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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]

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.

A letter to the editor is presented in response to the article "Hypoxic pulmonary vasoconstriction is/is not mediated by increased production of reactive oxygen species," in the September 2006 issue.

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]

Carotid body (CB) type I cells sense the blood Po 2 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 O 2 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 p47 phox 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/p47 phox tandem to alter type I cell potassium channels and therewith CND.

Carotid body (CB) type I cell hypoxia-sensing function is assumed to be based on potassium channel inhibition. Subsequent membrane depolarization initiates an intracellular calcium increase followed by transmitter release for excitation of synapses with linked nerve endings. Several reports, however, contradict this generally accepted concept by showing that type I cell oxygen-sensing properties vary significantly depending on the method of their isolation. We report therefore for the first time noninvasive mapping of the oxygen-sensing properties of type I cells within the intact adult mouse CB ex vivo by using multifocal Nipkow disk-based imaging of oxygen-, calcium- and potential-sensitive cellular dyes. Characteristic type I cell clusters were identified in the compact tissue by immunohistochemistry because of their large cell nuclei combined with positive tyrosine hydroxylase staining. The cellular calcium concentrations in these cell clusters either increased or decreased in response to reduced tissue oxygen concentrations. Under control conditions, cellular potential oscillations were uniform at ∼0.02 Hz. Under hypoxia-induced membrane depolarization, these oscillations ceased. Simultaneous increases and decreases in potential of these cell clusters resulted from spontaneous burstlike activities lasting ∼1.5 s. type I cells, identified during the experiments by cluster formation in combination with large cell nuclei, seem to respond to hypoxia with heterogeneous kinetics.

Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.