Carotid Body Chemosensory Research Articles

The Accelerator and Brake Modulatory System of Carotid Body Sensitivity

The carotid body (CB) is the primary sensor of arterial gases in the human body that through reflex pathways maintains cardiovascular homeostasis. It is known that CB chemosensitivity increases upon repeated stimulation as well as disease states; however, the molecular basis for this remains elusive. Objectives: To investigate the effect of excitatory and inhibitory amino acids in mediating CB afferent activity. Hypothesis: We hypothesised that glutamate and γ-aminobutyric acid (GABA) act to modulate CB sensitivity. Methods: Electrophysiological recordings of carotid sinus nerve (CSN) afferent discharge were made from an ex vivo arterially perfused common carotid artery bifurcation preparation. Effects of agents on CB sensitivity were assessed from responses evoked by cyanide (CN−) (1.23 μmol bolus). Results: The CSN response to CN− consisted of a rapid high amplitude discharge that was quenched abruptly, leading to a low amplitude tail. Glutamate administration augmented the CSN response to CN− by 2-fold compared to baseline ( p=0.0005). In contrast, GABA administration suppressed the CN− response by 2-fold ( p = 3.71 x 10−5). An N-methyl-D-aspartic acid (NMDA) receptor antagonist (MK801; 100 μM) increased the response amplitude resulting in one single high amplitude discharge (160% increase from baseline), abolishing the low amplitude tail. Such an effect was also observed following GABAA receptor blockade with bicuculline (500 μM), when an 80% increase was observed. The CN− response was blocked by PPADS (a P2X receptor antagonist; 100 μM) (242 ± 154 vs 4.82 ± 2.23 % change from baseline; p=0.03), suggesting it to be solely mediated by purinergic transmission. Summary of the data: We propose that CN-evoked excitation of CB chemosensory cells leads to the co-release of ATP and glutamate. ATP activates directly P2X receptors on petrosal chemosensory afferents, while glutamate acting via NMDA receptors on chemosensory cells leads to activity-induced potentiation of ATP release and co-release of GABA to quench ATP-mediated CSN discharge in a time-controlled manner. This is supported by our finding that blockade of either excitatory (NMDAR) or inhibitory (GABAA) receptors both heighten the CN− evoked CB response. A statement of the conclusions: Our results suggest an intrinsic ‘accelerator and brake’ paracrine mechanism within the CB involving glutamate and GABA controlling CB afferent sensitivity. Research was supported by the Health Research Council of New Zealand and the Sidney Taylor Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanisms Underlying Long-Term Facilitation in the Carotid Body

Glutamate and γ-aminobutyric acid (GABA) are major modulators of excitatory and inhibitory transmission in the mammalian brain. Glutamate transmission is critical for neural plasticity, learning and memory, whereas GABA plays a fundamental role in regulating neuronal excitability and facilitating the generation of neural oscillations. Our data indicates that both glutamate and GABA are released in the carotid body (CB) to modulates its response to hypoxia. Objectives: We set out to investigate whether repeated stimulation with glutamate and GABA induces lasting effects on CB afferent discharge. Hypothesis: We hypothesised that glutamate/GABA mediates long-term facilitation (LTF) of CB afferent activity. Methods: We mined high-throughput RNA sequence data and used immunohistochemistry to map components underlying neuroplasticity in the CB in Wistar and Spontaneously-hypertensive rats (SHR). CSN discharge was measured as a functional readout of CB function from an ex vivo arterially perfused carotid artery-CB preparation. Data: We catalogue the expression of glutamate receptors (R) and transporters in the rat and mouse CB. We found N-methyl-D-aspartic acid receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and transporters to be localised to the CB chemosensory cells. Targeted administration of 15 mM glutamate activated the carotid sinus nerve ( p=0.003). Repetitive [5 times, 2-minute duration, 5-minute intervals] application of 15 mM glutamate evoked LTF of CB afferent discharge as demonstrated by both a 12-fold increase in basal firing frequency (0.78 ± 0.37 vs 1232 ± 596 spikes 1s−1; p = 0.004) over 60 minutes ( p = 0.004) and amplification of the evoked response to chemical hypoxia (CN−; 1.23 μmol bolus) by 2-fold ( p = 0.009). The stimulation paradigm was specific as neither application of glutamate, 5 times for 1-minute at 5-minute intervals, nor a single glutamate exposure evoked LTF. In contrast, repeated application of 15mM GABA [3 times for 1-minute at 5-minute intervals] evoked long-term depression (LTD) of CB afferent activity as seen by a reduced basal discharge from 10.5 to 3.5 spikes 1s−1 ( p = 0.0004) and a 2-fold suppression of the response to chemical hypoxia ( p = 3.71 x 10−5). Summary of the data: Repetitive exposure to glutamate using a specific protocol increased basal discharge of CB afferent output akin to LTF and caused sensitisation of evoked responses to CN−. Repeated exposures of GABA depressed the CB basal discharge akin to LTD. A statement of the conclusions: It is conceivable that processes such as LTP and LTD operate in the CB to modulate the set point of peripheral chemoreceptor sensitivity and its state-dependent tonicity; the role of LTP in peripheral chemoreflex sensitisation in disease remains to be determined. Research was supported by the Health Research Council of New Zealand and the Sidney Taylor Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition.

Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.

Carotid Body Inflammation: Role in Hypoxia and in the Anti-inflammatory Reflex.

Emergent evidence indicates that the carotid body (CB) chemoreceptor may sense systemic inflammatory molecules and is an afferent arm of the anti-inflammatory reflex. Moreover, a proinflammatory milieu within the CB is involved in the enhanced CB chemosensory responsiveness to oxygen following sustained and intermittent hypoxia. In this review, we focus on the physiopathological participation of CBs in inflammatory diseases, such as sepsis and intermittent hypoxia.

Histochemical and immunohistochemical localization of nitrergic structures in the carotid body of spontaneously hypertensive rats

The carotid body (CB) is a multipurpose metabolic sensor that acts to initiate cardiorespiratory reflex adjustments to maintain homeostasis of blood-borne chemicals. Emerging evidence suggests that nitric oxide increases the CB chemosensory activity and this enhanced peripheral chemoreflex sensitivity contributes to sympathoexcitation and consequent pathology. The aim of this study was to examine by means of NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry the presence and distribution of nitrergic structures in the CB of spontaneously hypertensive rats (SHRs) and to compare their expression patterns to that of age-matched normotensive Wistar rats (NWRs). Histochemistry revealed that the chemosensory glomus cells were NADPH-d-negative but were encircled by fine positive varicosities, which were also dispersed in the stroma around the glomeruli. The NADPH-d-reactive fibers showed the same distributional pattern in the CB of SHRs, however their staining activity was weaker when compared with NWRs. Thin periglomerular, intraglomerular and perivascular varicose fibers, but not glomus or sustentacular cells in the hypertensive CB, constitutively expressed two isoforms of NOS, nNOS and eNOS. In addition, clusters of glomus cells and blood vessels in the CB of SHRs exhibited moderate immunoreactivity for the third known NOS isoenzyme, iNOS. The present study demonstrates that in the hypertensive CB nNOS and eNOS protein expression shows statistically significant down-regulation whereas iNOS expression is up-regulated in the glomic tissue compared to normotensive controls. Our results suggest that impaired NO synthesis could contribute to elevated blood pressure in rats via an increase in chemoexcitation and sympathetic nerve activity in the CB.

Potential Contribution of Carotid Body-Induced Sympathetic and Renin-Angiotensin System Overflow to Pulmonary Hypertension in Intermittent Hypoxia.

Obstructive sleep apnea (OSA), featured by chronic intermittent hypoxia (CIH), is an independent risk for systemic hypertension (HTN) and is associated with pulmonary hypertension (PH). The precise mechanisms underlying pulmonary vascular remodeling and PH in OSA are not fully understood. However, it has been suggested that lung tissue hypoxia, oxidative stress, and pro-inflammatory mediators following CIH exposure may contribute to PH. New evidences obtained in preclinical OSA models support that an enhanced carotid body (CB) chemosensory reactiveness to oxygen elicits sympathetic and renin-angiotensin system (RAS) overflow, which contributes to HTN. Moreover, the ablation of the CBs abolished the sympathetic hyperactivity and HTN in rodents exposed to CIH. Accordingly, it is plausible that the enhanced CB chemosensory reactivity may contribute to the pulmonary vascular remodeling and PH through the overactivation of the sympathetic-RAS axis. This hypothesis is supported by the facts that (i) CB stimulation increases pulmonary arterial pressure, (ii) denervation of sympathetic fibers in pulmonary arteries reduces pulmonary remodeling and pulmonary arterial hypertension (PAH) in humans, and (iii) administration of angiotensin-converting enzyme (ACE) or blockers of Ang II type 1 receptor (ATR1) ameliorates pulmonary remodeling and PH in animal models. In this review, we will discuss the supporting evidence for a plausible contribution of the CB-induced sympathetic-RAS axis overflow on pulmonary vascular remodeling and PH induced by CIH, the main characteristic of OSA.

