Gayatri Raghuraman

H2S mediates O2 sensing in the carotid body

Gaseous messengers, nitric oxide and carbon monoxide, have been implicated in O2 sensing by the carotid body, a sensory organ that monitors arterial blood O2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H2S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O2 sensing in the carotid body, express cystathionine γ-lyase (CSE), an H2S-generating enzyme, with hypoxia increasing H2S 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 H2S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H2S 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.

Protein kinase G–regulated production of H2S governs oxygen sensing

Complex interplay between three gases—oxygen, carbon monoxide, and hydrogen sulfide—is necessary to control breathing. Signaling when to breathe When oxygen concentrations in the blood are low, the carotid body triggers breathing reflexes, a response that requires the gasotransmitter hydrogen sulfide, which is generated by the enzyme CSE. When blood is adequately oxygenated, the enzyme HO-2 generates carbon monoxide, which inhibits CSE and decreases neural activity in the carotid body. Yuan et al. identified two cysteine residues that enabled HO-2 to generate carbon monoxide in an oxygen-sensitive manner. They found that carbon monoxide triggered an increase of the second messenger cGMP, which stimulated protein kinase G to phosphorylate CSE, thereby inhibiting this enzyme and suppressing carotid body activity. Reflexes initiated by the carotid body, the principal O2-sensing organ, are critical for maintaining cardiorespiratory 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.

Endogenous H2S is required for Hypoxic Sensing by Carotid Body Glomus Cells

H(2)S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O(2) sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H(2)S in an O(2)-sensitive manner and whether endogenous H(2)S is required for O(2) sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE(+/+) and CSE(-/-) mice. Physiological levels of hypoxia (Po(2) ∼30 mmHg) increased H(2)S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE(-/-) mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H(2)S donor (50 μM NaHS) increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca(2+)](i) responses to NaHS and hypoxia were markedly attenuated in the presence of Ca(2+)-free medium or cadmium chloride, a pan voltage-gated Ca(2+) channel blocker, or nifedipine, an L-type Ca(2+) channel inhibitor, suggesting that both hypoxia and H(2)S share common Ca(2+)-activating mechanisms. These results demonstrate that H(2)S generated by CSE is a physiologic mediator of the glomus cells response to hypoxia.

IFN-β-Mediated Up-Regulation of CD1d in Bacteria-Infected APCs

The expression of CD1d molecules is essential for the selection and activation of a unique subset of T cells, invariant NKT cells, which express limited TCR diversity and have been demonstrated to function in both regulatory and antimicrobial immune responses. Although it has been reported that the levels of CD1d expression can be modulated during infection, the mechanisms that mediate this effect are poorly defined. In this study, we show that infection of dendritic cells and macrophages both in vitro and in vivo with the intracellular pathogen Listeria monocytogenes leads to up-regulation of CD1d. IFN-β is required to mediate this up-regulation in L. monocytogenes infection, as well as being sufficient to up-regulate CD1d expression in vitro. Unlike MHC class I molecules, the increased surface expression of CD1d by IFN-β is not regulated at the transcriptional level. Confocal microscopy and metabolic labeling experiments show that the total pool of CD1d protein is increased in IFN-β-treated cells and that increased surface expression of CD1d is not due to the redistribution of the intracellular pool of CD1d. IFN-β treatment increases the de novo synthesis of CD1d. This change in surface CD1d expression was functionally relevant, as IFN-β-treated dendritic cells are more efficient in stimulating invariant NKT cells than untreated controls. Taken together, these data support a role for early IFN-β-mediated up-regulation of CD1d in NKT cell activation during infection.

Inherent variations in CO-H2S-mediated carotid body O2 sensing mediate hypertension and pulmonary edema

Significance The carotid body chemosensory reflex is a principal regulator of breathing and blood pressure. Humans and experimental animals display marked interindividual variation in the carotid body chemosensory reflex; however, the underlying mechanisms are not known. Here, we demonstrate differences in carotid body O2 sensing to be mediated by inherent variations in carbon monoxide-sensitive hydrogen sulfide signaling in three distinct rat strains. Hyposensitivity of the carotid body to hypoxia was associated with higher CO and lower H2S levels, poor ventilatory adaptation to hypobaric hypoxia, and pulmonary edema. Hypersensitivity of the carotid body to low O2 was accompanied by reduced CO, greater H2S generation, and hypertension. 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.

