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Chronic intermittent hypoxia (CIH) is a hallmark manifestation of obstructive sleep apnea (OSA), a widespread breathing disorder. CIH-treated rodents exhibit activation of the sympathetic nervous system and hypertension. Heightened carotid body (CB) activity has been implicated in CIH-induced hypertension. CB expresses high abundance of olfactory receptor (Olfr) 78, a G-protein coupled receptor. Olfr 78 null mice exhibit impaired CB sensory nerve response to acute hypoxia. Present study examined whether Olfr78 participates in CB-dependent activation of the sympathetic nervous system and hypertension in CIH-treated mice and in hemeoxygenase (HO)-2 null mice experiencing CIH as a consequence of naturally occurring OSA. CIH-treated wild-type (WT) mice showed hypertension, biomarkers of sympathetic nerve activation, and enhanced CB sensory nerve response to hypoxia and sensory long-term facilitation (sLTF), and these responses were absent in CIH-treated Olfr78 null mice. HO-2 null mice showed higher apnea index (AI) (58 ± 1.2 apneas/h) than WT mice (AI = 8 ± 0.8 apneas/h) and exhibited elevated blood pressure (BP), elevated plasma norepinephrine (NE) levels, and heightened CB sensory nerve response to hypoxia and sLTF. The magnitude of hypertension correlated with AI in HO-2 null mice. In contrast, HO-2/Olfr78 double null mice showed absence of elevated BP and plasma NE levels and augmented CB response to hypoxia and sLTF. These results demonstrate that Olfr78 participates in sympathetic nerve activation and hypertension and heightened CB activity in two murine models of CIH. NEW & NOTEWORTHY Carotid body (CB) sensory nerve activation is essential for sympathetic nerve excitation and hypertension in rodents treated with chronic intermittent hypoxia (CIH) simulating blood O(2) profiles during obstructive sleep apnea (OSA). Here, we report that CIH-treated mice and hemeoxygenase (HO)-2-deficient mice, which show OSA phenotype, exhibit sympathetic excitation, hypertension, and CB activation. These effects are absent in Olfr78 null and Olfr78/HO-2 double null mice.

Naked mole rats (NMRs) exhibit blunted hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). The mechanism(s) underlying these responses are largely unknown. We hypothesized that attenuated carotid body (CB) sensitivity to hypoxia and hypercapnia contributes to the near absence of ventilatory responses to hypoxia and COWe measured ex vivo CB sensory nerve activity, phrenic nerve activity (an estimation of ventilation), and blood gases in urethane-anesthetized NMRs and C57BL/6 mice breathing normoxic, hypoxic, or hypercapnic gases. CB morphology, carbon monoxide, and HRelative to mice, NMRs had blunted CB and HVR. Morphologically, NMRs have larger CBs, which contained more glomus cells than in mice. Furthermore, NMR glomus cells form a dispersed pattern compared to a clustered pattern in mice. Hemeoxygenase (HO)-1 mRNA was elevated in NMR CBs, and an HO inhibitor increased CB sensitivity to hypoxia in NMRs. This increase was blocked by an HConsistent with our hypothesis, impaired CB responses to hypoxia contribute in part to the blunted HVR in NMRs. Conversely, the HCVR and CB are more sensitive to CO

The role of olfactory receptor 78 (Olfr78) in carotid body (CB) response to hypoxia was examined. BL6 mice with global deletion of Olfr78 manifested an impaired hypoxic ventilatory response (HVR), a hallmark of the CB chemosensory reflex, CB sensory nerve activity, and reduced intracellular Ca2+ concentration ([Ca2+]i) response of glomus cells to hypoxia (Po2 ~ 40 mmHg). In contrast, severe hypoxia (Po2 ~ 10 mmHg) depressed breathing and produced a very weak CB sensory nerve excitation but robust elevation of [Ca2+]i in Olfr78 null glomus cells. CB sensory nerve excitation evoked by Olfr78 ligands, lactate, propionate, acetate, and butyrate were unaffected in mutant mice and were smaller than that evoked by hypoxia (Po2 ~ 40mmHg). Similar results were obtained in Olfr78 null mice on a JAX genetic background. These results demonstrate a role for Olfr78 in CB responses to a wide range of hypoxia, but not severe hypoxia, and do not require either lactate or any other short-chain fatty acids.NEW & NOTEWORTHY The current study demonstrates that olfactory receptor 78 (Olfr78), a G protein-coupled receptor, is an integral component of the hypoxic sensing mechanism of the carotid body to a wide range of low oxygen levels, but not severe hypoxia, and that Olfr78 participation does not require either lactate or any other short-chain fatty acids, proposed ligands of Olfr78.

Olfactory receptor (Olfr) 78 is expressed in the carotid bodies (CB) and participates in CB responses to acute hypoxia. Olfr78 is also expressed in the kidney, which is a major site of erythropoietin (Epo) production by hypoxia. The present study examined the role of Olfr78 in cardiorespiratory and renal Epo gene responses to hypobaric hypoxia (HH), simulating low O(2) condition experienced at high altitude. Studies were performed on adult, male wild-type (WT) and Olfr78 null mice treated with 18 h of HH (0.4 atmospheres). HH-treated WT mice exhibited increased baseline breathing, augmented hypoxic ventilatory response, elevated blood pressure, and plasma norepinephrine (NE) levels. These effects were associated with increased baseline CB sensory nerve activity and augmented CB sensory nerve response to subsequent acute hypoxia. In contrast, HH-treated Olfr78 null mice showed an absence of cardiorespiratory and CB sensory nerve responses, suggesting impaired CB-dependent cardiorespiratory adaptations. WT mice responded to HH with activation of the renal Epo gene expression and elevated plasma Epo levels, and these effects were attenuated or absent in Olfr78 null mice. The attenuated Epo activation by HH was accompanied with markedly reduced hypoxia-inducible factor (HIF)-2α protein and reduced activation of HIF-2 target gene Sod-1 in Olfr78 null mice, suggesting impaired transcriptional activation of HIF-2 contributes to attenuated Epo responses to HH. These results demonstrate a hitherto uncharacterized role for Olfr78 in cardiorespiratory adaptations and renal Epo gene activation by HH such as that experienced at high altitude. NEW & NOTEWORTHY In this study, we delineated a previously uncharacterized role for olfactory receptor 78 (Olfr78), a G-protein-coupled receptor in regulation of erythropoietin and cardiorespiratory responses to hypobaric hypoxia. Our results demonstrate a striking loss of cardiorespiratory adaptations accompanied by an equally striking absence of carotid body sensory nerve responses to hypobaric hypoxia in Olfr78 null mice. We further demonstrate a hitherto uncharacterized role for Olfr78 in erythropoietin activation by hypobaric hypoxia.

