RESUMEN
Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H2S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H2S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca2+]i by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po2 30-38 mmHg) evoked CA release and elevated [Ca2+]i. The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H2S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H2S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H2S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H2S.NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H2S production through soluble guanylyl cyclase-protein kinase G signaling cascade.
Asunto(s)
Monóxido de Carbono/metabolismo , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Cistationina gamma-Liasa/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Sulfuro de Hidrógeno/metabolismo , Hipoxia/metabolismo , Transducción de Señal/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
The role of hypoxia-inducible factor (HIF)-1 in pancreatic ß-cell response to intermittent hypoxia (IH) was examined. Studies were performed on adult wild-type (WT), HIF-1α heterozygous (HET), ß-cell-specific HIF-1-/- mice and mouse insulinoma (MIN6) cells exposed to IH patterned after blood O2 profiles during obstructive sleep apnea. WT mice treated with IH showed insulin resistance, and pancreatic ß-cell dysfunction manifested as augmented basal insulin secretion, and impaired glucose-stimulated insulin secretion and these effects were absent in HIF-1α HET mice. IH increased HIF-1α expression and elevated reactive oxygen species (ROS) levels in ß-cells of WT mice. The elevated ROS levels were due to transcriptional upregulation of NADPH oxidase (NOX)-4 mRNA, protein and enzymatic activity, and these responses were absent in HIF-1α HET mice as well as in ß-HIF-1-/- mice. IH-evoked ß-cell responses were absent in adult WT mice treated with digoxin, an inhibitor of HIF-1α. MIN6 cells treated with in vitro IH showed enhanced basal insulin release and elevated HIF-1α protein expression, and these effects were abolished with genetic silencing of HIF-1α. IH increased NOX4 mRNA, protein, and enzyme activity in MIN6 cells and disruption of NOX4 function by siRNA or scavenging H2O2 with polyethylene glycol catalase blocked IH-evoked enhanced basal insulin secretion. These results demonstrate that HIF-1-mediated transcriptional activation of NOX4 and the ensuing increase in H2O2 contribute to IH-induced pancreatic ß-cell dysfunction.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Hipoxia/genética , NADPH Oxidasa 4/genética , Oxígeno/farmacología , Apnea Obstructiva del Sueño/genética , Animales , Digoxina/farmacología , Modelos Animales de Enfermedad , Glucosa/metabolismo , Heterocigoto , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/metabolismo , Hipoxia/metabolismo , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/genética , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 4/antagonistas & inhibidores , NADPH Oxidasa 4/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Apnea Obstructiva del Sueño/metabolismo , Apnea Obstructiva del Sueño/patología , Activación TranscripcionalRESUMEN
In neonates, catecholamine (CA) secretion from adrenal medullary chromaffin cells (AMC) is an important mechanism for maintaining homeostasis during hypoxia. Nearly 90% of premature infants experience chronic intermittent hypoxia (IH) because of high incidence of apnea of prematurity, which is characterized by periodic stoppage of breathing. The present study examined the effects of repetitive hypoxia, designed to mimic apnea of prematurity, on CA release from AMC of neonatal rats. Neonatal rats were exposed to either control conditions or chronic intermittent hypoxia (IH) from ages postnatal days 0-5 (P0-P5), and CA release from adrenal medullary slices was measured after challenge with repetitive hypoxia (5 episodes of 30-s hypoxia, Po2 ~35 mmHg). In response to repetitive hypoxia, chronic IH-treated AMC exhibited sustained CA release, and this phenotype was not seen in control AMC. The sustained CA release was associated with long-lasting elevation of intracellular Ca2+ concentration ([Ca2+]i), which was due to store-operated Ca2+ entry (SOCE). 2-Aminoethoxydiphenyl borate, an inhibitor of SOCE, prevented the long-lasting [Ca2+]i elevation and CA release. Repetitive hypoxia increased H2O2 abundance, and polyethylene glycol (PEG)-catalase, a scavenger of H2O2 blocked this effect. PEG-catalase also prevented repetitive hypoxia-induced SOCE activation, sustained [Ca2+]i elevation, and CA release. These results demonstrate that repetitive hypoxia induces long-term facilitation of CA release in chronic IH-treated neonatal rat AMC through sustained Ca2+ influx mediated by SOCE.NEW & NOTEWORTHY Apnea of prematurity and the resulting chronic intermittent hypoxia are major clinical problems in neonates born preterm. Catecholamine release from adrenal medullary chromaffin cells maintains homeostasis during hypoxia in neonates. Our results demonstrate that chronic intermittent hypoxia induces a hitherto uncharacterized long-term facilitation of catecholamine secretion from neonatal rat chromaffin cells in response to repetitive hypoxia, simulating hypoxic episodes encountered during apnea of prematurity. The sustained catecholamine secretion might contribute to cardiovascular morbidities in infants with apnea of prematurity.
