Redox signaling in acute oxygen sensing

Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Review article Redox signaling in acute oxygen sensing Lin Gao a,b,c,⁎ , Patricia González-Rodríguez a,b,c,1 , Patricia Ortega-Sáenz a,b,c , José López-Barneo a,b,c,⁎ a Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain b Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, Seville, Spain c Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Seville, Spain ARTICLE INFO Keywords: Hypoxia Acute oxygen sensing Peripheral chemoreceptors Carotid body Adrenal medulla Mitochondrial complex I Reactive oxygen species (ROS) Pyridine nucleotides ABSTRACT Acute oxygen (O 2 ) sensing is essential for individuals to survive under hypoxic conditions. The carotid body (CB) is the main peripheral chemoreceptor, which contains excitable and O 2 -sensitive glomus cells with O 2 -regulated ion channels. Upon exposure to acute hypoxia, inhibition of K + channels is the signal that triggers cell depolarization, transmitter release and activation of sensory fibers that stimulate the brainstem respiratory center to produce hyperventilation. The molecular mechanisms underlying O 2 sensing by glomus cells have, however, remained elusive. Here we discuss recent data demonstrating that ablation of mitochondrial Ndufs2 gene selectively abolishes sensitivity of glomus cells to hypoxia, maintaining responsiveness to hypercapnia or hypoglycemia. These data suggest that reactive oxygen species and NADH generated in mitochondrial complex I during hypoxia are signaling molecules that modulate membrane K + channels. We propose that the structural substrates for acute O 2 sensing in CB glomus cells are “O 2 -sensing microdomains”formed by mitochondria and neighboring K + channels in the plasma membrane. 1. Introduction Oxygen (O 2 ) is necessary for oxidative phosphorylation, the major source of energy for the cells; therefore, the provision of sufficient O 2 to the tissues is a fundamental physiological challenge. Deficiency of O 2 (hypoxia), even if only transient, can have detrimental effects and critically contribute to the pathogenesis of severe and highly prevalent diseases in the human population. Adaptive responses, which can be acute or chronic, have evolved to minimize the effect of hypoxia on cells. During chronic (sustained) hypoxia, the hypoxia inducible factorproline hydroxylase pathway is activated to regulate the transcription of numerous “O 2 -sensitive”genes. As a result of this process, in the time course of hours or days cell aerobic metabolism is switched to nonaerobic, to decrease cellular needs of O 2 , and the number of red blood cells and blood vessels are increased to improve O 2 transport and distribution to the tissues [54,57]. In mammals, hypoxia also triggers fast (in seconds) life-saving cardiorespiratory reflexes (hyperventilation and sympathetic activation) (Fig. 1A) to increase gas exchange in the lungs and rapid delivery of O 2 to critical organs, such as the brain and heart. These acute responses to hypoxia are mediated by the carotid body (CB), the main arterial chemoreceptor, which is strategically located in the carotid bifurcation (Fig. 1B) [35]. In addition to the CB, there are other peripheral chemoreceptor tissues/organs acutely sensing hypoxia (e.g. pulmonary arteries, ductus arteriosus, adrenal medulla (AM) or neuroepithelial bodies in the lung), which, together, constitute a “homeostatic acute O 2 -sensing system”of fundamental biological and medical relevance (see [71]). 2. Membrane model of CB oxygen sensing The CB is composed of clusters of cells (glomeruli), which contain neural crest-derived O 2 -sensitive glomus cells (also called type I cells) in close contact with capillaries and afferent sensory nerve fibers (Fig. 1C) (see for a review [37]). Glomus cells are presynaptic-like elements containing neurosecretory vesicles filled with neurotransmitters (ATP, acetylcholine and dopamine, among others) that are released in response to hypoxia to activate the afferent fibers connected with the brainstem respiratory center. The CB is the main responsible for the http://dx.doi.org/10.1016/j.redox.2017.04.033 Received 8 February 2017; Received in revised form 24 March 2017; Accepted 24 April 2017 ⁎ Correspondence to: Instituto de Biomedicina de Sevilla (IBiS), Campus Hospital Universitario Virgen del Rocío, Avenida Manuel Siurot, s/n, 41013 Seville, Spain. 