The role of cystathionine-γ-lyase (CSE) derived H2S in the hypoxic and anoxic responses of the carotid body (CB) were examined. Experiments were performed on Sprague-Dawley rats, wild type and CSE knockout mice on C57BL/6 J background. Hypoxia (pO2 = 37 ± 3 mmHg) increased the CB sensory nerve activity and elevated H2S levels in rats. In contrast, anoxia (pO2 = 5 ± 4 mmHg) produced only a modest CB sensory excitation with no change in H2S levels. DL-propargylglycine (DL-PAG), a blocker of CSE, inhibited hypoxia but not anoxia-evoked CB sensory excitation and [Ca2+]i elevation of glomus cells. The inhibitory effects of DL-PAG on hypoxia were seen: a) when it is dissolved in saline but not in dimethyl sulfoxide (DMSO), and b) in glomus cells cultured for18 h but not in cells either soon after isolation or after prolonged culturing (72 h) requiring 1–3 h of incubation. On the other hand, anoxia-induced [Ca2+]i responses of glomus cell were blocked by high concentration of DL-PAG (300μM) either alone or in combination with aminooxyacetic acid (AOAA; 300μM) with a decreased cell viability. Anoxia produced a weak CB sensory excitation and robust [Ca2+]i elevation in glomus cells of both wild-type and CSE null mice. As compared to wild-type, CSE null mice exhibited impaired CB chemo reflex as evidenced by attenuated efferent phrenic nerve responses to brief hyperoxia (Dejours test), and hypoxia. Inhalation of 100% N2 (anoxia) depressed breathing in both CSE null and wild-type mice. These observations demonstrate that a) hypoxia and anoxia are not analogous stimuli for studying CB physiology and b) CSE-derived H2S contributes to CB response to hypoxia but not to that of anoxia.

The role of olfactory receptor 78 (Olfr78) in carotid body (CB) response to hypoxia was examined. BL6 mice with global deletion of Olfr78 manifested an impaired hypoxic ventilatory response (HVR), a hallmark of the CB chemosensory reflex, CB sensory nerve activity, and reduced intracellular Ca(2+) concentration ([Ca(2+)](i)) response of glomus cells to hypoxia (Po(2) ~ 40 mmHg). In contrast, severe hypoxia (Po(2) ~ 10 mmHg) depressed breathing and produced a very weak CB sensory nerve excitation but robust elevation of [Ca(2+)](i) in Olfr78 null glomus cells. CB sensory nerve excitation evoked by Olfr78 ligands, lactate, propionate, acetate, and butyrate were unaffected in mutant mice and were smaller than that evoked by hypoxia (Po(2) ~ 40mmHg). Similar results were obtained in Olfr78 null mice on a JAX genetic background. These results demonstrate a role for Olfr78 in CB responses to a wide range of hypoxia, but not severe hypoxia, and do not require either lactate or any other short-chain fatty acids. NEW & NOTEWORTHY The current study demonstrates that olfactory receptor 78 (Olfr78), a G protein-coupled receptor, is an integral component of the hypoxic sensing mechanism of the carotid body to a wide range of low oxygen levels, but not severe hypoxia, and that Olfr78 participation does not require either lactate or any other short-chain fatty acids, proposed ligands of Olfr78.

Hypoxia-inducible factors mediate adaptive responses to reduced O2 availability. In patients with obstructive sleep apnoea, repeated episodes of hypoxaemia and reoxygenation (intermittent hypoxia) are sensed by the carotid body (CB). The ensuing CB chemosensory reflex activates the sympathetic nervous system and increased secretion of catecholamines by the adrenal medulla, resulting in hypertension and breathing abnormalities. In the CB, intermittent hypoxia induces the formation of reactive oxygen species (ROS) and increased intracellular Ca2+ levels, which drive increased expression of hypoxia-inducible factor (HIF) 1α and a decrease in the levels of HIF-2α. Intermittent hypoxia increases HIF-1α-dependent expression of Nox2, encoding the pro-oxidant enzyme NADPH oxidase 2, and decreased HIF-2α-dependent expression of Sod2, encoding the anti-oxidant enzyme superoxide dismutase 2. These changes in gene expression drive persistently elevated ROS levels in the CB, brainstem, and adrenal medulla that are required for the development of hypertension and breathing abnormalities. The ROS generated by dysregulated HIF activity in the CB results in oxidation and inhibition of haem oxygenase 2, and the resulting reduction in the levels of carbon monoxide leads to increased hydrogen sulfide production, triggering glomus cell depolarization. Thus, the pathophysiology of obstructive sleep apnoea involves the dysregulation of O2 -regulated transcription factors, gasotransmitters, and sympathetic outflow that affects blood pressure and breathing.

Long-term exposure to intermittent hypoxia (LT-IH; 30 days), simulating blood O2 profiles during sleep apnea, has been shown to repress anti-oxidant enzyme (AOE) gene expression by DNA methylation in the carotid body (CB) reflex pathway, resulting in persistent elevation of plasma catecholamine levels and blood pressure. The present study examined the mechanisms by which LT-IH induces DNA methylation. Adult rats exposed to LT-IH showed elevated reactive oxygen species (ROS) in the CB, nucleus tractus solitarius (nTS) and rostroventrolateral medulla (RVLM) and adrenal medulla (AM), which represent the central and efferent limbs of the CB reflex, respectively. ROS scavenger treatment during the first ten days of IH exposure prevented ROS accumulation, blocked DNA methylation, and normalized AOE gene expression, suggesting that ROS generated during the early stages of IH activate DNA methylation. CB ablation prevented the ROS accumulation, normalized AOE gene expression in the nTS, RVLM, and AM and blocked DNA methylation, suggesting that LT-IH-induced DNA methylation in the central and efferent limbs of the CB reflex is indirect and requires CB neural activity. LT-IH increased DNA methyl transferase (Dnmt) activity through upregulation of Dnmt1 and 3b protein expression due to ROS-dependent inactivation of glycogen synthase kinase 3β (GSK3β) by protein kinase B (Akt). Treating rats with the pan-Akt inhibitor GSK690693 blocked the induction of Dnmt activity, Dnmt protein expression, and DNA methylation, leading to normalization of AOE gene expression as well as plasma catecholamine levels and blood pressure. This article is protected by copyright. All rights reserved

We examined the contribution of the carotid chemoreceptors to insulin-mediated increases in muscle sympathetic nerve activity (MSNA) in healthy humans. We hypothesized that reductions in carotid chemoreceptor activity would attenuate the sympathoexcitatory response to hyperinsulinemia. Young, healthy adults (9 male/9 female, 28 ± 1 yr, 24 ± 1 kg/m2) completed a 30-min euglycemic baseline followed by a 90-min hyperinsulinemic (1 mU·kg fat-free mass−1·min−1), euglycemic infusion. MSNA (microneurography of the peroneal nerve) was continuously measured. The role of the carotid chemoreceptors was assessed at baseline and during hyperinsulinemia via 1) acute hyperoxia, 2) low-dose dopamine (1–4 µg·kg−1·min−1), and 3) acute hyperoxia + low-dose dopamine. MSNA burst frequency increased from baseline during hyperinsulinemia ( P < 0.01). Acute hyperoxia had no effect on MSNA burst frequency at rest ( P = 0.74) or during hyperinsulinemia ( P = 0.83). The insulin-mediated increase in MSNA burst frequency ( P = 0.02) was unaffected by low-dose dopamine ( P = 0.60). When combined with low-dose dopamine, acute hyperoxia had no effect on MSNA burst frequency at rest ( P = 0.17) or during hyperinsulinemia ( P = 0.85). Carotid chemoreceptor desensitization in young, healthy men and women does not attenuate the sympathoexcitatory response to hyperinsulinemia. Our data suggest that the carotid chemoreceptors do not contribute to acute insulin-mediated increases in MSNA in young, healthy adults.