Contribution of adenosine and ATP to the carotid body chemosensory activity in ageing.

Adenosine and ATP are excitatory neurotransmitters involved in the carotid body (CB) response to hypoxia. During ageing the CB exhibits a decline in its functionality, demonstrated by decreased hypoxic responses. In aged rats (20-24months old) there is a decrease in: basal and hypoxic release of adenosine and ATP from the CB; expression of adenosine and ATP receptors in the petrosal ganglion; carotid sinus nerve (CSN) activity in response to hypoxia; and ventilatory responses to ischaemic hypoxia. There is also an increase in SNAP25, ENT1 and CD73 expression. It is concluded that, although CSN activity and ventilatory responses to hypoxia decrease with age, adjustments in purinergic metabolism in the CB in aged animals are present aiming to maintain the contribution of adenosine and ATP. The possible significance of the findings in the context of ageing and in CB-associated pathologies is considered. During ageing the carotid body (CB) exhibits a decline in its functionality. Here we investigated the effect of ageing on functional CB characteristics as well as the contribution of adenosine and ATP to CB chemosensory activity. Experiments were performed in 3-month-old and 20- to 24-month-old male Wistar rats. Ageing decreased: the number of tyrosine hydroxylase immune-positive cells, but not typeII cells or nestin-positive cells in the CB; the expression of P2X2 and A2A receptors in the petrosal ganglion; and the basal and hypoxic release of adenosine and ATP from the CB. Ageing increased ecto-nucleotidase (CD73) immune-positive cells and the expression of synaptosome associated protein 25 (SNAP25) and equilibrative nucleoside transporter 1 (ENT1) in the CB. Additionally, ageing did not modify basal carotid sinus nerve (CSN) activity or the activity in response to hypercapnia, but decreased CSN activity in hypoxia. The contribution of adenosine and ATP to stimuli-evoked CSN chemosensory activity in aged animals followed the same pattern of 3-month-old animals. Bilateral common carotid occlusions during 5, 10 and 15s increased ventilation proportionally to the duration of ischaemia, an effect decreased by ageing. ATP contributed around 50% to ischaemic-ventilatory responses in young and aged rats; the contribution of adenosine was dependent on the intensity of ischaemia, being maximal in ischaemias of 5s (50%) and much smaller in 15s ischaemias. Our results demonstrate that both ATP and adenosine contribute to CB chemosensory activity in ageing. Though CB responses to hypoxia, but not to hypercapnia, decrease with age, the relative contribution of both ATP and adenosine for CB activity is maintained.

