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Carotid body physiology meets cytochrome c oxidase crystallography Commentary to Ortega-Sáenz P, López-Barneo J. Physiology of the Carotid Body: From Molecules to Disease. Changing gas composition of the super-fusion medium from 20% O SB 2 sb , 3% CO SB 2 sb , 77% N SB 2 sb (normoxia) to 97% N SB 2 sb , 3% CO SB 2 sb (full hypoxia) is followed by a characteristic hypoxia-induced increased chemoreceptor sinus nerve activity (registration in black) serving as control. 1 CcO crystallographic changes of about 3 Ångström are displayed by CB chemoreceptor discharge. a Chemoreceptor discharge under normoxic and hypoxic CO application (red registration line) The effect of CO is shown as a percentage of peak chemoreceptor activity induced by 4-min hypoxia. [Extracted from the article]

Carotid body (CB) type I cells sense the blood PO2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp-NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND. [ABSTRACT FROM AUTHOR]

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells. Microsc. Res. Tech., 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1α and its constitutive β subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophorelabeled HIF-1 subtmits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1α than of HIF-1β, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between α and β subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models. [ABSTRACT FROM AUTHOR]

Abstract: Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-α activity and plasma membrane potassium-channel (PMPC) conductivity. [Copyright &y& Elsevier]

Structural and functional integrity of brain function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of brain function. In particular, reductions in oxygen availability (hypoxia) caused by systemic or local blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply to the brain by anaerobic glycolysis. Hypoxia has been implicated in central nervous system pathology in a number of disorders including stroke, head trauma, neoplasia and neurodegenerative disease. Complex cellular oxygen sensing systems have evolved for tight regulation of oxygen homeostasis in the brain. In response to variations in oxygen partial pressure (PO2 these induce adaptive mechanisms to avoid or at least minimize brain damage. A significant advance in our understanding of the hypoxia response stems from the discovery of the hypoxia inducible factors (HIF), which act as key regulators of hypoxia-induced gene expression. Depending on the duration and severity of the oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy saving and cellular protection mechanisms. Hypoxic adaptation encompasses an immediate depolarization block by changing potassium, sodium and chloride ion fluxes across the cellular membrane, a general inhibition of protein synthesis, and HIF-mediated upregulation of gene expression of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival or neural stem cell growth. However, sustained and prolonged activation of the HIF pathway may lead to a transition from neuroprotective to cell death responses. This is reflected by the dual features of the HIF system that include both anti- and proapoptotic components. These various responses might be based on a range of oxygen-sensing signal cascades, including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low PO2 affinity cytochrome c oxidase (aa3) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF prolyl-hydroxylase (PHD) and HIF asparaginyl hydroxylase, known as factor-inhibiting HIF (FIH-1). Thus specific oxygen-sensing cascades, by means of their different oxygen sensitivities, cell-specific and subcellular localization, may help to tailor various adaptive responses according to differences in tissue oxygen availability. [ABSTRACT FROM AUTHOR]

Examines the hypoxia-inducible factor1 (HIF-1) as an essential transcription factor for cellular adaptation to decreased oxygen availability. Decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP; Activation of a contransfected hypoxia-responsive luciferase reporter gene; Use of three-dimensional 2-photon confocal fluorescence microscopy.

The effect of natural and recombinant interferons (nIFN, rIFN) on cell growth, apoptosis and the production of vascular endothelial growth factor (VEGF) was investigated in the human melanoma cell line IGR 1. We determined cell proliferation, cell vitality, DNA synthesis, apoptosis, intracellular oxygen radicals (ROS) and VEGF-mRNA as well as VEGF-protein levels. rIFN-γ significantly inhibited growth by decreasing DNA synthesis and increasing apoptosis. Less pronounced was the growth inhibitory effect of nIFN-β because an increased rate of apoptosis was outweighed by enhanced DNA synthesis. nIFN-α only had minor effects on cell growth parameters. Under long-term incubation (144 h) nIFN-β decreased, but rIFN-γ increased production of the angiogen VEGF. Our data underscore the multiple effects of IFNs on melanoma cells and may contribute to the understanding of ambivalent results of melanoma therapy by IFNs. Particularly, the increased VEGF production under long-term treatment with serum IFN levels between 100 and 1200 IU/ml should be kept in mind. [ABSTRACT FROM AUTHOR]