People living at sea level experience intermittent hypoxia (IH) as a consequence of sleep apnea, which is a highly prevalent respiratory disorder. Sleep apnea patients and rodents exposed to IH exhibit autonomic dysfunction manifested as increased sympathetic nerve activity and hypertension. This article highlights physiologic basis of autonomic disturbances by IH, which involves abnormal activation of the carotid body (CB) chemo reflex by reactive oxygen species (ROS).We further evaluate major molecular mechanisms underlying IH-induced ROS generation including transcriptional activation of genes encoding pro-oxidant enzymes by hypoxia-inducible factor (HIF)-1 and transcriptional repression of anti-oxidant enzyme genes by DNA methylation. Lastly, evidence is presented for CB neural activity as a major regulator of HIF-1 activation and DNA methylation by IH in the chemo reflex pathway.

Carotid bodies are the sensory organs for detecting hypoxemia (decreased arterial blood oxygen levels) and ensuing chemo reflex is a major regulator of breathing and blood pressure. Chang et al (2015) proposed that olfactory receptor 78 (Olfr78) plays a major role in hypoxic sensing by the carotid body. However, such a possibility was questioned by a subsequent study ((Torres-Torrelo et al. 2018). The discrepancy between the two reports prompted the present study to re-examine the role of Olfr78 in hypoxic sensing by the carotid body (CB). Studies were performed on age and gender matched Olfr78 knock out mice generated on BL6 and JAX backgrounds and corresponding wild type mice. Breathing was monitored by plethysmography in un-sedated and efferent phrenic nerve activity in anesthetized mice. Carotid body sensory nerve activity was determined ex vivo and [Ca2+]i responses were monitored in isolated glomus cells, the primary O2 sensing cells of the carotid body. Olfr78 null mice on both BL6 and JAX backgrounds exhibited attenuated hypoxic ventilatory response, whereas breathing responses to CO2 were unaffected. The magnitude of hyperoxia-induced depression of breathing (Dejour’s test), which is an indirect measure of carotid body hypoxic sensing, was markedly reduced in Olfr78 mutant mice on both background strains. Furthermore, carotid body sensory nerve and glomus cell [Ca2+]i responses to hypoxia were attenuated in BL6 and JAX Olfr78 null mice. These results suggest that Olfr78 plays an important role in hypoxic sensing by the carotid body.

Key points The effects of short-term (ST; 10 days) and long-term (LT; 30 days) intermittent hypoxia (IH) on blood pressure (BP), breathing and carotid body (CB) chemosensory reflex were examined in adult rats. ST- and LT-IH treated rats exhibited hypertension, irregular breathing with apnoea and augmented the CB chemosensory reflex, with all these responses becoming normalized during recovery from ST- but not from LT-IH. The persistent cardiorespiratory responses to LT-IH were associated with elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla, which were a result of DNA methylation-dependent suppression of genes encoding anti-oxidant enzymes (AOEs). Treating rats with decitabine either during LT-IH or during recovery from LT-IH prevented DNA methylation of AOE genes, normalized the expression of AOE genes and ROS levels, reversed the heightened CB chemosensory reflex and hypertension, and also stabilized breathing. Abstract Rodents exposed to chronic intermittent hypoxia (IH), simulating blood O2 saturation profiles during obstructive sleep apnoea (OSA), have been shown to exhibit a heightened carotid body (CB) chemosensory reflex and hypertension. CB chemosensory reflex activation also results in unstable breathing with apnoeas. However, the effect of chronic IH on breathing is not known. In the present study, we examined the effects of chronic IH on breathing along with blood pressure (BP) and assessed whether the autonomic responses are normalized after recovery from chronic IH. Studies were performed on adult, male, Sprague–Dawley rats exposed to either short-term (ST; 10 days) or long-term (LT, 30 days) IH. Rats exposed to either ST- or LT-IH exhibited hypertension, irregular breathing with apnoeas, an augmented CB chemosensory reflex as indicated by elevated CB neural activity and plasma catecholamine levels, and elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla (AM). All these effects were normalized after recovery from ST-IH but not from LT-IH. Analysis of the molecular mechanisms underlying the persistent effects of LT-IH revealed increased DNA methylation of genes encoding anti-oxidant enzymes (AOEs). Treatment with decitabine, a DNA methylation inhibitor, either during LT-IH or during recovery from LT-IH, prevented DNA methylation, normalized the expression of AOE genes, ROS levels, CB chemosensory reflex and BP, and also stabilized breathing. These results suggest that persistent cardiorespiratory abnormalities caused by LT-IH are mediated by epigenetic re-programming of the redox state in the CB chemosensory reflex pathway.

What is the topic of this review? This article presents emerging evidence for heightened carotid body chemoreflex activity as a major driver of sympathetic activation and hypertension in sleep apnoea patients. What advances does it heighlight? This article discusses the recent advances on cellular, molecular and epigenetic mechanisms underlying the exaggerated chemoreflex in experimental models of sleep apnoea. The carotid bodies are the principal peripheral chemoreceptors for detecting changes in arterial blood oxygen concentration, and the resulting chemoreflex is a potent regulator of the sympathetic tone, blood pressure and breathing. Sleep apnoea is a disease of the respiratory system that affects several million adult humans. Apnoeas occur during sleep, often as a result of obstruction of the upper airway (obstructive sleep apnoea) or because of defective respiratory rhythm generation by the CNS (central sleep apnoea). Patients with sleep apnoea exhibit several co-morbidities, with the most notable among them being heightened sympathetic nerve activity and hypertension. Emerging evidence suggests that intermittent hypoxia resulting from periodic apnoea stimulates the carotid body, and the ensuing chemoreflex mediates the increased sympathetic tone and hypertension in sleep apnoea patients. Rodent models of intermittent hypoxia that simulate the O2 saturation profiles encountered during sleep apnoea have provided important insights into the cellular and molecular mechanisms underlying the heightened carotid body chemoreflex. This article describes how intermittent hypoxia affects the carotid body function and discusses the cellular, molecular and epigenetic mechanisms underlying the exaggerated chemoreflex.