Enhanced Carotid Body Chemosensory Discharge is Essential for the Hypertension Induced by Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) discharges, leading to sympathetic overflow and hypertension. CIH produces oxidative stress (ROS) in the CB, which contributes to enhance the CB drive to the brainstem and hypothalamic nuclei related to the cardiorespiratory control, such as the nucleus of the tractus solitary (NTS), the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus (PVN). We hypothesized whether the enhanced CB chemosensory drive may increase ROS levels in central cardiovascular nuclei during CIH‐induced hypertension. Accordingly, we studied the effects of CB ablation on ROS formation in the NTS, PVN and RVLM of hypertensive CIH‐rats. Male Sprague‐Dawley rats (200 g) were exposed to CIH (5–6% inspired O2 for 20s, followed by room air for 280s, 12 times/h, 8 h/day), for 28 days. Arterial blood pressure (BP) was measured by radio‐telemetry (DSI, USA). Following 21 days of CIH exposure, rats were anesthetized with isofluorane in 2% O2, and the CBs were cryogenically destroyed bilaterally using a fine‐tipped forcep cooled in liquid N2. Then, rats were maintained in CIH conditions for one more week. Another group of rats were maintained in CIH or Sham conditions for 28 days. At the end of the experiments, all animals were euthanized (pentobarbital 120mg/kg i.p.) and brains were removed and frozen at −20°C. Coronal sections of the NTS, RVLM and PVN were cut and incubated for 30 min in the dark with the ROS sensitive fluorescent probe dihydroethidium (DHE 10μM, Invitrogen USA). DHE fluorescence was measured using epifluorescence microscopy (Olympus Optical, Japan) at an excitation wavelength of 543 nm. Data were analyzed by one‐way ANOVA followed by Bonferroni's test. Compared to Sham rats, CIH‐rats showed a significant increase in mean BP (□ 10 mmHg, p<0.05). The CIH‐induced hypertension was abolished by the ablation of the CBs (CBA). ROS formation was markedly reduced by CBA in the NTS, RVLM and PVN of CIH‐exposed rats (CIH vs. CIH‐CBA: NTS: 6.2±1.5 vs. 1.5±02 au, RVLM: 4.9± 1.5 vs. 2.1±1.6 au, PVN: 5.4±1.8 vs. 1.6±05 au, p <0.05, n= 5–6 rats). Results support the evidence for an essential role of the CBs in the development and maintenance of the CIH‐induced hypertension and oxidative stress present in the NTS, RVLM and PVNSupport or Funding InformationFONDECYT 1150040This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelin-1 enhanced carotid body chemosensory activity in chronic intermittent hypoxia through PLC, PKC and p38MAPK signaling pathways

Endothelin-1 (ET-1), as it functions as a neuromodulator, has been associated with hypertension in chronic intermittent hypoxia (CIH) which attribute to enhanced carotid body sensibility to hypoxia. However, the molecular mechanism of ET-1 on carotid body sensibility in CIH is still not clear. Here, effect of ET-1 on carotid body chemosensory stimulation in rats exposed to either CIH or room air (Normoxia) was explored. Furthermore, Phospholipase C (PLC), Protein kinase C (PKC) or p38 MAPK antagonists were adopted to clarify the signalling pathways involved. Results showed that ET-1 induced a higher increase of carotid sinus nerve activity (CSNA) in animals exposed to CIH. Both ETA and ETB receptor expression were up-regulated by CIH exposure, but only ETA is responsible for ET-1 induced CSNA increase. Additional, the increase was inhibited by PLC, PKC, p38 MAPK antagonists and calcium channel blocker. Our findings support that ETA receptor mediates ET-1-induced CSNA increase through PLC, PKC and p38 MAPK signalling pathways in chronic intermittent hypoxia. Also, our study indicated that calcium influx was necessary for enhancing effect of ET-1 on CSNA.

Endothelin-1-Mediated Mechanisms in the Carotid Body Modulates Cardiovascular Responses in Rats Exposed to Chronic Intermittent Hypoxia

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea (OSA), is associated with hypertension. The increased of carotid body (CB) sensitivity due to enhanced sympathetic efferent may be mainly responsible for the elevation of blood pressure. Accordingly, we studied this effect of Endothelin-1 (ET-1)-induced CB chemosensory response to CIH, as a vasoactive peptide expressed in CB. The purpose of this study was to investigate the mean arterial blood pressure (MAP) and renal sympathetic nerve activity (RSNA) responses in CIH group by injecting ET-1 to directly stimulate CB chemoreceptor. Furthermore, whether ET receptor-mediated PKC and p38MAPK signaling pathway was involved in CIH-induced CB activation was also studied. Male Sprague–Dawley rats were exposed to CIH (8 h/day for 3 weeks) and the MAP and RSNA were recorded in CIH rats and Sham rats. Our results demonstrated that ET-1-induced MAP and RSNA increase were mainly mediated by ETA receptor activation in CB chemosensory after CIH exposure. Moreover, P38MAPK and PKC signaling pathway might be involved in ET-1-induced increase of MAP and RSNA in CIH group, which provided a potential therapeutic target of OSA.

Carotid Body Type-I Cells Under Chronic Sustained Hypoxia: Focus on Metabolism and Membrane Excitability.

