Mitochondrial Redox Signaling in O 2 -Sensing Chemoreceptor Cells

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 oxygen (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 engineered redox-sensitive green fluorescent 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 the 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 the 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 implication of ROS in acute responses to hypoxia in O₂-sensing cells in other organs. Antioxid. Redox Signal. 37, 274–289.

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, and inflammation, among others (20, 55, 72, 111, 139).

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 (54, 141) and are also needed for physiological compensation in patients with dysfunctions of gas exchange in the lung (119).

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 (71, 86, 137). Most of the current knowledge on hypoxia-induced chemoreception was obtained from studies initially performed in the CB, the main arterial chemoreceptor and prototypical acute O₂-sensing organ (50, 91). 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) (122).

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 the cellular redox status.

CB Chemoreceptor Activation by Hypoxia and Mitochondrial Inhibitors

Electrical properties of CB 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 the glossopharyngeal nerve to the brain stem 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 (98). Type II cells also contribute to paracrine signaling during chemotransduction, potentiating activation of sensory fibers by glomus cells (87, 127). The CB is rich in blood vessels and is considered 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 (91, 124). In addition, the CB can be activated by circulating hormones and cytokines (50, 51).

FIG. 1.

Activation of chemosensory CB glomus cells by hypoxia. (A) Scheme illustrating CB location and structural organization of a CB glomerulus. [modified from Ortega-Saenz and Lopez-Barneo (91)]. (B) Top: Scheme illustrating the perforated-patch technique and voltage ramp protocol (from −70 to −10 mV) applied to dispersed mouse glomus cells studied. Bottom: Representative traces of currents recorded from a glomus cell exposed to normoxia (PO₂ ∼145 mmHg, red), hypoxia (PO₂ ∼15 mmHg, blue), and recovery in normoxia (gray) [modified from Fernandez-Aguera et al. (36)]. (C) Cytosolic Ca²⁺ levels recorded from rat glomus cell as a function of O₂ tension. The inset illustrates that the increase in cytosolic [Ca²⁺] induced by hypoxia is abolished upon removal of external Ca²⁺ [modified from Urena et al. (130)]. (D) Schematic representation of the main steps in acute O₂ sensing by CB glomus cells [modified from Lopez-Barneo (67)]. CB, carotid body; ECA, external carotid artery; ICA, internal carotid artery; O₂, oxygen; PO₂, O₂ partial pressure.

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 (98), are excitable and can generate action potentials repetitively (32, 70). These cells contain O₂-sensitive background and voltage-gated K⁺ channels, which are both inhibited by decreases in O₂ tension (Fig. 1B) (12, 41, 70). 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 brain stem respiratory and autonomic centers (14, 68, 79, 130).

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 (38). Therefore, the electrophysiological and O₂-sensing properties of single glomus cells, summarized in Figure 1D, are the major determinants of O₂ regulation of breathing in experimental mammals and in humans (79, 93).

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 the 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 the current review and has been addressed recently [for reviews see Iturriaga et al. (50), Lopez-Barneo et al. (69), Rakoczy and Wyatt (105), and Yoo and Kim (144)]. Mitochondria have classically been considered to be implicated in acute O₂ sensing (78, 140). 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) (82, 92, 143). 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, B) (92).

FIG. 2.

Activation of glomus cells and occlusion of the hypoxic response by rotenone. (A, B) Representative amperometric recordings of the exocytotic release of catecholamines from CB glomus cells induced by hypoxia (A) and rotenone (5 μM) (B). In both cases, the secretory activity is abolished by blockade of Ca²⁺ channels with cadmium (0.2 mM) [modified from Ortega-Saenz et al. (92)]. (C) Inhibition of responsiveness to hypoxia by rotenone.

Although all ETC inhibitors mimic and impair further hypoxic responses (92, 143), 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-dependent Ca²⁺ channels on the plasma membrane, indicating the dependence of extracellular Ca²⁺ (Fig. 2A, 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 (44).

