Unraveling the Nrf2-ARE Signaling Pathway in the DF-1 Chicken Fibroblast Cell Line: Insights into T-2 Toxin-Induced Oxidative Stress Regulation.

The T-2 toxin (T2) poses a major threat to the health and productivity of animals. The present study aimed to investigate the regulatory mechanism of Nrf2 derived from broilers against T2-induced oxidative damage. DF-1 cells, including those with normal characteristics, as well as those overexpressing or with a knockout of specific components, were exposed to a 24 h treatment of 50 nM T2. The primary objective was to evaluate the indicators associated with oxidative stress and the expression of downstream antioxidant factors regulated by the Nrf2-ARE signaling pathway, at both the mRNA and protein levels. The findings of this study demonstrated a noteworthy relationship between the up-regulation of the Nrf2 protein and a considerable reduction in the oxidative stress levels within DF-1 cells (p < 0.05). Furthermore, this up-regulation was associated with a notable increase in the mRNA and protein levels of antioxidant factors downstream of the Nrf2-ARE signaling pathway (p < 0.05). Conversely, the down-regulation of the Nrf2 protein was linked to a marked elevation in oxidative stress levels in DF-1 cells (p < 0.05). Additionally, this down-regulation resulted in a significant decrease in both the mRNA and protein expression of antioxidant factors (p < 0.05). This experiment lays a theoretical foundation for investigating the detrimental impacts of T2 on broiler chickens. It also establishes a research framework for employing the Nrf2 protein in broiler chicken production and breeding. Moreover, it introduces novel insights for the prospective management of oxidative stress-related ailments in the livestock and poultry industry.

Deciphering the Hazardous Effects of AFB1 and T-2 Toxins: Unveiling Toxicity and Oxidative Stress Mechanisms in PK15 Cells and Mouse Kidneys.

In China, animal feeds are frequently contaminated with a range of mycotoxins, with Aflatoxin B1 (AFB1) and T-2 toxin (T-2) being two highly toxic mycotoxins. This study investigates the combined nephrotoxicity of AFB1 and T-2 on PK15 cells and murine renal tissues and their related oxidative stress mechanisms. PK15 cells were treated with the respective toxin concentrations for 24 h, and oxidative stress-related indicators were assessed. The results showed that the combination of AFB1 and T-2 led to more severe cellular damage and oxidative stress compared to exposure to the individual toxins (p < 0.05). In the in vivo study, pathological examination revealed that the kidney tissue of mice exposed to the combined toxins showed signs of glomerular atrophy. The contents of oxidative stress-related indicators were significantly increased in the kidney tissue (p < 0.05). These findings suggest that the combined toxins cause significant oxidative damage to mouse kidneys. The study highlights the importance of considering the combined effects of mycotoxins in animal feed, particularly AFB1 and T-2, which can lead to severe nephrotoxicity and oxidative stress in PK15 cells and mouse kidneys. The findings have important implications for animal feed safety and regulatory policy.

COVID-19 plasma exosomes promote proinflammatory immune responses in peripheral blood mononuclear cells.

Elevated serum cytokine production in COVID-19 patients is associated with disease progression and severity. However, the stimuli that initiate cytokine production in patients remain to be fully revealed. Virus-infected cells release virus-associated exosomes, extracellular vesicles of endocytic origin, into the blood to deliver viral cargoes able to regulate immune responses. Here, we report that plasma exosomes of COVID-19 patients contain SARS-CoV-2 double stranded RNA (dsRNA) and stimulate robust production of interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and other inflammatory cytokines and chemokines by human peripheral mononuclear cells. Exosome depletion abolished these stimulated responses. COVID-19 plasma exosomes induced proinflammatory responses in CD4+ T cells, CD8+ T cells, and CD14+ monocytes but not significantly in regulatory T cells, Th17 T cells, or central memory T cells. COVID-19 plasma exosomes protect the SARS-CoV-2 dsRNA cargo from RNase and deliver the dsRNA into recipient cells. These exosomes significantly increase expression of endosomal toll-like receptor 3 (TLR3), TLR7, TLR8, and TLR9 in peripheral T cells and monocytes. A pharmacological inhibitor of TLR3 considerably reduced cytokine and chemokine production by CD4+ and CD8+ T cells but not by CD14+ monocytes, highlighting divergent signaling pathways of immune cells in response to COVID-19 plasma exosomes. Our results identify a novel model of intercellular crosstalk following SARS-CoV-2 infection that evoke immune responses positioned to contribute to elevated cytokine production associated with COVID-19 progression, severity, and long-haul symptoms.

Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi's Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi's sarcoma (KS), the most common malignancy in people living with human immunodeficiency virus (HIV)/AIDS. The oral cavity is a major route for KSHV infection and transmission. However, how KSHV breaches the oral epithelial barrier for spreading to the body is not clear. Here, we show that exosomes purified from either the saliva of HIV-positive individuals or the culture supernatants of HIV-1-infected T-cell lines promote KSHV infectivity in immortalized and primary human oral epithelial cells. HIV-associated saliva exosomes contain the HIV trans-activation response element (TAR), Tat, and Nef RNAs but do not express Tat and Nef proteins. The TAR RNA in HIV-associated exosomes contributes to enhancing KSHV infectivity through the epidermal growth factor receptor (EGFR). An inhibitory aptamer against TAR RNA reduces KSHV infection facilitated by the synthetic TAR RNA in oral epithelial cells. Cetuximab, a monoclonal neutralizing antibody against EGFR, blocks HIV-associated exosome-enhanced KSHV infection. Our findings reveal that saliva containing HIV-associated exosomes is a risk factor for the enhancement of KSHV infection and that the inhibition of EGFR serves as a novel strategy for preventing KSHV infection and transmission in the oral cavity.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi's sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans-activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population.

Reactive oxygen radicals and gaseous transmitters in carotid body activation by intermittent hypoxia.

Sleep apnea is a prevalent respiratory disease characterized by periodic cessation of breathing during sleep causing intermittent hypoxia (IH). Sleep apnea patients and rodents exposed to IH exhibit elevated sympathetic nerve activity and hypertension. A heightened carotid body (CB) chemoreflex has been implicated in causing autonomic abnormalities in IH-treated rodents and in sleep apnea patients. The purpose of this article is to review the emerging evidence showing that interactions between reactive oxygen species (ROS) and gaseous transmitters as a mechanism cause hyperactive CB by IH. Rodents treated with IH exhibit markedly elevated ROS in the CB, which is due to transcriptional upregulation of pro-oxidant enzymes by hypoxia-inducible factor (HIF)-1 and insufficient transcriptional regulation of anti-oxidant enzymes by HIF-2. ROS, in turn, increases cystathionine γ-lyase (CSE)-dependent H2S production in the CB. Blockade of H2S synthesis prevents IH-evoked CB activation. However, the effects of ROS on H2S production are not due to direct effects on CSE enzyme activity but rather due to inactivation of heme oxygenase-2 (HO-2), a carbon monoxide (CO) producing enzyme. CO inhibits H2S production through inactivation of CSE by PKG-dependent phosphorylation. During IH, reduced CO production resulting from inactivation of HO-2 by ROS releases the inhibition of CO on CSE thereby increasing H2S. Inhibiting H2S synthesis prevented IH-evoked sympathetic activation and hypertension.

H2S production by reactive oxygen species in the carotid body triggers hypertension in a rodent model of sleep apnea.

Sleep apnea is a prevalent respiratory disease in which episodic cessation of breathing causes intermittent hypoxia. Patients with sleep apnea and rodents exposed to intermittent hypoxia exhibit hypertension. The carotid body senses changes in blood O2 concentrations, and an enhanced carotid body chemosensory reflex contributes to hypertension in sleep apnea patients. A rodent model of intermittent hypoxia that mimics blood O2 saturation profiles of patients with sleep apnea has shown that increased generation of reactive oxygen species (ROS) in the carotid body enhances the chemosensory reflex and triggers hypertension. CO generated by heme oxygenase-2 (HO-2) induces a signaling pathway that inhibits hydrogen sulfide (H2S) production by cystathionine γ-lyase (CSE), leading to suppression of carotid body activity. We found that ROS inhibited CO generation by HO-2 in the carotid body and liver through a mechanism that required Cys(265) in the heme regulatory motif of heterologously expressed HO-2. We showed that ROS induced by intermittent hypoxia inhibited CO production and increased H2S concentrations in the carotid body, which stimulated its neural activity. In rodents, blockade of H2S synthesis by CSE, by either pharmacologic or genetic approaches, inhibited carotid body activation and hypertension induced by intermittent hypoxia. Thus, our results indicate that oxidant-induced inactivation of HO-2, which leads to increased CSE-dependent H2S production in the carotid body, is a critical trigger of hypertension in rodents exposed to intermittent hypoxia.

