Mitochondrial Redox Signaling in O2-Sensing Chemoreceptor Cells

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Antioxidants & Redox Signaling DOI: https://doi.org/10.1089/ars.2021.0255. El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is the original submission version (pre-peer review) of the following article: Mitochondrial Redox Signaling in O2-Sensing Chemoreceptor Cells, which has now been formally published in final form at Antioxidants & Redox Signaling at https://doi.org/10.1089/ars.2021.0255. This original submission version of the article may be used for non-commercial”. 1 Mitochondrial redox signaling in O2-sensing chemoreceptor cells Lin Gao1,2,3, Patricia Ortega-Sáenz1,2,3, Alejandro Moreno-Domínguez1,2,3, José López-Barneo1,2,3,* 1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013-Seville, Spain 2Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, 41013-Seville, Spain 3Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031-Madrid, Spain *Corresponding author: José López-Barneo, MD, PhD Instituto de Biomedicina de Sevilla (IBiS) Campus Hospital Universitario Virgen del Rocío Avenida Manuel Siurot s/n 41013 Seville, Spain Telephone number: +34 955 923032 Fax number: +34 955 923101 Email address: [email protected] Key words: acute oxygen sensing, hypoxia, carotid body, glomus cells, chemoreceptor cells, mitochondrial signaling, reactive oxygen species, mitochondrial mutations, ion channels 2 Abstract Significance: Acute responses to hypoxia are essential for the survival of mammals. The carotid body (CB), the main arterial chemoreceptor, contains glomus cells with O2-sensitive K+ channels, which are inhibited during hypoxia to trigger adaptive cardiorespiratory reflexes. Recent Advances: In this review, recent advances in molecular mechanisms of acute O2 sensing in CB glomus cells are discussed, with a special focus on the signaling role of mitochondria through regulating cellular redox status. These advances have been achieved thanks to the use of genetically engineering redox-sensitive protein roGFP probes, which allowed us to monitor rapid changes in ROS production in real time in different subcellular compartments during hypoxia. This methodology was used in combination with conditional knockout mice models, pharmacological approaches, and transcriptomic studies. We have proposed a mitochondria-to-membrane signaling model of acute O2 sensing in which H2O2 released in mitochondrial intermembrane space serves as a signaling molecule to inhibit K+ channels on the plasma membrane. Critical Issues: Changes in mitochondrial reactive oxygen species (ROS) production during acute hypoxia are highly compartmentalized in submitochondrial regions. The use of redox-sensitive probes targeted to specific compartments is essential to fully understand the role of mitochondrial ROS in acute O2 sensing. Future Directions: Further studies are needed to specify the ROS and to characterize the target(s) of ROS in chemoreceptor cells during acute hypoxia. These data may also contribute to a more complete understanding of the 3 implication of ROS in acute responses to hypoxia in O2-sensing cells in other organs. 4 Adaptive acute responses to hypoxia Oxygen (O2) is essential for most forms of life since it participates in numerous biochemical reactions and is required for ATP synthesis by oxidative phosphorylation. The lack of O2 (hypoxia) even if transient, can produce irreversible cellular damage. Therefore, supply of sufficient O2 to cells in the organism to meet their metabolic demands is a physiological challenge. Adaptive cellular and organismal responses have evolved to minimize the deleterious effect of hypoxia. Sustained hypoxia (hours or days) triggers a generalized transcriptional response, which stimulates glycolysis to increase energy production anaerobically, increases red blood cell number, and activates angiogenesis. The regulated expression of O2-sensitive genes depends on prolyl hydroxylases (PHDs), which are oxygenases that use O2 as a substrate to hydroxylate hypoxia inducible transcription factors (HIFs) and thereby inhibit their activity in normoxic conditions. In hypoxia, HIF stabilization induces the expression of a broad cohort of genes. The PHD-HIF pathway regulates more than 2500 transcripts, many of them critically involved in pathophysiological processes such as stem cell fate and differentiation, tissue regeneration, cancer, inflammation, among others (20,49,66,96,122). However, the survival of mammals under hypoxia depends on acute cardiorespiratory reflexes, such as hyperventilation and increase in cardiac output, which rapidly (in a few seconds) increase O2 uptake by the lungs and its distribution to the tissues. These responses play a fundamental role in adaptation to high altitude (48,124) and are also needed for physiological compensation in patients with dysfunctions of gas exchange in the lung (104). 