Sleep apnea with periodic cessation of breathing during sleep is a highly prevalent respiratory disorder affecting an estimated 10% of adults. Patients with sleep apnea exhibit several co-morbidities including hypertension, stroke, disrupted sleep, and neurocognitive and metabolic complications. Emerging evidence suggests that a hyperactive carotid body (CB) chemo reflex is an important driver of apneas in sleep apnea patients. Gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H

The role of cystathionine-γ-lyase (CSE) derived H(2)S in the hypoxic and anoxic responses of the carotid body (CB) were examined. Experiments were performed on Sprague-Dawley rats, wild type and CSE knockout mice on C57BL/6J background. Hypoxia (pO(2)=37±3 mmHg) increased the CB sensory nerve activity and elevated H(2)S levels in rats. In contrast, anoxia (pO(2)=5±4 mmHg) produced only a modest CB sensory excitation with no change in H(2)S levels. DL-propargylglycine (DL-PAG), a blocker of CSE, inhibited hypoxia but not anoxia-evoked CB sensory excitation and [Ca(2+)](i) elevation of glomus cells. The inhibitory effects of DL-PAG on hypoxia were seen: a) when it is dissolved in saline but not in dimethyl sulfoxide (DMSO), and in glomus cells cultured for18h but not in cells either soon after isolation or after prolonged culturing (72h) requiring 1–3h of incubation. On the other hand, anoxia-induced [Ca(2+)](i) responses of glomus cell were blocked by high concentration of DL-PAG (300μM) either alone or in combination with aminooxyacetic acid (AOAA; 300μM) with a decreased cell viability. Anoxia produced a weak CB sensory excitation and robust [Ca(2+)](i) elevation in glomus cells of both wild-type and CSE null mice. As compared to wild-type, CSE null mice exhibited impaired CB chemo reflex as evidenced by attenuated efferent phrenic nerve responses to brief hyperoxia (Dejours test), and hypoxia. Inhalation of 100% N(2) (anoxia) depressed breathing in both CSE null and wild-type mice. These observations demonstrate that a) hypoxia and anoxia are not analogous stimuli for studying CB physiology and b) CSE-derived H(2)S contributes to CB response to hypoxia but not to that of anoxia.

Sleep apnea is a prevalent respiratory disease characterized by periodic cessation of breathing during sleep causing intermittent hypoxia (IH). Sleep apnea patients and rodents exposed to IH exhibit elevated sympathetic nerve activity and hypertension. A heightened carotid body (CB) chemo reflex has been implicated in causing autonomic abnormalities in IH treated rodents and in sleep apnea patients. The purpose of this article is to review the emerging evidence showing that interactions between reactive oxygen species (ROS) and gaseous transmitters as a mechanism causing hyperactive CB by IH. Rodents treated with IH exhibit markedly elevated ROS in the CB, which is due to transcriptional upregulation of pro-oxidant enzymes by hypoxia-inducible factor (HIF)-1, and insufficient transcriptional regulation of anti-oxidant enzymes by HIF-2. ROS, in turn, increases cystathionine-γ-lyase (CSE)-dependent H(2)S production in the CB. Blockade of H(2)S synthesis prevents IH-evoked CB activation. However, the effects of ROS on H(2)S production is not due to direct effects on CSE enzyme activity, rather due to inactivation of heme oxygenase-2 (HO-2), a carbon monoxide (CO) producing enzyme. CO inhibits H(2)S production through inactivation of CSE by PKG-dependent phosphorylation. During IH, reduced CO production resulting from inactivation of HO-2 by ROS releases the inhibition of CO on CSE thereby increases H(2)S. Inhibiting H(2)S synthesis prevented IH-evoked sympathetic activation and hypertension.

Carotid bodies are sensory organs for monitoring chemical composition of the arterial blood, especially the O

Immunohistochemistry (IHC) enables the detection and distribution of proteins in cells of tissues. IHC has become an indispensable approach for studying oxygen sensing by the carotid body (CB). This chapter provides a detailed description of IHC of CB tissue and isolated CB cells.

Sleep apnea with periodic cessation of breathing during sleep is a highly prevalent respiratory disorder affecting an estimated 10% of adults. Patients with sleep apnea exhibit several co-morbidities including hypertension, stroke, disrupted sleep, and neurocognitive and metabolic complications. Emerging evidence suggests that a hyperactive carotid body (CB) chemo reflex is an important driver of apneas in sleep apnea patients. Gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H2S) play important roles in oxygen sensing by the CB. We tested the hypothesis that an augmented CB chemo reflex stemming from disrupted CO-H2S signaling may lead to sleep apnea. This possibility was tested in mice deficient in hemeoxygenase-2 (HO-2), an enzyme involved in CO synthesis, which were shown to exhibit hyperactive CB activity due to high H2S levels. We found that HO-2−/− mice exhibit a high incidence of apneas during sleep compared to wild type mice. Blocking the CB hyperactivity with L-propargylglycine, an inhibitor of cystathionine-γ-lyase (CSE), which catalyzes H2S synthesis, prevented apneas in HO-2−/− mice. These findings suggest that targeting CB with inhibitors of CSE might be a novel therapeutic strategy for preventing sleep apnea.

Carotid bodies are sensory organs for monitoring chemical composition of the arterial blood, especially the O2 levels. Carotid bodies are located bilaterally at the bifurcation of the common carotid artery. Hypoxia increases sensory nerve activity of the carotid body, which is transmitted to the brainstem neurons triggering reflex stimulation of breathing and blood pressure. Measurement of action potentials from the carotid sinus nerve is a widely used approach for understanding the mechanisms of hypoxic sensing by the carotid body. Here, we describe the detailed methodology for recording action potential signals from the carotid sinus nerve from in vivo and ex vivo carotid bodies from rats.

Arterial blood O2levels are detected by specialized sensory organs called carotid bodies. Voltage-gated Ca2+channels (VGCCs) are important for carotid body O2sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1Hprotein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca2+concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout ( Cacna1h−/−) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca2+concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h−/−mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia.

Hypoxia‐inducible factors mediate adaptive responses to reduced O(2) availability. In patients with obstructive sleep apnoea, repeated episodes of hypoxaemia and reoxygenation (intermittent hypoxia) are sensed by the carotid body (CB). The ensuing CB chemosensory reflex activates the sympathetic nervous system and increased secretion of catecholamines by the adrenal medulla, resulting in hypertension and breathing abnormalities. In the CB, intermittent hypoxia induces the formation of reactive oxygen species (ROS) and increased intracellular Ca(2+) levels, which drive increased expression of hypoxia‐inducible factor (HIF) 1α and a decrease in the levels of HIF‐2α. Intermittent hypoxia increases HIF‐1α‐dependent expression of Nox2, encoding the pro‐oxidant enzyme NADPH oxidase 2, and decreased HIF‐2α‐dependent expression of Sod2, encoding the anti‐oxidant enzyme superoxide dismutase 2. These changes in gene expression drive persistently elevated ROS levels in the CB, brainstem, and adrenal medulla that are required for the development of hypertension and breathing abnormalities. The ROS generated by dysregulated HIF activity in the CB results in oxidation and inhibition of haem oxygenase 2, and the resulting reduction in the levels of carbon monoxide leads to increased hydrogen sulfide production, triggering glomus cell depolarization. Thus, the pathophysiology of obstructive sleep apnoea involves the dysregulation of O(2)‐regulated transcription factors, gasotransmitters, and sympathetic outflow that affects blood pressure and breathing. [Image: see text]

It is now well established that carotid bodies are sensory organs for monitoring arterial blood oxygen levels and trigger reflexes that are critical for maintaining homeostasis during hypoxemia. This review article provides a brief account of the early studies leading to the discovery of the carotid body as a sensory receptor and addresses current views of O