Protein kinase G-regulated production of H2S governs oxygen sensing.

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 (Cys(265) and Cys(282)) of HO-2. CO stimulated protein kinase G (PKG)-dependent phosphorylation of Ser(377) 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.

Hypoxia-inducible factors and hypertension: lessons from sleep apnea syndrome.

Systemic hypertension is one of the most prevalent cardiovascular diseases. Sleep-disordered breathing (SDB) with recurrent apnea is a major risk factor for developing essential hypertension. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of recurrent apnea. Rodent models patterned after the O2 profiles seen with SDB patients showed that CIH is the major stimulus for causing systemic hypertension. This article reviews the physiological and molecular basis of CIH-induced hypertension. Physiological studies have identified that augmented carotid body chemosensory reflex and the resulting increase in sympathetic nerve activity are major contributors to CIH-induced hypertension. Analysis of molecular mechanisms revealed that CIH activates hypoxia-inducible factor (HIF)-1 and suppresses HIF-2-mediated transcription. Dysregulation of HIF-1- and HIF-2-mediated transcription leads to imbalance of pro-oxidant and anti-oxidant enzyme gene expression resulting in increased reactive oxygen species (ROS) generation in the chemosensory reflex which is central for developing hypertension.

CaV3.2 T-type Ca²âº channels in H2S-mediated hypoxic response of the carotid body.

Arterial blood O2 levels are detected by specialized sensory organs called carotid bodies. Voltage-gated Ca(2+) channels (VGCCs) are important for carotid body O2 sensing. Given that T-type VGCCs contribute to nociceptive sensation, we hypothesized that they participate in carotid body O2 sensing. The rat carotid body expresses high levels of mRNA encoding the α1H-subunit, and α1H protein is localized to glomus cells, the primary O2-sensing cells in the chemoreceptor tissue, suggesting that CaV3.2 is the major T-type VGCC isoform expressed in the carotid body. Mibefradil and TTA-A2, selective blockers of the T-type VGCC, markedly attenuated elevation of hypoxia-evoked intracellular Ca(2+) concentration, secretion of catecholamines from glomus cells, and sensory excitation of the rat carotid body. Similar results were obtained in the carotid body and glomus cells from CaV3.2 knockout (Cacna1h(-/-)) mice. Since cystathionine-γ-lyase (CSE)-derived H2S is a critical mediator of the carotid body response to hypoxia, the role of T-type VGCCs in H2S-mediated O2 sensing was examined. Like hypoxia, NaHS, a H2S donor, increased intracellular Ca(2+) concentration and augmented carotid body sensory nerve activity in wild-type mice, and these effects were markedly attenuated in Cacna1h(-/-) mice. In wild-type mice, TTA-A2 markedly attenuated glomus cell and carotid body sensory nerve responses to hypoxia, and these effects were absent in CSE knockout mice. These results demonstrate that CaV3.2 T-type VGCCs contribute to the H2S-mediated carotid body response to hypoxia.

Regulation of hypoxia-inducible factor-α isoforms and redox state by carotid body neural activity in rats.

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 and the redox state by CIH in the central and peripheral nervous systems associated with the chemoreflex.

Intermittent hypoxia-induced endothelial barrier dysfunction requires ROS-dependent MAP kinase activation.

The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung microvascular endothelial cells exposed to IH-consisting alternating cycles of 1.5% O2 for 30s followed by 20% O2 for 5 min. IH decreased transendothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus dependent and reversible after reoxygenation. IH-exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins, and zona occludens-1 junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) were phosphorylated in IH-exposed cells. Inhibiting either ERK or JNK prevented the IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels, and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant, prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to reorganization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.

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

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.

Mutual antagonism between hypoxia-inducible factors 1α and 2α regulates oxygen sensing and cardio-respiratory homeostasis.

