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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.

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.

In neonates, catecholamine (CA) secretion from adrenal medullary chromaffin cells (AMC) is an important mechanism for maintaining homeostasis during hypoxia. Nearly 90% of premature infants experience chronic intermittent hypoxia (IH) because of high incidence of apnea of prematurity, which is characterized by periodic stoppage of breathing. The present study examined the effects of repetitive hypoxia, designed to mimic apnea of prematurity, on CA release from AMC of neonatal rats. Neonatal rats were exposed to either control conditions or chronic intermittent hypoxia (IH) from ages postnatal days 0–5 (P0–P5), and CA release from adrenal medullary slices was measured after challenge with repetitive hypoxia (5 episodes of 30-s hypoxia, Po2~35 mmHg). In response to repetitive hypoxia, chronic IH-treated AMC exhibited sustained CA release, and this phenotype was not seen in control AMC. The sustained CA release was associated with long-lasting elevation of intracellular Ca2+concentration ([Ca2+]i), which was due to store-operated Ca2+entry (SOCE). 2-Aminoethoxydiphenyl borate, an inhibitor of SOCE, prevented the long-lasting [Ca2+]ielevation and CA release. Repetitive hypoxia increased H2O2abundance, and polyethylene glycol (PEG)-catalase, a scavenger of H2O2blocked this effect. PEG-catalase also prevented repetitive hypoxia-induced SOCE activation, sustained [Ca2+]ielevation, and CA release. These results demonstrate that repetitive hypoxia induces long-term facilitation of CA release in chronic IH-treated neonatal rat AMC through sustained Ca2+influx mediated by SOCE.NEW & NOTEWORTHY Apnea of prematurity and the resulting chronic intermittent hypoxia are major clinical problems in neonates born preterm. Catecholamine release from adrenal medullary chromaffin cells maintains homeostasis during hypoxia in neonates. Our results demonstrate that chronic intermittent hypoxia induces a hitherto uncharacterized long-term facilitation of catecholamine secretion from neonatal rat chromaffin cells in response to repetitive hypoxia, simulating hypoxic episodes encountered during apnea of prematurity. The sustained catecholamine secretion might contribute to cardiovascular morbidities in infants with apnea of prematurity.

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.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT-IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation in the carotid body chemo reflex pathway resulting in persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Histone modification by lysine acetylation is another major epigenetic mechanism associated with gene regulation. Equilibrium between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) determine the level of lysine acetylation. Here we report that exposure of rat pheochromocytoma (PC)-12 cells to IH in vitro exhibited reduced HDAC enzyme activity due to proteasomal degradation of HDAC3 and HDAC5 proteins. Mechanistic investigations showed that IH-evoked decrease in HDAC activity increases lysine acetylation of α subunit of hypoxia inducible factor (HIF)-1α as well as Histone (H3) protein resulting in increased HIF-1 transcriptional activity. Trichostatin A (TSA), an inhibitor of HDACs, mimicked the effects of IH. Studies on rats treated with 10 days of IH or TSA showed reduced HDAC activity, HDAC5 protein, and increased HIF-1 dependent NADPH oxidase (NOX)-4 transcription in adrenal medullae (AM) resulting in elevated plasma catecholamines and blood pressure. Likewise, heme oxygenase (HO)-2 null mice, which exhibit IH because of high incidence of spontaneous apneas (apnea index 72 ± 1.2 apnea/h), also showed decreased HDAC activity and HDAC5 protein in the AM along with elevated circulating norepinephrine levels. These findings demonstrate that lysine acetylation of histone and non-histone proteins is an early epigenetic mechanism associated with sympathetic nerve activation and hypertension in rodent models of IH.

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H(2)S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H(2)S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca(2+)](i) by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po(2) 30–38 mmHg) evoked CA release and elevated [Ca(2+)](i). The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H(2)S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H(2)S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H(2)S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H(2)S. NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H(2)S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H(2)S production through soluble guanylyl cyclase-protein kinase G signaling cascade.

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.

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

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.

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.

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.

