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 Sgenerating 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.I n adult mammals, carotid bodies are the sensory organs responsible for monitoring arterial blood O 2 concentrations and relay sensory information to the brainstem neurons associated with regulation of breathing and the cardiovascular system (1). Carotid bodies are comprised mainly of two cell types: glomus (also called type I) and sustanticular (or type II) cells. Glomus cells, of neuronal nature, are considered the main hypoxiasensing cells. The gaseous messengers, carbon monoxide (CO) and nitric oxide (NO), generated by hemeoxygenase-2 (HO-2) and neuronal nitric oxide synthase (nNOS), respectively, physiologically inhibit carotid body activity (2-4). Because HO-2 and nNOS require molecular O 2 for their activity, stimulation of carotid body activity by hypoxia may reflect in part reduced formation of CO and NO (5).Like NO and CO, hydrogen sulfide (H 2 S) is a gasotransmitter physiologically regulating neuronal transmission (6) and vascular tone (7). Cystathionine γ-lyase (CSE) (EC 4.4.1.1) and cystathionine β-synthase (CBS) (4.2.1.22) are the major enzymes associated with generation of endogenous H 2 S (8, 9). CBS is the predominant H 2 S-synthesizing enzyme in the brain, CSE preponderates in the peripheral tissues whose H 2 S levels are reduced 90% in CSE −/− mice (7-10). Given that carotid bodies are peripheral organs and that H 2 S is redox active, we hypothesized that CSE-derived H 2 S plays a role in hypoxic sensing by the carotid body. We examined carotid body response to hypoxia in wild-type (CSE +/+ ) and CSE −/− mice as well as in rats treated with CSE inhibitor. Genetic deletion or pharmacologic inhibition of CSE dramatically impairs hypoxic sensing by the carotid body as well as in neonatal adrenal medullary chromaffin cells (AMC). ResultsLoss of Carotid Body Response to Hypoxia in CSE −/− Mice. CSE immunoreactivity was seen in glomus cells of carotid bodies from CSE +/+ mice...

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O 2 . A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2␣ is an orthologue of HIF-1␣, we examined the effects of IH on HIF-2␣, the O2-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1␣, HIF-2␣ was down-regulated by IH. Similar down-regulation of HIF-2␣ was also seen in carotid bodies and adrenal medullae from IH-exposed rats. Inhibitors of calpain proteases (ALLM, ALLN) prevented IH-evoked degradation of HIF-2␣ whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2␣ degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2␣ prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2␣ degradation and restored SOD2 activity, thereby preventing oxidative stress and hypertension. These observations demonstrate that, unlike continuous hypoxia, IH leads to down-regulation of HIF-2␣ via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.calcium signaling ͉ hypoxia inducible factors S leep-disordered breathing with recurrent apneas is a leading cause of morbidity and mortality affecting an estimated 18 million people in the United States alone (1-4). Recurrent apneas are characterized by transient, repetitive cessations of breathing (Ϸ10 sec in adults) resulting in periodic decreases in arterial blood O 2 or intermittent hypoxia (IH). Patients with recurrent apneas are at risk for developing several comorbidities including hypertension, sympathetic activation, breathing abnormalities, atherosclerosis, and stroke (4-8). Exposure of rodents to IH alone induces several co-morbidities reported in patients with recurrent apnea (9-11). However, little information is available on the molecular mechanisms underlying the morbidities associated with IH.Hypoxia-inducible factors (HIFs) mediate transcriptional responses to low O 2 (12). HIF-1 is the prototypical member of the HIF family and comprises an O 2 -regulated ␣ subunit and a constitutive ␤ subunit (13). HIF-1 transcriptional activity is induced under continuous hypoxia (CH) as a result of HIF-1␣ protein accumulation resulting from decreased O 2 -dependent proline hydroxylation, ubiquitination, and proteasomal degradation (14). Recent studies showed that IH leads to HIF-1␣ accumulation and utilizes signaling pathways distinct from those identified with CH (15). The importance of HIF-1 to IHassociated physiological and pathophysiological responses was studied in mice with heterozygous deficiency of HIF-1␣. IHevoked cardio-respiratory and metabolic morbidities were absent ...

Chronic intermittent hypoxia (CIH) augments physiological responses to low partial pressures of O 2 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% O 2 followed by 5 min of 21% O 2 ; 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% O 2 followed by 20 h of 21% O 2 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% CO 2 ; 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.