Breathing and blood pressure are under constant homeostatic regulation to maintain optimal oxygen delivery to the tissues. Chemosensory reflexes initiated by the carotid body and catecholamine secretion from the adrenal medulla are the principal mechanisms for maintaining respiratory and cardiovascular homeostasis; however, the underlying molecular mechanisms are not known. Here, we report that balanced activity of hypoxia-inducible factor-1 (HIF-1) and HIF-2 is critical for oxygen sensing by the carotid body and adrenal medulla, and for their control of cardio-respiratory function. In Hif2α(+/-) mice, partial HIF-2α deficiency increased levels of HIF-1α and NADPH oxidase 2, leading to an oxidized intracellular redox state, exaggerated hypoxic sensitivity, and cardio-respiratory abnormalities, which were reversed by treatment with a HIF-1α inhibitor or a superoxide anion scavenger. Conversely, in Hif1α(+/-) mice, partial HIF-1α deficiency increased levels of HIF-2α and superoxide dismutase 2, leading to a reduced intracellular redox state, blunted oxygen sensing, and impaired carotid body and ventilatory responses to chronic hypoxia, which were corrected by treatment with a HIF-2α inhibitor. None of the abnormalities observed in Hif1α(+/-) mice or Hif2α(+/-) mice were observed in Hif1α(+/-);Hif2α(+/-) mice. These observations demonstrate that redox balance, which is determined by mutual antagonism between HIF-α isoforms, establishes the set point for hypoxic sensing by the carotid body and adrenal medulla, and is required for maintenance of cardio-respiratory homeostasis.

Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis.

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

Hypoxia-inducible factor 1 mediates increased expression of NADPH oxidase-2 in response to intermittent hypoxia.

Sleep-disordered breathing with recurrent apnea is associated with intermittent hypoxia (IH). Cardiovascular morbidities caused by IH are triggered by increased generation of reactive oxygen species (ROS) by pro-oxidant enzymes, especially NADPH oxidase-2 (Nox2). Previous studies showed that (i) IH activates hypoxia-inducible factor 1 (HIF-1) in a ROS-dependent manner and (ii) HIF-1 is required for IH-induced ROS generation, indicating the existence of a feed-forward mechanism. In the present study, using multiple pharmacological and genetic approaches, we investigated whether IH-induced expression of Nox2 is mediated by HIF-1 in the central and peripheral nervous system of mice as well as in cultured cells. IH increased Nox2 mRNA, protein, and enzyme activity in PC12 pheochromocytoma cells as well as in wild-type mouse embryonic fibroblasts (MEFs). This effect was abolished or attenuated by blocking HIF-1 activity through RNA interference or pharmacologic inhibition (digoxin or YC-1) or by genetic knockout of HIF-1α in MEFs. Increasing HIF-1α expression by treating PC 12 cells with the iron chelator deferoxamine for 20 h or by transfecting them with HIF-1alpha expression vector increased Nox2 expression and enzyme activity. Exposure of wild-type mice to IH (8 h/day for 10 days) up-regulated Nox2 mRNA expression in brain cortex, brain stem, and carotid body but not in cerebellum. IH did not induce Nox2 expression in cortex, brainstem, carotid body, or cerebellum of Hif1a(+/-) mice, which do not manifest increased ROS or cardiovascular morbidities in response to IH. These results establish a pathogenic mechanism linking HIF-1, ROS generation, and cardiovascular pathology in response to IH.

Hypoxia-inducible factor 2α (HIF-2α) heterozygous-null mice exhibit exaggerated carotid body sensitivity to hypoxia, breathing instability, and hypertension.

Cardiorespiratory functions in mammals are exquisitely sensitive to changes in arterial O(2) levels. Hypoxia-inducible factors (e.g., HIF-1 and HIF-2) mediate transcriptional responses to reduced oxygen availability. We demonstrate that haploinsufficiency for the O(2)-regulated HIF-2α subunit results in augmented carotid body sensitivity to hypoxia, irregular breathing, apneas, hypertension, and elevated plasma norepinephrine levels in adult Hif-2α(+/-) mice. These dysregulated autonomic responses were associated with increased oxidative stress and decreased mitochondrial electron transport chain complex I activity in adrenal medullae as a result of decreased expression of major cytosolic and mitochondrial antioxidant enzymes. Systemic administration of a membrane-permeable antioxidant prevented oxidative stress, normalized hypoxic sensitivity of the carotid body, and restored autonomic functions in Hif-2α(+/-) mice. Thus, HIF-2α-dependent redox regulation is required for maintenance of carotid body function and cardiorespiratory homeostasis.