Asunto(s)
Médula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Hipoxia/metabolismo , Animales , Animales Recién Nacidos , Apnea/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Enfermedades del Prematuro/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
KEY POINTS: The mechanisms underlying long-term (30 days) intermittent hypoxia (LT-IH)-evoked DNA methylation of anti-oxidant enzyme (AOE) gene repression in the carotid body (CB) reflex pathway were examined. LT-IH-treated rats showed increased reactive oxygen species (ROS) levels in the CB reflex pathway. Administration of a ROS scavenger or CB ablation blocked LT-IH-evoked DNA methylation and AOE gene repression in the central and efferent limbs of the CB reflex. LT-IH increased DNA methyltransferase (Dnmt) activity through upregulation of Dnmt1 and 3b proteins by ROS-dependent inactivation of glycogen synthase kinase 3ß (GSK3ß) by Akt. A pan-Akt inhibitor prevented LT-IH-induced GSK3ß inactivation, elevated Dnmt protein expression and activity, AOE gene methylation, sympathetic activation and hypertension. ABSTRACT: Long-term exposure to intermittent hypoxia (LT-IH; 30 days), simulating blood O2 profiles during sleep apnoea, has been shown to repress anti-oxidant enzyme (AOE) gene expression by DNA methylation in the carotid body (CB) reflex pathway, resulting in persistent elevation of plasma catecholamine levels and blood pressure. The present study examined the mechanisms by which LT-IH induces DNA methylation. Adult rats exposed to LT-IH showed elevated reactive oxygen species (ROS) in the CB, nucleus tractus solitarius (nTS) and rostroventrolateral medulla (RVLM) and adrenal medulla (AM), which represent the central and efferent limbs of the CB reflex, respectively. ROS scavenger treatment during the first ten days of IH exposure prevented ROS accumulation, blocked DNA methylation, and normalized AOE gene expression, suggesting that ROS generated during the early stages of IH activate DNA methylation. CB ablation prevented the ROS accumulation, normalized AOE gene expression in the nTS, RVLM, and AM and blocked DNA methylation, suggesting that LT-IH-induced DNA methylation in the central and efferent limbs of the CB reflex is indirect and requires CB neural activity. LT-IH increased DNA methyl transferase (Dnmt) activity through upregulation of Dnmt1 and 3b protein expression due to ROS-dependent inactivation of glycogen synthase kinase 3ß (GSK3ß) by protein kinase B (Akt). Treating rats with the pan-Akt inhibitor GSK690693 blocked the induction of Dnmt activity, Dnmt protein expression, and DNA methylation, leading to normalization of AOE gene expression as well as plasma catecholamine levels and blood pressure.