In addition to CB glomus cells, rotenone also blocks the sensitivity to hypoxia in rat (121) and ovine (56) AM chromaffin cells. These results suggest that the inhibitory effect of rotenone on acute O₂ sensing is not limited to the 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⁺ (92). Both rotenone and MPP⁺ bind to a distal site (Q site) of MCI, which is highly conserved from bacteria to mammals (5, 37, 147, 148), 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, 36).

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 be involved in hypoxia-induced chemotransduction are NADH, reactive oxygen species (ROS), and ATP. The potential implication of MCI in acute O₂ sensing suggests that NADH, the MCI substrate, or NAD⁺/NADH ratio could signal changes in O₂ tension from mitochondria to plasma membrane of chemoreceptor cells. Pyridine nucleotides have been reported to bind to voltage-gated K⁺ channel β subunits and modulate their function (57, 123). In addition, cytosolic NADH has also been suggested to modulate cationic channels in glomus cells (124).

Several groups have observed a reversible increase in NAD(P)H autofluorescence induced by hypoxia in glomus cells, which is proportional to decreases in O₂ tension (4, 13, 31, 36). This hypoxic signal is absent in conditional knockout mice lacking NDUFS2, a conserved subunit participating in ubiquinone binding, which is essential for MCI structure and function (4, 36). Similar to hypoxia, MCI inhibitor rotenone induces an increase in NAD(P)H autofluorescence (4). Incubation of glomus cells with intracellular NADH prevents further modulation of voltage-gated K⁺ current by hypoxia (36). Taking together, these studies support the role of NADH as a signaling molecule in glomus cells during acute O₂ sensing.

Bioenergetic deficiency has also been proposed to address mitochondrial function in acute O₂ sensing, due to the contribution of mitochondria in energy production. A decrease in cytosolic ATP concentration or a decrease in ATP/ADP ratio in glomus cells during hypoxia has been considered a signal to regulate the function of K⁺ channels in the plasma membrane (131, 143). The possible role of ATP on acute O₂ sensing in CB glomus cells as well as in pulmonary myocytes and chromaffin cells has been a matter of debate (15, 35, 48, 88, 136).

Data on single glomus cells point against a relevant role of ATP. Hypoxia-induced inhibition of voltage-gated K⁺ channels has been reported in glomus cells dialyzed with 3–5 mM MgATP (73, 102). Moreover, the effects of hypoxia and rotenone (or genetic MCI deficiency), both of which inhibit the ETC and presumably ATP synthesis, should be additive but they are not. In addition, rotenone blocks the effect of hypoxia but does not alter glomus cell activation by hypoglycemia (44). Glomus cells are activated by hypoglycemia and by lactate, which are conditions expected to have opposite effects on ATP production (36, 44, 124). Therefore, a signaling role of ATP under hypoxia is unlikely, although it cannot be discarded completely until more definitive experiments, including precise measurements of ATP changes in glomus cells, are performed.

A mitochondrial-based redox sensor of acute hypoxia was proposed back in the 1990s, especially in hypoxia-induced pulmonary artery vasoconstriction (1, 64, 117, 118, 133). ROS represent collectively a group of molecules derived from O₂, which are formed by redox reactions or electronic excitations. Overproduction of ROS may lead to oxidative damage, such as chronic inflammation and other diseases (11, 22, 115). On the contrary, controlled ROS production in a low-concentration range plays a signaling role in a variety of physiological processes, which have been discussed extensively in the literature (11, 112, 115). Intracellular hydrogen peroxide (H₂O₂), with an overall concentration between 1 and 100 nM, is the major ROS in regulating intracellular redox status, and therefore, biological functions (115).

Mitochondria are the major source of ROS production, first identified almost 50 years ago (8, 17, 52). Electrons leaked from ETC reduce O₂ to superoxide radical anion (O₂ ⁻), or to a lesser extent, directly to H₂O₂. O₂ ⁻ can be converted to H₂O₂ by spontaneous dismutation or catalyzed by superoxide dismutases. Rotenone, which mimics hypoxic secretory effect, is also known to increase ROS production from MCI (129).