Sleep disordered breathing (SDB) with recurrent apnea is a major health problem affecting several million adult men and women. Humans with SDB are prone to develop hypertension. Studies on rodents established that exposure to chronic intermittent hypoxia (CIH) alone is sufficient to induce hypertension similar to that seen in patients with SDB. Available evidence from studies on experimental animals suggests that catecholamines secreted from adrenal medulla (AM), an end-organ of the sympathetic nervous system is a major contributor to CIH-induced hypertension. In this article, we present an overview of our current understanding on how CIH reconfigures AM function and highlight recent findings on the underlying cellular and molecular mechanisms.

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.

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.

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.

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.

Chronic intermittent hypoxia (CIH) leads to remodelling of the carotid body function, manifested by an augmented sensory response to hypoxia and induction of sensory long-term facilitation (LTF). It was proposed that endothelin-1 (ET-1) contributes to CIH-induced hypoxic hypersensitivity of the carotid body. The objectives of the present study were as follows: (i) to delineate the mechanisms by which CIH upregulates ET-1 expression in the carotid body; and (ii) to assess whether ET-1 also contributes to sensory LTF. Experiments were performed on adult, male rats exposed to alternating cycles of 5% O2 (15 s) and room air (5 min), nine episodes per hour and 8 h per day for 10 days. Chronic intermittent hypoxia increased ET-1 levels in glomus cells without significantly altering prepro-endothelin-1 mRNA levels. The activity of endothelin-converting enzyme increased with concomitant elevation of ET-1 levels in CIH-exposed carotid bodies, and MnTMPyP, a membrane-permeable antioxidant, prevented these effects. Hypoxia facilitated ET-1 release from CIH-treated carotid bodies, which is a prerequisite for activation of ET receptors; however, hypoxia had no effect on ET-1 release from control carotid bodies. In CIH-exposed carotid bodies, mRNAs encoding ETA receptor were upregulated, and an ETA receptor-specific antagonist abolished CIH-induced hypersensitivity of the hypoxic response, whereas it had no effect on the sensory LTF. These results suggest that ECE-dependent increased production of ET-1 coupled with hypoxia-evoked ET-1 release and the ensuing ETA receptor activation mediate the CIH-induced carotid body hypersensitivity to hypoxia, but the ETA signalling pathway is not associated with sensory LTF elicited by CIH.

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.

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.

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant 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 intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

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 investigated the role of protein phosphatases (PP) and protein kinases in tyrosine hydroxylase (TH) activation by two patterns of intermittent hypoxia (IH) in rat brainstem. Rats exposed to either IH15s (15 s, 5% O2; 5 min, 21%O2) or IH90s (90 s each of 10% O2 & 21%O2) for 10 days were used. IH15s but not IH90s caused a robust increase in TH activity, dopamine (DA) level, and TH phosphorylation at Ser-31 and Ser-40 in the medulla but not in the pons. Likewise, IH15s but not IH90s decreased activity and expression of protein phosphatase 2A (PP2A) and increased activity of multiple protein kinases. In vitro dephosphorylation with PP2A nearly abolished IH15s-induced increase in TH activity. IH15s increased generation of reactive oxygen species (ROS) in brainstem medullary regions which was nearly threefold higher than that evoked by IH90s. Antioxidants prevented IH15s-induced downregulation of PP2A and increases in multiple protein kinase activity with subsequent reversal of serine phosphorylation of TH, TH activity, and DA to control levels. These findings demonstrate that IH in a pattern-specific manner activates TH involving ROS-mediated sustained increase in TH phosphorylation via downregulation of PP2A and upregulation of protein kinases. Antioxid Redox Signal 11, 1777–1789.

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.

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.

Reflexes initiated by the carotid body, the principal O 2 -sensing organ, are critical for maintaining cardiorespiratory homeostasis during hypoxia. O 2 sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H 2 S) synthesis by cystathionine-γ-lyase (CSE). We report that O 2 stimulated the generation of CO, but not that of H 2 S, and required two cysteine residues in the heme regulatory motif (Cys 265 and Cys 282 ) of HO-2. CO stimulated protein kinase G (PKG)–dependent phosphorylation of Ser 377 of CSE, inhibiting the production of H 2 S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H 2 S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O 2 sensing through PKG-dependent regulation of H 2 S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing.

