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.

Souvannakitti D, Kumar GK, Fox A, Prabhakar NR. Neonatal intermittent hypoxia leads to long-lasting facilitation of acute hypoxia-evoked catecholamine secretion from rat chromaffin cells. J Neurophysiol 101: 2837-2846, 2009. First published April 1, 2009 doi:10.1152/jn.00036.2009. The objective of the present study was to examine the effects of intermittent hypoxia (IH) and sustained hypoxia (SH) on hypoxia-evoked catecholamine (CA) secretion from chromaffin cells in neonatal rats and assess the underlying mechanism(s). Experiments were performed on rat pups exposed to either IH (15-s hypoxia/5-min normoxia; 8 h/day) or SH (hypobaric hypoxia, 0.4 atm) or normoxia (controls) from P0 to P5. IH treatment facilitated hypoxia-evoked CA secretion and elevations in the intracellular calcium ion concentration ([Ca 2ϩ ] i ) and these responses were attenuated, but not abolished, by treatments designed to eliminate Ca 2ϩ flux into cells (Ca 2ϩ -free medium or Cd 2ϩ ), indicating that intracellular Ca 2ϩ stores were augmented by IH. Norepinephrine (NE) and epinephrine (E) levels of adrenal medullae were elevated in IH-treated pups. IH treatment increased reactive oxygen species (ROS) production in adrenal medullae and antioxidant treatment prevented IH-induced facilitation of CA secretion, elevations in [Ca 2ϩ ] i by hypoxia, and the up-regulation of NE and E. The effects of neonatal IH treatment on hypoxia-induced CA secretion and elevation in [Ca 2ϩ ] i , CA, and ROS levels persisted in rats reared under normoxia for Ͼ30 days. In striking contrast, chromaffin cells from SH-treated animals exhibited attenuated hypoxia-evoked CA secretion. In SH-treated cells hypoxiaevoked elevations in [Ca 2ϩ ] i , NE and E contents, and ROS levels were comparable with controls. These observations demonstrate that: 1) neonatal IH and SH evoke opposite effects on hypoxia-evoked CA secretion from chromaffin cells, 2) ROS signaling mediates the faciltatory effects of IH, and 3) the effects of neonatal IH on chromaffin cells persist into adult life. I N T R O D U C T I O NCatecholamine (CA) secretion from the adrenal medulla is critical for maintaining homeostasis under a variety of stress conditions including hypoxia (Lagercrantz and Bistoletti 1977;Seidler and Slotkin 1985). In adult animals, hypoxia-evoked CA secretion from chromaffin cells is neurogenic and requires activation of the sympathetic nervous system (Seidler and Slotkin 1986;Yokotani et al. 2002). In neonates the sympathetic nervous system is not well developed (Seidler and Slotkin 1985). In this case, hypoxia still evokes CA secretion from neonatal chromaffin cells by directly affecting their excitability and elevating intracellular calcium ion concentration ( [Ca 2ϩ ] i ; Takeuchi et al. 2001;Thompson et al. 1997). These studies demonstrate that neonatal chromaffin cells have the ability to sense acute hypoxia similar to the glomus cells of the carotid bodies of adult mammals.Chronic perturbations in environmental O 2 profoundly influence carotid body sensory respo...