NADPH oxidase 2 mediates intermittent hypoxia-induced mitochondrial complex I inhibition: relevance to blood pressure changes in rats.

Previous studies identified NADPH oxidases (Nox) and mitochondrial electron transport chain at complex I as major cellular sources of reactive oxygen species (ROS) mediating systemic and cellular responses to intermittent hypoxia (IH). In the present study, we investigated potential interactions between Nox and the mitochondrial complex I and assessed the contribution of mitochondrial ROS in IH-evoked elevation in blood pressure. IH treatment led to stimulus-dependent activation of Nox and inhibition of complex I activity in rat pheochromocytoma (PC)12 cells. After re-oxygenation, Nox activity returned to baseline values within 3 h, whereas the complex I activity remained downregulated even after 24 h. IH-induced complex I inhibition was prevented by Nox inhibitors, Nox2 but not Nox 4 siRNA, in cell cultures and was absent in gp91(phox-/Y) (Nox2 knock-out; KO) mice. Using pharmacological inhibitors, we show that ROS generated by Nox activation mobilizes Ca(2+) flux from the cytosol to mitochondria, leading to S-glutathionylation of 75- and 50-kDa proteins of the complex I and inhibition of complex I activity, which results in elevated mitochondrial ROS. Systemic administration of mito-tempol prevented the sustained but not the acute elevations of blood pressure in IH-treated rats, suggesting that mitochondrial-derived ROS contribute to sustained elevation of blood pressure.

NADPH oxidase-dependent regulation of T-type Ca2+ channels and ryanodine receptors mediate the augmented exocytosis of catecholamines from intermittent hypoxia-treated neonatal rat chromaffin cells.

Nearly 90% of premature infants experience the stress of intermittent hypoxia (IH) as a consequence of recurrent apneas (periodic cessation of breathing). In neonates, catecholamine secretion from the adrenal medulla is critical for maintaining homeostasis under hypoxic stress. We recently reported that IH treatment enhanced hypoxia-evoked catecholamine secretion and [Ca2+]i responses in neonatal rat adrenal chromaffin cells and involves reactive oxygen species (ROS). The purpose of the present study was to identify the source(s) of ROS generation and examine the mechanisms underlying the enhanced catecholamine secretion by IH. Neonatal rats of either sex (postal day 0-5) were exposed to either IH or normoxia. IH treatment increased NADPH oxidase (NOX) activity, upregulated NOX2 and NOX4 transcription in adrenal medullae, and a NOX inhibitor prevented the effects of IH on hypoxia-evoked chromaffin cell secretion. IH upregulated Cav3.1 and Cav3.2 T-type Ca2+ channel mRNAs via NOX/ROS signaling and augmented T-type Ca2+ current in IH-treated chromaffin cells. Mibefradil, a blocker of T-type Ca2+ channels attenuated the effects of hypoxia on [Ca2+]i and catecholamine secretion in IH-treated cells. In Ca2+-free medium, IH-treated cells exhibited higher basal [Ca2+]i levels and more pronounced [Ca2+]i responses to hypoxia compared with controls, and blockade of ryanodine receptors (RyRs) prevented these effects. RyR2 and RyR3 mRNAs were upregulated, RyR2 was S-glutathionylated in IH-treated adrenal medullae, and NOX/ROS inhibitors prevented these effects. These results demonstrate that neonatal IH treatment leads to NOX/ROS-dependent recruitment of T-type Ca2+ channels and RyRs, resulting in augmented [Ca2+]i mobilization and catecholamine secretion.

Neonatal intermittent hypoxia impairs neuronal nicotinic receptor expression and function in adrenal chromaffin cells.

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: 2837-2846, 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 alpha(3-), alpha(5-), alpha(7-), and beta(2-) and beta(4-)nicotinic acetylcholine receptor (nAChR) subunits in adrenal medullae from control P5 rats. Nicotine-elevated intracellular Ca(2+) concentration ([Ca(2+)](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(2+)](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.

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