Asunto(s)
Cuerpo Carotídeo/fisiología , Metilación de ADN , Animales , Encéfalo/fisiología , Cuerpo Carotídeo/cirugía , Hipoxia/metabolismo , Hipoxia/fisiopatología , Masculino , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
KEY POINTS: The effects of short-term (ST; 10 days) and long-term (LT; 30 days) intermittent hypoxia (IH) on blood pressure (BP), breathing and carotid body (CB) chemosensory reflex were examined in adult rats. ST- and LT-IH treated rats exhibited hypertension, irregular breathing with apnoea and augmented the CB chemosensory reflex, with all these responses becoming normalized during recovery from ST- but not from LT-IH. The persistent cardiorespiratory responses to LT-IH were associated with elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla, which were a result of DNA methylation-dependent suppression of genes encoding anti-oxidant enzymes (AOEs). Treating rats with decitabine either during LT-IH or during recovery from LT-IH prevented DNA methylation of AOE genes, normalized the expression of AOE genes and ROS levels, reversed the heightened CB chemosensory reflex and hypertension, and also stabilized breathing. ABSTRACT: Rodents exposed to chronic intermittent hypoxia (IH), simulating blood O2 saturation profiles during obstructive sleep apnoea (OSA), have been shown to exhibit a heightened carotid body (CB) chemosensory reflex and hypertension. CB chemosensory reflex activation also results in unstable breathing with apnoeas. However, the effect of chronic IH on breathing is not known. In the present study, we examined the effects of chronic IH on breathing along with blood pressure (BP) and assessed whether the autonomic responses are normalized after recovery from chronic IH. Studies were performed on adult, male, Sprague-Dawley rats exposed to either short-term (ST; 10 days) or long-term (LT, 30 days) IH. Rats exposed to either ST- or LT-IH exhibited hypertension, irregular breathing with apnoeas, an augmented CB chemosensory reflex as indicated by elevated CB neural activity and plasma catecholamine levels, and elevated reactive oxygen species (ROS) levels in the CB and adrenal medulla (AM). All these effects were normalized after recovery from ST-IH but not from LT-IH. Analysis of the molecular mechanisms underlying the persistent effects of LT-IH revealed increased DNA methylation of genes encoding anti-oxidant enzymes (AOEs). Treatment with decitabine, a DNA methylation inhibitor, either during LT-IH or during recovery from LT-IH, prevented DNA methylation, normalized the expression of AOE genes, ROS levels, CB chemosensory reflex and BP, and also stabilized breathing. These results suggest that persistent cardiorespiratory abnormalities caused by LT-IH are mediated by epigenetic re-programming of the redox state in the CB chemosensory reflex pathway.
Asunto(s)
Hipertensión/fisiopatología , Hipoxia/fisiopatología , Trastornos Respiratorios/fisiopatología , Aconitato Hidratasa/metabolismo , Médula Suprarrenal/metabolismo , Animales , Presión Sanguínea , Cuerpo Carotídeo/metabolismo , Cuerpo Carotídeo/fisiología , Catalasa/genética , Metilación de ADN , Epigénesis Genética , Expresión Génica , Glutatión Peroxidasa/genética , Hipertensión/sangre , Hipertensión/genética , Hipertensión/metabolismo , Hipoxia/sangre , Hipoxia/genética , Hipoxia/metabolismo , Masculino , Malondialdehído/metabolismo , Norepinefrina/sangre , Oxidación-Reducción , Peroxirredoxinas/genética , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Trastornos Respiratorios/sangre , Trastornos Respiratorios/genética , Trastornos Respiratorios/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