1 Present address: Department of Physiology, Northwestern University, 303 E. Superior Street, Lurie 8-250, Chicago, IL 60611, USA. E-mail addresses: [email protected] (L. Gao), [email protected] (J. López-Barneo). Abbreviations: AM, adrenal medulla; CB, carotid body; ETC, electron transport chain; H 2 O 2 , hydrogen peroxide; MCI, mitochondrial complex I; MCII, mitochondrial complex II; MCIII, mitochondrial complex III; MCIV, Mitochondrial complex IV; MPP + , 1-methyl-4-phenylpyridinium; O 2 , oxygen; PO 2 , oxygen tension; QH 2 , ubiquinol; ROS, reactive oxygen species; TH, tyrosine hydroxylase Redox Biology 12 (2017) 908–915 Available online 26 April 2017 2213-2317/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). T acute hypoxic ventilatory response (HVR) (Fig. 1A), as this reflex is practically abolished after CB resection [63] or in animal models with CB atrophy (decrease in size and cell number) [39]. At the cellular level, the HVR is represented by the secretory response to hypoxia characteristic of single glomus cells, which can be monitored by amperometry (Fig. 1D). Genetic or pharmacological alterations that result in elimination of glomus cell responsiveness to hypoxia also abolish the HVR ([14]; see below). CB glomus cells are excitable cells that contain a variety of voltagegated and background ion channels (see for review [37]). It has been more than two decades since a “membrane model”of glomus cell chemotransduction was established (see [36,49,60]). The model is based on the existence in glomus cells of K + channels reversibly inhibited by the decrease in oxygen tension (PO 2 ), resulting in cell depolarization (see for a recent review [38]). This leads to an influx of extracellular Ca 2+ , which triggers the release of neurotransmitters from secretory vesicles in order to activate afferent sensory nerve fibers [5,66]. As a consequence, the respiratory center in the brainstem is activated leading to hyperventilation (Fig. 1E). In addition to hypoxia, CB glomus cells are activated by other chemosensory stimuli, including hypercapnia, decrease in extracellular pH, and hypoglycemia [16,19,47,75].O 2 -sensitive ion channels have also been reported in other organs of the “homeostatic acute O 2 -sensing system”, such as the AM [26,32,42,56,61], resistance pulmonary arteries [52,74], neuroepithelial bodies in the lung airway [43,73], or the ductus arteriosus [40]. Fig. 1. Membrane model of acute O 2 sensing by arterial chemoreceptor cells. A. Plethysmographic recording of hypoxic ventilatory response from a wildtype mouse. Note the increase in breathing frequency (breaths/min) as the % O 2 in air decreases. B. Photograph of the human carotid artery (CA) bifurcation after cleaning the surrounding fat and connective tissue. The arrow indicates the carotid body (scale bar: 1cm). C. Carotid body glomerulus with indication of glomus (type I) cells and sustentacular (type II) cells in contact with capillaries and afferent sensory fibers. V. vessel. D. Amperometric recording of the secretory response to hypoxia from carotid body glomus cells of a wildtype mouse. Each individual spike-like signal indicates a quantum of dopamine released from a secretory granule (pA, picoAmperes). The red color trace, in picoCoulombs (pC), represents the cumulative secretion signal (time integral of the recordings show at the top). E. Membrane model of acute O 2 sensing by arterial chemoreceptor cells. See text for details. (Modified from [14,34,46]). L. Gao et al. Redox Biology 12 (2017) 908–915 909 Although the “membrane model”explains the basic sensory function of the CB, the underlying molecular mechanisms have remained poorly understood and a subject of debate [30,37,38,50]. This partially results from the small tissue size of the CB, which makes it technically difficult to study, and the fact that the response to hypoxia can be easily lost in apparently healthy CB glomus cells. Although several hypotheses have been proposed to explain how changes in blood PO 2 lead to modulation of K + channel function (mitochondrial energy metabolism, redox signaling, gas transmitters, etc.), none of them has gained broad acceptance due to the lack of consensus among the workers in the field (see for updated reviews [37,38]). Recently, production of reactive oxygen species (ROS) by mitochondrial complex I (MCI) has been reported to play a critical role in CB acute O 2 sensing [14]. In the next section, we discuss the role of MCI signaling in acute O 2 sensing, with special attention to the roles of ROS and NADH in the signaling pathway. Although we focus on the CB, many of the concepts and processes discussed may be also applicable to cells in other organs acutely responding to hypoxia. 3. Mitochondria complex I signaling during hypoxia is mediated by ROS and pyridine nucleotides Mitochondria have long been considered a potential candidate in the signaling pathway of O 2 sensing due to the high O 2 consumption of the CB [10,59] and the fact that this organ is highly sensitive to mitochondrial poisons [44,72]. Similar to hypoxia (see Fig. 1E), glomus cell activation by mitochondrial inhibitors requires extracellular Ca 2+ influx [44]; hence hypoxic mitochondria could generate signaling molecules to regulate the function of K + channels in the plasma membrane. Indeed, it has been proposed that a decrease in cytosolic ATP concentration in glomus cells during hypoxia can modulate membrane K + channels [67,72]. Mitochondrial complex IV (MCIV) is the natural O 2 -dependent site in the electron transport chain (ETC). Therefore, changes