As discussed later in this review, recent studies support the signaling role of mitochondrial ROS during hypoxia in CB glomus cells, pulmonary artery myocytes, and neonatal AM chromaffin cells (1, 4, 36, 63, 121, 133 –135). In glomus cells, hypoxia induces a dose-dependent increase in ROS at the intermembrane space (IMS) and the cytosol (4, 36). In addition, intracellular dialysis of glomus cells with H₂O₂ increases input resistance (an electrical change compatible with the inhibition of background K⁺ channels) and blunts any further effect of hypoxia (36).

Mitochondrial ROS Signaling in CB Chemoreceptor Cells During Acute Hypoxia

Sites of mitochondrial ROS production

O₂ ⁻ and H₂O₂ are the two primary ROS produced in mitochondria. Most O₂ ⁻ generated in the matrix is rapidly converted to H₂O₂ by mitochondrial superoxide dismutase (SOD2). In the IMS, the conversion of O₂ ⁻ to H₂O₂ is catalyzed by cytosolic superoxide dismutase (SOD1), which easily crosses the mitochondrial outer membrane. Mitochondrial H₂O₂ can diffuse to the cytosol, modulating cytosolic redox status and physiological processes, such as acute O₂ sensing.

Until now, at least 11 specific sites in the ETC and associated dehydrogenases have been identified to produce and release O₂ ⁻ (and H₂O₂) in either the mitochondrial matrix or the IMS [for detailed reviews see Brand (9 –11), Murphy (83), Waypa et al. (136), and Wong et al. (142)]. Quinone binding sites at MCI and mitochondrial complex III (MCIII) (I_Q and III_Q sites, respectively) contribute to the majority of ROS production from the mitochondrial ETC. O₂ ⁻ produced from the I_Q site is believed to be released to matrix (11).

However, as discussed later in this review, in O₂-sensitive glomus cells, hypoxia-induced reversal electron transport (RET) of MCI may cause O₂ ⁻ produced at the I_Q site to be released to the IMS (4, 36). O₂ ⁻ produced in the Q binding sites of MCIII can be delivered to both IMS and matrix (11, 136). O₂ ⁻ produced at the FMN site in the peripheral arm of MCI (I_F site), as well as in other NADH-related dehydrogenases, is released to mitochondrial matrix.

In addition, electrons leaked from the flavin site in mitochondrial complex II (MCII) (II_F site) and other FADH₂-related dehydrogenases contribute to a small amount of O₂ ⁻ delivered to mitochondrial matrix. Most H₂O₂ in the matrix is degraded by peroxiredoxin-3 (PRDX3) and glutathione peroxidases, such as glutathione peroxidase 1 (GPX1). Only a small percentage (∼10%) of H₂O₂ is either converted to other ROS or diffused to IMS and cytosol (11, 21). Inhibition of MCI by rotenone results in O₂ ⁻ production from the I_F site and other associated mitochondrial dehydrogenases (9, 10, 89, 125).

Real-time measurement of mitochondrial ROS with genetic probes

Monitoring changes in real-time ROS production during acute hypoxia has been a technical challenge, as most traditional techniques are not sensitive enough to record small transient variations in ROS levels in single chemoreceptor cells. The commonly used techniques either require ROS accumulation or detect ROS-induced modifications of lipids, proteins, and DNA (84). As a result, these techniques detect oxidative damages rather than rapid and subtle changes in ROS production during physiological processes, such as acute hypoxia.

To circumvent these limitations, redox-sensitive fluorescent probes (dichlorodihydrofluorescein, lucigenin, superoxide-selective hydroethidine and its mitochondria-targeted derivative MitoSOX, and Amplex Red, among others) have been developed (2, 3, 84, 136). However, these probes also have serious drawbacks due to their high susceptibility to artifactual side reactions, limitation in location, lack of specificity to specific oxidant, and modification of plasma membrane and mitochondrial membrane potentials.