Chronic sustained hypoxia (CSH) evokes ventilatory acclimatization characterized by a progressive hyperventilation due to a potentiation of the carotid body (CB) chemosensory response to hypoxia. The transduction of the hypoxic stimulus in the CB begins with the inhibition of K+ currents in the chemosensory (type-I) cells, which in turn leads to membrane depolarization, Ca2+ entry and the subsequent release of one- or more-excitatory neurotransmitters. Several studies have shown that CSH modifies both the level of transmitters and chemoreceptor cell metabolism within the CB. Most of these studies have been focused on the role played by such putative transmitters and modulators of CB chemoreception, but less is known about the effect of CSH on metabolism and membrane excitability of type-I cells. In this mini-review, we will examine the effects of CSH on the ion channels activity and excitability of type-I cell, with a particular focus on the effects of CSH on the TASK-like background K+ channel. We propose that changes on TASK-like channel activity induced by CSH may contribute to explain the potentiation of CB chemosensory activity.

Translating carotid body function into clinical medicine.

The carotid body (CB) is considered the main O2 chemoreceptor, which contributes to cardiorespiratory homeostasis and ventilatory acclimatization. In clinical medicine, the most common pathologies associated with the CB are tumours. However, a growing body of evidence supports the novel idea that an enhanced CB chemosensory discharge contributes to the autonomic dysfunction and pathological consequences in obstructive sleep apnoea (OSA), hypertension, systolic heart failure (HF) and cardiometabolic diseases. Heightened CB chemosensory reactivity elicited by oxidative stress has been involved in sympathetic hyperactivity, cardiorespiratory instability, hypertension and insulin resistance. CB ablation, which reduces sympathetic hyperactivity, decreases hypertension in animal models of OSA and hypertension, eliminates breathing instability and improves animal survival in HF, and restores insulin tolerance in cardiometabolic models. Thus, data obtained from preclinical studies highlight the importance of the CB in the progression of sympathetic-related diseases, supporting the idea that appeasing the enhanced CB chemosensory drive may be useful in improving cardiovascular, respiratory and endocrine alterations. Accordingly, CB ablation has been proposed and used as a treatment for moderating resistant hypertension and HF-induced sympathetic hyperactivity in humans. First-in-human studies have shown that CB ablation reduces sympathetic overactivity, transiently reduces severe hypertension and improves quality of life in HF patients. Thus, CB ablation would be a useful therapy to reverse sympathetic overactivation in HF and severe hypertension, but caution is required before it is widely used due to the crucial physiological function played by the CB. Further studies in preclinical models are required to assess side-effects of CB ablation.

Adenosine and dopamine oppositely modulate a hyperpolarization-activated current Ih in chemosensory neurons of the rat carotid body in co-culture.

Adenosine and dopamine (DA) are neuromodulators in the carotid body (CB) chemoafferent pathway, but their mechanisms of action are incompletely understood. Using functional co-cultures of rat CB chemoreceptor (type I) cells and sensory petrosal neurons (PNs), we show that adenosine enhanced a hyperpolarization-activated cation current Ih in chemosensory PNs via A2a receptors, whereas DA had the opposite effect via D2 receptors. Adenosine caused a depolarizing shift in the Ih activation curve and increased firing frequency, whereas DA caused a hyperpolarizing shift in the curve and decreased firing frequency. Acute hypoxia and isohydric hypercapnia depolarized type I cells concomitant with increased excitation of adjacent PNs; the A2a receptor blocker SCH58261 inhibited both type I and PN responses during hypoxia, but only the PN response during isohydric hypercapnia. We propose that adenosine and DA control firing frequency in chemosensory PNs via their opposing actions on Ih . Adenosine and dopamine (DA) act as neurotransmitters or neuromodulators at the carotid body (CB) chemosensory synapse, but their mechanisms of action are not fully understood. Using a functional co-culture model of rat CB chemoreceptor (type I) cell clusters and juxtaposed afferent petrosal neurons (PNs), we tested the hypothesis that adenosine and DA act postsynaptically to modulate a hyperpolarization-activated, cyclic nucleotide-gated (HCN) cation current (Ih ). In whole-cell recordings from hypoxia-responsive PNs, cAMP mimetics enhanced Ih whereas the HCN blocker ZD7288 (2μm) reversibly inhibited Ih . Adenosine caused a potentiation of Ih (EC50 ∼ 35nm) that was sensitive to the A2a blocker SCH58261 (5nm), and an ∼16mV depolarizing shift in V½ for voltage dependence of Ih activation. By contrast, DA (10μm) caused an inhibition of Ih that was sensitive to the D2 blocker sulpiride (1-10μm), and an ∼11mV hyperpolarizing shift in V½ . Sulpiride potentiated Ih in neurons adjacent to, but not distant from, type I cell clusters. DA also decreased PN action potential frequency whereas adenosine had the opposite effect. During simultaneous paired recordings, SCH58261 inhibited both the presynaptic hypoxia-induced receptor potential in type I cells and the postsynaptic PN response. By contrast, SCH58261 inhibited only the postsynaptic PN response induced by isohydric hypercapnia. Confocal immunofluorescence confirmed the localization of HCN4 subunits in tyrosine hydroxylase-positive chemoafferent neurons in tissue sections of rat petrosal ganglia. These data suggest that adenosine and DA, acting through A2a and D2 receptors respectively, regulate PN excitability via their opposing actions on Ih .