in MCIV cytochrome c oxidase activity or its affinity to O 2 could also affect the sensibility to hypoxia. Although a lowaffinity cytochrome c oxidase has been suggested to exist in the CB, this hypothesis has not been proven experimentally (see below) [13,41,6]. Recent interest on the role of mitochondria in acute O 2 -sensing is based on studies performed using blockers of mitochondrial ETC [19,44]. It was shown that the response to acute hypoxia in rat CB glomus cells is efficiently blocked by MCI inhibitors, such as rotenone or 1-methyl-4-phenylpyridinium (MPP + ), acting on the quinone binding site. In contrast, MCI proximal inhibitors (e.g. diphenyliodonium), acting outside the distal ubiquinone-binding site, appeared to have much less effect [44]. Interestingly, mitochondrial inhibitors that abolish responsiveness to hypoxia do not alter sensitivity to hypoglycemia in glomus cells [19]. In agreement with the data obtained in glomus cells, it has been shown that functional mitochondria are required for O 2 sensing in immortalized AM chromaffin cells [7].In addition, the inhibition of sensitivity to hypoxia by rotenone was also observed in ovine [27] and rat [62] chromaffin cells. These findings led to the hypothesis that a rotenone-binding molecule is involved in acute Fig. 2. Loss of mitochondrial complex I (MCI) signaling during hypoxia in Ndufs2-deficient mice. A. Plethysmographic recording (left) and quantification (right) showing selective loss of hypoxic ventilatory response in Ndufs2 knockout mice. Responsiveness to hypercapnia (Hc, CO 2 ) remained unaltered. Nx, normoxia; Hx, hypoxia. B. Amperometric recording (left) and quantification (right) demonstrating selective loss of secretory response to hypoxia in Ndufs2-deficient glomus cells (pA, picoAmperes; pC, picoCoulombs). C, control; KO, knockout. C. Changes in NAD(P)H auto-fluorescence during hypoxia in control and Ndufs2-deficient carotid body glomus cells. D. Changes in ROS levels in the cytosol (left) and intermembrane mitochondrial space (IMS, right) during hypoxia in control and Ndufs2-deficient glomus cells infected with redox-sensitive fluorescent probes (roGFP). a.u., arbitrary units; **, p< 0.01. (Modified from [14]). L. Gao et al. Redox Biology 12 (2017) 908–915 910 O 2 sensing by peripheral chemoreceptors. Rotenone is known to block MCI by preventing ubiquinone binding to a site that is highly conserved from bacteria to mammals [15,4,77,78]. To test the involvement of MCI in acute O 2 -sensing, we generated mice with conditional ablation of the Ndufs2 gene. This nuclear gene encodes a 49 kDa protein that contributes to the ubiquinone-binding site, located at the junction between the peripheral and transmembrane arms of MCI near the most distal Fe/S cluster (N2 site) from where electrons are transferred to ubiquinone [15,25,4,77,78].In our initial experiments, the Ndufs2 gene was deleted in tyrosine hydroxylase (TH)-positive catecholaminergic cells, such as O 2 -sensitive CB glomus cells and AM chromaffin cells (TH-NDUFS2 mice; [14]). THNDUFS2 mice show a complete abolition of the HVR while maintaining normal responsiveness to hypercapnia (Fig. 2A). In fair correspondence with this observation at the whole animal level, hypoxia-induced catecholamine secretion is also abolished in single Ndufs2-deficient glomus cells, although they show normal responses to hypercapnia (Fig. 2B) and hypoglycemia [14]. The selective loss of responsiveness to hypoxia is also observed in ESR-NDUFS2 mice, in which the floxed Ndufs2 alleles are deleted after the administration of tamoxifen, once the mice have reached adulthood [14]. In addition, AMs from Ndufs2- deficient mice (TH-NDUFS2 and ESR-NDUFS2 models) also fail to respond to hypoxia. Interestingly, CB glomus cells in Ndufs2-null mice appear in normal number and have normal morphology, ATP levels, and density of voltage-dependent ion channels [14]. This suggests that Ndufs2-deficient glomus cells, without MCI function, rely on succinatedependent mitochondrial complex II (MCII) activity for oxidative phosphorylation. In agreement with this finding, abundant glomus cell loss has been observed in MCII-deficient mice [11,51]. MCI catalyzes the oxidation of NADH to NAD + and the reduction of ubiquinone to ubiquinol (QH 2 ). Therefore, pyridine nucleotides could be potential mitochondria signaling molecules to modulate membrane ion channels. This hypothesis is supported by early reports showing that the level of pyridine nucleotides (NADH and NADPH) in glomus cells, as determined by microfluorimetry, is reversibly increased during hypoxia [13,6]. In addition, we have shown that increases in NAD(P) H induced by hypoxia are abolished in Ndufs2-deficient glomus cells (Fig. 2C). Rotenone mimics hypoxia, as it induces external Ca 2+ -dependent secretion from glomus cells, and is also known to increase ROS production in MCI [44,65]. Therefore, mitochondria ROS production during hypoxia could also play a signaling role. It is long