Chronic intermittent hypoxia (CIH) is a hallmark manifestation of sleep apnea. A heightened carotid body activity and the resulting chemosensory reflex mediate increased sympathetic nerve activity by CIH. However, the mechanisms underlying heightened carotid body activity by CIH are not known. An elevation of intracellular calcium ion concentration ([Ca(2+)]i) in glomus cells, the primary oxygen-sensing cells, is an essential step for carotid body activation by hypoxia. In the present study, we examined the effects of CIH on the glomus cell [Ca(2+)]i response to hypoxia and assessed the underlying mechanisms. Glomus cells were harvested from adult rats or wild-type mice treated with 10 days of either room air (control) or CIH (alternating cycles of 15 s of hypoxia and 5 min of room air; 9 episodes/h; 8 h/day). CIH-treated glomus cells exhibited an enhanced [Ca(2+)]i response to hypoxia, and this effect was absent in the presence of 2-(4-cyclopropylphenyl)-N-((1R)-1-[5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl]ethyl)acetamide (TTA-A2), a specific inhibitor of T-type Ca(2+) channels, and in voltage-gated calcium channel, type 3.2 (CaV3.2), null glomus cells. CaV3.2 knockout mice exhibited an absence of CIH-induced hypersensitivity of the carotid body. CIH increased reactive oxygen species (ROS) levels in glomus cells. A ROS scavenger prevented the exaggerated TTA-A2-sensitive [Ca(2+)]i response to hypoxia. CIH had no effect on CaV3.2 mRNA levels. CIH augmented Ca(2+) currents and increased CaV3.2 protein in plasma membrane fractions of human embryonic kidney-293 cells stably expressing CaV3.2, and either a ROS scavenger or brefeldin-A, an inhibitor of protein trafficking, prevented these effects. These findings suggest that CIH leads to an augmented Ca(2+) influx via ROS-dependent facilitation of CaV3.2 protein trafficking to the plasma membrane.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Cuerpo Carotídeo/metabolismo , Hipoxia/metabolismo , Animales , Bencenoacetamidas/administración & dosificación , Canales de Calcio Tipo T/fisiología , Cuerpo Carotídeo/efectos de los fármacos , Hipoxia de la Célula , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas , Piridinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cuerpo Carotídeo/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Oxígeno/metabolismo , Médula Suprarrenal/fisiología , Animales , Presión Sanguínea , Sistema Cardiovascular , Cuerpo Carotídeo/metabolismo , Catecolaminas/metabolismo , Heterocigoto , Homeostasis , Hipoxia , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Células PC12 , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
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.
Asunto(s)
Epigénesis Genética , Corazón/fisiología , Homeostasis , Hipoxia/fisiopatología , Pulmón/fisiología , Animales , Animales Recién Nacidos , Islas de CpG , Metilación de ADN , RatasRESUMEN
Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and ß-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed ß-adrenergic inotropic responses that were associated with impaired ß-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.
Asunto(s)
Fenómenos Fisiológicos Cardiovasculares , Glutatión Reductasa/metabolismo , Alcohol Deshidrogenasa , Animales , Calcio/metabolismo , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Diástole/efectos de los fármacos , Femenino , Glutatión Reductasa/deficiencia , Hemodinámica/efectos de los fármacos , Isoproterenol/farmacología , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/efectos de los fármacos , Miocardio/citología , Miocardio/enzimología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Óxido Nítrico Sintasa/metabolismo , Nitrosación , Transporte de Proteínas/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
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.
Asunto(s)
Médula Suprarrenal/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cuerpo Carotídeo/fisiología , Hipertensión/fisiopatología , Hipoxia/fisiopatología , Estrés Oxidativo/fisiología , Mecánica Respiratoria/fisiología , Análisis de Varianza , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Presión Sanguínea , Western Blotting , Complejo I de Transporte de Electrón/metabolismo , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Noqueados , Norepinefrina/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Pletismografía Total , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0215905.].