Role of Carotid Body in Intermittent Hypoxia-Related Hypertension.

Obstructive sleep apnea (OSA), a common breathing disorder, is recognized as an independent risk factor for systemic hypertension. Among the alterations induced by OSA, the chronic intermittent hypoxia (CIH) is considered the main factor for the hypertension. Exposure of rodents to CIH is the gold-standard method to study the mechanisms involved in the cardiovascular alterations induced by OSA. Although it is well known that CIH produces hypertension, the underlying mechanisms are not totally elucidated. It is likely that the CIH-induced systemic oxidative stress and inflammation may elicit endothelial dysfunction and increase the arterial blood pressure. In addition, OSA patients and animals exposed to CIH show sympathetic hyperactivity and potentiated cardiorespiratory responses to acute hypoxia, suggesting that CIH enhances the peripheral hypoxic chemoreflex. Recent experimental evidences support the proposal that CIH selectively enhances carotid body (CB) chemosensory reactivity to oxygen, which in turn increases sympathetic outflow leading to neurogenic hypertension. In this review, we will discuss the supporting evidence for a critical role of the CB in the generation and maintenance of the hypertension induced by CIH, also, the contribution of oxidative stress to enhance CB chemosensory drive and the activation of sympathetic-related centers in the brain.

Ventilatory and Carotid Body Chemosensory Responses In Rats Exposed To Short‐Term Sustained Hypoxia

Previous studies performed in in situ rat preparations reported that the exposure to short‐term (24 h) sustained hypoxia (SH) generates active expiration at rest, increases baseline sympathetic activity during expiration and enhances sympathetic responses to peripheral chemoreceptor stimulation. In the present study, we evaluated basal and hypoxic‐induced changes in ventilatory parameters of unanesthetized juvenile male Holtzman rats (80–90 g) previously exposed to SH (10% O2, 24 hs). In addition, we examined the chemosensory response of carotid body chemoreceptors of decerebrated, artificially‐perfused in situ preparations of juvenile rats (60–80 g) submitted to SH. At resting conditions, SH rats (n=12) exhibited augmented (P<0.05) respiratory frequency (109±2 vs 97±1 cpm), tidal volume (14±1 vs 10±1 ml/kg) and minute ventilation (1550±137 vs 979±114 ml/kg.min) compared to control rats (n=12). SH rats also showed greater tachypneic and hyperventilatory responses (P<0.05) to a new hypoxia episode (7% O2, 20 min) in comparison to control rats. The magnitude of increase in tidal volume was similar between groups. The in situ preparations of SH rats (n=8), but not controls (n=8), exhibited additional bursts in baseline abdominal motor activity, indicating the presence of active expiration at rest. Interestingly, basal afferent activity of carotid sinus nerve (CSN) was similar (0.67±0.18 vs 0.65±0.18 μV) between SH and control rats. Moreover, the CSN excitatory response to stimulation of peripheral chemoreceptors with potassium cyanide was not different between groups (ΔCSN: 1235±372 vs 922±278 %), although the in situ preparations of SH rats showed greater sympathoexcitatory reflex responses (P<0.05). Our findings indicate that augmented basal ventilation and enhanced ventilatory and sympathetic responses to hypoxia in rats exposed to SH are not dependent on increased basal and sensorial activity of carotid body chemoreceptors, but may rely on central mechanisms.Support or Funding InformationFAPESP (2013/17.251‐6) and CNPq

Carotid Body Ablation Abrogates Hypertension and Autonomic Alterations Induced by Intermittent Hypoxia in Rats.

