Chronic Metformin Administration Does Not Alter Carotid Sinus Nerve Activity in Control Rats.

Metformin is a glucose-lowering, insulin-sensitizing drug that is commonly used in the treatment of type 2 diabetes (T2D). In the last decade, the carotid body (CB) has been described as a metabolic sensor implicated in the regulation of glucose homeostasis, being CB dysfunction crucial for the development of metabolic diseases, such as T2D. Knowing that metformin could activate AMP-activated protein kinase (AMPK) and that AMPK has been described to have an important role in CB hypoxic chemotransduction, herein we have investigated the effect of chronic metformin administration on carotid sinus nerve (CSN) chemosensory activity in basal and hypoxic and hypercapnic conditions in control animals. Experiments were performed in male Wistar rats subjected to 3 weeks of metformin (200 mg/kg) administration in the drinking water. The effect of chronic metformin administration was tested in spontaneous and hypoxic (0% and 5% O2) and hypercapnic (10% CO2) evoked CSN chemosensory activity. Metformin administration for 3 weeks did not modify the basal CSN chemosensory activity in control animals. Moreover, the CSN chemosensory response to intense and moderate hypoxia and hypercapnia was not altered by the chronic metformin administration. In conclusion, chronic metformin administration did not modify chemosensory activity in control animals.

Intermittent Hypoxia and Diet-Induced Obesity on the Intestinal Wall Morphology in a Murine Model of Sleep Apnea.

This work analyzes the impact of two conditions, intermittent hypoxia exposure and high-fat diet in rats as models of sleep apnea. We studied the autonomic activity and histological structure of the rat jejunum and whether the overlapping of both conditions, as often observed in patients, induces more deleterious effects on the intestinal barrier. We found alterations in jejunum wall histology, predominantly in HF rats, based on increased crypt depth and submucosal thickness, as well as decreased muscularis propria thickness. These alterations were maintained with the IH and HF overlap. An increase in the number and size of goblet cells in the villi and crypts and the infiltration of eosinophils and lymphocytes in the lamina propria suggest an inflammatory status, confirmed by the increase in plasma CRP levels in all experimental groups. Regarding the CAs analysis, IH, alone or combined with HF, causes a preferential accumulation of NE in the catecholaminergic nerve fibers of the jejunum. In contrast, serotonin increases in all three experimental conditions, with the highest level in the HF group. It remains to be elucidated whether the alterations found in the present work could affect the permeability of the intestinal barrier, promoting sleep apnea-induced morbidities.

Effect of Carotid Body Denervation on Systemic Endothelial Function in a Diabetic Animal Model.

Endothelial dysfunction is an essential intermediary for development of cardiovascular diseases associated with diabetes and hypertension (HT). The carotid body (CB) dysfunction contributes to dysmetabolic states, and the resection of carotid sinus nerve (CSN) prevents and reverts dysmetabolism and HT. Herein, we investigated if CSN denervation ameliorates systemic endothelial dysfunction in an animal model of type 2 diabetes mellitus (T2DM).We used Wistar male rats submitted to HFHSu diet during 25 weeks and the correspondent age-matched controls fed with a standard diet. CSN resection was performed in half of the groups after 14 weeks of diet. In vivo insulin sensitivity, glucose tolerance and blood pressure, ex vivo aortic artery contraction and relaxation and nitric oxide (NO) levels in plasma and aorta, aorta nitric oxide synthase (NOS) isoforms, and PGF2αR levels were evaluated.We demonstrated that, alongside to dysmetabolism and HT reversion, CSN resection restores endothelial function in the aorta and decreases the NO levels in plasma and aorta at the same time that restores normal levels of iNOS in aorta without changing eNOS or PGF2αR levels.These results suggest that the modulation of CB activity can be important for the treatment of HT and endothelial dysfunction related with T2DM.

Effects of Gestational Intermittent Hypoxia on Placental Morphology and Fetal Development in a Murine Model of Sleep Apnea.