ago that a mitochondrial redox-based sensor was proposed to mediate hypoxic pulmonary artery vasoconstriction [2,31,69], although whether cytosolic ROS increases or decreases during hypoxia in pulmonary artery myocytes and other cell types has been a matter of controversy [20,23,70,71]. In the pulmonary artery, the main source of ROS generated during hypoxia has been suggested to be mitochondrial complex III (MCIII) [70]. On the other hand, non-mitochondrial ROS generated in NADPH oxidases have been reported to participate in acute O 2 sensing in several tissues [17,33,9]. In the CB setting, however, this proposal has not received strong experimental support [22]. In addition, ROS have traditionally been considered not involved in CB chemotransduction [1,72]. ROS are promiscuous agents and their determination in small cells is a long-standing methodological challenge. In recent years, a ratiometric redox probe, based on a genetically encoded green fluorescent protein (roGFP), has been developed [12,21,55]. Using roGFP targeted to either the cytosol or mitochondria intermembrane space we have been able to detect robust reversible increases in ROS in CB glomus cells exposed to hypoxia, which are absent, or drastically reduced in amplitude, in Ndufs2-deficient cells (Fig. 2D). Taken together, these results demonstrate that in Ndufs2-null mice the response to hypoxia is specifically lost and MCI is essential for acute O 2 sensing by peripheral chemoreceptors. Based on the data obtained from Ndufs2-null mice, we have proposed a mechanistic model of O 2 sensing in arterial chemoreceptor, which relies on the generation of MCI signaling molecules (ROS and NADH) during hypoxia capable of modulating membrane ion channels (see [14,38]). We suggest the existence in glomus cell mitochondria of a special form of MCIV whose enzymatic activity is decreased during hypoxia. This would slow down ETC and lead to accumulation of reduced quinone (QH 2 ), which in turn would cause an increase in NADH and in ROS production due to backlog accumulation of electrons in the iron/sulfur clusters of MCI (Fig. 3). When QH 2 reaches sufficiently high value, reversal of MCI (a state favored by high succinate-dependent quinone reduction [53,68,8] may potentiate the generation of ROS and NADH [14,38]. Recent data have shown that glomus cells express atypical mitochondrial subunits (Ndufa4l2 and Cox4i2), which could account for the high sensitivity of CB cells to hypoxia ([76]; our unpublished data). Cox4i2 is induced by low PO 2 in some cells to optimize O 2 consumption during hypoxia [18]. However, the role of Cox4i2 may change in different tissues (see [24]) and in the CB it may be its co-expression with Ndufa4l2, which confers upon MCIV special sensitivity to PO 2 . Indeed, Ndufa4 (the predominantly expressed isoform of Ndufa4l2) is expressed in close association with MCIV subunits [3,24]. In addition, we have shown that succinate concentration is higher in the normoxic CB than in other neural (central and peripheral) tissues studied [14], thus supporting the continuous production of QH 2 . This proposal is also compatible with the fact, mentioned before, that glomus cell survival strongly depends on succinate dehydrogenase (MCII) activity [11,51]. Although the model is compatible with the concomitant production of ROS in MCI and MCIII, the experimental data suggest that in glomus cells ROS produced outside MCI do not efficiently signal the plasma membrane to produce rise of cytosolic Ca 2+ or cell secretion. Blockade of glomus cell sensitivity to hypoxia by rotenone is not reverted by incubation of Fig. 3. Model of mitochondrial complex I signaling during hypoxia in carotid body glomus cells mediated by ROS and pyridine nucleotides (NADH). See text for description. Q, ubiquinone; QH 2 , ubiquinol; MCI, MCII, MCIII, MCIV, mitochondrial complex I, II, III, IV, respectively. (Modified from [38]). L. Gao et al. Redox Biology 12 (2017) 908–915 911 the cells with membrane permeable methyl succinate to favor respiration through the MCII-III-IV pathway [44]. Similarly, although methyl succinate can support respiration (O 2 consumption) in cells from THNDUFS2 mice, this treatment fails to rescue hypoxia sensitivity in Ndufs2-deficient glomus cells [14]. 