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) chemosensory responses to hypoxia and produces autonomic dysfunction, cardiac arrhythmias, and hypertension. We tested whether autonomic alterations, arrhythmogenesis, and the progression of hypertension induced by CIH depend on the enhanced CB chemosensory drive, by ablation of the CB chemoreceptors. Male Sprague-Dawley rats were exposed to control (Sham) conditions for 7 days and then to CIH (5% O2, 12/h 8 h/d) for a total of 28 days. At 21 days of CIH exposure, rats underwent bilateral CB ablation and then exposed to CIH for 7 additional days. Arterial blood pressure and ventilatory chemoreflex response to hypoxia were measured in conscious rats. In addition, cardiac autonomic imbalance, cardiac baroreflex gain, and arrhythmia score were assessed during the length of the experiments. In separate experimental series, we measured extracellular matrix remodeling content in cardiac atrial tissue and systemic oxidative stress. CIH induced hypertension, enhanced ventilatory response to hypoxia, induced autonomic imbalance toward sympathetic preponderance, reduced baroreflex gain, and increased arrhythmias and atrial fibrosis. CB ablation normalized blood pressure, reduced ventilatory response to hypoxia, and restored cardiac autonomic and baroreflex function. In addition, CB ablation reduced the number of arrhythmias, but not extracellular matrix remodeling or systemic oxidative stress, suggesting that reductions in arrhythmia incidence during CIH were related to normalization of cardiac autonomic balance. Present results show that autonomic alterations induced by CIH are critically dependent on the CB and support a main role for the CB in the CIH-induced hypertension.

Single cell transcriptome analysis of mouse carotid body glomus cells.

Carotid body (CB) glomus cells mediate acute oxygen sensing and the initiation of the hypoxic ventilatory response, yet the gene expression profile of these cells is not available. We demonstrate that the single cell RNA-Seq method is a powerful tool for identifying highly expressed genes in CB glomus cells. Our single cell RNA-Seq results characterized novel CB glomus cell genes, including members of the G protein-coupled receptor signalling pathway, ion channels and atypical mitochondrial electron transport chain subunits. A heterologous cell-based screening identified acetate (which is known to affect CB glomus cell activity) as an agonist for the most highly abundant G protein-coupled receptor (Olfr78) in CB glomus cells. These data established the first transcriptome profile of CB glomus cells, highlighting genes with potential implications in CB chemosensory function. The carotid body (CB) is a major arterial chemoreceptor containing glomus cells whose activities are regulated by changes in arterial blood content, including oxygen. Despite significant advancements in the characterization of their physiological properties, our understanding of the underlying molecular machinery and signalling pathway in CB glomus cells is still limited. To overcome this, we employed the single cell RNA-Seq method by performing next-generation sequencing on single glomus cell-derived cDNAs to eliminate contamination of genes derived from other cell types present in the CB. Using this method, we identified a set of genes abundantly expressed in glomus cells, which contained novel glomus cell-specific genes. Transcriptome and subsequent in situ hybridization and immunohistochemistry analyses identified abundant G protein-coupled receptor signalling pathway components and various types of ion channels, as well as members of the hypoxia-inducible factors pathway. A short-chain fatty acid olfactory receptor Olfr78, recently implicated in CB function, was the most abundant G protein-coupled receptor. Two atypical mitochondrial electron transport chain subunits (Ndufa4l2 and Cox4i2) were among the most specifically expressed genes in CB glomus cells, highlighting their potential roles in mitochondria-mediated oxygen sensing. The wealth of information provided by the present study offers a valuable foundation for identifying molecules functioning in the CB.

Intermittent Hypoxia-Induced Carotid Body Chemosensory Potentiation and Hypertension Are Critically Dependent on Peroxynitrite Formation.

Oxidative stress is involved in the development of carotid body (CB) chemosensory potentiation and systemic hypertension induced by chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea. We tested whether peroxynitrite (ONOO−), a highly reactive nitrogen species, is involved in the enhanced CB oxygen chemosensitivity and the hypertension during CIH. Accordingly, we studied effects of Ebselen, an ONOO− scavenger, on 3-nitrotyrosine immunoreactivity (3-NT-ir) in the CB, the CB chemosensory discharge, and arterial blood pressure (BP) in rats exposed to CIH. Male Sprague-Dawley rats were exposed to CIH (5% O2, 12 times/h, 8 h/day) for 7 days. Ebselen (10 mg/kg/day) was administrated using osmotic minipumps and BP measured with radiotelemetry. Compared to the sham animals, CIH-treated rats showed increased 3-NT-ir within the CB, enhanced CB chemosensory responses to hypoxia, increased BP response to acute hypoxia, and hypertension. Rats treated with Ebselen and exposed to CIH displayed a significant reduction in 3-NT-ir levels (60.8 ± 14.9 versus 22.9 ± 4.2 a.u.), reduced CB chemosensory response to 5% O2 (266.5 ± 13.4 versus 168.6 ± 16.8 Hz), and decreased mean BP (116.9 ± 13.2 versus 82.1 ± 5.1 mmHg). Our results suggest that CIH-induced CB chemosensory potentiation and hypertension are critically dependent on ONOO− formation.

