Key pointsr Rats exposed to chronic intermittent hypoxia (CIH) exhibited imbalanced expression of hypoxia-inducible factor (HIF)-α isoforms and oxidative stress in brainstem regions associated with the carotid body (CB) chemoreflex, and in the adrenal medulla, an end organ of the sympathetic nervous system. r Selective ablation of the CB abolished the effects of CIH on HIF-α isoform expression and oxidative stress.r In the adrenal medulla, chemoreflex-mediated sympathetic activation regulates HIF-α isoform expression via muscarinic acetylcholine receptor-mediated Ca 2+ influx and the resultant activation of the mammalian target of rapamycin pathway and calpain proteases.r Thus, CB neural activity regulates HIF-α isoform expressions and redox state in the central and peripheral nervous system associated with the chemoreflex pathway under the setting of CIH.Abstract 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 Abbreviations Ac-LLM-CHO, N-acetyl-leucine-leucine-methionine-aldehyde; AChR, acetylcholine receptor; AIH, acute intermittent hypoxia; AM, adrenal medulla; ASA, adrenal sympathetic ablation; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid; BP, blood pressure; CA, catecholamines; CB, carotid body; CBA, carotid body ablation; CIH, chronic intermittent hypoxia; HIF-α, hypoxia-inducible factor alpha; HR, heart rate; mAChR, muscarinic ACh receptor; MDA, malondialdehyde; mTOR, mammalian target of rapamycin; nAChR, nicotinic ACh receptor; Nox2, NADPH oxidase 2; nTS, nucleus tractus solitarious; PC12, phe...

Reflexes initiated by the carotid body, the principal O2-sensing organ, are critical for maintaining cardio-respiratory homeostasis during hypoxia. O2 sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H2S) synthesis by cystathionine-γ-lyase (CSE). We report that O2 stimulated the generation of CO, but not that of H2S, and required two cysteine residues in the heme regulatory motif (Cys265 and Cys282) of HO-2. CO stimulated protein kinase G (PKG)–dependent phosphorylation of Ser377 of CSE, inhibiting the production of H2S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H2S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O2 sensing through PKG-dependent regulation of H2S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing.

Patients suffering from cholestasis, the slowing or stoppage of bile flow, commonly report experiencing an intense, chronic itch. Numerous pruritogens are up-regulated in cholestatic patient sera, including bile acids (BAs). Acute injection of BAs results in itch in both mice and humans, and BA-modulating therapy is effective in controlling patient itch. Here, we present evidence that human sensory neuron-expressed Mas-related G protein-coupled receptor X4 (MRGPRX4), an orphan member of the Mrgpr family of GPCRs, is a BA receptor. Using Ca2+ imaging, we determined that pathophysiologically relevant levels of numerous BAs activated MRGPRX4. No mouse Mrgpr orthologs were activated by BAs. To assess the in vivo relevance of BA activation of MRGPRX4, we generated a humanized mouse with targeted expression of MRGPRX4 in itch-encoding sensory neurons. BAs activated MRGPRX4+ sensory neurons at higher levels compared with WT neurons. Compared with control animals, MRGPRX4+ mice scratched more upon acute injection of BAs and in a model of cholestatic itch. Overall, these data suggest that targeting MRGPRX4 is a promising strategy for alleviating cholestatic itch.

Oxygen (O 2 ) 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 O 2 sensing by carbon monoxide (CO)-sensitive hydrogen sulfide (H 2 S) signaling contribute to reflex variation in three genetically distinct rat strains. Compared with SpragueDawley (SD) rats, Brown-Norway (BN) rats exhibit impaired carotid body O 2 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 H 2 S levels than SD rat, whereas SH carotid bodies have reduced CO and greater H 2 S 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 H 2 S generation, restoring O 2 sensing and preventing hypoxia-induced pulmonary edema. Increasing CO levels in SH carotid bodies reduced H 2 S generation, preventing hypersensitivity to hypoxia and controlling hypertension in SH rats.high-altitude hypoxia | gasotransmitters | sympathetic tone | cystathionine-γ-lyase O xygen, an essential substrate for generating ATP, is vital for sustaining much of life on earth. A low availability of oxygen directs vertebrates' complex respiratory and cardiovascular systems to maintain optimal oxygenation of tissues by increasing ventilation and blood pressure. Interestingly, ventilatory responses to hypoxia are not uniform but, instead, exhibit substantial variation among humans (1). These varied ventilatory responses to hypoxia result in dire physiological consequences: a diminished hypoxic ventilatory response can result in poor adaptation to low O 2 environments (2) and high-altitude pulmonary edema (3-5), whereas a heightened response is associated with essential hypertension (6). Similar variations in hypoxic response have also been documented in different strains of rodents (7-10). In comparison with Sprague-Dawley (SD) rats, Brown-Norway (BN) rats display a markedly reduced ventilatory response to hypoxia (8, 9), whereas Spontaneous Hypertensive (SH) rats exhibit an augmented response (11). SH rats also present enhanced sympathetic nerve activity and hypertension (7). Despite the physiological significance, the mechanisms underlying interindividual variation in systemic responses to hypoxia are not known.The carotid body is the key sensor of arterial blood oxygen, and its chemosensory reflex is a critical regulator of breathing, sympathetic tone, and blood pressure (12, 13). Differing responses in ventilation an...

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Highlights d The mammalian receptor Mrgprb2 and MRGPRX2 can detect bacterial QSMs d QSM detection by Mrgprb2 and MRGPRX2 in mast cells elicits antibacterial mediator release d Mrgprb2 recognition of QSMs is critical for an effective immune response to bacteria d Pharmacologic activation of Mrgprb2 and MRGPRX2 enhances bacterial clearance

Various pathologic conditions result in jaundice, a yellowing of the skin due to a buildup of bilirubin. Patients with jaundice commonly report experiencing an intense non-histaminergic itch. Despite this association, the pruritogenic capacity of bilirubin itself has not been described, and no bilirubin receptor has been identified. Here, we demonstrate that pathophysiologic levels of bilirubin excite peripheral itch sensory neurons and elicit pruritus through MRGPRs, a family of G-protein coupled receptors expressed in primary sensory neurons. Bilirubin binds and activates two MRGPRs, mouse MRGPRA1 and human MRGPRX4. In two mouse models of pathologic hyperbilirubinemia, we show that genetic deletion of either Mrgpra1 or Blvra, the gene that encodes the bilirubin-producing enzyme biliverdin reductase, attenuates itch. Similarly, plasma isolated from hyperbilirubinemic patients evoked itch in wild-type animals but not Mrgpra1-/- animals. Removing bilirubin decreased the pruritogenic capacity of patient plasma. Based on these data, targeting MRGPRs is a promising strategy for alleviating jaundice-associated itch.

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 (HS) as the major effector molecule driving apneas. Genetic ablation of the HS-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 HS signaling in the CB leads to apneas and suggest that CSE inhibition may be a useful therapeutic intervention for preventing CB-driven sleep apnea.