In recent years, a new generation of redox-sensitive genetically engineered protein probes have been developed to detect specific oxidants with high sensitivity (84, 134, 136). These probes include redox-sensitive green fluorescent protein (roGFP) (18, 27, 30, 134), redox-sensitive yellow fluorescent protein HyPer (6), redox-sensitive fluorescence resonance energy transfer-probe (45), and peroxiredoxin-based probe roGFP2-Tsa2ΔC_R (81). All these probes are specifically sensitive to changes in H₂O₂ levels. In addition to redox-sensitive fluorescent protein probes, other alternative probes have also been explored, such as small-molecule fluorescent probes with improved selectivity (28, 77, 116, 149). A dual-purpose mitochondrial O₂ ⁻ probe (MitoNeoD) has been developed, which permits monitoring changes in O₂ ⁻ production, especially in mitochondrial matrix, in vivo by mass spectrometry and in vitro by fluorescence (113).

Recently, we have set up a protocol to record in real time rapid changes in intracellular redox status of mouse glomus cells during acute hypoxia using roGFP probes in combination with microfluorimetric techniques (42). Redox-sensitive cysteine residues are incorporated in roGFP probes, which allow reciprocal changes in emission intensity when excited at two different wavelengths, and thus, a ratiometric quantification of redox status (Fig. 3A–D) (42). Genetically engineered roGFP probes can be designed to target specific subcellular compartments, such as cytosol, mitochondrial matrix, or intermembrane space (4, 36, 42, 134).

FIG. 3.

Measurement of ROS production by microfluorimetry using roGFP probes. (A) Schematic representation of the microfluorimetric set up. (B, C) Representative CB slice 48 h after adenoviral infection in Brightfield (B) and with excitation of 480 nm and emission of 535 nm (C), demonstrating green fluorescent signal due to roGFP expression. A CB glomerulus is highlighted with a white discontinuous line. Scale bar, 10 μm. (D) Recordings of fluorescent signals (green) recorded with an excitation at 400 or 480 nm and an emission at 535 nm. The 400/480 ratio is represented in black. Responses to oxidants (H₂O₂) and reductants (DTT) demonstrate the redox regulation of the roGFP signal. Modified from Gao et al. (42). CCD, charge-coupled device; DTT, dithiothreitol; H₂O₂, hydrogen peroxide; roGFP, redox-sensitive green fluorescent protein; ROS, reactive oxygen species.

Compartmentalized mitochondrial ROS changes during acute hypoxia

Reversible changes in ROS production during exposure of CB glomus cells to hypoxia have been measured using roGFP probes designed to target specific subcellular compartments (42, 134). Using roGFP targeted to cytosol (36), a transient reversible increase in the ratio of fluorescent signals excited at 400 and 484 nm (400/484 ratio) is detected upon hypoxic treatment, indicating a low O₂ partial pressure (PO₂)-induced increase in the cytosolic H₂O₂ level in glomus cells (Fig. 4A).

FIG. 4.

Compartmentalized changes in mitochondrial ROS elicited by hypoxia in CB glomus cells. (A–D) Representative recordings of changes in cytosolic (A), IMS (B), and matrix (D) ROS induced by hypoxia (H, PO₂ ∼15 mmHg). Changes in IMS ROS amplitude at different O₂ tensions are represented in (C). H₂O₂, 0.1 mM, was used as a probe control. (E, F) Representative recordings of the changes in matrix (E) and IMS (F) ROS production recorded from glomus cells in response to hypoxia and 5 μM rotenone. Modified from Arias-Mayenco et al. (4) and Fernandez-Aguera et al. (36). IMS, intermembrane space.