4. Intracellular redox regulation of ionic currents in glomus cells The primary effect of hypoxia in glomus cells is to inhibit membrane K + channels to produce depolarization. CB glomus cells express a variety of O 2 -sensitive K + channels, including voltage-dependent K + channels, maxi-K channels, and background K + channels ([45], see for reviews [37,38]). Therefore, it seems that rather than a specificK + channel type, hypoxia regulates numerous K + channels and other ion channels over a broad range of membrane potentials [35,58]. As shown in Fig. 4A, hypoxia inhibits background channels, causing an increase in input resistance, and voltage-gated K + channels in CB glomus cells, both effects being reversible upon re-oxygenation. Interestingly, similar to hypoxia, intracellular application of oxidants H 2 O 2 and diamide (a thiol-oxidizing agent) result in an increase in input resistance whereas N-acetylcysteine (a reducing agent) has no effect (Fig. 4B). In the presence of intracellular H 2 O 2 or diamide the inhibitory effect of hypoxia on background channels is blocked, leaving unaltered the modulation of the current mediated by voltage-gated channels (Fig. 4C,D). These results, suggesting that cysteine oxidation inhibits the O 2 sensitive K + current in glomus cells, are compatible with the signaling role of ROS on membrane channels, in particular on background channels, which are the most critical for the regulation of the cell resting potential (see preceding section). Task3 homomers or Task1/Task3 heteromers seem to be the most abundant ion channel subunits contributing to the O 2 -sensitive background K + currents in CB glomus cells [28,64]; although acute responsiveness to hypoxia is maintained in the Task1/Task3 knockout mouse [45]. Interestingly, Task 3 subunits contain several cysteine residues facing the cytosol [29], which is compatible with the effects of sulfhydryl reagents described in Fig. 4. To our knowledge, the modulation of Task 3 channels by internal redox reagents has not been studied in detail, however the closely related TREK2 channels are inhibited by the intracellular application of oxidizing agents [48]. It remains also the possibility that in addition to the pore-forming subunits, accessory Fig. 4. Redox regulation of ionic currents in carotid body glomus cells. A. Hypoxia-induced reversible inhibition of background and voltage-dependent K + currents recorded during depolarizing ramps in patch clamped glomus cells (perforated patch recording). The initial part of the recordings (between −90 and −40 mV) is shown at an expanded amplitude and time base on the right part of the panel. Nx, normoxia, Hx, hypoxia, R, re-oxygenation. B. Top. Inhibition of the background current in glomus cells following intracellular application of H 2 O 2 (15 µM). Bottom. Increase in input resistance (inversely proportional to the slope of the background current) in glomus cells recorded with intracellular solutions containing: H 2 O 2 (15–50 µM), N-acetylcysteine (25 µM), or diamide (50–60 μM). C. and D. Blockage of hypoxia-induced closure of background K + channels by H 2 O 2 (15 µM) or diamide (50–60 μM). Recordings between −90 and −40 mV are shown at an expanded amplitude and time base *, p< 0.05; **, p< 0.01. (Modified from [14]). L. Gao et al. Redox Biology 12 (2017) 908–915 912 subunits modulating ion channel function are susceptible to redox regulation during hypoxia. NADH, the other signaling molecule produced in hypoxic mitochondria, seems to modulate preferentially voltage-dependent K + channels (see [14]). This concept is, however, preliminary and must be further investigated in future experimental work. 5. Conclusions We have proposed a model of acute O 2 sensing by peripheral chemoreceptors, which combines the standard membrane model based on the modulation of O 2 -sensitive K + channels [36], and the production of signaling molecules (ROS and NADH) in mitochondria [14].CB glomus cells contain numerous mitochondria located near the plasma membrane [14,51], therefore it is conceivable that they form with neighboring K + channels “O 2 -sensing microdomains”(Fig. 5). However, specialized morphological structures supporting this concept have not yet been described. Glomus cells contain several subtypes of “O 2 - sensitive”K + channels, hence it is logical that chemosensory transduction relies on relatively promiscuous signals (such as ROS or NADH) rather than on an O 2 sensor molecule selectively associated with a specificK + channel class (see [45,14]). Although fundamental advances have recently been made regarding the mechanism underlying acute O 2 sensing by peripheral chemoreceptors, several questions remain to be clarified by further research. In the TH-NDUFS2 mice, failure to form MCI poses an abnormal challenge to cell intermediary metabolism [14]. A mouse model defective in ubiquinone binding without affecting the MCI formation and activity, if available, could critically improve our understanding of the relationship between MCI and O 2 sensing. ROS and NADH/NAD + metabolism, oxidative phosphorylation, and energy production are closely linked in mitochondria. Therefore analytical experimental tools that could separate these processes would be also of great value to discern their specific role in acute O 2 sensing. Superoxide and H 2 O 2 react with different molecules and cause distinct molecular modifications. The specific ROS implicated in acute O 2 sensing and their direct molecular targets are not known. In this regard, thiol redox proteomics in CB tissue could eventually help to identify the targets of hypoxia-induced ROS. In addition, it remains to be determined whether during hypoxia ROS are generated only in MCI or if MCIII also participates in acute O 2 sensing by CB cells, as it seems to do in pulmonary arteries [70]. Finally, it cannot be ruled out the possibility that in CB glomus cells, as well as in other cells of the homeostatic acute O 2 sensing system [71], multiple O 2 -sensing mechanisms work in coordination to ensure a fast response to hypoxia. Advances in these questions will also help us understanding the role of the CB in hypoxia-mediated diseases. