Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O₂ levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O₂ imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O₂ in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O₂ levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O₂ in 3D tissue models.     

Zur Temperaturaktivierung der Substratatmung tierischer Gewebe

Summary (1) The influence of temperature on tissue respiration of frog skin and rat liver, stimulated by succinate and pyruvate, was studied. The temperatures ranged from 17.5 to 47.5°C.(2) The activation-energy () of the substrate-stimulated respiration was significantly lower than that of the endogeneous O₂-uptake. Therefore, in the submaximal range of temperature, the relative activity of the stimulated respiration decreased with increasing temperatures.     

Increased mitochondrial palmitoylcarnitine/carnitine countertransport by flavone causes oxidative stress and apoptosis in colon cancer cells

Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria, which is followed by an increase in O₂^−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and the subsequent metabolic generation of O₂^−. in mitochondria is the initiating factor for the execution of apoptosis. Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005     

Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress

We investigated the effects of ischemia duration on the functional response of mitochondria to reperfusion and its relationship with changes in mitochondrial susceptibility to oxidative stress. Mitochondria were isolated from hearts perfused by the Langendorff technique immediately after different periods of global ischemia or reperfusion following such ischemia periods. Rates of O₂ consumption and H₂O₂ release with complex I- and complex II-linked substrates, lipid peroxidation, overall antioxidant capacity, capacity to remove H₂O₂, and susceptibility to oxidative stress were determined. The effects of ischemia on some parameters were time dependent so that the changes were greater after 45 than after 20 min of ischemia, or were significantly different to the nonischemic control only after 45 min of ischemia. Thus, succinate-supported state 3 respiration exhibited a significant decrease after 20 min of ischemia and a greater decrease after 45 min, while pyruvate malate-supported respiration showed a significant decrease only after 45 min of ischemia, indicating an ischemia-induced early inhibition of complex II and a late inhibition of complex I. Furthermore, both succinate and pyruvate malate-supported H₂O₂ release showed significant increases only after 45 min of ischemia. Similarly, whole antioxidant capacity significantly increased and susceptibility to oxidants significantly decreased after 45 min of ischemia. Such changes were likely due to the accumulation of reducing equivalents, which are able to remove peroxides and maintain thiols in a reduced state. This condition, which protects mitochondria against oxidants, increases mitochondrial production of oxyradicals and oxidative damage during reperfusion. This could explain the smaller functional recovery of the tissue and the further decline of the mitochondrial function after reperfusion following the longer period of oxygen deprivation. Received 18 May 2001; received after revision 17 July 2001; accepted 24 July 2001     

Imaging of oxygen and hypoxia in cell and tissue samples

Molecular oxygen (O₂) is a key player in cell mitochondrial function, redox balance and oxidative stress, normal tissue function and many common disease states. Various chemical, physical and biological methods have been proposed for measurement, real-time monitoring and imaging of O₂ concentration, state of decreased O₂ (hypoxia) and related parameters in cells and tissue. Here, we review the established and emerging optical microscopy techniques allowing to visualize O₂ levels in cells and tissue samples, mostly under in vitro and ex vivo, but also under in vivo settings. Particular examples include fluorescent hypoxia stains, fluorescent protein reporter systems, phosphorescent probes and nanosensors of different types. These techniques allow high-resolution mapping of O₂ gradients in live or post-mortem tissue, in 2D or 3D, qualitatively or quantitatively. They enable control and monitoring of oxygenation conditions and their correlation with other biomarkers of cell and tissue function. Comparison of these techniques and corresponding imaging setups, their analytical capabilities and typical applications are given.     

Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold-induced oxidative stress

We determined characteristics of rat liver mitochondrial fractions, resolved at 1000 (M₁), 3000 (M₃), and 10,000 g (M₁₀) after 2 and 10 days cold exposure. In all groups, the M₁ fraction exhibited the highest oxidative capacity, oxidative damage, H₂O₂ production rate, and susceptibility to stress conditions, and the lowest antioxidant levels. Cold exposure increased cytochrome oxidase activity in all fractions and succinate-supported O₂ consumption in the M₁ and M₁₀ fractions during state 3 and state 4 respiration, respectively. With succinate, the H₂O₂ release rate increased in all fractions during state 4 and state 3 respiration, whereas with pyruvate/malate, it increased only during state 4 respiration. Increases in tissue mitochondrial proteins caused a faster H₂O₂ flow from the mitochondrial to cytosolic compartment, which was limited by the reduction in the M₁ fraction. Despite increased liposoluble antioxidant levels, cold also caused enhanced oxidative damage and susceptibility to oxidative challenge and Ca²⁺-induced swelling in all fractions. These changes leading to elimination of H₂O₂-overproducing mitochondria avoided excessive tissue damage. We propose that triiodothyronine, whose levels increase in the cold environment, brings about the biochemical changes producing oxidative damage and those limiting its extent.Received 16 July 2004; received after revision 27 September 2004; accepted 18 October 2004     

Wip1 protects hydrogen peroxide-induced colonic epithelial barrier dysfunction

Tight junctions (TJs) create a paracellular permeability barrier. Although reactive oxygen species have been implicated as mediators of inflammation in inflammatory bowel diseases, their influence on the function of colonic epithelial TJs remains unknown. Oxidative stress-mediated colonic epithelial permeability was significantly attenuated by a p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Although the amount of TJ proteins was not altered, hydrogen peroxide (H₂O₂) changed the localization of claudin-4 protein from an NP-40 insoluble fraction to a soluble fraction and from an apical TJ to lateral membrane. The p38 MAP kinase inactivator Wip1 significantly attenuated phosphorylation of p38 MAP kinase, and oxidative stress mediated permeability. H₂O₂-induced changes in claudin-4 localization were abolished by SB203580 pretreatment as well as Wip1-expressing adenovirus infection. This is the first study to demonstrate that exogenous Wip1 functions to protect oxidative stress-mediated colonic mucosal permeability and that H₂O₂-induced claudin-4 dislocalization is abolished by Wip1. Received 14 June 2007; received after revision 8 October 2007; accepted 8 October 2007     

Superoxide dismutase activity during the plasmodial life cycle ofPhysarum polycephalum

Summary Superoxide dismutase activity was slow throughout the cell cycle of surface cultures ofPhysarum polycephalum. This activity increased markedly when the organism was induced to spherulate. Glutathione (GSH) and hydrogen peroxide (H₂O₂) concentrations changed very little during the cell cycle. During spherulation GSH decreased; H₂O₂ and the cyanide-resistant respiration of plasmodial homogenates increased.     

EP4 receptor stimulation down-regulates human eosinophil function

Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE₂ receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca²⁺ mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE₂ and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.     

Is there a critical tissue oxygen tension for bioenergetic status and cellular pH regulation in solid tumors?

Bioenergetic and metabolic status have been correlated with tissue oxygenation in murine fibrosarcomas (FSaII) of varying sizes (44–600 mm³). Ratios of -nucleoside triphosphates to inorganic phosphate (NTP/P_i) and phosphocreatine to inorganic phosphate (PCr/P_i) ratios derived from³¹P nuclear magnetic resonance spectroscopy (NMR) were positively correlated to median tissue O₂ tension (pO₂) values using O₂-sensitive needle electrodes. pH declined during growth with intracellular acidosis being evident in tumors >350 mm³. Whereas lactic acid formation greatly contributed to this decline in small and medium-sized tumors, adenosine triphosphate (ATP) hydrolysis and slowing down of the activities of pumps involved in cellular pH regulation seem to be major factors responsible for intracellular acidification in bulky tumors. PCr levels decreased at an early growth stage, whilst ATP concentrations dropped in bulky malignancies only, coinciding with a decrease in adenylate energy charge and a substantial rise in the levels of total P_i On average, median pO₂ values of ca. 10 mmHg represent a critical threshold for energy metabolism. At higher median O₂ tensions, levels of ATP, phosphomonoester (PME) and total P_i were relatively constant. This coincided with intracellular alkalosis or neutrality and stable adenylate ratios. On average, median pO₂ values <10 mmHg coincided with intracellular acidosis, ATP depletion, a drop in energy charge and rising P_i levels.     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2^+/+ and CerS2^?/? mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2^?/? mice developed more severe disease than CerS2^+/+ mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2^?/? mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2^?/? mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2^?/? mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4⁺ and Th17⁺ T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.     

