatment with antiserum against beta-endorphin or Met-enkephalin or the mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) or the delta-opioid receptor antagonist naltrindole but not with dynorphin A[1-17] antiserum or the kappa-opioid receptor antagonist nor-binaltorphimine. In addition, tail-flick inhibition produced by 14,15-EET treatment was blocked by intrathecal pretreatment with Met-enkephalin antiserum, naltrindole, or CTOP but not with beta-endorphin antiserum. It is concluded that 1) 14,15-EET itself does not have any affinity for mu- or delta-opioid receptors and 2) 14,15-EET activates beta-endorphin and Met-enkephalin, which subsequently act on mu- and delta-opioid receptors to produce antinociception.

Role of JNK in network formation of human lung microvascular endothelial cells.

The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells align into tubular networks in the three-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK, and p38 pathways, inhibited network formation of these cells. Adenoviral-mediated overexpression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel compared with negative controls. This result suggested that the ERK pathway may not be essential for tube assembly, a conclusion which was supported by the action of specific MEK inhibitor PD 184352, which also did not alter network formation. Inhibition of the JNK pathway using SP-600125 or l-stereoisomer (l-JNKI-1) blocked network formation, whereas the p38 MAPK blocker SB-203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo.

Noble gases without anesthetic properties protect myocardium against infarction by activating prosurvival signaling kinases and inhibiting mitochondrial permeability transition in vivo.

BACKGROUND: The anesthetic noble gas, xenon, produces cardioprotection. We hypothesized that other noble gases without anesthetic properties [helium (He), neon (Ne), argon (Ar)] also produce cardioprotection, and further hypothesized that this beneficial effect is mediated by activation of prosurvival signaling kinases [including phosphatidylinositol-3-kinase, extracellular signal-regulated kinase, and 70-kDa ribosomal protein s6 kinase] and inhibition of mitochondrial permeability transition pore (mPTP) opening in vivo. METHODS: Rabbits (n = 98) instrumented for hemodynamic measurement and subjected to a 30-min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion received 0.9% saline (control), three cycles of 70% He-, Ne-, or Ar-30% O2 administered for 5 min interspersed with 5 min of 70% N2-30% O2 before LAD occlusion, or three cycles of brief (5 min) ischemia interspersed with 5 min reperfusion before prolonged LAD occlusion and reperfusion (ischemic preconditioning). Additional groups of rabbits received selective inhibitors of phosphatidylinositol-3-kinase (wortmannin; 0.6 mg/kg), extracellular signal-regulated kinase (PD 098059; 2 mg/kg), or 70-kDa ribosomal protein s6 kinase (rapamycin; 0.25 mg/kg) or mPTP opener atractyloside (5 mg/kg) in the absence or presence of He pretreatment. RESULTS: He, Ne, Ar, and ischemic preconditioning significantly (P < 0.05) reduced myocardial infarct size [23% +/- 4%, 20% +/- 3%, 22% +/- 2%, 17% +/- 3% of the left ventricular area at risk (mean +/- sd); triphenyltetrazolium chloride staining] versus control (45% +/- 5%). Wortmannin, PD 098059, rapamycin, and atractyloside alone did not affect infarct size, but these drugs abolished He-induced cardioprotection. CONCLUSIONS: The results indicate that noble gases without anesthetic properties produce cardioprotection by activating prosurvival signaling kinases and inhibiting mPTP opening in rabbits.

Emerging mechanisms for growth and protection of the vasculature by cytochrome P450-derived products of arachidonic acid and other eicosanoids.

Arachidonic acid (AA) is an essential fatty acid that is metabolized by cyclooxygenase (COX), lipoxygenase (LOX) or cytochrome P450 (CYP) enzymes to generate eicosanoids which in turn mediate a number of biological activities including regulation of angiogenesis. While much information on the effects of COX and LOX products is known, the physiological relevance of the CYP-derived products of AA are less well understood. CYP enzymes are highly expressed in the liver and kidney, but have also been detected at lower levels in the brain, heart and vasculature. A number of these enzymes, including members of the CYP 4 family, predominantly catalyze conversion of AA to 20-hydroxyeicosatetraenoic acid (20-HETE) while the CYP epoxygenases generate mainly epoxyeicosatrienoic acids (EETs). This review will focus on the emerging roles of inhibitors of eicosanoid production with emphasis on the CYP pathways, in the regulation of angiogenesis and tumor growth. We also discuss current observations describing the protective effects of EETs for survival of the endothelium.

Inhibition of apoptotic protein p53 lowers the threshold of isoflurane-induced cardioprotection during early reperfusion in rabbits.

INTRODUCTION: Exposure to isoflurane before and during early reperfusion protects against myocardial infarction by activating phosphatidylinositol-3-kinase (PI3K)-mediated signaling. The apoptotic protein, p53, is regulated by PI3K, and inhibition of p53 protects against ischemic injury. We tested the hypothesis that p53 inhibition lowers the threshold of isoflurane-induced postconditioning in vivo. METHODS: Rabbits (n = 73) instrumented for hemodynamic measurement and subjected to a 30-min left anterior descending coronary artery occlusion and 3-h reperfusion received 0.9% saline (control), isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) administered for 3 min before and 2 min after reperfusion, the p53 inhibitor pifithrin-alpha (1.5 or 3.0 mg/kg), or 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha. Other rabbits received 3.0 mg/kg pifithrin-alpha or 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha after pretreatment with the selective PI3K inhibitor wortmannin (0.6 mg/kg) or the mitochondrial permeability transition pore opener atractyloside (5 mg/kg). RESULTS: Isoflurane (1.0 but not 0.5 MAC), pifithrin-alpha (3.0 but not 1.5 mg/kg), and the combination of 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha significantly (P < 0.05) reduced infarct size (21% +/- 4%, 43% +/- 7%, 22% +/- 4%, 45% +/- 4%, and 28% +/- 3% [mean +/- sd], respectively, of left ventricular area at risk; triphenyltetrazolium chloride staining) when compared with control (45% +/- 2%). Atractyloside, but not wortmannin, abolished 3.0 mg/kg pifithrin-alpha-induced cardioprotection, whereas atractyloside and wortmannin blocked reductions in infarct size produced by 0.5 MAC isoflurane plus 1.5 mg/kg pifithrin-alpha. CONCLUSION: The results indicate that inhibition of the apoptotic protein p53 lowers the threshold of isoflurane-induced cardioprotection during early reperfusion in vivo.

Reactive oxygen species cerebral autoregulation in health and disease.

Reactive oxygen species (ROS) are a family of oxygen-derived free radicals that are produced in mammalian cells under normal and pathologic conditions. Many ROS, such as the superoxide anion (O2-) and hydrogen peroxide (H2O2), act as cellular signaling molecules within blood vessels, altering mechanisms mediating mechanical signal transduction and autoregulation of cerebral blood flow. This article focuses on the actions of ROS, such as O2.- and H2O2, and how they influence mechanisms responsible for the modulation of pressure-induced myogenic tone in the cerebral circulation and blood flow autoregulation in response to elevated arterial pressure. ROS may be a key target for therapeutic interventions in pediatric patients who have hypoxic injury or altered cerebral metabolism induced by trauma or infection.