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by the Botín Foundation, Spanish Ministry of Economy, Industry, and Competitiveness (SAF2012-39343; PIE 13/ 0004), and the European Research Council (PRJ201502629). References [1] M.T. Agapito, G. Sanz-Alfayate, A. Gomez-Nino, C. Gonzalez, A. Obeso, General redox environment and carotid body chemoreceptor function, Am. J. Physiol. Cell Physiol. 296 (2009) C620–C631. [2] S.L. Archer, J. Huang, T. Henry, D. Peterson, E.K. Weir, A redox-based O 2 sensor in rat pulmonary vasculature, Circ. Res. 73 (1993) 1100–1112. [3] E. Balsa, R. Marco, E. Perales-Clemente, R. Szklarczyk, E. Calvo, M.O. Landazuri, J.A. Enriquez, NDUFA4 is a subunit of complex IV of the mammalian electron transport chain, Cell Metab. 16 (2012) 378–386. [4] R. Baradaran, J.M. Berrisford, G.S. Minhas, L.A. Sazanov, Crystal structure of the entire respiratory complex I, Nature 494 (2013) 443–448. [5] K.J. Buckler, R.D. Vaughan-Jones, Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells, J. Physiol. 476 (1994) 423–428. [6] K.J. Buckler, P.J. Turner, Oxygen sensitivity of mitochondrial function in rat arterial chemoreceptor cells, J. Physiol. 591 (2013) 3549–3563. [7] J. Buttigieg, S.T. Brown, M. Lowe, M. Zhang, C.A. Nurse, Functional mitochondria are required for O 2 but not CO 2 sensing in immortalized adrenomedullary chromaffin cells, Am. J. Physiol. Cell Physiol. 294 (2008) C945–C956. [8] E.T. Chouchani, et al., Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS, Nature 515 (2014) 431–435. [9] A.R. Cross, L. Henderson, O.T. Jones, M.A. Delpiano, J. Hentschel, H. Acker, Involvement of an NAD(P)H oxidase as a PO 2 sensor protein in the rat carotid body, Biochem. J. 272 (1990) 743–747. [10] Md.B. Daly, C.J. Lambertsen, A. Schweitzer, Observations on the volume of blood flow and oxygen utilization of the carotid body in the cat, J. Physiol. 125 (1954) 67–89. [11] B. Diaz-Castro, C.O. Pintado, P. Garcia-Flores, J. Lopez-Barneo, J.I. Piruat, Differential impairment of catecholaminergic cell maturation and survival by genetic mitochondrial complex II dysfunction, Mol. Cell Biol. 32 (2012) 3347–3357. [12] C.T. Dooley, T.M. Dore, G.T. Hanson, W.C. Jackson, S.J. Remington, R.Y. Tsien, Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators, J. Biol. Chem. 279 (2004) 22284–22293. [13] M.R. Duchen, T.J. Biscoe, Mitochondrial function in type I cells isolated from rabbit Fig. 5. Model of mitochondrial complex I-mediated acute O 2 sensing by arterial chemoreceptor cells. I, II, III, IV, mitochondrial complex I, II, III, IV, respectively; PO 2 , oxygen tension; QH 2 , ubiquinol; ΔVm, increase in membrane potential (depolarization). See text for details (Modified from [14]). L. Gao et al. Redox Biology 12 (2017) 908–915 913 arterial chemoreceptors, J. Physiol. 450 (1992) 13–31. [14] M.C. Fernandez-Aguera, L. Gao, P. Gonzalez-Rodriguez, C.O. Pintado, I. Arias- Mayenco, P. Garcia-Flores, A. Garcia-Perganeda, A. Pascual, P. Ortega-Saenz, J. Lopez-Barneo, Oxygen sensing by arterial chemoreceptors depends on mitochondrial complex I signaling, Cell Metab. 22 (2015) 825–837. [15] K. Fiedorczuk, J.A. Letts, G. Degliesposti, K. Kaszuba, M. Skehel, L.A. Sazanov, Atomic structure of the entire mammalian mitochondrial complex I, Nature 538 (2016) 406–410. [16] R.S. Fitzgerald, M. Shirahata, I. Chang, E. Kostuk, The impact of hypoxia and low glucose on the release of acetylcholine and ATP from the incubated cat carotid body, Brain Res. 1270 (2009) 39–44. [17] X.W. Fu, D. Wang, C.A. Nurse, M.C. Dinauer, E. Cutz, NADPH oxidase is an O 2 sensor in airway chemoreceptors: evidence from K + current modulation in wildtype and oxidase-deficient mice, Proc. Natl. Acad. Sci. USA 97 (2000) 4374–4379. [18] R. Fukuda, H. Zhang, J.W. Kim, L. Shimoda, C.V. Dang, G.L. Semenza, HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells, Cell 129 (2007) 111–122. [19] M. Garcia-Fernandez, P. Ortega-Saenz, A. Castellano, J. Lopez-Barneo, Mechanisms of low-glucose sensitivity in carotid body glomus cells, Diabetes 56 (2007) 2893–2900. [20] R.B. Hamanaka, N.S. Chandel, Mitochondrial reactive oxygen species regulate hypoxic signaling, Curr. Opin. Cell Biol. 21 (2009) 894–899. [21] G.T. Hanson, R. Aggeler, D. Oglesbee, M. Cannon, R.A. Capaldi, R.Y. Tsien, S.J. Remington, Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators, J. Biol. Chem. 279 (2004) 13044–13053. [22] L. He, J. Chen, B. Dinger, K. Sanders, K. Sundar, J. Hoidal, S. Fidone, Characteristics of carotid body chemosensitivity in NADPH oxidase-deficient mice, Am. J. Physiol. Cell Physiol. 