A similar fluorescent signal can also be recorded using the roGFP probe targeted to mitochondrial IMS (Fig. 4B). Hypoxia-induced increases in 400/484 ratio in both the cytosol and mitochondrial IMS occur with a time course of seconds to minutes, which is similar to the dynamic range of cellular and systemic responses to hypoxia. The intensity of hypoxia-induced ROS signal in the IMS is proportional to the decrease in PO₂ (Fig. 4B).

The relationship between IMS ROS production and PO₂ follows a hyperbolic curve (Fig. 4C) that is similar to the increase in cytosolic Ca²⁺ (Fig. 1C) or secretory activity (79) induced by hypoxia in glomus cells, therefore suggesting the participation of the ROS signal in chemosensory transduction. However, in glomus cells transfected with a roGFP probe targeted to the mitochondrial matrix, hypoxia systematically induces a decrease in the 400/480 ratio, suggesting a decrease in H₂O₂ production. Treatment with exogenous H₂O₂ results in an increased 400/484 ratio in the matrix, indicating that the roGFP probe targeted to matrix is sensitive to H₂O₂ (Fig. 4D). Hypoxia-induced decrease in ROS production in mitochondrial matrix was also confirmed using MitoNeoD, an O₂ ⁻ probe targeted to mitochondrial matrix (4, 113).

Previous pharmacological studies demonstrate that the MCI inhibitor rotenone blocks hypoxia-induced secretory activity in glomus cells (92). 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 I_F 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, 62, 83, 89, 125, 132).

As expected, in mouse glomus cells, rotenone stimulates a reversible increase in H₂O₂ in mitochondrial matrix (Fig. 4E) (4), which seems to diffuse to the IMS (Fig. 4F). Rotenone treatment strongly inhibits a further increase in H₂O₂ in IMS during hypoxia, but has no effect on hypoxia-induced decrease in H₂O₂ in mitochondrial matrix (Fig. 4E, 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 I_Q site. The hypoxic increase in IMS ROS contrasts with the decrease in matrix ROS during hypoxia, a phenomenon that is probably nonspecific 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 MCI together with NDUFS7 and ND1 (5, 106). In mice and glomus cells deficient in NDUFS2, hypoxic responses are selectively abolished, whereas responses to hypercapnia are maintained (4, 36). In addition, hypoxia-induced mitochondrial signals (NADH and ROS) are also lost in NDUFS2-deficient glomus cells (4, 36). Whereas a hypoxia-induced increase in H₂O₂ in the cytosol (36) and mitochondrial IMS is strongly reduced in amplitude in glomus cells lacking NDUFS2 (Fig. 5A), a hypoxia-mediated decrease in H₂O₂ in mitochondrial matrix is maintained (Fig. 5B).

FIG. 5.

Hypoxia-induced compartmentalized changes in mitochondrial ROS production in mitochondrial complex I (NDUFS2)- or HIF2α-deficient mice. (A, B) Recordings of changes in IMS (A) and matrix (B) ROS production induced by hypoxia (H, PO₂ ∼15 mmHg) in glomus cells from wild-type and NDUFS2-deficient mice. (C, D) Changes in IMS (C) and matrix (D) ROS production induced by hypoxia (H, PO₂ ∼15 mmHg) in glomus cells from wild-type and HIF2α-deficient mice. Signals induced by exogenous H₂O₂ are shown. Modified from Arias-Mayenco et al. (4), Fernandez-Aguera et al. (36), and Moreno-Dominguez et al. (80). HIF2α, hypoxia inducible factor 2α.

These results are in agreement with those obtained from rotenone-treated wild-type glomus cells (Fig. 4E, 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 hypoxia inducible factor 2α [HIF2α]) and two atypical subunit isoforms of the catalytic core of mitochondrial complex IV (MCIV) (Cox4i2 and Cox8b) in glomus cells (43, 146). Although HIF2α is classically considered a transcription factor regulating gene expression during chronic hypoxia, these transcriptomic data along with studies using conditional knockout mice deficient in HIF2α (47, 80) demonstrate an essential role of this factor in acute O₂ sensing by glomus cells. HVRs are diminished significantly in HIF2α-deficient mice. In addition, these mice show a loss of glomus cell and mitochondrial responsiveness to hypoxia (80).