Systemic hypertension is one of the most prevalent cardiovascular diseases. Sleep-disordered breathing (SDB) with recurrent apnea is a major risk factor for developing essential hypertension. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of recurrent apnea. Rodent models patterned after the O2 profiles seen with SDB patients showed that CIH is the major stimulus for causing systemic hypertension. This article reviews the physiological and molecular basis of CIH-induced hypertension. Physiological studies have identified that augmented carotid body chemosensory reflex and the resulting increase in sympathetic nerve activity are major contributors to CIH-induced hypertension. Analysis of molecular mechanisms revealed that CIH activates hypoxia-inducible factor (HIF)-1 and suppresses HIF-2-mediated transcription. Dysregulation of HIF-1- and HIF-2-mediated transcription leads to imbalance of pro-oxidant and anti-oxidant enzyme gene expression resulting in increased reactive oxygen species (ROS) generation in the chemosensory reflex which is central for developing hypertension.

Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH-evoked cardio-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia-inducible factor-1 (HIF-1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF-1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF-1 and if so whether it contributes to cardio-respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild-type (WT) and heterozygous (HET) mice partially deficient in HIF-1α, the O2 regulated subunit of the HIF-1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h−1, 8 h day−1) or to 10 days of 21% O2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long-term facilitation (sLTF) of the chemoreceptor activity in CIH-exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH-exposed HET mice. Analysis of cardio-respiratory responses in CIH-exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH-exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH-induced increases in ROS but also CIH-evoked HIF-1α up-regulation in WT mice. These results indicate that: (a) HIF-1 activation is critical for eliciting CIH-induced carotid body-mediated cardio-respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF-1 and ROS.

Chronic intermittent hypoxia (CIH) augments physiological responses to low partial pressures of O2 in the arterial blood. Adrenal medullae from adult rats, however, are insensitive to direct effects of acute hypoxia. In the present study, we examined whether CIH induces hypoxic sensitivity in the adult rat adrenal medulla and, if so, by what mechanism(s). Experiments were performed on adult male rats exposed to CIH (15 s of 5% O2 followed by 5 min of 21% O2; 9 episodes h−1; 8 h d−1; for 3 or 10 days) or to comparable, cumulative durations of continuous hypoxia (CH; 4 h of 7% O2 followed by 20 h of 21% O2 for 1 or 10 days). Noradrenaline (NA) and adrenaline (ADR) effluxes were monitored from ex vivo adrenal medullae. In adrenal medullae of rats exposed to CIH, acute hypoxia evoked robust NA and ADR effluxes, whereas these responses were absent in control rats or in those exposed to CH for 1 or 10 days. Hypercapnia (10% CO2; either acidic, pH 6.8, or isohydric, pH 7.4) was ineffective in eliciting catecholamine (CA) efflux from control, CIH or CH rats. Nicotine (100 μm) evoked NA and ADR effluxes in control rats, and this response was abolished in CIH but not in CH rats. Systemic administration of 2-deoxyglucose depleted ADR content in control rats, and CIH attenuated this response, indicating downregulation of neurally regulated CA secretion. Cytosolic and mitochondrial aconitase enzyme activities decreased in CIH adrenal medullae, suggesting increased generation of superoxide anions. Systemic administration of antioxidants reversed the effect of CIH on the adrenal medulla. Rats exposed to CIH exhibited increased blood pressures and elevated plasma CA, and antioxidants abolished these responses. These observations demonstrate that CIH induces hypoxic sensing in the adult rat adrenal medulla via mechanisms involving increased generation of superoxide anions and suggest that hypoxia-evoked CA efflux from the adrenal medulla contributes, in part, to elevated blood pressure and plasma CA.