282 (2002) C27–C33. [23] P. Hernansanz-Agustin, A. Izquierdo-Alvarez, F.J. Sanchez-Gomez, E. Ramos, T. Villa-Pina, S. Lamas, A. Bogdanova, A. Martinez-Ruiz, Acute hypoxia produces a superoxide burst in cells, Free Radic. Biol. Med. 71 (2014) 146–156. [24] B. Kadenbach, M. Huttemann, The subunit composition and function of mammalian cytochrome c oxidase, Mitochondrion 24 (2015) 64–76. [25] N. Kashani-Poor, K. Zwicker, S. Kerscher, U. Brandt, A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I, J. Biol. Chem. 276 (2001) 24082–24087. [26] D.J. Keating, G.Y. Rychkov, M.L. Roberts, Oxygen sensitivity in the sheep adrenal medulla: role of SK channels, Am. J. Physiol. Cell Physiol. 281 (2001) C1434–C1441. [27] D.J. Keating, G.Y. Rychkov, P. Giacomin, M.L. Roberts, Oxygen-sensing pathway for SK channels in the ovine adrenal medulla, Clin. Exp. Pharmacol. Physiol. 32 (2005) 882–887. [28] D. Kim, E.J. Cavanaugh, I. Kim, J.L. Carroll, Heteromeric TASK-1/TASK-3 is the major oxygen-sensitive background K+ channel in rat carotid body glomus cells, J. Physiol. 587 (2009) 2963–2975. [29] Y. Kim, H. Bang, D. Kim, TASK-3, a new member of the tandem pore K(+) channel family, J. Biol. Chem. 275 (2000) 9340–9347. [30] P. Kumar, N.R. Prabhakar, Peripheral chemoreceptors: function and plasticity of the carotid body, Compr. Physiol. 2 (2012) 141–219. [31] R.M. Leach, H.M. Hill, V.A. Snetkov, T.P. Robertson, J.P. Ward, Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor, J. Physiol. 536 (2001) 211–224. [32] J. Lee, W. Lim, S.Y. Eun, S.J. Kim, J. Kim, Inhibition of apamin-sensitive K + current by hypoxia in adult rat adrenal chromaffin cells, Pflug. Arch. 439 (2000) 700–704. [33] Y.M. Lee, B.J. Kim, Y.S. Chun, I. So, H. Choi, M.S. Kim, J.W. Park, NOX4 as an oxygen sensor to regulate TASK-1 activity, Cell Signal. 18 (2006) 499–507. [34] J. Lopez-Barneo, Oxygen and glucose sensing by carotid body glomus cells, Curr. Opin. Neurobiol. 13 (2003) 493–499. [35] J. Lopez-Barneo, R. Pardal, P. Ortega-Saenz, Cellular mechanism of oxygen sensing, Annu Rev. Physiol. 63 (2001) 259–287. [36] J. Lopez-Barneo, J.R. Lopez-Lopez, J. Urena, C. Gonzalez, Chemotransduction in the carotid body: K + current modulated by PO 2 in type I chemoreceptor cells, Science 241 (1988) 580–582. [37] J. Lopez-Barneo, P. Gonzalez-Rodriguez, L. Gao, M.C. Fernandez-Aguera, R. Pardal, P. Ortega-Saenz, Oxygen sensing by the carotid body: mechanisms and role in adaptation to hypoxia, Am. J. Physiol. Cell Physiol. 310 (2016) C629–C642. [38] J. Lopez-Barneo, P. Ortega-Saenz, P. Gonzalez-Rodriguez, M.C. Fernandez-Aguera, D. Macias, R. Pardal, L. Gao, Oxygen-sensing by arterial chemoreceptors: mechanisms and medical translation, Mol. Asp. Med. 47–48 (2016) 90–108. [39] D. Macias, M.C. Fernandez-Aguera, V. Bonilla-Henao, J. Lopez-Barneo, Deletion of the von Hippel-Lindau gene causes sympathoadrenal cell death and impairs chemoreceptor-mediated adaptation to hypoxia, EMBO Mol. Med. 6 (2014) 1577–1592. [40] E.D. Michelakis, I. Rebeyka, X. Wu, A. Nsair, B. Thebaud, K. Hashimoto, J.R. Dyck, A. Haromy, G. Harry, A. Barr, S.L. Archer, O 2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor, Circ. Res. 91 (2002) 478–486. [41] E. Mills, F.F. Jobsis, Mitochondrial respiratory chain of carotid body and chemoreceptor response to changes in oxygen tension, J. Neurophysiol. 35 (1972) 405–428. [42] N. Mochizuki-Oda, Y. Takeuchi, K. Matsumura, Y. Oosawa, Y. Watanabe, Hypoxiainduced catecholamine release and intracellular Ca 2+ increase via suppression of K + channels in cultured rat adrenal chromaffin cells, J. Neurochem. 69 (1997) 377–387. [43] I. O'Kelly, R.H. Stephens, C. Peers, P.J. Kemp, Potential identification of the O 2 - sensitive K+ current in a human neuroepithelial body-derived cell line, Am. J. Physiol. 276 (1999) L96–L104. [44] P. Ortega-Saenz, R. Pardal, M. Garcia-Fernandez, J. Lopez-Barneo, Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells, J. Physiol. 548 (2003) 789–800. [45] P. Ortega-Saenz, K.L. Levitsky, M.T. Marcos-Almaraz, V. Bonilla-Henao, A. Pascual, J. Lopez-Barneo, Carotid body chemosensory responses in mice deficient of TASK channels, J. Gen. Physiol. 135 (2010) 379–392. [46] P. Ortega-Saenz, R. Pardal, K. Levitsky, J. Villadiego, A.B. Munoz-Manchado, R. Duran, V. Bonilla-Henao, I. Arias-Mayenco, V. Sobrino, A. Ordonez, M. Oliver, J.J. Toledo-Aral, J. Lopez-Barneo, Cellular properties and chemosensory responses of the human carotid body, J. Physiol. 591 (2013) 6157–6173. [47] R. Pardal, J. Lopez-Barneo, Low glucose-sensing cells in the carotid body, Nat. Neurosci. 5 (2002) 197–198. [48] K.S. Park, H. Bang, E.Y. Shin, C.H. Kim, Y. Kim, The Inhibition of TREK2 channel by an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid), via interaction with the C- terminus distal to the 353rd amino acid, Korean J. Physiol. Pharmacol. 