Previous studies reported that chronic intermittent hypoxia (CIH) results in an imbalanced expression of hypoxia-inducible factor-α (HIF-α) isoforms and oxidative stress in rodents, which may be due either to the direct effect of CIH or indirectly via hitherto uncharacterized mechanism(s). As neural activity is a potent regulator of gene transcription, we hypothesized that carotid body (CB) neural activity contributes to CIH-induced HIF-α isoform expression and oxidative stress in the chemoreflex pathway. Experiments were performed on adult rats exposed to CIH for 10 days. Rats exposed to CIH exhibited: increased HIF-1α and decreased HIF-2α expression; increased NADPH oxidase 2 and decreased superoxide dismutase 2 expression; and oxidative stress in the nucleus tractus solitarius and rostral ventrolateral medulla as well as in the adrenal medulla (AM), a major end organ of the sympathetic nervous system. Selective ablation of the CB abolished these effects. In the AM, sympathetic activation by the CB chemoreflex mediates CIH-induced HIF-α isoform imbalance via muscarinic acetylcholine receptor-mediated Ca(2+) influx, and the resultant activation of mammalian target of rapamycin pathway and calpain proteases. Rats exposed to CIH presented with hypertension, elevated sympathetic activity and increased circulating catecholamines. Selective ablation of either the CB (afferent pathway) or sympathetic innervation to the AM (efferent pathway) abolished these effects. These observations uncover CB neural activity-dependent regulation of HIF-α isoforms and the redox state by CIH in the central and peripheral nervous systems associated with the chemoreflex.

Studies on experimental animals established that the carotid bodies are sensory organs for detecting arterial blood O2 levels and that the ensuing chemosensory reflex is a major regulator of cardiorespiratory functions during hypoxia. However, little information is available on the human carotid body responses to hypoxia. The present study was performed on human carotid bodies obtained from surgical patients undergoing elective head and neck cancer surgery. Our results show that exposing carotid body slices to hypoxia for a period as brief as 5 min markedly facilitates the release of ACh and ATP. Furthermore, prolonged hypoxia for 1 h induces an increased release of interleukin (IL)-1β, IL-4, IL-6, IL-8 and IL-10. Immunohistochemical analysis revealed that type 1 cells of the human carotid body express an array of cytokine receptors as well as hypoxia-inducible factor-1α and hypoxia-inducible factor-2α. Taken together, these results demonstrate that ACh and ATP are released from the human carotid body in response to hypoxia, suggesting that these neurotransmitters, as in several experimental animal models, play a role in hypoxic signalling also in the human carotid body. The finding that the human carotid body releases cytokines in response to hypoxia adds to the growing body of information suggesting that the carotid body may play a role in detecting inflammation, providing a link between the immune system and the nervous system.

The sympathoexcitatory effects of insulin are well-established, although the exact mechanisms by which insulin stimulates the sympathetic nervous system are not completely understood. The majority of research supports a primary role for the central nervous system in the gradual and sustained rise in muscle sympathetic nerve activity (MSNA) in response to hyperinsulinemia; in addition, recent studies in animals suggests carotid body chemoreceptors respond to increases in systemic insulin levels. Intermittent activation of the carotid chemoreceptors, similar to that seen in patients with sleep apnea, can result in sensory long term facilitation and may contribute to the observed rise in baseline MSNA in this population. Consistent with this idea, insulin exposure results in sustained increases in MSNA that persist even when plasma insulin levels return to baseline. We propose the carotid chemoreceptors contribute to insulin-mediated sympathoexcitation and the persistent rise in MSNA in patients with sustained hyperinsulinemia. If the carotid chemoreceptors sense and respond to changes in systemic insulin levels, these organs may provide a viable target for the treatment of disorders known to exhibit sustained hyperinsulinemia and sympathoexcitation including, but not limited to, obesity, hypertension, sleep apnea, metabolic syndrome, cardiovascular disease, and diabetes.

Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Humans and animals exhibit substantial interindividual variation in this chemosensory reflex response, with profound effects on cardiorespiratory functions. However, the underlying mechanisms are not known. Here, we report that inherent variations in carotid body O2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O2 sensing and develop pulmonary edema as a consequence of poor ventilatory adaptation to hypobaric hypoxia. Spontaneous Hypertensive (SH) rat carotid bodies display inherent hypersensitivity to hypoxia and develop hypertension. BN rat carotid bodies have naturally higher CO and lower H2S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H2S generation. Higher CO levels in BN rats were associated with higher substrate affinity of the enzyme heme oxygenase 2, whereas SH rats present lower substrate affinity and, thus, reduced CO generation. Reducing CO levels in BN rat carotid bodies increased H2S generation, restoring O2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H2S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats.

Carotid bodies detect hypoxia in arterial blood, translating this stimulus into physiological responses via the CNS. It is long established that ion channels are critical to this process. More recent evidence indicates that gasotransmitters exert powerful influences on O2sensing by the carotid body. Here, we review current understanding of hypoxia-dependent production of gasotransmitters, how they regulate ion channels in the carotid body, and how this impacts carotid body function.

The carotid body is a sensory organ for detecting arterial blood O2 levels and reflexly mediates systemic cardiac, vascular and respiratory responses to hypoxia. This article presents a brief review of the roles of gaseous messengers in the sensory transduction at the carotid body, genetic and epigenetic influences on hypoxic sensing and the role of the carotid body chemoreflex in cardiorespiratory diseases. Type I (also called glomus) cells, the site of O2 sensing in the carotid body, express haem oxygenase-2 and cystathionine-γ-lyase, the enzymes which catalyse the generation of CO and H2S, respectively. Physiological studies have shown that CO is an inhibitory gas messenger, which contributes to the low sensory activity during normoxia, whereas H2S is excitatory and mediates sensory stimulation by hypoxia. Hypoxia-evoked H2S generation in the carotid body requires the interaction of cystathionine-γ-lyase with haem oxygenase-2, which generates CO. Hypoxia-inducible factors 1 and 2 constitute important components of the genetic make-up in the carotid body, which influence hypoxic sensing by regulating the intracellular redox state via transcriptional regulation of pro- and antioxidant enzymes. Recent studies suggest that developmental programming of the carotid body response to hypoxia involves epigenetic changes, e.g. DNA methylation of genes encoding redox-regulating enzymes. Emerging evidence implicates heightened carotid body chemoreflex in the progression of autonomic morbidities associated with cardiorespiratory diseases, such as sleep-disordered breathing with apnoea, congestive heart failure and essential hypertension.

Sleep apnea, which is the periodic cessation of breathing during sleep, is a major health problem affecting over 10 million people in the United States and is associated with several sequelae, including hypertension and stroke. Clinical studies suggest that abnormal carotid body (CB) activity may be a driver of sleep apnea. Because gaseous molecules are important determinants of CB activity, aberrations in their signaling could lead to sleep apnea. Here, we report that mice deficient in heme oxygenase-2 (HO-2), which generates the gaseous molecule carbon monoxide (CO), exhibit sleep apnea characterized by high apnea and hypopnea indices during rapid eye movement (REM) sleep. Similar high apnea and hypopnea indices were also noted in prehypertensive spontaneously hypertensive (SH) rats, which are known to exhibit CB hyperactivity. We identified the gaseous molecule hydrogen sulfide (H2S) as the major effector molecule driving apneas. Genetic ablation of the H2S-synthesizing enzyme cystathionine-γ-lyase (CSE) normalized breathing in HO-2-/- mice. Pharmacologic inhibition of CSE with l-propargyl glycine prevented apneas in both HO-2-/- mice and SH rats. These observations demonstrate that dysregulated CO and H2S signaling in the CB leads to apneas and suggest that CSE inhibition may be a useful therapeutic intervention for preventing CB-driven sleep apnea.