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We recently reported that adrenomedullary chromaffin cells (AMC) from neonatal rats treated with intermittent hypoxia (IH) exhibit enhanced catecholamine secretion by hypoxia (Souvannakitti D, Kumar GK, Fox A, Prabhakar NR. J Neurophysiol 101: [2837][2838][2839][2840][2841][2842][2843][2844][2845][2846] 2009). In the present study, we examined whether neonatal IH also facilitate AMC responses to nicotine, a potent stimulus to chromaffin cells. Experiments were performed on rats exposed to either IH (15-s hypoxia-5-min normoxia; 8 h/day) or to room air (normoxia; controls) from ages postnatal day 0 (P0) to P5. Quantitative RT-PCR analysis revealed expression of mRNAs encoding ␣ 3-, ␣5-, ␣7-, and ␤2-and ␤4-nicotinic acetylcholine receptor (nAChR) subunits in adrenal medullae from control P5 rats. Nicotine-elevated intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) in AMC and nAChR antagonists prevented this response, suggesting that nAChRs are functional in neonatal AMC. In IH-treated rats, nAChR mRNAs were downregulated in AMC, which resulted in a markedly attenuated nicotine-evoked elevation in [Ca 2ϩ ]i and subsequent catecholamine secretion. Systemic administration of antioxidant prevented IH-evoked downregulation of nAChR expression and function. P35 rats treated with neonatal IH exhibited reduced nAChR mRNA expression in adrenal medullae, attenuated AMC responses to nicotine, and impaired neurogenic catecholamine secretion. Thus the response to neonatal IH lasts for at least 30 days. These observations demonstrate that neonatal IH downregulates nAChR expression and function in AMC via reactive oxygen species signaling, and the effects of neonatal IH persist at least into juvenile life, leading to impaired neurogenic catecholamine secretion from AMC.adrenal medullary chromaffin cells; nicotinic cholinergic receptors; recurrent apneas; reactive oxygen species; catecholamine secretion ADRENAL MEDULLARY CHROMAFFIN cells (AMC) are sensitive to hypoxia in neonates, and low O 2 stimulates catecholamine secretion (2,10,21,31,33,35,37). Hypoxia-evoked catecholamine secretion from AMC involves inhibition of various types of K ϩ channels, leading to depolarization (10,15,17,20,38) Acetylcholine released from the splanchnic nerve activates neuronal nicotinic acetylcholine receptors (nAChRs) on AMC and evokes catecholamine secretion. Although sympathetic innervation to target organs is incomplete in neonates (10, 34), previous studies reported nAChR expression in neonatal AMC (19,32). Given that nicotine is a potent excitatory stimulus to AMC (32), in the present study, we tested the hypothesis that exposing neonatal rats to IH enhances the AMC response to nicotine in a manner similar to that reported for hypoxia. MATERIALS AND METHODSExperimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Chicago. Experiments were performed on neonatal Sprague-Dawley rats between ages postnatal day 0 (P0) and P35.Exposure to IH. Rat pups (P0), along with their mothers, were exposed to...

In the present study we examined the effects of intermittent (IH) and sustained hypoxia (SH) on low PO(2)-evoked catecholamine (CA) secretion from neonatal rat chromaffin cells. Experiments were performed on chromaffin cells isolated from rat pups exposed to either IH (P0-P5; 15 s hypoxia-5 min normoxia;8 h/day) or SH (hypobaric hypoxia; 0.4ATM). CA secretion from chromaffin cells was monitored by amperometry. Control chromaffin cells, from P5 rat pups, exhibited robust CA secretion in response to acute hypoxia. IH facilitated whereas SH attenuated hypoxia-evoked CA secretion. IH increased the epinephrine and norepinephrine content of the adrenal medulla whereas SH had no effect. These results demonstrate that neonatal exposures IH and SH exert diametrically opposed effects on acute hypoxia-evoked CA secretion from chromaffin cells and CA contents.

Acute mountain sickness (AMS) can cause capillary hyper-permeability and vasogenic edema. However, its underlying mechanisms remained unclear and there is no previous in vitro study on AMS. We therefore conducted an in vitro study and examined whether continuous hypobaric hypoxia (CHH) could alter expression of junctional protein complex of vascular endothelial cells, causing hyper-permeabilization. EA.hy926 human endothelial cells were exposed to either CHH or normoxia for up to 24 h. Flow cytometry using annexin V/propidium iodide co-staining demonstrated that cell death had no significant difference at 12-h, but was increased by CHH at 24-h. Transendothelial resistance (TER) of endothelial cell monolayer was progressively decreased by CHH from 1-h to 24-h. Western blot analysis and immunofluorescence study demonstrated decreased expression levels of VE-cadherin, PECAM-1 and ZO-1 junctional proteins at both 12-h and 24-h exposure time-points. Interestingly, while the main form of ZO-1 (220 kDa) was decreased, its degraded form (100 kDa) was increased by 24-h CHH that might be linked to the increased cell death. Our data have demonstrated that CHH caused vascular endothelial hyper-permeability and defective junctional protein complex by reducing expression levels of VE-cadherin, PECAM-1, and ZO-1. Taken together, these data may explain pathophysiology underlying vascular hyperpermeability in AMS.