Detecting Validated Intracellular ROS Generation with 18F-dihydroethidine-Based PET.

PURPOSE: To determine the sensitivity of the 18F-radiolabelled dihydroethidine analogue ([18F]DHE) to ROS in a validated ex vivo model of tissue oxidative stress. PROCEDURES: The sensitivity of [18F]DHE to various ROS-generating systems was first established in vitro. Then, isolated rat hearts were perfused under constant flow, with contractile function monitored by intraventricular balloon. Cardiac uptake of infused [18F]DHE (50-150 kBq.min-1) was monitored by γ-detection, while ROS generation was invoked by menadione infusion (0, 10, or 50 µm), validated by parallel measures of cardiac oxidative stress. RESULTS: [18F]DHE was most sensitive to oxidation by superoxide and hydroxyl radicals. Normalised [18F]DHE uptake was significantly greater in menadione-treated hearts (1.44 ± 0.27) versus control (0.81 ± 0.07) (p < 0.05, n = 4/group), associated with concomitant cardiac contractile dysfunction, glutathione depletion, and PKG1α dimerisation. CONCLUSION: [18F]DHE reports on ROS in a validated model of oxidative stress where perfusion (and tracer delivery) is unlikely to impact its pharmacokinetics.

Live-Cell Assessment of Reactive Oxygen Species Levels Using Dihydroethidine.

Reactive oxygen species (ROS) play an important role in cellular (patho)physiology. Empirical evidence suggests that mitochondria are an important source of ROS, especially under pathological conditions. Here, we describe a method for ROS measurement using dihydroethidium (HEt) and live-cell microscopy.

Measurement of Superoxide Production in Acute Hypoxia by Fixed-Cell Microscopy.

Redox signaling implication in cell adaptation to hypoxia has been studied for a long time, both in long-term and acute responses. However, measurement of superoxide and other reactive oxygen species (ROS) in acute hypoxia is technically challenging, for example, because of the need to overcome the effect of cell reoxygenation before measurement.Here we describe a method we have developed for measuring superoxide production in acute hypoxia using the fluorescent probe dihydroethidine in fixed-cell microscopy. The method allows measuring the kinetics of superoxide production (or other ROS with the appropriate probes) by incubating the probe in different time windows during hypoxia incubation.

The effect of dihydropyrazines on lipopolysaccharide-stimulated human hepatoma HepG2 cells via regulating the TLR4-MyD88-mediated NF-κB signaling pathway.

Dihydropyrazines (DHPs), including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are glycation products that are spontaneously generated in vivo and ingested via food. DHPs generate various radicals and reactive oxygen species (ROS), which can induce the expression of several antioxidant genes in HepG2 cells. However, detailed information on DHP-response pathways remains elusive. To address this issue, we investigated the effects of DHP-3 on the nuclear factor-κB (NF-κB) pathway, a ROS-sensitive signaling pathway. In lipopolysaccharide-stimulated (LPS-stimulated) HepG2 cells, DHP-3 decreased phosphorylation levels of inhibitor of NF-κB (IκB) and NF-κB p65, and nuclear translocation of NF-κB p65. In addition, DHP-3 reduced the expression of Toll-like receptor 4 (TLR4) and the adaptor protein myeloid differentiation primary response gene 88 (MyD88). Moreover, DHP-3 suppressed the mRNA expression of tumor necrosis factor-alpha (TNFα), and interleukin-1 beta (IL-1ß). Taken together, these results suggest that DHP-3 acts as a negative regulator of the TLR4-MyD88-mediated NF-κB signaling pathway.

Getting to the Heart of the Matter: Migraine, Triptans, DHE, Ditans, CGRP Antibodies, First/Second-Generation Gepants, and Cardiovascular Risk.