It is now well established that carotid bodies are sensory organs for monitoring arterial blood oxygen levels and trigger reflexes that are critical for maintaining homeostasis during hypoxemia. This review article provides a brief account of the early studies leading to the discovery of the carotid body as a sensory receptor and addresses current views of O2 sensing mechanism(s) in the carotid body and their physiological importance.

Sleep apnea is a prevalent respiratory disease in which episodic cessation of breathing causes intermittent hypoxia. Patients with sleep apnea and rodents exposed to intermittent hypoxia exhibit hypertension. The carotid body senses changes in blood O 2 concentrations, and an enhanced carotid body chemosensory reflex contributes to hypertension in sleep apnea patients. A rodent model of intermittent hypoxia that mimics blood O 2 saturation profiles of patients with sleep apnea has shown that increased generation of reactive oxygen species (ROS) in the carotid body enhances the chemosensory reflex and triggers hypertension. CO generated by heme oxygenase-2 (HO-2) induces a signaling pathway that inhibits hydrogen sulfide (H 2 S) production by cystathionine γ-lyase (CSE), leading to suppression of carotid body activity. We found that ROS inhibited CO generation by HO-2 in the carotid body and liver through a mechanism that required Cys 265 in the heme regulatory motif of heterologously expressed HO-2. We showed that ROS induced by intermittent hypoxia inhibited CO production and increased H 2 S concentrations in the carotid body, which stimulated its neural activity. In rodents, blockade of H 2 S synthesis by CSE, by either pharmacologic or genetic approaches, inhibited carotid body activation and hypertension induced by intermittent hypoxia. Thus, our results indicate that oxidant-induced inactivation of HO-2, which leads to increased CSE-dependent H 2 S production in the carotid body, is a critical trigger of hypertension in rodents exposed to intermittent hypoxia.

Carotid bodies are sensory organs for monitoring arterial blood oxygen (O 2 ) levels, and the ensuing reflexes maintain cardio-respiratory homeostasis during hypoxia. This article provides a brief update of the role of carbon monoxide (CO) and hydrogen sulfide (H 2 S) in hypoxic sensing by the carotid body. Glomus cells, the primary site of O 2 sensing in the carotid body express heme oxygenase-2 (HO-2), a CO catalyzing enzyme. HO-2 is a heme containing enzyme and has high affinity for O 2 . Hypoxia inhibits HO-2 activity and reduces CO generation. Pharmacological and genetic approaches suggest that CO inhibits carotid body sensory activity. Stimulation of carotid body activity by hypoxia may reflect reduced formation of CO. Glomus cells also express cystathionine γ-lyase (CSE), an H 2 S generating enzyme. Exogenous application of H 2 S donors, like hypoxia, stimulate the carotid body activity and CSE knockout mice exhibit severely impaired sensory excitation by hypoxia, suggesting that CSE catalyzed H 2 S is an excitatory gas messenger. Hypoxia increases H 2 S generation in the carotid body, and this response was attenuated or absent in CSE knockout mice. HO inhibitor increased and CO donor inhibited H 2 S generation. It is proposed that carotid body response to hypoxia requires interactions between HO-2–CO and CSE–H 2 S systems.

Recurrent apnea with chronic intermittent hypoxia (CIH) is a major clinical problem in adult humans and infants born preterm. Patients with recurrent apnea exhibit heightened sympathetic activity as well as elevated plasma catecholamine levels, and these phenotypes are effectively recapitulated in rodent models of CIH. This article summarizes findings from studies addressing sympathetic activation in recurrent apnea patients and rodent models of CIH and the underlying cellular and molecular mechanisms. Available evidence suggests that augmented chemoreflex and attenuated baroreflex contribute to sympathetic activation by CIH. Studies on rodents showed that CIH augments the carotid body response to hypoxia and attenuates the carotid baroreceptor response to increased sinus pressures. Processing of afferent information from chemoreceptors at the central nervous system is also facilitated by CIH. Adult and neonatal rats exposed to CIH exhibit augmented catecholamine secretion from the adrenal medulla. Adrenal demedullation prevents the elevation of circulating catecholamines in CIH-exposed rodents. Reactive oxygen species (ROS)-mediated signaling is emerging as the major cellular mechanism triggering sympatho-adrenal activation by CIH. Molecular mechanisms underlying increased ROS generation by CIH seem to involve transcriptional dysregulation of genes encoding pro-and antioxidant enzymes by hypoxia-inducible factor-1 and -2, respectively.

The carotid body is a sensory organ that detects acute changes in arterial blood oxygen (O2) levels and reflexly mediates systemic cardiac, vascular, and respiratory responses to hypoxia. This article provides a brief update of the roles of gas messengers as well as redox homeostasis by hypoxia-inducible factors (HIFs) in hypoxic sensing by the carotid body. Carbon monoxide (CO) and nitric oxide (NO), generated by heme oxygenase-2 (HO-2) and neuronal nitric oxide synthase (nNOS), respectively, inhibit carotid body activity. Molecular O2 is a required substrate for the enzymatic activities of HO-2 and nNOS. Stimulation of carotid body activity by hypoxia may reflect reduced formation of CO and NO. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine γ-lyase (CSE), an H2S generating enzyme. Cth −/− mice, which lack CSE, exhibit severely impaired hypoxia-induced H2S generation, sensory excitation, and stimulation of breathing in response to low O2. Hypoxia-evoked H2S generation in the carotid body requires the interaction of CSE with HO-2, which generates CO. Carotid bodies from Hif1a +/− mice with partial HIF-1α deficiency do not respond to hypoxia, whereas carotid bodies from mice with partial HIF-2α deficiency are hyper-responsive to hypoxia. The opposing roles of HIF-1α and HIF-2α in the carotid body have provided novel insight into molecular mechanisms of redox homeostasis and its role in hypoxia sensing. Heightened carotid body activity has been implicated in the pathogenesis of autonomic morbidities associated with sleep-disordered breathing, congestive heart failure, and essential hypertension. The enzymes that generate gas messengers and redox regulation by HIFs represent potential therapeutic targets for normalizing carotid body function and downstream autonomic output in these disease states.