In HIF2α-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, D). In HIF2α-deficient mice, expression of atypical isoforms of MCIV subunits Cox4i2 and Cox8b is greatly suppressed. In addition, the hypoxic response is lost in conditional knockout mice deficient in COX4I2 (80). These results demonstrate that the enrichment of HIF2α 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 (43, 80, 146). As mentioned above, genetic ablation of the gene coding COX4I2 strongly decreases sensitivity to hypoxia in glomus cells (80). 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) (96).

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 with the physiological range of O₂ that modulates glomus cells (Figs. 1C and 4C) (4). Therefore, it seems that other factors (e.g., expression of COX8B or pyruvate carboxylase-dependent Kreb's cycle anaplerosis) may influence the 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 I_Q 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 as well (Fig. 6A).

FIG. 6.

Changes in glomus cell mitochondrial ROS production during acute hypoxia. (A) Scheme illustrating conventional electron transport chain function (black lines) and reversal of electron transport in hypoxia (red lines). Accumulation of reduced ubiquinone (QH₂) and production of NADH and ROS are indicated. (B) Schematic model of the compartmentalized ROS production during acute exposure to hypoxia (red lines) or rotenone (light brown lines). DH, dehydrogenases; Hx, hypoxia; IMS, intermembrane space; IM, inner mitochondrial membrane; MCI, MCII, MCIII, and MCIV, mitochondrial complexes I, II, III, and IV, respectively; OM, outer mitochondrial membrane; QH₂, ubiquinol; SOD1 and SOD2, superoxide dismutase 1 and 2, respectively.

Reverse MCI function is an intriguing property of MCI and is associated with a succinate-dependent high level of ubiquinol, as it occurs in glomus cells (36, 43, 62, 66, 104, 132). The RET results in a burst of O₂ ⁻ production at the I_Q site and the reduction of NAD⁺ to NADH at the FMN site (19, 61, 83, 104). O₂ ⁻ produced at the I_Q 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), which locally accumulate in the cytosol, near the plasma membrane, to inhibit neighboring K⁺ channels and trigger adaptive responses. The IMS hypoxic ROS signal may have also a component due to O₂ ⁻ production at a highly reduced outer III_Q site (Fig. 6A, B) (134).

In normoxic conditions, rotenone blocks electron transport at MCI and produces ROS at the I_F site directed to the matrix (Fig. 4E). In the presence of rotenone, the IMS ROS signal induced by hypoxia is greatly suppressed due to blockade of MCI RET (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; Fig. 4E).

Modulation of membrane ion channels by ROS in CB glomus cells

The mitochondria-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 (36). On the contrary, modulation of ion conductance by intracellular redox status has been reported previously in a variety of ion channels on the plasma membrane, in which channel inhibition is frequently associated with cysteine oxidation (7, 16, 60, 97, 107, 108).

In CB glomus cells, intracellular dialysis with H₂O₂ (15–50 μM) or a thiol-oxidizing agent diamide (50–60 μM) 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) (36). N-acetyl-cysteine, a reducing agent, has no effect on the basal level of K⁺ current (Fig. 7D). This suggests that in normoxic conditions, glomus cells are in a relatively reduced state, as determined by the cytosolic roGFP probe (36). 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.

FIG. 7.

Effect of cytosolic redox reagents on the electrical response to hypoxia in glomus cells. (A) Representative current traces recorded from a voltage-clamped dispersed glomus cell (perforated-patch mode) internally dialyzed with H₂O₂ (15 μM) during exposure to normoxic (PO₂ ∼145 mmHg, red and gray, control and recovery, respectively) and hypoxic (PO₂ ∼15 mmHg, blue) solutions. The voltage pulse protocol is similar to that in Figure 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 μM). (C) Comparison of currents' traces elicited by the indicated voltage-clamp protocol in control glomus cells (blue) and in cells dialyzed with H₂O₂ (15 μM). (D) Values of input resistance (mean ± SEM) in glomus cells dialyzed with the indicated redox reagents. *p < 0.05; **p < 0.01. Modified from Fernandez-Aguera et al. (36).