Obstructive sleep apnea (OSA) during pregnancy is characterized by episodes of intermittent hypoxia (IH) during sleep, resulting in adverse health outcomes for mother and offspring. Despite a prevalence of 8-20% in pregnant women, this disorder is often underdiagnosed.We have developed a murine model of gestational OSA to study IH effects on pregnant mothers, placentas, fetuses, and offspring. One group of pregnant rats was exposed to IH during the last 2 weeks of gestation (GIH). One day before the delivery date, a cesarean section was performed. Other group of pregnant rats was allowed to give birth at term to study offspring's evolution.Preliminary results showed no significant weight differences in mothers and fetuses. However, the weight of GIH male offspring was significantly lower than the controls at 14 days (p < 0.01). The morphological study of the placentas showed an increase in fetal capillary branching, expansion of maternal blood spaces, and number of cells of the external trophectoderm in the tissues from GIH-exposed mothers. Additionally, the placentas from the experimental males were enlarged (p < 0.05). Further studies are needed to follow the long-term evolution of these changes to relate the histological findings of the placentas with functional development of the offspring in adulthood.

Analysis of Bone Histomorphometry in Rat and Guinea Pig Animal Models Subject to Hypoxia.

Hypoxia may be associated with alterations in bone remodeling, but the published results are contradictory. The aim of this study was to characterize the bone morphometry changes subject to hypoxia for a better understanding of the bone response to hypoxia and its possible clinical consequences on the bone metabolism. This study analyzed the bone morphometry parameters by micro-computed tomography (µCT) in rat and guinea pig normobaric hypoxia models. Adult male and female Wistar rats were exposed to chronic hypoxia for 7 and 15 days. Additionally, adult male guinea pigs were exposed to chronic hypoxia for 15 days. The results showed that rats exposed to chronic constant and intermittent hypoxic conditions had a worse trabecular and cortical bone health than control rats (under a normoxic condition). Rats under chronic constant hypoxia were associated with a more deteriorated cortical tibia thickness, trabecular femur and tibia bone volume over the total volume (BV/TV), tibia trabecular number (Tb.N), and trabecular femur and tibia bone mineral density (BMD). In the case of chronic intermittent hypoxia, rats subjected to intermittent hypoxia had a lower cortical femur tissue mineral density (TMD), lower trabecular tibia BV/TV, and lower trabecular thickness (Tb.Th) of the tibia and lower tibia Tb.N. The results also showed that obese rats under a hypoxic condition had worse values for the femur and tibia BV/TV, tibia trabecular separation (Tb.Sp), femur and tibia Tb.N, and BMD for the femur and tibia than normoweight rats under a hypoxic condition. In conclusion, hypoxia and obesity may modify bone remodeling, and thus bone microarchitecture, and they might lead to reductions in the bone strength and therefore increase the risk of fragility fracture.

Contribution of adenosine and ATP to the carotid body chemosensory activity in ageing.

KEY POINTS: Adenosine and ATP are excitatory neurotransmitters involved in the carotid body (CB) response to hypoxia. During ageing the CB exhibits a decline in its functionality, demonstrated by decreased hypoxic responses. In aged rats (20-24 months old) there is a decrease in: basal and hypoxic release of adenosine and ATP from the CB; expression of adenosine and ATP receptors in the petrosal ganglion; carotid sinus nerve (CSN) activity in response to hypoxia; and ventilatory responses to ischaemic hypoxia. There is also an increase in SNAP25, ENT1 and CD73 expression. It is concluded that, although CSN activity and ventilatory responses to hypoxia decrease with age, adjustments in purinergic metabolism in the CB in aged animals are present aiming to maintain the contribution of adenosine and ATP. The possible significance of the findings in the context of ageing and in CB-associated pathologies is considered. ABSTRACT: During ageing the carotid body (CB) exhibits a decline in its functionality. Here we investigated the effect of ageing on functional CB characteristics as well as the contribution of adenosine and ATP to CB chemosensory activity. Experiments were performed in 3-month-old and 20- to 24-month-old male Wistar rats. Ageing decreased: the number of tyrosine hydroxylase immune-positive cells, but not type II cells or nestin-positive cells in the CB; the expression of P2X2 and A2A receptors in the petrosal ganglion; and the basal and hypoxic release of adenosine and ATP from the CB. Ageing increased ecto-nucleotidase (CD73) immune-positive cells and the expression of synaptosome associated protein 25 (SNAP25) and equilibrative nucleoside transporter 1 (ENT1) in the CB. Additionally, ageing did not modify basal carotid sinus nerve (CSN) activity or the activity in response to hypercapnia, but decreased CSN activity in hypoxia. The contribution of adenosine and ATP to stimuli-evoked CSN chemosensory activity in aged animals followed the same pattern of 3-month-old animals. Bilateral common carotid occlusions during 5, 10 and 15 s increased ventilation proportionally to the duration of ischaemia, an effect decreased by ageing. ATP contributed around 50% to ischaemic-ventilatory responses in young and aged rats; the contribution of adenosine was dependent on the intensity of ischaemia, being maximal in ischaemias of 5 s (50%) and much smaller in 15 s ischaemias. Our results demonstrate that both ATP and adenosine contribute to CB chemosensory activity in ageing. Though CB responses to hypoxia, but not to hypercapnia, decrease with age, the relative contribution of both ATP and adenosine for CB activity is maintained.