RESUMEN
NEW FINDINGS: What is the central question of this study? Periodic decreases in arterial blood O2 or chronic intermittent hypoxia (CIH) is a hallmark feature of sleep apnoea patients. Despite a large body of clinical evidence linking sleep disordered breathing with apnoeas to diabetes, the causal relationships between CIH and ß-cell function and the underlying molecular mechanisms have not been established. What is the main finding and its importance? In a rodent model, we show that mitochondrial oxidative stress generated by CIH leads to pancreatic ß-cell dysfunction manifested by augmented basal insulin secretion, insulin resistance, defective proinsulin processing and impaired glucose-stimulated insulin secretion. The results of the present study provide evidence for direct effects of CIH on ß-cell function, which may be an underlying molecular mechanism contributing to the development of type 2 diabetes among sleep apnoea patients. Breathing disorders with recurrent apnoea produce periodic decreases in arterial blood O2, i.e. chronic intermittent hypoxia (CIH). Recurrent apnoea patients and CIH-exposed rodents exhibit several co-morbidities, including diabetes. However, the effects of CIH on pancreatic ß-cell function are not known. In the present study, we investigated pancreatic ß-cell function in C57BL6 mice exposed to 30 days of CIH. Compared with control animals, the CIH-exposed mice exhibited elevated levels of fasting plasma insulin but comparable glucose levels and higher homeostasis model assessment, indicating insulin resistance. Pancreatic ß-cell morphology was unaltered in CIH-exposed mice. Insulin content was decreased in CIH-exposed ß-cells, and this effect was associated with increased proinsulin levels. The mRNA and protein levels of the enzyme prohormone convertase 1, which converts proinsulin to insulin, were downregulated in CIH-treated islets. More importantly, glucose-stimulated insulin secretion was impaired in CIH-exposed mice and in isolated islets. Mitochondrial levels of reactive oxygen species (ROS) were elevated in CIH-exposed pancreatic islets. Treatment of mice with mito-tempol, a scavenger of mitochondrial ROS, during exposure to CIH prevented the augmented insulin secretion and restored the proinsulin and homeostasis model assessment values to control levels. These results demonstrate that CIH leads to pancreatic ß-cell dysfunction, manifested by augmented basal insulin secretion, insulin resistance, defective proinsulin processing, impaired glucose-stimulated insulin secretion and increased mitochondrial ROS, which mediate the effects of CIH on pancreatic ß-cell function.
Asunto(s)
Hipoxia/fisiopatología , Células Secretoras de Insulina/metabolismo , Animales , Glucemia/metabolismo , Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/etiología , Glucagón/sangre , Homeostasis , Humanos , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Secreción de Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Proinsulina/sangre , Proinsulina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Síndromes de la Apnea del Sueño/complicacionesRESUMEN
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.
Asunto(s)
Apnea/enzimología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Calpaína/metabolismo , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Animales , Apnea/mortalidad , Sistema Nervioso Autónomo/efectos de los fármacos , Sistema Nervioso Autónomo/patología , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Calpaína/antagonistas & inhibidores , Hipoxia de la Célula/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/antagonistas & inhibidoresRESUMEN
AIM: Naked mole rats (NMRs) exhibit blunted hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). The mechanism(s) underlying these responses are largely unknown. We hypothesized that attenuated carotid body (CB) sensitivity to hypoxia and hypercapnia contributes to the near absence of ventilatory responses to hypoxia and CO2 in NMRs. METHODS: We measured ex vivo CB sensory nerve activity, phrenic nerve activity (an estimation of ventilation), and blood gases in urethane-anesthetized NMRs and C57BL/6 mice breathing normoxic, hypoxic, or hypercapnic gases. CB morphology, carbon monoxide, and H2 S levels were also determined. RESULTS: Relative to mice, NMRs had blunted CB and HVR. Morphologically, NMRs have larger CBs, which contained more glomus cells than in mice. Furthermore, NMR glomus cells form a dispersed pattern compared to a clustered pattern in mice. Hemeoxygenase (HO)-1 mRNA was elevated in NMR CBs, and an HO inhibitor increased CB sensitivity to hypoxia in NMRs. This increase was blocked by an H2 S synthesis inhibitor, suggesting that interrupted gas messenger signaling contributes to the blunted CB responses and HVR in NMRs. Regarding hypercapnia, CB and ventilatory responses to CO2 in NMRs were larger than in mice. Carbonic anhydrase (CA)-2 mRNA is elevated in NMR CBs, and a CA inhibitor blocked the augmented CB response to CO2 in NMRs, indicating CA activity regulates augmented CB response to CO2 . CONCLUSIONS: Consistent with our hypothesis, impaired CB responses to hypoxia contribute in part to the blunted HVR in NMRs. Conversely, the HCVR and CB are more sensitive to CO2 in NMRs.