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 (43, 58, 128, 146). TASK3 subunits contain cysteine residues, which are susceptible to cytosolic redox modulation by H₂O₂ (59). TREK2, K⁺ background channels closely related to TASK channels, are also inhibited by intracellular oxidants (99).

On the contrary, acute responses to hypoxia are intact in double knockout mice deficient in both TASK1 and TASK3 (90), indicating redundant functions among K⁺ background channels in CB glomus cells. In addition to acting directly on pore-forming subunits, ROS also interact with accessory β subunits of voltage-gated K⁺ channels (108). Moreover, redox regulation has been reported to modulate phosphatase and protein kinase activities (34, 114, 126). It is unclear whether this redox-dependent phosphorylation/dephosphorylation balance could indirectly regulate ion channel activities and, therefore, their responsiveness to changes in O₂.

Role of ROS in Other Acute O₂ Sensing Tissues

The mitochondria-to-membrane signaling model, summarized in Figure 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 (121). Hypoxia-induced secretory activity is also abolished in chromaffin cells of adult mice deficient in MCI subunit NDUFS2 (36). ROS are also implicated in the chemotransduction pathway leading to the inhibition of K⁺ current in postnatal chromaffin cells.

FIG. 8.

Schematic representation of the mitochondria-to-membrane signaling model of acute O₂ sensing by CB glomus cells. MCIV is the oxygen sensor; MCI is the effector; and NADH and ROS are signaling molecules that modulate ion channels in the plasma membrane. IMS, intermembrane space; COX4I2, cytochrome c oxidase subunit IV isoform 2; COX8B, cytochrome c oxidase subunit VIII isoform b. Modified from Moreno-Dominguez et al. (80).

However, a hypoxia-induced decrease in ROS production, rather than an increase in ROS production, is proposed, based on the measurement of luminol chemiluminescence and treatments with exogenous H₂O₂, antioxidants, and antioxidant enzymes (121). 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 to divert blood to better ventilated regions to maximize gas exchange. Although it is generally accepted that changes in mitochondrial ROS production are implicated in the hypoxic response of pulmonary arteries, it has been under debate since the 1990s whether ROS production is increased or decreased during hypoxia in these tissues (1, 10, 46, 75, 76, 83, 133 –136). 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 (134). This increase in H₂O₂ production appears to be dependent on MCIII, as it is absent in mice deficient in MCIII subunit RISP (135). On the contrary, using H₂O₂-specific probes (HyPer) targeted to the 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 (33). Dysfunction of hypoxia-induced pulmonary vasoconstriction is also observed in mice with deficient MCII (95), further demonstrating the implication of mitochondrial ETC in acute O₂ sensing in the lung.

In addition to mitochondrial origin, ROS produced from NADPH oxidase have also been proposed in pulmonary arterial vasoconstriction (39, 40, 65, 138), which may amplify the mitochondrial ROS signal.

CB ROS Production and Mechanisms of Disease

The CB has classically attracted clinical interest due to its critical role in adaptation of the organism to high altitude (acclimatization to hypoxia) or lung diseases characterized by gas exchange restrictions (54, 119, 141). More recently, the CB has regained medical attention due to its involvement in the pathogenesis of diseases prevalent in the human population. The CB chemoreflex has a strong autonomic component, and therefore, it has been suggested that the exaggerated sympathetic outflow characteristic of obstructive sleep apnea (OSA) or chronic heart failure (CHF) is due to CB overactivation secondary to chronic intermittent hypoxia (CIH) and hypoperfusion, respectively [see for reviews Iturriaga (49), Nanduri et al. (85), Schultz et al. (110), Zera et al. (145)].