The present experiment was designed to investigate the possible involvement of glutamate and taurine in the depressor response produced by angiotensin (Ang)-(1-7) at the caudal ventrolateral medulla (CVLM) in rats anesthetized with urethane and alpha-chloralose. Microinjection of Ang-(1-7) into the CVLM elicited a depressor response which was partially blocked by nonselective glutamate receptors antagonist kynurenic acid, whereas selective Ang-(1-7) antagonist Ang779 produced a pressor response which was significantly attenuated by taurine receptors antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide. Release of glutamate and taurine in the CVLM was evaluated with microdialysis, and the contents of these amino acids were measured with high performance liquid chromatography-fluorescent detection. The depressor response to Ang-(1-7) was accompanied by an increased release of glutamate and a decrease of taurine at the CVLM, whereas the pressor response to Ang779 was associated with a decreased release of glutamate and an increase of taurine. These results suggest that Ang-(1-7) and its antagonist Ang779 modulate the release of glutamate and taurine at the CVLM, which in turn contributes at least in part to the blood pressure response to Ang-(1-7) and Ang779.

Mutations in the potassium channel gene Kv1.1 are associated with human episodic ataxia type 1 (EA-1) syndrome characterized by movement disorders and epilepsy. Ataxic episodes in EA-1 patients are often associated with exercise or emotional stress, which suggests a prominent role for the autonomic nervous system. Many of these alterations are reproduced in the Kv1.1-null mouse. Kv1.1 also regulates excitability of sensory neurons essential in cardiovascular and respiratory reflexes. We examined the neural control of the respiratory system of littermate wild-type (control) and Kv1.1-null mice during low O2(hypoxia). Immunohistochemical studies demonstrated Kv1.1 in the afferent limb of the carotid body chemoreflex (the major regulator in the response to hypoxia), consisting of the carotid body, petrosal ganglion, and nucleus of the solitary tract (NTS). Respiration was examined by plethysmography. Null mice exhibited a greater increase in respiration during hypoxia compared with controls.In vitrocarotid body sensory discharge during hypoxia was greater in null than control mice. In the caudal NTS, evoked EPSCs in brainstem slices were similar between control and null mice. However, the frequency of spontaneous and miniature EPSCs was greater in null mice. Null mice also exhibited more asynchronous release after a stimulus train. These results demonstrate the important role of Kv1.1 in afferent chemosensory activity and suggest that mutations in the human Kv1.1 gene have functional consequences during stress responses that involve respiratory reflexes.

Earlier studies on cell culture models suggested that immediate early genes (IEGs) play an important role in cellular adaptations to hypoxia. Whether IEGs are also necessary for hypoxic adaptations in intact animals is not known. In the present study we examined the potential importance of fos B, an IEG in ventilatory acclimatization to hypoxia. Experiments were performed on wild type and mutant mice lacking the fos B gene. Ventilation was monitored by whole body plethysmography in awake animals. Baseline ventilation under normoxia, and ventilatory response to acute hypoxia and hypercapnia were comparable between wild type and mutant mice. Hypobaric hypoxia (0.4 atm; 3 days) resulted in a significant elevation of baseline ventilation in wild type but not in mutant mice. Wild type mice exposed to hypobaric hypoxia manifested an enhanced hypoxic ventilatory response compared to pre-hypobaric hypoxia. In contrast, hypobaric hypoxia had no effect on the hypoxic ventilatory response in mutant mice. Hypercapnic ventilatory responses, however, were unaffected by hypobaric hypoxia in both groups of mice. These results suggest that the fos B, an immediate early gene, plays an important role in ventilatory acclimatization to hypoxia in mice.

Carotid bodies are functionally immature at birth and exhibit poor sensitivity to hypoxia. Previous studies have shown that continuous hypoxia at birth impairs hypoxic sensing at the carotid body. Intermittent hypoxia (IH) is more frequently experienced in neonatal life. Previous studies on adult animals have shown that IH facilitates hypoxic sensing at the carotid bodies. On the basis of these studies, in the present study we tested the hypothesis that neonatal IH facilitates hypoxic sensing of the carotid body and augments ventilatory response to hypoxia. Experiments were performed on 2-day-old rat pups that were exposed to 16 h of IH soon after the birth. The IH paradigm consisted of 15 s of 5% O2 (nadir) followed by 5 min of 21% O2 (9 episodes/h). In one group of experiments (IH and control, n = 6 pups each), sensory activity was recorded from ex vivo carotid bodies, and in the other (IH and control, n = 7 pups each) ventilation was monitored in unanesthetized pups by plethysmography. In control pups, sensory response of the carotid body was weak and was slow in onset (∼100 s). In contrast, carotid body sensory response to hypoxia was greater and the time course of the response was faster (∼30 s) in IH compared with control pups. The magnitude of the hypoxic ventilatory response was greater in IH compared with control pups, whereas changes in O2 consumption and CO2 production during hypoxia were comparable between both groups. The magnitude of ventilatory stimulation by hyperoxic hypercapnia (7% CO2-balance O2), however, was the same between both groups of pups. These results demonstrate that neonatal IH facilitates carotid body sensory response to hypoxia and augments hypoxic ventilatory chemoreflex.