Asunto(s)
Anhidrasas Carbónicas , Cuerpo Carotídeo , Animales , Dióxido de Carbono , Monóxido de Carbono , Hipercapnia , Hipoxia , Ratones , Ratones Endogámicos C57BL , Ratas Topo , Oxígeno , ARN Mensajero , Respiración , UretanoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Síndromes de la Apnea del Sueño/metabolismo , Animales , Cuerpo Carotídeo/efectos de los fármacos , Cuerpo Carotídeo/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/enzimología , Deferoxamina/farmacología , Digoxina/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Furanos/farmacología , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Indazoles/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , NADPH Oxidasa 2 , Células PC12 , Sistema Nervioso Periférico/efectos de los fármacos , Sistema Nervioso Periférico/enzimología , Ratas , Sideróforos/farmacología , Síndromes de la Apnea del Sueño/genéticaRESUMEN
Reactive oxygen species (ROS) modulate neuronal excitability. In the present study we examined the effects of hydrogen peroxide (H(2)O(2)), a well established ROS, on neuronal activity from two neonatal mouse brain regions, i.e., the pre-Bötzinger complex (preBötC) within the ventral respiratory column (VRC) and the CA1 area of the hippocampus. In the preBötC, 2.2 mM H(2)O(2) evoked a transient depression followed by augmentation of neuronal activity. The iron chelator deferoxamine (500 µM) did not prevent H(2)O(2)-mediated neuronal augmentation but prevented the initial depression. Combined application of Fe(2+) and H(2)O(2) only caused depression of the preBötC rhythm. In contrast, H(2)O(2) suppressed neuronal activity in the CA1 region, and this effect was accentuated by coapplication of Fe(2+) and H(2)O(2), suggesting that hydroxyl radical generated by Fenton reaction mediates the effects of H(2)O(2) on CA1 neuronal activity. Malondialdehyde (MDA) levels were monitored as an index of lipid peroxidation in H(2)O(2)-treated preBötC and CA1 areas. MDA levels were unaltered in H(2)O(2)-treated preBötC, whereas MDA levels were markedly elevated in the CA1 region. These findings suggest that 1) exogenous administration of H(2)O(2) exerts differential effects on neuronal activities of preBötC versus CA1 neuronal populations and 2) H(2)O(2) is a potent modulator of respiratory rhythmogenesis from the preBötC without affecting global oxidative status.
Asunto(s)
Hipocampo/citología , Peróxido de Hidrógeno/farmacología , Neuronas/efectos de los fármacos , Oxidantes/farmacología , Centro Respiratorio/citología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Animales , Animales Recién Nacidos , Biofisica , Deferoxamina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Compuestos Ferrosos/farmacología , Antagonistas del GABA/farmacología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/metabolismo , Ratones , Técnicas de Placa-Clamp , Picrotoxina/farmacología , Piperazinas/farmacología , Centro Respiratorio/efectos de los fármacos , Sideróforos/farmacologíaRESUMEN
ROS, including (.)OH, is now recognized as the hallmark of inflammation and damage to the anti-proteinase barrier has been implicated in a number of pathophysiological conditions. We have previously shown that O2( -) and H2O2/HOCl are physiologically relevant inactivators of alpha 2M, a key anti-proteinase. Here, we show that (.)OH disrupted caprine alpha2M structure and antiproteolytic potential in vitro, suggesting that its function could be compromised via oxidative modification.
