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Effect of p47^phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells

¹Department of Physiology and ²Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah; and ³Departamento de Bioquimica y Biologia Molecular y Fisiologia/Instituto de Biología y Genética Molecular Facultad de Medicina, Universidad de Valladolid y Consejo Superior de Investigaciones Científicas, Valladolid, Spain

Submitted 11 January 2005 ; accepted in final form 12 July 2005

Membrane potential in oxygen-sensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K⁺ channels. Coupling of PO₂ to the open-closed state of channels may involve production of reactive oxygen species (ROS) by NADPH oxidase. One hypothesis suggests that ROS are produced in proportion to the prevailing PO₂ and a subset of K⁺ channels closes as ROS levels decrease. We evaluated ROS levels in normal and p47^phox gene-deleted [NADPH oxidase knockout (KO)] type I cells using the ROS-sensitive dye dihydroethidium (DHE). In normal cells, hypoxia elicited an increase in ROS, which was blocked by the specific NADPH oxidase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF, 3 mM). KO type I cells did not respond to hypoxia, but the mitochondrial uncoupler azide (5 µM) elicited increased fluorescence in both normal and KO cells. Hypoxia had no effect on ROS production in sensory and sympathetic neurons. Methodological control experiments showed that stimulation of neutrophils with a cocktail containing the chemotactic peptide N-formyl-Met-Leu-Phe (1 µM), arachidonic acid (10 µM), and cytochalasin B (5 µg/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 µM). KO neutrophils did not respond; however, azide (5 µM) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K⁺ current and increased intracellular Ca²⁺ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca²⁺ and enhanced the depression of K⁺ current in low O₂. Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K⁺ channel activity in hypoxia.

reactive oxygen species; potassium channels; cellular redox; dihydroethidium; cell calcium

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