Sleep disordered breathing with recurrent apnea (transient, repetitive cessation of breathing) is a major cause of morbidity and mortality in adult humans (Nieto et al., 2000) and pre-term infants (Poets et al., 1994). Recurrent apneas are associated with periodic decreases in arterial blood oxygen or intermittent hypoxia (IH). Humans with recurrent apnea and rodents exposed to IH exhibit autonomic morbidities including persistent sympathetic activation, hypertension, and elevated circulating catecholamines (Narkiewicz and Somers, 1997; Peppard et al., 2000; Prabhakar and Kumar, 2010). Studies on humans (Narkiewicz and Somers, 1997) and rodent models of IH (Fletcher et al., 1992; Peng et al., 2006) have shown that the carotid body, which is the primary chemoreceptor for detecting changes in arterial PO2, responds to IH by triggering reflex activation of the sympathetic nervous system and enhanced catecholamine secretion from adrenal medullary chromaffin cells (AMC), leading to elevated circulating catecholamines and elevated blood pressure (Bao et al., 1997; Kumar et al., 2006; Souvannakitti et al., 2009). IH increases the levels of reactive oxygen species (ROS) in the carotid body (Peng et al., 2003) and adrenal medulla (Kumar et al., 2006; Souvannakitti et al., 2009). Anti-oxidant treatment prevents increased carotid body function (Peng et al., 2003; Peng and Prabhakar, 2004; Del Rio et al., 2010), augmented catecholamine secretion from AMC (Kumar et al., 2006; Kuri et al., 2007; Souvannakitti et al., 2009), and elevated blood pressure (Peng et al., 2006; Troncoso Brindeiro et al., 2007), indicating that ROS signaling is a critical cellular mechanism underlying IH-evoked autonomic morbidities. NADPH oxidase (Nox) activity is a major source of cellular ROS (Bedard and Krause, 2007). IH leads to increased expression of several Nox isoforms in the carotid body (Peng et al., 2009), AMC (Souvannakitti et al., 2010), and central nervous system (CNS; Zhan et al., 2005) as well as in cultured PC12 rat pheochromocytoma cells, which are derived from AMC (Yuan et al., 2008). Of the various Nox isoforms, Nox2 has been implicated in mediating the effects of IH on carotid body function (Peng et al., 2009), catecholamine secretion from AMC (Souvannakitti et al., 2010), and sleep behavior (Zhan et al., 2005). However, mechanisms underlying increased Nox2 gene expression in response to IH have not been elucidated. The transcriptional activator hypoxia-inducible factor 1 (HIF-1) is a master regulator of O2 homeostasis that controls multiple physiological processes by regulating the expression of hundreds of genes (Mole et al., 2009; Semenza, 2009). HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1β subunit and an O2-regulated HIF-1α subunit (Wang et al., 1995). We previously reported that IH increases HIF-1α protein levels in PC12 cell cultures (Yuan et al., 2005, 2008) and in mice (Peng et al., 2006). In catecholamine-producing PC12 cells, the induction of HIF-1α protein levels by IH requires ROS-dependent activation of phospholipase Cγ (PLCγ), protein kinase C (PKC), and mammalian target of rapamycin (mTOR; Yuan et al., 2008). Physiological studies of wild-type (WT) mice exposed to IH revealed striking autonomic morbidities, including heightened carotid body activity, elevated plasma catecholamines, exaggerated hypoxic ventilatory response, and hypertension (Peng et al., 2006). In contrast, littermate Hif1a+/− mice, which are heterozygous for a null [knockout (KO)] allele at the locus encoding HIF-1α, do not display any of these IH-induced autonomic responses (Peng et al., 2006). Furthermore, ROS levels were significantly increased in the CNS of IH-exposed WT mice, but not in Hif1a+/− mice (Peng et al., 2006). Taken together, these results indicated that IH-induced ROS leads to HIF-1 activation, which leads to further ROS generation, thereby creating a feed-forward mechanism that is essential for the pathogenesis of cardiovascular and respiratory abnormalities. Establishing the molecular mechanism by which HIF-1 increases ROS generation is the critical next step in understanding the pathobiology of IH. Based on the observations described above, we tested the hypothesis that in response to IH, HIF-1 increases ROS generation by activation of the Nox2 gene.

Gaseous messengers, nitric oxide and carbon monoxide, have been implicated in O 2 sensing by the carotid body, a sensory organ that monitors arterial blood O 2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H 2 S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O 2 sensing in the carotid body, express cystathionine γ-lyase (CSE), an H 2 S-generating enzyme, with hypoxia increasing H 2 S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H 2 S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H 2 S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.

We previously reported that intermittent hypoxia (IH) augments hypoxic sensory response (HSR) and increases the number of glomus cells in neonatal carotid bodies. In the present study, we tested the hypothesis that recruitment of endothelin-1 (ET-1) signaling by reactive oxygen species (ROS) plays a critical role in IH-evoked changes in neonatal carotid bodies. Experiments were performed on neonatal rats exposed either to 10 days of IH (P0–P10; 8 h/day) or to normoxia. IH augmented HSR of the carotid bodies ex vivo and resulted in hyperplasia of glomus cells. The effects of IH were associated with enhanced basal release of ET-1 under normoxia, sensitization of carotid body response to exogenous ET-1, and upregulation of ETA but not an ETB receptor mRNA without altering the ET-1 content. An ETA but not ETB receptor antagonist prevented augmented HSR by IH. ROS levels were elevated in carotid bodies from IH-treated rat pups as evidenced by increased levels of malondialdehyde. Systemic administration of manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 5 mg/kg ip), a scavenger of O2•−, prevented IH-induced elevation of ROS, basal release of ET-1, upregulation of ETA mRNA, and augmented HSR. In striking contrast, MnTMPyP treatment had no significant effect on IH-induced hyperplasia of glomus cells. These results demonstrate that IH-evoked increase in HSR involve a ROS-mediated increase in basal ET-1 release and upregulation of ETA receptor mRNA.

The effects of short-term (ST; 10 days) and long-term (LT; 30 days) intermittent hypoxia (IH) on blood pressure (BP), breathing and carotid body (CB) chemosensory reflex were examined in adult rats. ST- and LT-IH treated rats exhibited hypertension, irregular breathing with apnoea and augmented the CB chemosensory reflex, with all these responses becoming normalized during recovery from ST- but not from LT-IH. The persistent cardiorespiratory responses to LT-IH were associated with elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla, which were a result of DNA methylation-dependent suppression of genes encoding anti-oxidant enzymes (AOEs). Treating rats with decitabine either during LT-IH or during recovery from LT-IH prevented DNA methylation of AOE genes, normalized the expression of AOE genes and ROS levels, reversed the heightened CB chemosensory reflex and hypertension, and also stabilized breathing.Rodents exposed to chronic intermittent hypoxia (IH), simulating blood O

Chronic intermittent hypoxia (CIH) is a common and life-threatening condition that occurs in many different diseases, including sleep-disordered breathing manifested as recurrent apneas. Reactive oxygen species (ROS) have been identified as one of the causative factors in a variety of morbidities. The purpose of this article is to present a brief overview of recent studies implicating a critical role of ROS in evoking phenotypic adverse effects in experimental models of CIH and in patients with recurrent apneas. In experimental models, CIH activates ROS signaling that contributes to several systemic and cellular responses that include (a) altered carotid body function, the primary chemoreceptor for sensing changes in arterial blood O2; (b) elevated blood pressures; (c) enhanced release of transmitters and neurotrophic factors; (d) altered sleep and cognitive behaviors; and (e) activation of second-messenger pathways and transcriptional factors. Considerable evidence indicates elevated ROS levels in patients experiencing CIH as a consequence of recurrent apneas. Antioxidants not only prevent many of the CIH-evoked physiologic and cellular responses in experimental settings, but more important, they also offer protection against certain phenotypic adverse effects in patients with recurrent apneas, suggesting their potential therapeutic value in alleviating certain morbidities associated with recurrent apneas.