PREMISE: The science of migraine pathophysiology has advanced significantly since the 1930's. Imaging techniques, neurochemical analysis, clinical trials, and the clinical experience of providers treating migraine patients have not only sharpened our understanding of the disease, but have also led to the development of novel neural-based targets. Targeted therapies such as calcitonin gene-related peptide (CGRP) antibodies and "Second Generation" CGRP receptor antagonists (Gepants) have not only demonstrated efficacy, but have not resulted in any significant cardiovascular nor other serious adverse events. "First Generation" Gepants were associated with liver toxicity. PROBLEM: Triptans and dihydroergotamine (DHE) are contraindicated in patients with hemiplegic and basilar migraine based on theories of migraine pathophysiology from the 1930s. While our understanding of migraine has evolved substantially, perceived concerns of safety from almost a century ago continue to preclude their use in certain patient populations. POTENTIAL SOLUTION: While migraine aura was once thought to be primarily due to vasoconstriction, current evidence debunks this concept. For instance, hemiplegic migraine is the consequence of genetic mutations resulting in channelopathies without evidence of cerebral ischemia or infarction. Evidence of basilar artery constriction as postulated in basilar migraine is also lacking. This recognition has led the International Headache Society to rename basilar-type migraine to migraine with brainstem aura. The following discussion reviews current literature with respect to migraine as a neuronal disorder, as well as the published data on the safety of triptans, DHE, Ditans (a novel class of 5-HT1f receptor agonists), CGRP antibodies, and Gepants.

Multiorgan Development of Oxidative and Nitrosative Stress in LPS-Induced Endotoxemia in C57Bl/6 Mice: DHE-Based In Vivo Approach.

Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O2 ∙-) abundance in vivo by quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E+), as well as ethidium (E+) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide (∙NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E+ varied in the liver, lung, and kidney; however, the ratios of 2-OH-E+/E+ and 2-OH-E+/DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of ∙NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of ∙NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased ∙NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E2). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O2 ∙- production in vivo. Furthermore, the ratio of 2-OH-E+/E+ oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O2 ∙--mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.

Protective effect of herbal medicine Huangqi decoction against chronic cholestatic liver injury by inhibiting bile acid-stimulated inflammation in DDC-induced mice.

BACKGROUND: Huangqi decoction (HQD), a classic traditional herbal medicine, has been used for liver fibrosis, but its effect on intrahepatic chronic cholestatic liver injury remains unknown. PURPOSE: In the present study, we investigated the hepatoprotective effect of HQD and the underlying molecular mechanisms in 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine (DDC)-induced chronic cholestatic mice. METHODS: The DDC-induced cholestatic mice were administrated HQD for 4 or 8 weeks. Serum biochemistry and morphology were investigated. The serum and liver bile acid (BA) levels were detected by ultra performance liquid chromatography-tandem mass spectrometry. The liver expression of BA metabolizing enzymes and transporters, and inflammatory and fibrotic markers was measured by real-time polymerase chain reaction, western blotting, and immunohistochemistry. RESULTS: HQD treatment for 4 or 8 weeks ameliorated DDC-induced liver injury by improving impaired hepatic function and tissue damage. HQD treatment for 8 weeks further decreased the liver expression of cytokeratin 19, tumor growth factor (TGF)-ß, collagen I, and α-smooth muscle actin, and ameliorated ductular reaction and liver fibrosis. HQD markedly decreased the accumulation of serum and liver BA. The expression of BA-metabolizing enzymes, cytochrome P450 2b10 and UDP glucuronosyltransferase 1 A1, and multidrug resistance-associated protein 2, Mrp3, and Mrp4 involved in BA homeostasis was increased by 4 weeks of HQD treatment. The expression of BA uptake transporter Na+-taurocholate cotransporting polypeptide was decreased and that of Mrp4 was increased after 8 weeks of HQD treatment. Nuclear factor-E2-related factor-2 (Nrf2) was remarkably induced by HQD treatment. Additionally, HQD treatment for 8 weeks decreased the liver expression of inflammatory factors, interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, monocyte chemoattractant protein-1, and intracellular adhesion molecule-1. HQD suppressed the nuclear factor (NF)-κB pathway. CONCLUSION: HQD protected mice against chronic cholestatic liver injury and biliary fibrosis, which may be associated with the induction of the Nrf2 pathway and inhibition of the NF-κB pathway, ameliorating BA-stimulated inflammation.

Nrf2 Activation Ameliorates Hepatotoxicity Induced by a Heme Synthesis Inhibitor.

