Ganesh K. Kumar

Induction of sensory long-term facilitation in the carotid body by intermittent hypoxia: Implications for recurrent apneas

Reflexes from the carotid body have been implicated in cardiorespiratory disorders associated with chronic intermittent hypoxia (CIH). To investigate whether CIH causes functional and/or structural plasticity in the carotid body, rats were subjected to 10 days of recurrent hypoxia or normoxia. Acute exposures to 10 episodes of hypoxia evoked long-term facilitation (LTF) of carotid body sensory activity in CIH-conditioned but not in control animals. The magnitude of sensory LTF depended on the length of CIH conditioning and was completely reversible and unique to CIH, because conditioning with a comparable duration of sustained hypoxia was ineffective. Histological analysis revealed no differences in carotid body morphology between control and CIH animals. Previous treatment with superoxide anion (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\cdot}-}\end{equation*}\end{document}) scavenger prevented sensory LTF. In the CIH-conditioned animals, carotid body aconitase enzyme activity decreased compared with controls. These observations suggest that increased generation of reactive oxygen species contribute to sensory LTF. In CIH animals, carotid body complex I activity of the mitochondrial electron transport is inhibited, suggesting mitochondria as one source of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\cdot}-}\end{equation*}\end{document} generation. These observations demonstrate that CIH induces a previously uncharacterized form of reactive oxygen species-dependent, reversible, functional plasticity in carotid body sensory activity. The sensory LTF may contribute to persistent reflex activation of sympathetic nerve activity and blood pressure in recurrent apnea patients experiencing CIH.

Heterozygous HIF-1α deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia

Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH‐evoked cardio‐respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia‐inducible factor‐1 (HIF‐1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF‐1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF‐1 and if so whether it contributes to cardio‐respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild‐type (WT) and heterozygous (HET) mice partially deficient in HIF‐1α, the O2 regulated subunit of the HIF‐1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h−1, 8 h day−1) or to 10 days of 21% O2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long‐term facilitation (sLTF) of the chemoreceptor activity in CIH‐exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH‐exposed HET mice. Analysis of cardio‐respiratory responses in CIH‐exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH‐exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1‐methyl‐4‐pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH‐induced increases in ROS but also CIH‐evoked HIF‐1α up‐regulation in WT mice. These results indicate that: (a) HIF‐1 activation is critical for eliciting CIH‐induced carotid body‐mediated cardio‐respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF‐1 and ROS.

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.

Chronic intermittent hypoxia induces hypoxia-evoked catecholamine efflux in adult rat adrenal medulla via oxidative stress

Chronic intermittent hypoxia (CIH) augments physiological responses to low partial pressures of O2 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% O2 followed by 5 min of 21% O2; 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% O2 followed by 20 h of 21% O2 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% CO2; 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.

Nitric oxide in the sensory function of the carotid body

Recent studies suggest that nitric oxide (NO) may act as a chemical messenger in the nervous system. Since neurotransmitters are considered necessary for the sensory function of the carotid body, and molecular O2 is a co-factor for NO synthesis, we examined whether (a) chemoreceptor tissue also synthesizes NO and if so, (b) does endogenous NO affect chemosensory activity. Experiments were performed on carotid bodies obtained from anesthetized cats (n = 20). Distribution of nitric oxide synthase (NOS), an enzyme that catalyzes the formation of NO was examined using NADPH-diaphorase histochemistry. Many nerve plexuses innervating the chemoreceptor tissue were positive for NADPH-diaphorase, indicating that the nerve fibers are the primary source of NO production in the carotid body. Radiometric analysis of NOS activity of the chemoreceptor tissue averaged 1.94 pmol [3H]citrulline/min/mg protein. NOS activity was significantly less in low pO2 reaction medium than in room air controls. Chemosensory activity in vitro increased in a dose-dependent manner in response to L-omega-nitro arginine (L-NNA), an inhibitor of NOS activity. The effects of NOS inhibitor were enantiomer selective as evidenced by reversal of the responses by L- but not D-arginine. These observations imply that endogenous NO is inhibitory to carotid body sensory activity. cGMP levels of L-NNA-treated carotid bodies were significantly less than untreated controls, suggesting that the actions of NO are coupled to the cGMP second messenger system, as elsewhere in the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

NADPH Oxidase Is Required for the Sensory Plasticity of the Carotid Body by Chronic Intermittent Hypoxia

Respiratory motoneuron response to hypoxia is reflex in nature and carotid body sensory receptor constitutes the afferent limb of this reflex. Recent studies showed that repetitive exposures to hypoxia evokes long term facilitation of sensory nerve discharge (sLTF) of the carotid body in rodents exposed to chronic intermittent hypoxia (CIH). Although studies with anti-oxidants suggested the involvement of reactive oxygen species (ROS)-mediated signaling in eliciting sLTF, the source of and the mechanisms associated with ROS generation have not yet been investigated. We tested the hypothesis that ROS generated by NADPH oxidase (NOX) mediate CIH-evoked sLTF. Experiments were performed on ex vivo carotid bodies from rats and mice exposed either to 10 d of CIH or normoxia. Acute repetitive hypoxia evoked a ∼12-fold increase in NOX activity in CIH but not in control carotid bodies, and this effect was associated with upregulation of NOX2 mRNA and protein, which was primarily localized to glomus cells of the carotid body. sLTF was prevented by NOX inhibitors and was absent in mice deficient in NOX2. NOX activation by CIH required 5-HT release and activation of 5-HT2 receptors coupled to PKC signaling. Studies with ROS scavengers revealed that H2O2 generated from O2·− contributes to sLTF. Priming with H2O2 elicited sLTF of carotid bodies from normoxic control rats and mice, similar to that seen in CIH-treated animals. These observations reveal a novel role for NOX-induced ROS signaling in mediating sensory plasticity of the carotid body.