Although the molecular bases for these maladaptive processes are still poorly understood, available data suggest that cumulative ROS production by CB cells may have a critical role. Several groups have shown that hypoxia/reoxygenation cycles, the main feature of OSA, produce oxidative and nitro-oxidative stress in the CB, which in turn enhances the CB chemosensory responses to hypoxia leading to cardiovascular and autonomic alterations. ROS may increase the expression of signaling pathways such as proinflammatory cytokines and ET-1, which may contribute to potentiating the CB chemosensory responses to hypoxia and other stimuli.

In addition, ROS could modify the O₂-sensitive K⁺ channels, increasing the intracellular Ca²⁺ levels, which in turn evokes the release of excitatory transmitters (27, 94, 100, 101). CHF has also been shown to produce oxidative stress in the CB (29). In glomus cells from CHF rabbits, the outward voltage-gated K⁺ current has a smaller amplitude and its sensitivity to hypoxic inhibition is enhanced, resulting in the discharge of chemoreceptor afferents under normoxic state and an increase in discharge responsiveness to hypoxia (109). It has been shown that CB resection or deafferentation restores the sympathetic tone and improves associated cardiovascular alterations in an animal model of CIH and CHF (25, 26, 74).

However, the translation of these procedures to the clinical setting requires strict monitoring because bilateral CB removal may cause cardiovascular events, particularly during episodes of hypoxia and hypercapnia (49, 53, 103). In this scenario, antioxidants are potential therapeutic options to pharmacological modulation of CB overactivation. Systemic administration of low-molecular-mass antioxidant compounds has been shown to be ineffective to eliminate ROS in clinical trials. However, a more refined redox pharmacology could be developed to target specific signaling pathways in dysfunctional CB cells (23, 115). In this regard, it is noteworthy to stress a recent report indicating that mitochondrial antioxidants decrease CB afferent discharges in hypoxia and blunt the HVR in awake rats (120).

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 the survival of mammals in hypoxic environments. In acute hypoxia, ROS produced in mitochondria diffuse to the cytosol and inhibit K⁺ channels in the 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 the 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 the mitochondrial H₂O₂ level, such as modification of SOD1 or SOD2 expression, and 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 overexpressing recombinant K⁺ channels and thiol-redox proteomics may help us identify the direct target(s) of ROS during acute hypoxia.

Footnotes

Acknowledgments

The authors would like to thank all members of the laboratory who contributed to the experiments discussed in this review.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This research was supported by grants from the 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-1255654), all cofunded by FEDER, and the European Research Council (ERC-ADGPRJ201502629).

Abbreviations Used

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Mitochondrial Redox Signaling in O 2 -Sensing Chemoreceptor Cells

Abstract

Significance:

Recent Advances:

Critical Issues:

Future Directions:

Adaptive Acute Responses to Hypoxia

CB Chemoreceptor Activation by Hypoxia and Mitochondrial Inhibitors

Electrical properties of CB cells and mechanism of chemotransduction

Occlusion of responsiveness to hypoxia by rotenone

O2-sensitive mitochondrial signaling

Mitochondrial ROS Signaling in CB Chemoreceptor Cells During Acute Hypoxia

Sites of mitochondrial ROS production

Real-time measurement of mitochondrial ROS with genetic probes

Compartmentalized mitochondrial ROS changes during acute hypoxia

Loss of ROS signaling and responsiveness to hypoxia in genetically modified mice

Modulation of membrane ion channels by ROS in CB glomus cells

Role of ROS in Other Acute O2 Sensing Tissues

CB ROS Production and Mechanisms of Disease

Concluding Remarks

Footnotes

Acknowledgments

Author Disclosure Statement

Funding Information

Abbreviations Used

References

O₂-sensitive mitochondrial signaling

Role of ROS in Other Acute O₂ Sensing Tissues