Bioelectronic modulation of carotid sinus nerve activity in the rat: a potential therapeutic approach for type 2 diabetes.

AIMS/HYPOTHESIS: A new class of treatments termed bioelectronic medicines are now emerging that aim to target individual nerve fibres or specific brain circuits in pathological conditions to repair lost function and reinstate a healthy balance. Carotid sinus nerve (CSN) denervation has been shown to improve glucose homeostasis in insulin-resistant and glucose-intolerant rats; however, these positive effects from surgery appear to diminish over time and are heavily caveated by the severe adverse effects associated with permanent loss of chemosensory function. Herein we characterise the ability of a novel bioelectronic application, classified as kilohertz frequency alternating current (KHFAC) modulation, to suppress neural signals within the CSN of rodents. METHODS: Rats were fed either a chow or high-fat/high-sucrose (HFHSu) diet (60% lipid-rich diet plus 35% sucrose drinking water) over 14 weeks. Neural interfaces were bilaterally implanted in the CSNs and attached to an external pulse generator. The rats were then randomised to KHFAC or sham modulation groups. KHFAC modulation variables were defined acutely by respiratory and cardiac responses to hypoxia (10% O2 + 90% N2). Insulin sensitivity was evaluated periodically through an ITT and glucose tolerance by an OGTT. RESULTS: KHFAC modulation of the CSN, applied over 9 weeks, restored insulin sensitivity (constant of the insulin tolerance test [KITT] HFHSu sham, 2.56 ± 0.41% glucose/min; KITT HFHSu KHFAC, 5.01 ± 0.52% glucose/min) and glucose tolerance (AUC HFHSu sham, 1278 ± 20.36 mmol/l × min; AUC HFHSu KHFAC, 1054.15 ± 62.64 mmol/l × min) in rat models of type 2 diabetes. Upon cessation of KHFAC, insulin resistance and glucose intolerance returned to normal values within 5 weeks. CONCLUSIONS/INTERPRETATION: KHFAC modulation of the CSN improves metabolic control in rat models of type 2 diabetes. These positive outcomes have significant translational potential as a novel therapeutic modality for the purpose of treating metabolic diseases in humans.

Role of reactive oxygen species and sulfide-quinone oxoreductase in hydrogen sulfide-induced contraction of rat pulmonary arteries.

Application of H2S ("sulfide") elicits a complex contraction in rat pulmonary arteries (PAs) comprising a small transient contraction (phase 1; Ph1) followed by relaxation and then a second, larger, and more sustained contraction (phase 2; Ph2). We investigated the mechanisms causing this response using isometric myography in rat second-order PAs, with Na2S as a sulfide donor. Both phases of contraction to 1,000 µM Na2S were attenuated by the pan-PKC inhibitor Gö6983 (3 µM) and by 50 µM ryanodine; the Ca2+ channel blocker nifedipine (1 µM) was without effect. Ph2 was attenuated by the mitochondrial complex III blocker myxothiazol (1 µM), the NADPH oxidase (NOX) blocker VAS2870 (10 µM), and the antioxidant TEMPOL (3 mM) but was unaffected by the complex I blocker rotenone (1 µM). The bath sulfide concentration, measured using an amperometric sensor, decreased rapidly following Na2S application, and the peak of Ph2 occurred when this had fallen to ~50 µM. Sulfide caused a transient increase in NAD(P)H autofluorescence, the offset of which coincided with development of the Ph2 contraction. Sulfide also caused a brief mitochondrial hyperpolarization (assessed using tetramethylrhodamine ethyl ester), followed immediately by depolarization and then a second more prolonged hyperpolarization, the onset of which was temporally correlated with the Ph2 contraction. Sulfide application to cultured PA smooth muscle cells increased reactive oxygen species (ROS) production (recorded using L012); this was absent when the mitochondrial flavoprotein sulfide-quinone oxoreductase (SQR) was knocked down using small interfering RNA. We propose that the Ph2 contraction is largely caused by SQR-mediated sulfide metabolism, which, by donating electrons to ubiquinone, increases electron production by complex III and thereby ROS production.