Role of oxidative stress in intermittent hypoxia‐induced immediate early gene activation in rat PC12 cells

Intermittent hypoxia (IH) occurs in many pathophysiological conditions. The molecular mechanisms associated with IH, however, have received little attention. Previous studies have reported that the c‐fos gene via formation of activator protein‐1 (AP‐1) transcription factor contributes to adaptive responses to continuous hypoxia. In the present study, using a cell culture model we examined whether IH activates c‐fos and AP‐1 and if so, by what mechanisms. Experiments were performed on rat phaeochromocytoma cells exposed to 21% O2 (normoxia) or 60 and 120 cycles of IH, each cycle consisting 15 s of hypoxia followed by 4 min of normoxia. IH resulted in a significant elevation of c‐fos mRNA as well as transcriptional activation. IH was more potent and induced a longer lasting activation of c‐fos than comparable cumulative duration of continuous hypoxia. IH increased AP‐1 activity and tyrosine hydroxylase (TH) mRNA, an AP‐1‐regulated downstream gene, and these effects were prevented by antisense c‐fos. Superoxide dismutase mimetic, a potent scavenger of superoxide anions, prevented IH‐induced c‐fos, AP‐1 and TH activations. IH increased superoxide anion levels in mitochondria as evidenced by decreased aconitase enzyme activity and increased levels of hydrogen peroxide, a stable dismutated product of superoxide anions. Complex I of the mitochondrial electron transport chain was markedly inhibited in IH exposed cells. Pharmacological inhibitors of complex I mimicked the effects of IH during normoxia and occluded the effects of IH on c‐fos activation, suggesting the involvement of the mitochondrial electron transport chain in the generation of superoxide anions during IH. These results suggest IH‐induced c‐fos‐mediated transcriptional activation involves oxidative stress.

Cardiovascular alterations by chronic intermittent hypoxia: Importance of carotid body chemoreflexes

1. Humans experiencing intermittent hypoxia (IH) owing to recurrent apnoea syndromes exhibit serious cardiovascular morbidity, including high blood pressure, increased sympathetic nerve activity, cardiac arrhythmia and myocardial infarction. Although apnoeas are accompanied by a simultaneous decrease in arterial O2 (hypoxia) and an increase in CO2 (hypercapnia), studies on experimental animals suggest that hypoxia, rather than hypercapnia, is the primary stimulus for developing hypertension and enhanced sympathetic nerve activity. Enhanced hypoxic‐sensing ability of the carotid bodies and the ensuing reflex activation of the sympathetic nervous system have been suggested to play a critical role in cardiorespiratory alterations resulting from recurrent apnoeas.

Intermittent hypoxia degrades HIF-2α via calpains resulting in oxidative stress: Implications for recurrent apnea-induced morbidities

Intermittent hypoxia (IH) occurs in many pathological conditions including recurrent apneas. Hypoxia-inducible factors (HIFs) 1 and 2 mediate transcriptional responses to low O2. A previous study showed that HIF-1 mediates some of the IH-evoked physiological responses. Because HIF-2α is an orthologue of HIF-1α, we examined the effects of IH on HIF-2α, the O2-regulated subunit expression, in pheochromocytoma 12 cell cultures. In contrast to the up-regulation of HIF-1α, HIF-2α was down-regulated by IH. Similar down-regulation of HIF-2α 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-2α whereas inhibitors of prolyl hydroxylases or proteosome were ineffective. IH activated calpain proteases and down-regulated the endogenous calpain inhibitor calpastatin. IH-evoked HIF-2α degradation led to inhibition of SOD2 transcription, resulting in oxidative stress. Over-expression of transcriptionally active HIF-2α prevented IH-evoked oxidative stress and restored SOD2 activity. Systemic treatment of IH-exposed rats with ALLM rescued HIF-2α 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-2α via a calpain-dependent signaling pathway and results in oxidative stress as well as autonomic morbidities.

Oxidative stress in the systemic and cellular responses to intermittent hypoxia

Abstract Patients with chronic intermittent hypoxia (IH) caused by recurrent apneas have a greatly increased risk for developing hypertension, myocardial infarctions, and stroke. The purpose of this article is to highlight some of the recent studies focusing on the mechanisms associated with systemic and cellular effects of IH in experimental animals and cell culture models. Rats exposed to chronic IH exhibited elevated blood pressures and increased sympathetic nerve activity, partly due to enhanced reflexes arising from carotid bodies. Direct recordings of the carotid body sensory activity showed that chronic IH selectively augmented hypoxic sensitivity, and induced a novel form of functional plasticity manifested as sensory longterm facilitation. In cell culture models, prior exposure to IH resulted in facilitation of hypoxiainduced transmitter release and activation of several protein kinases. IH caused activation of cFos and activator protein-1 (AP-1) transcription factor and tyrosine hydroxylase, an APregulated downstream gene. For a given duration and intensity of hypoxia, IH was more potent and caused longerlasting activation than continuous hypoxia. Scavengers of reactive oxygen species (ROS) prevented IHinduced systemic and cellular responses. Inhibition of complex I of the mitochondrial electron transport chain appears to be one of the sources for IHinduced generation of ROS. The persistent oxidative stress may contribute to the progression of morbidity associated with chronic IH caused by recurrent apneas, and antioxidants might be of considerable therapeutic value in preventing the progression of disease associated with chronic IH.