Sleep-disordered breathing with recurrent apnea is a major cause of morbidity and mortality. Affected individuals have increased risk of systemic hypertension. Sleep apnea results in chronic intermittent hypoxia (CIH). Exposure of rodents to CIH is sufficient to induce hypertension by activation of the carotid body and sympathetic nervous system, leading to increased levels of circulating catecholamines. CIH induces increased levels of reactive oxygen species (ROS), and antioxidant treatment blocks CIH-induced hypertension. The transcriptional activator hypoxia-inducible factor 1 (HIF-1) plays an essential role in O2 homeostasis. HIF-1 activity is induced when mice or cultured cells are subjected to CIH, an effect that is blocked by antioxidants. The carotid bodies from mice that are heterozygous for a null (knockout) allele at the locus encoding HIF-1 appear histologically normal but do not respond to continuous hypoxia or CIH. In contrast to wild-type littermates, when heterozygous-null mice are subjected to CIH, they do not develop hypertension or increased levels of HIF-1, catecholamines, or ROS. The data suggest the existence of a feed-forward mechanism in which CIH-induced ROS activate HIF-1, which then promotes persistent oxidative stress, which may further amplify HIF-1 activation, with its consequent effects on gene expression.

Patients with recurrent apnoeas exhibit autonomic abnormalities manifested as persistent increase in sympathetic nerve activity (SNA). Several studies suggest that chronic intermittent hypoxia (CIH) resulting from recurrent apnoeas is a major stimulus for evoking autonomic morbidity. Although it has been proposed that CIH, by way of activating the chemoreceptor reflex, leads to sympathetic excitation, the underlying mechanisms are incompletely understood. Studies on experimental models have provided new insights into the mechanisms associated with CIH-evoked sympathoexcitation. The purpose of this article is to highlight recent information on systemic, cellular and molecular analysis of the effects of CIH on chemoreceptor-mediated sympathoexcitation. Chronic intermittent hypoxia exerts two major effects on the chemoreceptor reflex: (a) augmentation of the carotid body and sympathetic effector responses to acute hypoxia; and (b) induction of long-lasting activation of both the sensor and the effector that persists several hours after termination of CIH. Available evidence indicates that CIH may facilitate processing of chemoreceptor afferent information at the central nervous system. Recent studies suggest that reactive oxygen species-mediated signalling is a major cellular mechanism, and transcriptional activation by hypoxia-inducible factor-1 is one of the critical molecular mechanisms underlying chemoreceptor-mediated sympathoexcitation by CIH.

Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. A heightened carotid body activity and the resulting chemosensory reflex mediate increased sympathetic nerve activity by CIH. However, the mechanisms underlying heightened carotid body activity by CIH are not known. An elevation of intracellular calcium ion concentration ([Ca2+]i) in glomus cells, the primary oxygen-sensing cells, is an essential step for carotid body activation by hypoxia. In the present study, we examined the effects of CIH on the glomus cell [Ca2+]iresponse to hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats or wild-type mice treated with 10 days of either room air (control) or CIH (alternating cycles of 15 s of hypoxia and 5 min of room air; 9 episodes/h; 8 h/day). CIH-treated glomus cells exhibited an enhanced [Ca2+]iresponse to hypoxia, and this effect was absent in the presence of 2-(4-cyclopropylphenyl)- N-((1R)-1-[5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl]ethyl)acetamide (TTA-A2), a specific inhibitor of T-type Ca2+channels, and in voltage-gated calcium channel, type 3.2 (CaV3.2), null glomus cells. CaV3.2 knockout mice exhibited an absence of CIH-induced hypersensitivity of the carotid body. CIH increased reactive oxygen species (ROS) levels in glomus cells. A ROS scavenger prevented the exaggerated TTA-A2-sensitive [Ca2+]iresponse to hypoxia. CIH had no effect on CaV3.2 mRNA levels. CIH augmented Ca2+currents and increased CaV3.2 protein in plasma membrane fractions of human embryonic kidney-293 cells stably expressing CaV3.2, and either a ROS scavenger or brefeldin-A, an inhibitor of protein trafficking, prevented these effects. These findings suggest that CIH leads to an augmented Ca2+influx via ROS-dependent facilitation of CaV3.2 protein trafficking to the plasma membrane.

The discovery of carotid bodies as sensory receptors for detecting arterial blood oxygen levels, and the identification and elucidation of the roles of hypoxia-inducible factors (HIFs) in oxygen homeostasis have propelled the field of oxygen biology. This review highlights the gas-messenger signaling mechanisms associated with oxygen sensing, as well as transcriptional and non-transcriptional mechanisms underlying the maintenance of oxygen homeostasis by HIFs and their relevance to physiology and pathology.

Intermittent hypoxia (IH) increases reactive oxygen species generation resulting in oxidative stress in the adrenal medulla (AM), a major end-organ of the sympathetic nervous system which facilitates catecholamine secretion by hypoxia. Here, we show that carotid body chemoreflex contributes to IH-induced oxidative stress in the AM. Carotid bodies were ablated by cryocoagulation of glomus cells, the putative O(2) sensing cells. Carotid body ablated (CBA) and control rats were exposed to IH and the redox state of the AM was assessed biochemically. We found that IH raised reactive oxygen species levels along with an increase in NADPH oxidase (Nox), a pro-oxidant enzyme and a decrease in superoxide dismutase-2 (SOD2), an anti-oxidant enzyme. Further, IH increased hypoxia-inducible factor (HIF)-1α, whereas decreased HIF-2α, the transcriptional regulator of Nox and SOD-2, respectively. These IH-induced changes in the AM were absent in CBA rats. Moreover, IH increased splanchnic nerve activity and facilitated hypoxia-evoked catecholamine efflux from the AM and CBA prevented these effects. These findings suggest that IH-induced oxidative stress and catecholamine efflux in the AM occurs via carotid body chemoreflex involving HIF α isoform mediated imbalance in pro-, and anti-oxidant enzymes.

Recurrent apnea with intermittent hypoxia (IH) is a major clinical problem in infants born preterm. Recent epidemiological studies showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension. Thus, apnea of prematurity predisposes individuals to autonomic dysfunction in adulthood. Experimental studies showed that adult rats exposed to IH as neonates exhibit augmented carotid body and adrenal chromaffin cells (AMC) response to hypoxia and irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity of the carotid body and AMC in adult rats exposed to neonatal IH was associated with increased oxidative stress, decreased expression of genes encoding anti-oxidant enzymes, and increased expression of pro-oxidant enzymes. Epigenetic mechanisms including DNA methylation leads to long-term changes in gene expression. The decreased expression of the Sod2 gene, which encodes the anti-oxidant enzyme, superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during IH exposure prevented the oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction in adult rats. These findings suggest that epigenetic mechanisms, especially DNA methylation contributes to neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.