Transcription factor Nrf2 protects hepatocytes against various toxicants by upregulating cytoprotective genes. The heme synthesis inhibitor 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) leads to liver injury around the portal vein, unlike other groups of toxicants that cause hemorrhage and necrosis in the centrilobular area. To examine whether and how Nrf2 protects livers from the injury, we fed DDC to Nrf2 knockout (Nrf2KO), wild-type (WT), Keap1flox/flox (Keap1-knockdown; Keap1KD), and liver-specific Keap1 knockout (Keap1-Alb) mice, as these lines of mice exhibit stepwise increases in Nrf2 protein expression levels. Liver-specific Keap1::Nrf2 double-knockout (Keap1::Nrf2-Alb) mice were also exploited to examine the contribution of Nrf2. Two weeks after DDC feeding, Keap1-Alb mice were fully recovered from body weight loss, but the WT and Nrf2KO mice were not. The liver-to-body-weight ratio of Keap1-Alb mice was significantly larger than that of WT and Nrf2KO mice. Two indicators of hepatotoxicity, alanine aminotransferase and bilirubin in plasma, were both elevated in WT mice, but downregulated in Keap1-Alb mice after the DDC-feeding. DDC-induced porphyrin accumulation was reduced in the livers of Keap1-Alb and Keap1KD mice compared with that of WT mice. When assessed by the Nqo1 level, Nrf2 expression was further enhanced by DDC in Keap1-Alb mice, suggesting that DDC may have a Keap1 independent potential to activate Nrf2. Genetic activation of Nrf2 in Keap1-Alb mice increased the extracellular excretion of porphyrins, but contrary to our expectation, hepatic damages in Nrf2KO mice appeared to be similar to that of WT mice. Based on these observations, we conclude that Nrf2 activation protects livers against DDC-elicited hepatotoxicity.

Nuclear Translocation of RELB Is Increased in Diseased Human Liver and Promotes Ductular Reaction and Biliary Fibrosis in Mice.

BACKGROUND & AIMS: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction. METHODS: We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin ß (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (CyldΔLPC mice and Cyld/RelbΔLPC mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl4 to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB. RESULTS: In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of CyldΔLPC mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/RelbΔLPC mice. Compared with livers from control mice, livers from CyldΔLPC mice (but not Cyld/RelbΔLPC mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl4 exposure. CyldΔLPC mice and Cyld/RelbΔLPC mice had improved liver function on the DDC diet compared with control mice fed the DDC diet. CONCLUSION: Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in CyldΔLPC mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.

Smooth muscle glucose metabolism promotes monocyte recruitment and atherosclerosis in a mouse model of metabolic syndrome.

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor-deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.

Sarcopenia in a mice model of chronic liver disease: role of the ubiquitin-proteasome system and oxidative stress.

Sarcopenia is the loss of muscle mass and strength produced by aging or secondary to chronic diseases such as chronic liver disease (CLD). Although not all types of sarcopenia involve the same features, the most common are decreased fiber diameter and myosin heavy chain (MHC) levels, increased activity of ubiquitin-proteasome system (UPS) and reactive oxygen species (ROS). In this study, we aim to characterize the development of sarcopenia secondary to CLD induced by the hepatotoxin 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). For this purpose, four-months-old male C57BL6 mice were fed with normal diet or DDC supplemented diet for 6 weeks. Functional tests to evaluate muscle strength, mobility, and motor skills were performed in alive mice. The muscle strength in isolated gastrocnemius was also assayed via electrophysiological measurements. Morphometric measures of fibers' diameter, total and ubiquitinated protein levels of myosin heavy chain (MHC), E3 ubiquitin ligases, ROS, and oxidation-dependent modified proteins in gastrocnemius tissue were also determined. Our results demonstrated that mice fed the DDC diet developed muscle wasting as evidenced by a loss of muscle mass and decreased muscle strength. The muscles of mice fed with DDC diet have a decreased diameter of fibers and MHC levels, also as increased MuRF-1 and atrogin-1 protein levels, ROS levels, and oxidation-modified protein levels. Additionally, control and DDC mice have the same food and water intake as well as mobility. Our results demonstrate mice with CLD develop sarcopenia involving decreased levels of myofibrillar proteins, increased UPS, and oxidative stress, but not for impaired caloric intake or immobility.

Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP.

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIß triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.

Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis.

Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

Live-cell assessment of mitochondrial reactive oxygen species using dihydroethidine.