Confocal microscopy, image deconvolution and computer-assisted methods have been used to reconstruct the three-dimensional (3-D) distribution of tubulin in cells. The techniques were applied to tumor cells growing under regular culture conditions (planar cultivation) and those penetrating into reconstituted collagen matrices (spatial cultivation). As expected the application of deconvolution algorithms enhanced the resolution of results. The deconvolution using the maximum likelihood estimation included the measurement of the point spread function of the optical setup. The data visualization of the resulting data sets uses volume as well as surface rendering approaches. The 3-D reconstruction gives a clear image of the spatial arrangement of tubulin fibers in relation to cell shape and position of other cellular organelles, particularly the nucleus. The tubulin forms an intricate network of fibers of variable thickness. The highest tubulin concentrations appear in the cell periphery and particularly in pseudopodia/invadopodia. This is indicative of an enhanced transport of intracellular material facilitating cell movement and lysis of the extracellular matrix. The investigation is assumed to stimulate further experiments using a variety of techniques leading to the complete understanding of the spatial architecture of living cells.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cobalt and nickel stimulate, as does hypoxia, the production of erythropoietin (EPO) in HepG2 cells. Under hypoxic conditions, a decrease in the level of intracellular reactive oxygen species (ROS) is thought to stimulate EPO expression. Cobalt and nickel may interact with the putative oxygen sensor by changing the redox state of the central iron atom of heme proteins, similar to the effects of hypoxia. It was investigated, therefore, whether cobalt and nickel interact with hemeproteins or ROS scavenging systems in the control of intracellular ROS level. Cobalt chloride (100 μM, 24 h) oxidized non respiratory as well respiratory hemeproteins and increased the oxygen consumption. In contrast, nickel chloride (300 μM, 24 h) primarily reduced respiratory hemeproteins and decreased the oxygen consumption. In HepG2 cells treated with CoCl2, iron and cobalt were localized in cytosolic granules close to the cell nucleus and in mitochondria at concentrations up to 12 mM or 41 mM, respectively. Intracellular nickel was not measurable. Three-dimensional reconstruction of confocal laser microscopy images revealed hot spots of hydroxyl radical generation by a Fenton reaction at the sites of cytosolic iron accumulation. The OH levels decreased in cobalt-treated (to 81 %) as well as in nickel-treated (to 67 %) HepG2 cells, accompanied by an increase of EPO expression to 167 % and 150 %, respectively. Our results underline the importance of OH formed by a Fenton reaction for triggerimg EPO production. Identification of the primary hemeprotein being the oxygen sensor was not possible due to the antagonistic effects of cobalt and nickel on the redox state of detectable hemeproteins. [ABSTRACT FROM AUTHOR]

Small intensely fluorescent (SIF) cells are paraganglionic cells derived from sympathicoblasts which may serve as interneurons, endo-/paracrine cells or arterial chemoreceptors within sympathetic ganglia. Like paraganglionic cells of other locations, e.g., carotid body glomus cells, they are responsive to hypoxia. Recent studies on glomus cells and other hypoxia-sensing cells suggested the involvement of a b 558-type cytochrome and intracellular generation of H2O2 in the process of oxygen sensing. In the present study, we demonstrate the occurrence of the small subunit of cytochrome b 558, p22phox, in SIF cells of guinea-pig sympathetic ganglia by immunohistochemistry using two different antisera. H2O2 production was monitored in explanted intact superior cervical ganglia of 2-day-old rats by confocal laser scanning analysis of rhodamine 123 fluorescence generated due to oxidation of dihydrorhodamine 123 by H2O2. Using this technique, SIF cell clusters appeared as sites of highest H2O2 production within the ganglia. Thus, SIF cells exhibit two key features of an oxidase system generating reactive oxygen species. This may be involved in the proposed chain of events in oxygen sensing, but alternative cellular functions of this system have also to be considered. [ABSTRACT FROM AUTHOR]

A PCO electrode working on the principle of electrical conductivity is described. The calibration curve can be linearized according to the formula $$G = G_0 + b\sqrt {P{\text{CO}}_2 } $$ . This linearity has been tested in the PCO range of 0.93-9.33 kPa (7-70 Torr). For the experiments electrodes are used which have conductivity values of about 50 nS and drifts of maximally 5%/h at a PCO of 5.33 kPa (40 Torr). The response time ( T) is about 20 s. The temperature sensitivity is 2.4 nS/1 K between 298K-310K. The standard error of the measurements is σ=0.33 nS. With these electrodes tissue PCO can be measured on the surface of various organs. [ABSTRACT FROM AUTHOR]

The NAD(P)H cytochrome b5 oxidoreductase, Ncb5or (previously named b5+b5R), is widely expressed in human tissues and broadly distributed among the animal kingdom. NCB5OR is the first example of an animal flavohemoprotein containing cytochrome b5 and chrome b5 reductase cytodomains. We initially reported human NCB5OR to be a 487-residue soluble protein that reduces cytochrome c, methemoglobin, ferricyanide, and molecular oxygen in vitro. Bioinformatic analysis of genomic sequences suggested the presence of an upstream start codon. We confirm that endogenous NCB5OR indeed has additional NH2-terminal residues. By performing fractionation of subcellular organelles and confocal microscopy, we show that NCB5OR colocalizes with calreticulin, a marker for endoplasmic reticulum. Recombinant NCB5OR is soluble and has stoichiometric amounts of heme and flavin adenine dinucleotide. Resonance Raman spectroscopy of NCB5OR presents typical signatures of a six-coordinate low-spin heme similar to those found in other cytochrome b5 proteins. Kinetic measurements showed that full-length and truncated NCB5OR reduce cytochrome c actively in vitro. However, both full-length and truncated NCB5OR produce superoxide from oxygen with slow turnover rates: kcat = ∼0.05 and ∼1 s-1, respectively. The redox potential at the heme center of NCB5OR is -108 mV, as determined by potentiometric titrations. Taken together, these data suggest that endogenous NCB5OR is a soluble NAD(P)H reductase preferentially reducing substrate(s) rather than transferring electrons to molecular oxygen and therefore not an NAD(P)H oxidase for superoxide production. The subcellular localization and redox properties of NCB5OR provide important insights into the biology of NCB5OR and the phenotype of the Ncb5or-null mouse. [ABSTRACT FROM AUTHOR]