Mitochondrial Complex I Dysfunction and Peripheral Chemoreflex Sensitivity in a FASTK-Deficient Mice Model.

The molecular mechanisms underlying O2-sensing by carotid body (CB) chemoreceptors remain undetermined. Mitochondria have been implicated, due to the sensitivity of CB response to electron transport chain (ETC) blockers. ETC is one of the major sources of reactive oxygen species, proposed as mediators in oxygen sensing. Fas-activated serine/threonine phosphoprotein is a sensor of mitochondrial stress that modulates protein translation to promote survival of cells exposed to adverse conditions. A translational variant of Fas-activated serine/threonine kinase (FASTK) is required for the biogenesis of ND6 mRNA, the mitochondrial encoded subunit 6 of the NADH dehydrogenase complex (Complex I). Ablating FASTK expression reduced Complex I activity in vivo by about 50%. We have tested the hypothesis of Complex I participation in O2-sensing structures by studying the effect of hypoxia in FASTK-/- knockout mice. Ventilatory response to acute hypoxia and hypercapnia tests showed similar sensitivity and CB catecholaminergic activity in knockout and wild type mice; hypoxic pulmonary vasoconstriction response also was similar. Pulmonary artery contractility in vitro, using small vessel myography, showed a significantly decreased relaxation to rotenone in knockout mice pre-constricted vessels with PGF2α. In conclusion, FASTK-/- knockout mice maintain respiratory chemoreflex under hypoxia and hypercapnia stress suggesting that completely functional Complex I ND6 protein is not required for these responses.

Hydrogen Sulfide as an O2 Sensor: A Critical Analysis.

There is increasing interest in the physiological actions and therapeutic potential of the gasotransmitter hydrogen sulfide (H2S). In addition to exerting antihypertensive, anti-inflammatory, antioxidant, and pro-angiogenic effects, H2S has been suggested to play a central and ubiquitous role in O2 sensing. According to this concept, because H2S is metabolized by oxidation, its cellular concentration varies inversely with the ambient pO2 such that hypoxia causes a rise in intracellular [H2S]; this then acts to induce appropriate cellular responses. In particular, it has been proposed that H2S underpins O2 sensing in the carotid body, which triggers increases in ventilation in response to hypoxemia, and also in pulmonary arteries, which constrict in response to local alveolar hypoxia. This process, termed hypoxic pulmonary vasoconstriction (HPV), acts to divert blood to better-oxygenated regions of the lung, thereby maintaining the ventilation-perfusion ratio and minimizing hypoxia-induced falls in blood O2 saturation. In this chapter, we present a critical review of the evidence supporting and questioning this model in both HPV and the carotid body.

Hypoxic pulmonary vasoconstriction in isolated rat pulmonary arteries is not inhibited by antagonists of H2 S-synthesizing pathways.