5 The detection of hypoxia and the initiation of adaptive acute reflexes depend on a series of specialized tissues or organs, such as the carotid body (CB), adrenal medulla (AM), pulmonary/systemic arteries, and neuroepithelial bodies in the lung airways, which together form a “homeostatic acute O2- sensing system” (65,78,120). Most of the current knowledge on hypoxiainduced chemoreception was obtained from studies initially performed in the CB, the main arterial chemoreceptor and prototypical acute O2-sensing organ (45,82). The CB is essential for O2 regulation of breathing, as its surgical removal results in individuals who are unaware of hypoxemia and thus exhibit a complete absence of hypoxic ventilatory response (HVR) (106). In this review, recent advances in molecular mechanisms of acute O2 sensing by CB glomus cells are discussed, with a special focus on the signaling role of mitochondria through regulating cellular redox status. Carotid body chemoreceptor activation by hypoxia and mitochondrial inhibitors Electrical properties of carotid body cells and mechanism of chemotransduction The CB is a paired organ located at the carotid artery bifurcation that is organized in clusters of cells, denominated as glomeruli. Each glomerulus contains a few O2-sensitive glomus cells (also called type I cells), which establish chemosensory synapses with afferent fibers transmitting sensory information via glossopharyngeal nerve to the brainstem respiratory and autonomic centers (Fig. 1A). Glomus cells are surrounded by type II cells and immature progenitors, which provide structural support and contribute to the hypertrophy of the organ during acclimatization to hypoxia (88). The CB is also 6 rich in blood vessels and is considered as one of the most irrigated organs in the body (24). The CB functions as a multimodal sensory organ, which in addition to changes in O2 tension, can detect changes in CO2 tension, pH, glucose, and lactate in the blood (82,108). In addition, the CB can be activated by circulating hormones and cytokines (45,46). The basic cellular mechanisms underlying the O2 sensing properties of CB glomus cells were discovered several decades ago. It was shown that glomus cells, which derive from the neural crest (88), are excitable and can generate action potentials repetitively (28,64). These cells contain O2-sensitive background and voltage-gated K+ channels, which are both inhibited by decreases in O2 tension (Fig. 1B) (12,37,64). The inhibition of these K+ channels leads to a depolarization of the plasma membrane, which in turn activates voltage-dependent Ca2+ channels causing Ca2+ influx. The increased cytosolic Ca2+ triggers neurotransmitter release by exocytosis, to stimulate afferent sensory fibers transferring information to the brainstem respiratory and autonomic centers (14,62,72,113). The rise in cytosolic Ca2+ required for glomus cell secretion under hypoxia depends on extracellular Ca2+, since it can be abolished with either Ca2+ free recording solution or addition of Ca2+ chelator EGTA to the recording solution (Fig. 1C). In glomus cells, the changes in intracellular Ca2+ induced by O2 tension follow a hyperbolic curve (Fig. 1C) similar to the relationship existing between ventilation and the level of O2 tension in arterial blood (34). Therefore, the electrophysiological and O2- sensing properties of single glomus cells, summarized in Fig. 1D, are the major determinants of the O2 regulation of breathing in experimental mammals and in humans (72,84). 7 Occlusion of responsiveness to hypoxia by rotenone Although there is a consensus regarding the basic process of sensory function in chemoreceptor cells during hypoxia, the molecular mechanisms underlying this process have remained not fully understood. Since late 1990s, several hypotheses have been proposed to address the nature of the molecules that detect hypoxia and the signaling pathway leading to inhibition of K+ channels on the plasma membrane. However, most hypotheses were discarded eventually due to lack of solid support from experimental data. A detailed discussion about these hypotheses is beyond the scope of current review and has been addressed recently (for reviews see 45,63,92,127). Mitochondria have classically been considered to be implicated in acute O2 sensing (71,123). These organelles are the largest consumer of cellular O2 for energy production by oxidative phosphorylation, which makes them a natural candidate to detect changes in O2 tension and to initiate adaptive responses to hypoxia. Supporting