Reactive oxygen species (ROS) play an important role in both physiology and pathology. Mitochondria are an important source of the primary ROS superoxide. However, accurate detection of mitochondrial superoxide especially in living cells remains a difficult task. Here, we describe a method and the pitfalls to detect superoxide in both mitochondria and the entire cell using dihydroethidium (HEt) and live-cell microscopy.

Microporation is an efficient method for siRNA-induced knockdown of PEX5 in HepG2 cells: evaluation of the transfection efficiency, the PEX5 mRNA and protein levels and induction of peroxisomal deficiency.

The pathomechanism of peroxisomal biogenesis disorders (PBDs), a group of inherited autosomal recessive diseases with mutations of peroxin (PEX) genes, is not yet fully understood. Therefore, several knockout models, e.g., the PEX5 knockout mouse, have been generated exhibiting a complete loss of peroxisomal function. In this study, we wanted to knockdown PEX5 using the siRNA technology (1) to mimic milder forms of PBDs in which the mutated peroxin has some residual function and (2) to analyze the cellular consequences of a reduction of the PEX5 protein without adaption during the development as it is the case in a knockout animal. First, we tried to optimize the transfection of the hepatoma cell line HepG2 with PEX5 siRNA using different commercially available liposomal and non-liposomal transfection reagents (Lipofectamine(®) 2000, FuGENE 6, HiPerFect(®), INTERFERin™, RiboJuice™) as well as microporation using the Neon™ Transfection system. Microporation was found to be superior to the transfection reagents with respect to the transfection efficiency (100 vs. 0-70%), to the reduction of PEX5 mRNA (by 90 vs. 0-50%) and PEX5 protein levels (by 70 vs. 0-50%). Interestingly, we detected that a part of the cleaved PEX5 mRNA still existed as 3' fragment (15%) 24 h after microporation. Using microporation, we further analyzed whether the reduced PEX5 protein level impaired peroxisomal function. We indeed detected a reduced targeting of SKL-tagged proteins into peroxisomes as well as an increased oxidative stress as found in PBD patients and respective knockout mouse models. Knockdown of the PEX5 protein and functional consequences were at a maximum 48 h after microporation. Thereafter, the PEX5 protein was resynthesized, which may allow the temporal analysis of the loss as well as the reconstitution of peroxisomes in the future. In conclusion, we propose microporation as an efficient and reproducible method to transfect HepG2 cells with PEX5 siRNA. We succeeded to transiently knockdown PEX5 mRNA and its protein level leading to functional consequences similar as observed in peroxisome deficiencies.

FIP200 is required for maintenance and differentiation of postnatal neural stem cells.

Despite recent studies showing that inhibition of autophagy depletes the hematopoietic stem cell pool and increases intracellular reactive oxygen species (ROS), it remains unknown whether autophagy is essential in the maintenance of other stem cells. Moreover, it is unclear whether and how the aberrant ROS increase causes depletion of stem cells. Here we report that ablation of FIP200 (also known as Rb1cc1), a gene essential for autophagy induction in mammalian cells, results in a progressive loss of neural stem cells (NSCs) and impairment in neuronal differentiation specifically in the postnatal brain, but not the embryonic brain, in mice. The defect in maintaining the postnatal NSC pool was caused by p53-dependent apoptotic responses and cell cycle arrest. However, the impaired neuronal differentiation was rescued by treatment with the antioxidant N-acetylcysteine but not by p53 inactivation. These data reveal that FIP200-mediated autophagy contributes to the maintenance and functions of NSCs through regulation of oxidative state.

The physiological response of the marine platyhelminth Macrostomum lignano to different environmental oxygen concentrations.