Stimulation by D-glucose of mitochondrial respiration

Summary D-glucose increases O₂ uptake by cerebellum mitochondria. This effect is abolished by D-glucose-6-phosphate and D-mannoheptulose. It is proposed that the phosphorylation of D-glucose as catalyzed by bound hexokinase directly affects mitochondrial respiration.     

Ein Beitrag zur irreversiblen Temperaturinaktivierung der Gewebsatmung

Summary (1) Tissue slices of the liver and pieces of the skin of summer-frogs were incubated at 47.5°C for 30–150 min. Their respiration was measured during this incubation period and thereafter at a temperature of 37.5°C.(2) As a sign of an irreversible inactivation of the enzymes, the average respiration was inhibited. However, after certain periods of incubation at 47.5°C, over-shoot phenomena in the O₂-uptake at the temperature of 37.5°C were seen.     

The oxygen consumption of some tissues from hypophysectomized goldfish,Carassius auratus L.

Summary Hypophysectomy has no effect on the O₂ consumption of minced brain and white muscle tissue, while liver tissue shows a marked reduction. This reduction in liver O₂ consumption is attributed to the increased glycogen content that follows hypophysectomy which has the effect of increasing the nonmetabolizing dry weight component of the cells.Supported by the National Research Council of Canada, Grant Number A-3744 to P. H. J.     

ATP-induced Ca<Superscript>2+</Superscript>-signalling mechanisms in the regulation of mesenchymal stem cell migration

The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca²⁺ is a primary second messenger in the control and regulation of a wide range of cell functions including cell migration. Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca²⁺ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca²⁺ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca²⁺ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), albeit with different kinetics and capacity. Reduction in the ER Ca²⁺ level following the P2Y receptor activation can further induce store-operated Ca²⁺ entry as a distinct Ca²⁺ influx pathway that contributes in ATP-induced increase in the [Ca²⁺]_c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca²⁺ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.     

Tagesperiodik des O2-Umsatzes der Rattenleber

Summary There is a 24 h periodicity of the rat liver tissue respiration (Qo₂) with maximal values at about 07.30 h and minimal values at about 17.00 h.

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Auslandsstipendiat der Schweizerischen Regierung (1963/64).

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Die Arbeit wurde unterstützt von der Max-Planck-Gesellschaft zur Förderung der Wissenschaften, Deutschland.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production inTrypanosoma cruzi andCrithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis inTrypanosoma cruzi and stimulated O₂ uptake and generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H₂O₂ production were obtained withCrithidia fasciculata despite the presence of catalase in this organism.Acknowledgments. This work was aided by grants from the University of Buenos Aires, the Scientific Office of the American States Organization and CEDIQUIFA (Buenos Aires). S.G.G. and M.P.M.P. are Research Fellows and A.O.M.S. is Career Investigator of CONICET (Argentina). L. T. Viñas and M. G. Gutierrez lent able technical assistance.     

Thiamine transport by human intestine in vitro

Summary Surgical specimens of human gastrointestinal mucosa and muscle were incubated in vitro with thiaminethiazole-2-¹⁴C. Labelled thiamine uptake was uphill in mucosal tissues and downhill in muscle. Small intestinal mucosa accumulated labelled thiamine in a phosphorylated form, while gastric and colonic mucosa, as well as muscular layers of all gastrointestinal segments studied, did not.This work was supported in part by a grant from the Italian Research Council, Rome, Italy.     

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H₂O₂). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H₂O₂ applied. Moderate doses of H₂O₂ enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H₂O₂-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H₂O₂-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H₂O₂-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H₂O₂. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H₂O₂, while all H₂O₂-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H₂O₂ plays an important role in the induced tolerance against oxidative stress. Received 11 December 2001; received after revision 25 January 2002; accepted 4 February 2002