this hypothesis are the high O2 consumption (24) and the sensitivity of CB glomus cells to inhibitors of mitochondrial electron transport chain (ETC) (75,83,126). Similar to hypoxia, inhibition of ETC complexes also stimulates secretory activity and Ca2+ influx from extracellular space, and inhibits K+ channels in glomus cells (Fig. 2A and B) (83). Although all ETC inhibitors mimic and impair further hypoxic responses (83,126), rotenone, a mitochondrial complex I (MCI) inhibitor, is particularly efficient in blocking hypoxic responses in rat CB glomus cells. Treatment with rotenone induces catecholamine secretion from glomus cells, which, similar to the response elicited by hypoxia, is inhibited by cadmium, a blocker of voltage- 8 dependent Ca2+ channel on the plasma membrane, indicating the dependence of extracellular Ca2+ (Fig. 2A and B). Incubation with rotenone abolishes catecholamine secretion from glomus cells during hypoxia (Fig. 2C). This inhibitory effect of rotenone is specific to hypoxia, since rotenone has no effect on hypoglycemia-induced catecholamine secretion in glomus cells (40). In addition to CB glomus cells, rotenone also blocks the sensitivity to hypoxia in rat (105) and ovine (50) AM chromaffin cells. These results suggest that the inhibitory effect of rotenone on acute O2 sensing is not limited to CB, but it is a rather general phenomenon in chemoreceptor cells. Another MCI inhibitor 1- methyl-4-phenylpyridinium (MPP+) also blocks hypoxic responses in glomus cells, whereas MCI proximal inhibitors, such as diphenyliodonium, present less effect than rotenone and MPP+ (83). Both rotenone and MPP+ bind to a distal site (Q site) of MCI, which is highly conserved from bacteria to mammals (5, 33,129,130), thereby preventing the binding of ubiquinone and inhibiting the MCI NADH/quinone oxidoreductase activity. These data suggest that mitochondria in chemoreceptor cells are highly sensitive to hypoxia and that a signaling pathway from mitochondria to plasma membrane exits during acute O2 sensing, in which MCI, especially the rotenone and ubiquinone-binding site, may have a special role. As shown below, this idea has been demonstrated in mice with complete genetic disruption of MCI activity (4,32). O2-sensitive mitochondrial signaling The implication of mitochondria in acute O2 sensing implies that one or several classes of mitochondria-derived molecules must be generated during hypoxia to signal membrane ion channels. The signaling molecules proposed to 15 Hypoxia-induced decrease in ROS production in mitochondrial matrix was also confirmed using MitoNeoD, an O2- probe targeted to mitochondrial matrix (4,98). Previous pharmacological studies demonstrate that MCI inhibitor rotenone blocks hypoxia-induced secretory activity in glomus cells (83). Rotenone binds to the ubiquinone binding site (Q site) at MCI and blocks both forward and reverse reactions of MCI. Rotenone is also known to increase ROS production from the IF site (the peripheral arm of MCI) and upstream dehydrogenases due to the backlog of electrons in MCI, with O2- (and H2O2) being released to mitochondrial matrix (10,11,56,76,80,109,115). As expected, in mouse glomus cells rotenone stimulates a transient reversible increase in H2O2 in mitochondrial matrix (Fig. 4E) (4), which seems to diffuse to the IMS (Fig. 4F). Rotenone treatment strongly inhibits further increase in H2O2 in IMS during hypoxia, but has no effect on hypoxia-induced decrease in H2O2 in mitochondrial matrix (Fig. 4E and F) (4). These data suggest that an increase in mitochondria IMS ROS is associated with sensing hypoxia by glomus cells. This signal is strongly inhibited by rotenone, indicating that it is mainly originated at the MCI IQ site. The hypoxic increase in IMS ROS contrasts with the decrease in matrix ROS during hypoxia, a phenomenon that is probably non-specific and is due to the generalized lack of O2. Loss of ROS signaling and responsiveness to hypoxia in genetically modified mice The generation of conditional knockout mice deficient in MCI subunit NDUFS2 has allowed us to better understand the signaling role of ROS derived from MCI in acute O2 sensing. NDUFS2 forms the ubiquinone binding site of 16 MCI together with NDUFS7 and ND1 (5,93). In mice and glomus cells deficient in NDUFS2, hypoxic responses are selectively abolished, whereas responses to hypercapnia are maintained (4,32). In addition, hypoxia-induced mitochondrial signals (NADH and ROS) are also lost in NDUFS2-deficient glomus cells (4,32). Whereas hypoxia-induced increase in H2O2 in cytosol (32) and mitochondrial IMS are strongly reduced in amplitude in glomus cells lacking NDUFS2 (Fig. 5A), hypoxia-mediated decrease in H2O2 in mitochondrial