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

Abstract

. Effect of p47 phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells. Am J Physiol Lung Cell Mol Physiol 289: L916 -L924, 2005; doi:10.1152/ajplung.00015.2005.-Membrane potential in oxygensensitive type I cells in carotid body is controlled by diverse sets of voltage-dependent and -independent K ϩ 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 PO 2 and a subset of K ϩ 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 M) 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-formylMet-Leu-Phe (1 M), arachidonic acid (10 M), and cytochalasin B (5 g/ml) elicited a rapid increase in DHE fluorescence. This response was blocked by the NADPH oxidase inhibitor diphenyleneiodonium (10 M). KO neutrophils did not respond; however, azide (5 M) elicited a rapid increase in fluorescence. Physiological studies in type I cells demonstrated that hypoxia evoked an enhanced depression of K ϩ current and increased intracellular Ca 2ϩ levels in KO vs. normal cells. Moreover, AEBSF potentiated hypoxia-induced increases in intracellular Ca 2ϩ and enhanced the depression of K ϩ current in low O 2. Our findings suggest that local compartmental increases in oxidase activity and ROS production inhibit the activity of type I cells by facilitating K ϩ channel activity in hypoxia.reactive oxygen species; potassium channels; cellular redox; dihydroethidium; cell calcium EXPOSURE OF CAROTID BODY ARTERIAL chemoreceptors to lowered PO 2 elicits increased neural activity in the carotid sinus nerve (CSN) and reflex cardiopulmonary adjustments that mitigate the adverse effects of hypoxemia. Increased carotid body activity occurs at relatively moderate PO 2 levels, in contrast to the severe hypoxia required to elicit metabolic and functional adjustments in non-O 2 -sensing tissues (19,20,23). Efforts to understand the molecular physiology of hypoxic chemotransduction in the carotid body have focused primarily on the relationship between PO 2 and the activity of K ϩ channels in neuroectoderm-derived type I cells (23,40). In the rat, multiple studies have demonstrated that hypoxia inhibits two types of K ϩ channels: 1) a large-conductance, voltage-and calciumdependent channel [maxiK (39, 50)] and 2) a voltage-independent TASK-type channel that me...

Pharmacological inhibition of neuronal NADPH oxidase protects against 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells

“…In these studies, the neuroprotective properties of apocynin and DPI were associated with inhibition of microglial NADPH oxidase activity, but the role of neuronal NADPH oxidase was not investigated. The neuroprotective effects observed in this study with AEBSF, another NADPH oxidase inhibitor, are also in agreement with studies demonstrating AEBSF prevents NADPH oxidase-induced ROS generation, and cytotoxic and apoptotic cell death in non-neuronal tissues (He et al, 2005;Polytarchou and Papadimitriou, 2005).…”

Chemoreception in the context of the general biology of ROS

“…Nonetheless, there are some observations in our previous experiments (He et al, 2005) and in those of Kevin et al (2003) that do not fit the reliability concept, i.e., that dihydroethidium, upon its reaction with ROS (mostly O 2 •− ), generates fluorescent oxidized products which remain trapped inside the cells and therefore quantification of the fluorescence yields a measurement of the amount of ROS produced. In both of these studies it has been found that short pulses of sodium azide or full ischemia generated fluorescent signals that disappear on the elimination of the sodium azide or on restitution of the flow.…”

“…First, based in preliminary evidence (He et al, 2004, also unpublished), we have included CB chemoreceptor cells as a specific place of expression of Nox4 because we have immunocytochemically located it in chemoreceptor cells and found that its transcription rate increases in animals exposed to a 10% atmosphere for 7 days. Second, we ascribe a role to Nox4 in oxygen chemoreception because we have provided evidence that NADPH oxidase-derived (Nox4) derived ROS act as negative second messengers of the hypoxic transduction cascade in chemoreceptor cells and the overall oxygen chemoreception process (He et al, 2005; see article by Dinger et al, 2007). It should be mentioned that in addition to Nox4 we have searched for Nox2 in CB sections and found that it does not co-localize with the chemoreceptor cell marker tyrosine hydroxylase, its expression being restricted to the stroma of the organ.…”

“…In this regard, there are two groups of recent data that deserve some comments. The first group of data was obtained by comparing responses in chemoreceptor cells from control and knockout mice for the p47phox subunit of NADPH oxidase (He et al, 2005). Mice CB chemoreceptor cell K + currents are in a large percentage sensitive to iberiotoxin, indicating that they are carried through maxi-K channels, although other Kv channels, some of them sensitive to hypoxia, have also been described in mice cells (Perez-Garcia et al, 2004).…”

“…In chemoreceptor cells, the only measurement of ROS production has been performed by He et al (2005;see Fig. 7 in Dinger et al, 2007).…”