Carotid Body Ablation Reverses the Hypertension and Autonomic Changes Induced by Intermittent Hypoxia

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea syndrome, enhances carotid body (CB) chemosensory discharges in normoxia, eliciting sympathetic overactivation and hypertension. We propose that the enhanced CB chemosensory drive plays a crucial role in the generation of the hypertension during CIH. To study the origin of higher blood pressures during CIH, we performed selective ablation of the CB once animals became hypertensive following CIH exposure. Male Sprague‐Dawley rats (200 g) were exposed to CIH (5% O2, 12 times/h, 8 h/day) and arterial blood pressure (BP) was measured with radiotelemetry. After 3 weeks of CIH, under anesthesia both CBs were cryogenically destroyed with an ice cold needle, and rats were kept one more week in CIH. The effectiveness of this maneuver was confirmed by the disappearance of the ventilatory response to NaCN. After 3 weeks of CIH exposure, rats displayed hypertension (BP~10 mmHg), enhanced hypoxic ventilatory response and reduced baroreflex sensitivity (BRS) from 2.28±0.05 to 1.76±0.10 ms/mmHg. The ablation of the CBs markedly decreases the elevated BP, reduced the enhanced ventilatory response to hypoxia and normalized BRS to 2.52±0.12 ms/mmHg. We found that the cardiorespiratory alterations induced by CIH are critically dependent on functional CB chemoreceptors. Also, our results showed that the CIH‐induced hypertension was abolished by ablation of the CB despite the continuous exposure to CIH stimulation. Thus, our results indicate that the CB plays a pivotal role in the progression and maintenance of the CIH‐induced hypertension.Supported by Puente 28/2014 PUC

Inflammation and oxidative stress during intermittent hypoxia: the impact on chemoreception.

What is the topic of this review? This article describes the contribution of oxidative stress and pro-inflammatory cytokines to the enhanced carotid body chemosensory responsiveness to the hypoxia and systemic hypertension induced by chronic intermittent hypoxia. What advances does it highlight? Chronic intermittent hypoxia enhances the carotid body chemosensory discharge during normoxia and hypoxia, leading to sympathetic overactivity and hypertension. New evidence suggests that chronic intermittent hypoxia increases pro-inflammatory cytokines. Here, we discuss the role of inflammation in the alterations of the carotid chemoreceptor function as well as the cardiorespiratory alterations following chronic intermittent hypoxia. Chronic intermittent hypoxia (CIH), the main characteristic of obstructive sleep apnoea, enhances carotid body (CB) chemosensory discharges during normoxia and hypoxia and elicits hypertension. These alterations are attributed to oxidative stress, because antioxidants prevent the enhanced CB chemosensory discharges and the hypertension. In this report, we discuss new evidence supporting the suggestion that oxidative stress-induced upregulation of pro-inflammatory cytokines (i.e. tumour necrosis factor-α and interleukin-1β) in the CB is involved in the chemosensory potentiation and the hypertension following CIH. Anti-inflammatory treatment with ibuprofen prevents the increased tumour necrosis factor-α and interleukin-1β levels in the CB and the hypertension, but does not reduce the enhanced chemosensory hypoxic response and the local oxidative stress in the CB. In contrast, antioxidant treatment with ascorbic acid prevents the increase in cytokine concentrations and CB oxidative stress, the chemosensory potentiation and the hypertension. Thus, the enhanced CB chemosensory responses to hypoxia depend critically on the oxidative stress, but not on the increased tumour necrosis factor-α and interleukin-1β in the CB. We discuss a possible role for pro-inflammatory cytokines in development of the hypertension produced by CIH, acting on cardiorespiratory centres located in the CNS.