Angiotensin II evokes sensory long-term facilitation of the carotid body via NADPH oxidase

We previously reported that reactive oxygen species generated by NADPH oxidase 2 (Nox2) induces sensory plasticity of the carotid body, manifested as a progressive increase in baseline sensory activity or sensory long-term facilitation (sLTF). ANG II, a peptide generated within the carotid body, is a potent activator of Nox2. In the present study, we tested the hypothesis that ANG II evokes sLTF of the carotid body via Nox2 activation. Experiments were performed on carotid bodies ex vivo from adult rats and mice. Sensory activity was recorded from the carotid sinus nerve. Repetitive (5 times for 30 s each at 5-min intervals), but not continuous (for 150 s), application of 60 pM ANG II evoked robust sLTF of the carotid body. ACh, ATP, substance P, and KCl, when applied repetitively, stimulated the carotid body but did not evoke sLTF. Reactive oxygen species levels increased in response to repetitive applications of ANG II, and this effect was blocked by apocynin, an inhibitor of Nox2, as well as losartan, an angiotensin type 1 (AT(1)) receptor antagonist. Losartan, apocynin, and 4-(2-aminoethyl)benzenesulfonyl fluoride prevented ANG II-induced sLTF, which was absent in mice deficient in gp91(phox), the catalytic subunit of the Nox2 complex. These results demonstrate that repetitive application of ANG II induces sLTF of the carotid body via activation of Nox2 by AT(1) receptors.

Endothelin-1 mediates attenuated carotid baroreceptor activity by intermittent hypoxia

The objectives of the present study were to examine the effects of intermittent hypoxia (IH) on arterial baroreflex function and assess the underlying mechanism(s). Experiments were performed on adult male rats treated with 14 days of IH (15 s of hypoxia, 5 min of normoxia; 8 h/day) or normoxia (control). Arterial blood pressures were elevated in IH-treated rats, and this effect was associated with attenuated heart rate and splanchnic sympathetic nerve responses to arterial baroreflex activation. In IH-treated rats, carotid baroreceptor responses to elevated sinus pressures were attenuated. Endothelin-1 (ET-1) levels were elevated in the carotid sinus region of IH-treated rats, and this effect was associated with increased endothelin converting enzyme (ECE) activity, which generates biologically active ET-1. ET(A) receptor antagonist prevented the effects of IH on carotid baroreceptor activity. In IH-treated rats, reactive oxygen species (ROS) levels were elevated in the carotid sinus region, and antioxidant treatment prevented the effects of IH on ET-1 levels, ECE activity, carotid baroreceptor activity, and baroreflex function. These results demonstrate that 1) IH attenuates arterial baroreflex function, which is in part due to reduced carotid baroreceptor responses to elevated carotid sinus pressure, and 2) IH-induced carotid baroreceptor dysfunction involves reactive oxygen species-dependent upregulation of ET-1 signaling in the carotid sinus region.

Intermittent hypoxia activates peptidylglycine α-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.

Role of oxidative stress-induced endothelin-converting enzyme activity in the alteration of carotid body function by chronic intermittent hypoxia

•  What is the central question of this study?  What mechanisms mediate chronic intermittent hypoxia‐induced increases in endothelin‐1 in the carotid body? •  What is the main finding and its importance?  Upregulation of endothelin‐1 by chronic intermittent hypoxia results from reactive oxygen species‐dependent activation of endothelin‐converting enzyme and is not the result of augmented endothelin‐1 gene transcriptional activity. The resultant increased endothelin‐1 acts via endothelin‐1A receptors to induce hypoxic hypersensitivity of the carotid body but does not contribute to the sensory long‐term facilitation observed in this condition. These findings provide mechanistic insight into how chronic intermittent hypoxia alters carotid body function.

Enhanced Neuropeptide Y Synthesis During Intermittent Hypoxia in the Rat Adrenal Medulla: Role of Reactive Oxygen Species–Dependent Alterations in Precursor Peptide Processing

Intermittent hypoxia (IH) associated with recurrent apneas often leads to cardiovascular abnormalities. Previously, we showed that IH treatment elevates blood pressure and increases plasma catecholamines (CAs) in rats via reactive oxygen species (ROS)-dependent enhanced synthesis and secretion from the adrenal medulla (AM). Neuropeptide Y (NPY), a sympathetic neurotransmitter that colocalizes with CA in the AM, has been implicated in blood pressure regulation during persistent stress. Here, we investigated whether IH facilitates NPY synthesis in the rat AM and assessed the role of ROS signaling. IH increased NPY-like immunoreactivity in many dopamine-β-hydroxylase-expressing chromaffin cells with a parallel increase in preproNPY mRNA and protein. IH increased the activities of proNPY-processing enzymes, which were due, in part, to elevated protein expression and increased proteolytic processing. IH increased ROS generation, and antioxidants reversed IH-induced increases in ROS, preproNPY, and its processing to bioactive NPY in the AM. IH treatment increased blood pressure and antioxidants and inhibition of NPY amidation prevented this response. These findings suggest that IH-induced elevation in NPY expression in the rat AM is mediated by ROS-dependent augmentation of preproNPY mRNA expression and proNPY-processing enzyme activities and contributes to IH-induced elevation of blood pressure.