The respiration rate of meiofauna is difficult to measure, and the response to variations in the environmental oxygen concentration has so far been mainly addressed through behavioral investigation. We investigated the effect of different oxygen concentrations on the physiology of the marine platyhelminth Macrostomum lignano. Respiration was measured using batches of 20 animals in a glass microtiter plate equipped with optical oxygen sensor spots. At higher oxygen saturations (>12 kPa), the animals showed a clear oxyconforming behavior. However, below this value, the flatworms kept respiration rates constant at 0.064±0.001 nmol O2 l(-1) h(-1) individual(-1) down to 3 kPa PO2, and this rate was increased by 30% in animals that were reoxygenated after enduring a period of 1.5 h in anoxia. Physiological changes related to tissue oxygenation were assessed using live imaging techniques with different fluorophores in animals maintained in normoxic (21 kPa), hyperoxic (40 kPa) or near-anoxic (~0 kPa) conditions and subjected to anoxia-reoxygenation. The pH-sensitive dyes Ageladine-A and BCECF both indicated that pHi under near-anoxia increases by about 0.07-0.10 units. Mitochondrial membrane potential, Δψm, was higher in anoxic and hyperoxic than in normoxic conditions (JC1 dye data). Staining with ROS-sensitive dyes - DHE for detection of superoxide anion (O2•(-)) formation and C-H DFFDA for other ROS species aside from O2•(-) (H2O2, HOO• and ONOO) - showed increased ROS formation following anoxia-reoxygenation treatment. Animals exposed to hyperoxic, normoxic and anoxic treatments displayed no significant differences in O2•(-) formation, whereas mitochondrial ROS formation as detected by C-H2DFFDA was higher after hyperoxic exposure and lowest under near-anoxia conditions compared with the normoxic control group. Macrostomum lignano seems to be a species that is tolerant of a wide range of oxygen concentrations (being able to maintain aerobic metabolism from extremely low PO2 up to hyperoxic conditions), which is an essential prerequisite for successfully dealing with the drastic environmental oxygen variations that occur within intertidal sediments.

Iodinated contrast media differentially affect afferent and efferent arteriolar tone and reactivity in mice: a possible explanation for reduced glomerular filtration rate.

PURPOSE: To determine the effect of the iodinated contrast medium iodixanol on arteriolar tone in afferent and efferent arterioles of the glomerulus and the functional interactions with the major modulators of arteriolar tone, angiotensin II and nitric oxide, in mice. MATERIALS AND METHODS: Animal handling conformed to the ethics guidelines of the Office for Health and Social Matters of Berlin. Arterioles were isolated from 136 C57BL/6 mice, perfused with either vehicle solution or iodixanol (23 mg of iodine per milliliter) for 20 minutes, followed by angiotensin II administration. Fluorescence of 3-amino-4-(N-methylamino)-2',7'-difluorofluorescein (DAF-FM) and dihydroethidium (DHE) were used for quantification of nitric oxide bioavailability and superoxide concentration, respectively. Statistical analysis of time- and dose-dependent data was performed by using the nonparametric test for repeated measurements. RESULTS: With iodixanol, afferent arteriole diameters were significantly reduced from 9.2 µm to 8.3 µm; in control group, the diameters were increased from 8.7 µm to 9.3 µm (P = .008). Nitric oxide synthase inhibition augmented iodixanol-induced constriction, with diameters reduced from 9.9 µm to 5.8 µm (P < .0001). DAF-FM fluorescence increased less during iodixanol treatment and nitric oxide synthase inhibition (3.6% and 3.7% vs 10.7% in control group, P = .009 and P = .049, respectively), indicating impaired nitric oxide bioavailability. With iodixanol, DHE fluorescence ratio was increased by 12% (P < .0001). Angiotensin II responses were enhanced by iodixanol and by nitric oxide synthase inhibition after perfusion with iodixanol (3.3 µm and 4.3 µm vs 7.5 µm [control group] with 1 × 10(-6)/mol/L angiotensin II, P = .03 for both). In contrast, in efferent arterioles, neither their basal diameters nor the responses to angiotensin II were significantly affected by iodixanol. CONCLUSION: A more pronounced effect of iodixanol on afferent than on efferent arterioles may contribute to the reduction of glomerular filtration rate in contrast medium-induced acute kidney injury. Decreased nitric oxide bioavailability and increased concentration of superoxide explain the increased tone and reactivity in afferent arterioles perfused with iodixanol.

Superoxide production during ischemia-reperfusion in the perfused rat heart: a comparison of two methods of measurement.

Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia-reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223-38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal.

4-Isoxazolyl-1,4-dihydropyridines exhibit binding at the multidrug-resistance transporter.

The 4-isoxazolyl-dihydropyridines (IDHPs) exhibit inhibition of the multidrug-resistance transporter (MDR-1), and exhibit an SAR distinct from their activity at voltage gated calcium channels (VGCC). Among the four most active IDHPs, three were branched at C-5 of the isoxazole, including the most active analog, 1k.