matrix is maintained (Fig 5B). These results are in agreement with those obtained from rotenonetreated wild-type glomus cells (see Fig. 4E and F), and further suggest that the increase in H2O2 production in mitochondrial IMS is a signal associated with responsiveness to acute hypoxia. CB glomus cells have intrinsic metabolic properties, which make their mitochondria highly sensitive to hypoxia. Studies of gene expression profiling demonstrate a constitutive high level of expression of Epas1 (the gene coding HIF2) and atypical isoforms of mitochondrial complex IV (MCIV) subunits (Cox4i2, Cox8b, and Ndufa4l2) in glomus cells (39,128). Although HIF2 is classically considered as a transcription factor regulating gene expression during chronic hypoxia, these transcriptomic data along with studies using conditional knockout mice deficient in HIF2 (43,73) demonstrate an essential role of this factor in acute O2 sensing by glomus cells. HVRs are diminished significantly in HIF2-deficient mice. In addition, these mice show a loss of glomus cell and mitochondrial responsiveness to hypoxia (73). In HIF2- deficient glomus cells, the hypoxia-induced IMS ROS signal is inhibited whereas the decrease in ROS production in mitochondrial matrix is not affected (Fig. 5C and D). In HIF2-deficient mice, expression of atypical isoforms of 17 MCIV subunits Cox4i2, Cox8b, and Ndufa4l2 is greatly suppressed. In addition, the hypoxic response is lost in conditional knockout mice deficient in COX4I2 (73). These results demonstrate that the enrichment of HIF2 results in an induction of gene expression of atypical isoforms of MCIV subunits, thus making glomus cells highly sensitive to hypoxia. The data obtained from wild-type mice and mice with mitochondrial mutations have led us to propose a mitochondria-to-membrane signaling model of acute O2 sensing in CB glomus cells. In this model MCIV functions as an O2 sensor with a low apparent affinity for O2 due to the atypical isoforms expressed in glomus cell cytochrome c oxidase (39,73,128). As mentioned above, genetic ablation of the gene coding COX4I2 strongly decreases sensitivity to hypoxia in glomus cells (73). Regarding this subunit isoform, it has recently been reported that genetically engineered HEK293 cells present a COX4I2-dependent MCIV affinity for O2 (P50 about 1 mmHg) lower than that with COX4I1 (P50 about 0.5 mmHg) (86). Although this study supports our hypothesis of COX4I2-dependent low affinity for O2, the levels of O2 tension that modulate HEK293 cell mitochondria are too low compared to the physiological range of O2 that modulates glomus cells (see Fig. 1C and Fig. 4C) (4). Therefore, it seems that other factors (e.g. expression of COX8B or pyruvate carboxylase dependent Kreb’s cycle anaplerosis) may influence sensitivity of glomus cell mitochondria to O2. During hypoxia, the decrease in MCIV activity causes a backlog of electrons along the ETC and accumulation of ubiquinol (QH2) at the IQ site of MCI. This leads to not only a slowdown of MCI reaction in the forward direction, but most likely a switch to the reverse direction (Fig. 6A). Reverse MCI function is an intriguing property of MCI and is associated with succinate-dependent high 18 level of ubiquinol, as it occurs in glomus cells (32,39,56,60,91,115). The reverse electron transport results in a burst of O2- production at the IQ site and the reduction of NAD+ to NADH at the FMN site (19,55,76,91). O2- produced at the IQ site may be electrostatically repelled by the negative potential in the mitochondrial matrix and directly channeled to the IMS, where it is converted to H2O2 by SOD1. As a result, MCI functions as an effector, releasing mitochondrial signals (NADH and H2O2) that inhibit K+ channels on the plasma membrane to trigger adaptive responses. The IMS hypoxic ROS signal may have also a component due to O2- production at a highly reduced outer IIIQ site (Fig. 6A and B) (117). In normoxic conditions, rotenone blocks electron transport at MCI and produces ROS at the IF site directed to the matrix (see Fig. 4E). In the presence of rotenone the IMS ROS signal induced by hypoxia is greatly suppressed due to blockade of MCI RET (see Fig. 4F). However, hypoxia-induced decrease in ROS production in the matrix, possibly due to the inhibited activity of ETC and associated dehydrogenases, is unaffected by rotenone (Fig. 6B; see Fig. 4E). Modulation of membrane ion channels by ROS in carotid body glomus cells The mitochondrial-to-membrane signaling model predicts that mitochondria-originated NADH and H2O2 regulate K+ channels on the plasma membrane during acute hypoxia. Indeed, dialysis of glomus cells with NADH inhibits modulation of voltage-dependent K+ channels by hypoxia (32). On the other hand, modulation of ion conductance by intracellular redox status have been reported previously in a variety of ion channels on the plasma membrane, in which channel inhibition is frequently associated with cysteine oxidation 19 (7,16,54,87,94,95). In CB glomus cells, intracellular dialysis with H2O2 (15-50 M) or a thiol-oxidizing agent diamide (50-60 M) inhibits background K+ channels, as demonstrated by an increase in input resistance, without affecting voltage-dependent K+ channels. These oxidants mimic the hypoxic effect and prevent further modulation of these channels by hypoxia (Fig. 7A-D) (32). N- acetyl-cysteine, a reducing agent, has no effect on basal level of K+ current (Fig. 7D). This suggests that in normoxic conditions glomus cells are in a relatively reduced state, as determined by cytosolic roGFP probe (32). Taken together, these data support that during hypoxia, mitochondria-released H2O2 may inhibit background K+ channels on the plasma membrane of glomus cells to initiate cell depolarization. The molecular mechanisms underlying the redox regulation of K+ channels in glomus cells are not yet fully known. Background K+ channels TASK1 and TASK3 are highly expressed in CB glomus cells, which form mainly TASK3 homomers or TASK1/TASK3 heteromers (39,52,111,128). TASK3 subunits contain cysteine residues, which are susceptible to cytosolic redox modulation by H2O2 (53). TREK2, K+ background channels closely related to TASK channels, are also inhibited by intracellular oxidants (89). On the other hand, acute responses to hypoxia are intact in double knockout mice deficient in both TASK1 and TASK3 (81), indicating redundant functions among K+ background channels in CB glomus cells. In addition to acting directly on poreforming subunits, ROS also interact with accessory  subunits of voltage-gated K+ channels (95). Moreover, redox regulation has been reported to modulate phosphatase and protein kinase activities (30,99,110). It is unclear whether this redox-dependent phosphorylation-dephosphorylation balance could indirectly 20 regulate ion channel activities and, therefore, their responsiveness to changes in O2. Role of ROS in other acute O2 sensing tissues The mitochondrial-to-membrane signaling model, summarized in Fig 8, which is established mainly based on results obtained from CB glomus cells, may also apply to other chemoreceptor cells. Chromaffin cells in AM are highly sensitive to hypoxia, especially in neonates, although its sensitivity decreases in adult animals. O2 sensitivity in chromaffin cells depends on MCI function, as rotenone mimics hypoxia-induced inhibition of K+ current and prevents further inhibition by hypoxia in rat chromaffin cells (105). Hypoxia-induced secretory activity is also abolished in chromaffin cells of adult mice deficient in MCI subunit NDUFS2 (32). ROS is also implicated in the chemotransduction pathway leading to the inhibition of K+ current in postnatal chromaffin cells. However, a hypoxia-induced decrease in ROS production, rather than increase in ROS production, is proposed, based on the measurement of luminol chemiluminescence and treatments with exogenous H2O2, antioxidants and antioxidant enzymes (105). It is technically difficult to measure ROS production using roGFP probes in primary AM cultures due to the short survival time of chromaffin cells in these cultures. Further studies are necessary to clarify the changes of ROS production in AM cells during hypoxia. Pulmonary resistance artery smooth muscle cells are sensitive to O2 tension, which produce Ca2+-dependent vasoconstriction during hypoxia in order to divert blood to better ventilated regions to maximize gas exchange. Although it is generally accepted that changes in mitochondrial ROS production 21 is implicated in the hypoxic response of pulmonary arteries, it has been under debate since 1990s whether ROS production is increased or decreased during hypoxia in these tissues (1,10,42,68,69,76,116,117,118,119). Part of the dispute results from the difference in detection probes used and the location of measurement systems applied in each study. Using roGFP probes, an increase in H2O2 production is detected in the IMS upon treatment of hypoxia for 10-30 minutes (117). This increase in H2O2 production appears to be dependent on MCIII, as it is absent in mice deficient in MCIII subunit RISP (118). On the other hand, using H2O2-specific probes (HyPer) targeted to cytosol and mitochondria, a hypoxia-induced decrease in H2O2 production has been reported in pulmonary smooth muscle cells, which is associated with MCI based on results using siNdufs2 (29). Dysfunction of hypoxia-induced pulmonary vasoconstriction is also observed in mice with deficient