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Radical Hidroxilo/química , alfa-Macroglobulinas/química , Animales , Antioxidantes/farmacología , Cabras , Oxidación-Reducción , Inhibidores de Proteasas/química , Tripsina/metabolismo , alfa-Macroglobulinas/metabolismoRESUMEN
The alpha subunit of the voltage gated human ether-a-go-go-related (hERG) potassium channel regulates cell excitability in a broad range of cell lines. HERG channels are also expressed in a variety of cancer cells and control cell proliferation and apoptosis. Hypoxia, a common feature of tumors, alters gating properties of hERG currents in SH-SY5Y neuroblastoma cells. In the present study, we examined the molecular mechanisms and physiological significance underlying hypoxia-altered hERG currents in SH-SY5Y neuroblastoma cells. Hypoxia reduced the surface expression of 150kDa form and increased 125kDa form of hERG protein expression in the endoplasmic reticulum (ER). The changes in protein expression were associated with ~50% decrease in hERG potassium conductance. ER retention of hERG 125kDa form by CH was due to defective trafficking and was rescued by exposing cells to hypoxia at low temperatures or treatment with E-4031, a hERG channel blocker. Prolonged association of hERG with molecular chaperone Hsp90 resulting in complex oligomeric insoluble aggregates contributed to ER accumulation and trafficking defect. Hypoxia increased reactive oxygen species (ROS) levels and manganese (111) tetrakis (1methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant prevented hypoxia-induced degradation of 150kDa and accumulation of 125kDa forms. Impaired trafficking of hERG by hypoxia was associated with reduced cell proliferation and this effect was prevented by antioxidant treatment. These results demonstrate that hypoxia through increased oxidative stress impairs hERG trafficking, leading to decreased K+ currents resulting in cell cycle arrest in SH-SY5Y cells.
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Puntos de Control del Ciclo Celular , Canales de Potasio Éter-A-Go-Go/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Proliferación Celular , Retículo Endoplásmico/metabolismo , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Transporte de Proteínas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The mechanisms by which nitric oxide (NO) influences myocardial Ca2+ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca2+ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dtmax normalized to end-diastolic volume; dP/dt-EDV) by 51+/-5% (P<0.001) and lusitropy (tau; tau) by 20.3+/-2.0% (P<0.05). These responses were markedly attenuated in mice lacking NOS1 (NOS1-/-) (15+/-2% increase in dP/dt-EDV; P<0.001 versus WT; and no change in tau; P<0.01 versus WT). Isolated myocytes from NOS1-/- (approximately 2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca2+ transients ([Ca2+]i) compared with WT. SR Ca2+ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca2+]i at 4 Hz increased 107+/-23% above 1 Hz response) but not in NOS1-/- (13+/-26%; P<0.01 versus WT). In contrast, mice lacking NOS3 (NOS3-/-) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca2+]i, and SR Ca2+ stores. NOS1-/- had increased SR Ca2+ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca2+ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.
Asunto(s)
Calcio/metabolismo , Contracción Miocárdica/fisiología , Óxido Nítrico/fisiología , Animales , Presión Sanguínea/fisiología , Western Blotting , Tamaño de la Célula/fisiología , Estimulación Eléctrica , Femenino , Genotipo , Hemodinámica/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Sarcómeros/fisiología , Retículo Sarcoplasmático/metabolismo , Función Ventricular Izquierda/fisiologíaRESUMEN
Chronic intermittent hypoxia (CIH) is a common state experienced in several breathing disorders, including obstructive sleep apnea (OSA) and apneas of prematurity. Unraveling how CIH affects the CNS, and in turn how the CNS contributes to apneas is perhaps the most challenging task. The preBötzinger complex (preBötC) is a pre-motor respiratory network critical for inspiratory rhythm generation. Here, we test the hypothesis that CIH increases irregular output from the isolated preBötC, which can be mitigated by antioxidant treatment. Electrophysiological recordings from brainstem slices revealed that CIH enhanced burst-to-burst irregularity in period and/or amplitude. Irregularities represented a change in individual fidelity among preBötC neurons, and changed transmission from preBötC to the hypoglossal motor nucleus (XIIn), which resulted in increased transmission failure to XIIn. CIH increased the degree of lipid peroxidation in the preBötC and treatment with the antioxidant, 5,10,15,20-Tetrakis (1-methylpyridinium-4-yl)-21H,23H-porphyrin manganese(III) pentachloride (MnTMPyP), reduced CIH-mediated irregularities on the network rhythm and improved transmission of preBötC to the XIIn. These findings suggest that CIH promotes a pro-oxidant state that destabilizes rhythmogenesis originating from the preBötC and changes the local rhythm generating circuit which in turn, can lead to intermittent transmission failure to the XIIn. We propose that these CIH-mediated effects represent a part of the central mechanism that may perpetuate apneas and respiratory instability, which are hallmark traits in several dysautonomic conditions.