12 (2008) 211–216. [49] C. Peers, Hypoxic suppression of K+ currents in type I carotid body cells: selective effect on the Ca2(+)-activated K+ current, Neurosci. Lett. 119 (1990) 253–256. [50] C. Peers, Acute oxygen sensing–Inching ever closer to an elusive mechanism, Cell Metab. 22 (2015) 753–754. [51] A. Platero-Luengo, S. Gonzalez-Granero, R. Duran, B. Diaz-Castro, J.I. Piruat, J.M. Garcia-Verdugo, R. Pardal, J. Lopez-Barneo, An O 2 -sensitive glomus cell-stem cell synapse induces carotid body growth in chronic hypoxia, Cell 156 (2014) 291–303. [52] J.M. Post, J.R. Hume, S.L. Archer, E.K. Weir, Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction, Am. J. Physiol. 262 (1992) C882–C890. [53] K.R. Pryde, J. Hirst, Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer, J. Biol. Chem. 286 (2011) 18056–18065. [54] P.J. Ratcliffe, Oxygen sensing and hypoxia signalling pathways in animals: the implications of physiology for cancer, J. Physiol. 591 (2013) 2027–2042. [55] S.J. Remington, Fluorescent proteins: maturation, photochemistry and photophysics, Curr. Opin. Struct. Biol. 16 (2006) 714–721. [56] G.Y. Rychkov, M.B. Adams, I.C. McMillen, M.L. Roberts, Oxygen-sensing mechanisms are present in the chromaffin cells of the sheep adrenal medulla before birth, J. Physiol. 509 (Pt 3) (1998) 887–893. [57] G.L. Semenza, Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology, Annu. Rev. Pathol. 9 (2014) 47–71. [58] L.A. Shimoda, J. Polak, Hypoxia. 4. Hypoxia and ion channel function, Am. J. Physiol. Cell Physiol. 300 (2011) C951–C967. [59] H. Starlinger, D.W. Lubbers, Oxygen consumption of the isolated carotid body tissue (cat), Pflug. Arch. 366 (1976) 61–66. [60] A. Stea, C.A. Nurse, Whole-cell and perforated-patch recordings from O2-sensitive rat carotid body cells grown in short- and long-term culture, Pflug. Arch. 418 (1991) 93–101. [61] R.J. Thompson, A. Jackson, C.A. Nurse, Developmental loss of hypoxic chemosensitivity in rat adrenomedullary chromaffin cells, J. Physiol. 498 (Pt 2) (1997) 503–510. [62] R.J. Thompson, J. Buttigieg, M. Zhang, C.A. Nurse, A rotenone-sensitive site and H 2 O 2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells, Neuroscience 145 (2007) 130–141. [63] H.J. Timmers, J.M. Karemaker, W. Wieling, H.A. Marres, H.T. Folgering, J.W. Lenders, Baroreflex and chemoreflex function after bilateral carotid body tumor resection, J. Hypertens. 21 (2003) 591–599. [64] P.J. Turner, K.J. Buckler, Oxygen and mitochondrial inhibitors modulate both monomeric and heteromeric TASK-1 and TASK-3 channels in mouse carotid body type-1 cells, J. Physiol. 591 (2013) 5977–5998. [65] J.F. Turrens, A. Boveris, Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria, Biochem. J. 191 (1980) 421–427. [66] J. Urena, R. Fernandez-Chacon, A.R. Benot, G.A. Alvarez de Toledo, J. Lopez- Barneo, Hypoxia induces voltage-dependent Ca2+ entry and quantal dopamine secretion in carotid body glomus cells, Proc. Natl. Acad. Sci. USA 91 (1994) 10208–10211. [67] R. Varas, C.N. Wyatt, K.J. Buckler, Modulation of TASK-like background potassium channels in rat arterial chemoreceptor cells by intracellular ATP and other nucleotides, J. Physiol. 583 (2007) 521–536. [68] T.V. Votyakova, I.J. Reynolds, DeltaPsi(m)-dependent and -independent production of reactive oxygen species by rat brain mitochondria, J. Neurochem. 79 (2001) 266–277. [69] G.B. Waypa, N.S. Chandel, P.T. Schumacker, Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing, Circ. Res. 88 (2001) 1259–1266. [70] G.B. Waypa, J.D. Marks, R.D. Guzy, P.T. Mungai, J.M. Schriewer, D. Dokic, M.K. Ball, P.T. Schumacker, Superoxide generated at mitochondrial complex III triggers acute responses to hypoxia in the pulmonary circulation, Am. J. Respir. Crit. Care Med. 187 (2013) 424–432. [71] E.K. Weir, J. Lopez-Barneo, K.J. Buckler, S.L. Archer, Acute oxygen-sensing mechanisms, N. Engl. J. Med. 353 (2005) 2042–2055. [72] C.N. Wyatt, K.J. Buckler, The effect of mitochondrial inhibitors on membrane currents in isolated neonatal rat carotid body type I cells, J. Physiol. 556 (2004) 175–191. [73] C. Youngson, C. Nurse, H. Yeger, E. Cutz, Oxygen sensing in airway chemoreceptors, Nature 365 (1993) 153–155. L. Gao et al. Redox Biology 12 (2017) 908–915 914 [74] X.J. Yuan, W.F. Goldman, M.L. Tod, L.J. Rubin, M.P. Blaustein, Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes, Am. J. Physiol. 264 (1993) L116–L123. [75] M. Zhang, J. Buttigieg, C.A. Nurse, Neurotransmitter mechanisms mediating lowglucose signalling in cocultures and fresh tissue slices of rat carotid body, J. Physiol. 578 (2007) 735–750. [76] T. Zhou, M.S. Chien, S. Kaleem, H. Matsunami, Single cell transcriptome analysis of mouse carotid body glomus cells, J. Physiol. 594 (2016) 4225–4251. [77] J. Zhu, K.R. Vinothkumar, J. Hirst, Structure of mammalian respiratory complex I, Nature 536 (2016) 354–358. [78] V. Zickermann, C. Wirth, H. Nasiri, K. Siegmund, H. Schwalbe, C. Hunte, U. Brandt, Structural biology. Mechanistic insight from the crystal structure of mitochondrial complex I, Science 347 (2015) 44–49. L. Gao et al. Redox Biology 12 (2017) 908–915 915