MCII (85), further demonstrating the implication of mitochondrial ETC in acute O2 sensing in the lung. In addition to mitochondrial origin, ROS produced from NADPH oxidase has also been proposed in pulmonary arterial vasoconstriction (35,36,59,121), which may amplify the mitochondrial ROS signal. Concluding remarks The development of genetically engineered redox-sensitive protein probes has allowed us to monitor in real time rapid changes in ROS production in CB cells. This has helped us better understand the signaling role of ROS with low concentration in acute O2 sensing, a fundamental physiological process essential for survival of mammals in hypoxic environments. In acute hypoxia, ROS produced in mitochondria diffuse to cytosol and inhibit K+ channels in the 22 plasma membrane of glomus cells, triggering depolarization and transmitter release. This mitochondria-to-membrane signaling mechanism (Fig. 8) may also apply to other O2-sensing chemoreceptor cells. Several questions remain to be addressed in the chemotransduction pathway during acute hypoxia, such as the specific ROS implicated and their direct molecular target(s). During hypoxia, electron leakage in mitochondrial ETC could result in increases in O2- production, which may be converted rapidly to H2O2 by SOD2 in mitochondrial matrix or by SOD1 in IMS. It would be interesting to study whether manipulation of mitochondrial H2O2 level, such as modification of SOD1 or SOD2 expression, overexpression of peroxiredoxins (especially PRDX3), glutathione peroxidases (such as GPX1), or transgenic mitochondrial targeted catalase (mCAT) (22,23), affects acute response to hypoxia in glomus cells. PRDX3, GPX1 and mCAT all remove H2O2. It remains to be determined whether MCIII contributes to ROS signaling during acute hypoxia in glomus cells, as it happens in pulmonary artery smooth muscle cells. In vitro cell line models over-expressing recombinant K+ channels and thiol-redox proteomics may help us identify the direct target(s) of ROS during acute hypoxia. Acknowledgments The authors would like to thank all members of the laboratory who contributed to the experiments discussed in this review. Funding This research was supported by grants from Spanish Ministries of Science and Innovation and Health (SAF2016-74990-R and PID2019-106410RB-I00), from the Andalusian Government (FEDER Andalucía 2014-2020, 2018 Call, US- 23 1255654), all co-funded by FEDER, and the European Research Council (ERCADGPRJ201502629). References 1. Archer SL, Huang J, Henry T, Peterson D, Weir EK. A redox-based O2 sensor in rat pulmonary vasculature. Circ Res 73: 1100-1112, 1993. 2. Archer SL, Nelson DP, Weir EK. Detection of activated O2 species in vitro and in rat lungs by chemiluminescence. J Appl Physiol (1985) 67: 1912-1921, 1989. 3. Archer SL, Nelson DP, Weir EK. Simultaneous measurement of O2 radicals and pulmonary vascular reactivity in rat lung. J Appl Physiol (1985) 67: 1903-1911, 1989. 4. Arias-Mayenco I, Gonzalez-Rodriguez P, Torres-Torrelo H, Gao L, Fernandez- Aguera MC, Bonilla-Henao V, Ortega-Saenz P, Lopez-Barneo J. 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J Biol Chem 285: 14210-14216, 2010. 47 membrane; MCI, MCII, MCIII, and MCIV, mitochondrial complexes I, II, III, and IV, respectively; OM, outer mitochondrial membrane; SOD1 and SOD2, superoxide dismutase 1 and 2, respectively. 48 Figure 7. Effect of cytosolic redox reagents on the electrical response to hypoxia in glomus cells. A. Representative current traces recorded from a voltageclamped dispersed glomus cell (perforated-patch mode) internally dialyzed with H2O2 (15 M) during exposure to normoxic (PO2 ~145 mmHg, red and grey, control and recovery respectively) and hypoxic (PO2 ~15 mmHg, blue) solutions. The voltage pulse protocol is similar to that in Fig. 1B. B. Increase of input resistance (inhibition of background current amplitude) induced by hypoxia in normal glomus cells (control) and the disappearance of this phenomenon in cells dialyzed with H2O2 (15-30 M). C. Comparison of currents traces elicited by the indicated voltage-clamp protocol in control glomus cells (blue) and in cells dialyzed with H2O2 (15 M). D. Values of input 49 resistance (mean ± SEM) in glomus cells dialyzed with the indicated redox reagents. Modified from 32. 50 Figure 8. Schematic representation of the mitochondria to membrane signaling model of acute O2 sensing by carotid body glomus cells. MCIV is the oxygen sensor; MCI is the effector; and NADH and ROS are signalling molecules that modulate ion channels in the plasma membrane. IMS, mitochondrial intermembrane space; COX4I2, cytochrome c oxidase subunit IV isoform 2; COX8B, cytochrome c oxidase subunit VIII isoform b. Modified from 73.