Reflexes arising from the carotid bodies may play an important role in cardiorespiratory changes evoked by chronic intermittent hypoxia (CIH). In the present study, we examined whether CIH affects the hypoxic sensing ability of the carotid bodies and, if so, by what mechanisms. Experiments were performed on adult male rats (Sprague-Dawley, 250–300 g) exposed to two paradigms of CIH for 10 days: 1) multiple exposures to short durations of intermittent hypoxia per day (SDIH; 15sof5%O2 + 5 min of 21% O2, 9 episodes/h, 8 h/day) and 2) single exposure to longer durations of intermittent hypoxia per day [LDIH; 4 h of hypobaric hypoxia (0.4 atm/day) + 20 h of normoxia]. Carotid body sensory response to graded isocapnic hypoxia was examined in both groups of animals under anesthetized conditions. Hypoxic sensory response was significantly enhanced in SDIH but not in LDIH animals. Similar enhancement in hypoxic sensory response was also elicited in ex vivo carotid bodies from SDIH animals, suggesting that the effects were not secondary to cardiovascular changes. SDIH, however, had no significant effect on the hypercapnic sensory response. The effects of SDIH on the hypoxic sensory response completely reversed after SDIH animals were placed in a normoxic environment for an additional 10 days. Previous treatment with systemic administration of [Formula: see text] radical scavenger prevented SDIH-induced augmentation of the hypoxic sensory response. These results demonstrate that SDIH but not LDIH results in selective augmentation of the hypoxic response of the carotid body and [Formula: see text] radicals play an important role in SDIH-induced sensitization of the carotid body.

Long-term facilitation (LTF) of breathing elicited by episodic hypoxia (EH) is an extensively studied example of plasticity of respiratory motor behavior. Previous studies employed the paradigm of EH wherein each episode of hypoxia was 5 min. This paradigm is rarely encountered in nature. Brief episodes of hypoxia are encountered frequently with recurrent apneas, wherein hypoxic episodes last a few seconds only. Recent studies suggest that chronic intermittent hypoxia (CIH) represents a form of oxidative stress involving reactive O2species. The objectives of the present study were to determine 1) whether acute, repeated, brief EH (15 s) elicit LTF in breathing and 2) whether prior conditioning with CIH modulates acute EH-induced LTF of breathing, and if so whether reactive O2 species are involved. Experiments were performed on anesthetized, vagotomized, paralyzed, and mechanically ventilated rats, and efferent phrenic nerve activity was monitored as an index of respiratory motor output. In control animals, acute EH (15-s hypoxia; 10 episodes; n = 9) increased minute neural respiration, which persisted during 60 min of the posthypoxic period, suggesting LTF of breathing. EH-induced LTF of respiration was markedly augmented in CIH-conditioned animals (15-s hypoxia, 9 episodes/h, 8 h/day for 10 days; n = 9). By contrast, conditioning with a comparable, cumulative duration of sustained hypoxia (4-h hypoxia; n = 8) did not augment LTF elicited by acute EH. Systemic administration of manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (5 mg · kg−1 · day−1for 10 days), a potent scavenger of O[Formula: see text]·, prevented CIH-induced potentiation of LTF ( n = 9). These results demonstrate that 1) acute, brief EH elicits LTF in respiratory motor output; 2) prior conditioning with CIH, but not with comparable, cumulative duration of sustained hypoxia, augments LTF elicited by acute EH; and 3) O[Formula: see text]· radical scavenger prevents CIH-induced potentiation of LTF of respiration.

The carotid body (CB) chemosensory reflex regulates breathing and sympathetic activity during hypoxia. Here, we determined mechanisms underlying regulation of cardio-vascular function by chemosenso...