An increase in the H2 S (hydrogen sulphide, hereafter sulphide) concentration in pulmonary artery smooth muscle cells (PASMCs) has been proposed to mediate hypoxic pulmonary vasoconstriction (HPV). We evaluated this hypothesis in isolated rat intrapulmonary arteries (IPAs) by examining the effects of the sulphide precursor cysteine and sulphide-synthesis blockers on HPV and also on normoxic pulmonary vasoconstriction (NPV) stimulated by prostaglandin F2α (PGF2α ) and by the drug LY83583, which causes contraction in IPAs by increasing cellular reactive oxygen species levels. Experiments with several blockers of cystathionine γ-lyase (CSE), the enzyme responsible for sulphide synthesis in the vasculature, demonstrated that propargylglycine (PAG, 1 mm) had little or no effect on the NPV caused by PGF2α or LY83583. Conversely, other CSE antagonists tested, aminooxyacetic acid (AOAA, 100 µm), ß-cyanoalanine (BCA, 500 µm) and hydroxylamine (HA, 100 µm), altered the NPV to PGF2α (BCA increased, HA inhibited) and/or LY83583 (BCA increased, AOAA and HA inhibited). Preincubating IPAs in physiological saline solution (PSS) containing 1 mm cysteine increased the amplitude of the NPV to PGF2(α) by ∼50%, and had a similar effect on HPV elicited by hypoxic challenge with 0% O2 . The enhancement of both responses by cysteine was abolished by pretreatment with 1 mm PAG. Measurements carried out with an amperometric electrode demonstrated that incubation with 1 mm cysteine under anoxic conditions (to minimize sulphide oxidation) greatly potentiated the release of sulphide from pieces of rat liver and that this release was strongly antagonized by PAG, indicating that at this concentration PAG could enter cells intact and antagonize CSE. PAG at 1 mm had no effect on HPV recorded in control PSS, or in PSS supplemented with physiological concentrations of cysteine (10 µm), cystine (50 µm) and glutamate (100 µm) in order to prevent the possible depletion of intracellular cysteine during experiments. Application of a combination of 1 mm cysteine and 1 mm α-ketoglutarate to promote sulphide synthesis via the cysteine aminotransferase/mercaptopyruvate sulphurtransferase (CAT/MST) pathway caused an increase in HPV similar to that observed for cysteine. This was partially blocked by the CAT antagonist aspartate (1 mm) and also by PAG. However, HPV was not increased by 1 mm α-ketoglutarate alone, and HPV in the absence of α-ketoglutarate and cysteine was not attenuated by aspartate. Pretreatment of IPAs with dithiothreitol (DTT, 1 mm), proposed to promote the conversion of mitochondrial thiosulphate to sulphide, did not increase the release of sulphide from pieces of rat liver in either the presence or the absence of 1 mm cysteine, and virtually abolished HPV. The results provide evidence that the sulphide precursor cysteine can promote both NPV and HPV in rat IPA by generating sulphide via a PAG-sensitive pathway, presumably CSE. However, HPV evoked under control conditions was unaffected by the blockade of CSE. Moreover, HPV was not affected by the CAT antagonist aspartate and was blocked rather than enhanced by DTT. The data therefore indicate that sulphide generated by CSE or CAT/MST or from thiosulphate is unlikely to contribute to O2 sensing during HPV in these arteries.

Hypoxic pulmonary vasoconstriction, carotid body function and erythropoietin production in adult rats perinatally exposed to hyperoxia.

KEY POINTS: Adult animals that have been perinatally exposed to oxygen-rich atmospheres (hyperoxia), recalling those used for oxygen therapy in infants, exhibit a loss of hypoxic pulmonary vasoconstriction, whereas vasoconstriction elicited by depolarizing agents is maintained. Loss of pulmonary hypoxic vasoconstriction is not linked to alterations in oxygen-sensitive K(+) currents in pulmonary artery smooth muscle cells. Loss of hypoxic vasoconstriction is associated with early postnatal oxidative damage and corrected by an antioxidant diet. Perinatal hyperoxia damages carotid body chemoreceptor cell function and the antioxidant diet does not reverse it. The hypoxia-elicited increase in erythropoietin plasma levels is not affected by perinatal hyperoxia. The potential clinical significance of the findings in clinical situations such as pneumonia, chronic obstructive pulmonary disease or general anaesthesia is considered. ABSTRACT: Adult mammalians possess three cell systems that are activated by acute bodily hypoxia: pulmonary artery smooth muscle cells (PASMC), carotid body chemoreceptor cells (CBCC) and erythropoietin (EPO)-producing cells. In rats, chronic perinatal hyperoxia causes permanent carotid body (CB) atrophy and functional alterations of surviving CBCC. There are no studies on PASMC or EPO-producing cells. Our aim is to define possible long-lasting functional changes in PASMC or EPO-producing cells (measured as EPO plasma levels) and, further, to analyse CBCC functional alterations. We used 3- to 4-month-old rats born and reared in a normal atmosphere or exposed to perinatal hyperoxia (55-60% O2 for the last 5-6 days of pregnancy and 4 weeks after birth). Perinatal hyperoxia causes an almost complete loss of hypoxic pulmonary vasoconstriction (HPV), which was correlated with lung oxidative status in early postnatal life and prevented by antioxidant supplementation in the diet. O2 -sensitivity of K(+) currents in the PASMC of hyperoxic animals is normal, indicating that their inhibition is not sufficient to trigger HPV. Perinatal hyperoxia also abrogated responses elicited by hypoxia on catecholamine and cAMP metabolism in the CB. An increase in EPO plasma levels elicited by hypoxia was identical in hyperoxic and control animals, implying a normal functioning of EPO-producing cells. The loss of HPV observed in adult rats and caused by perinatal hyperoxia, comparable to oxygen therapy in premature infants, might represent a previously unrecognized complication of such a medical intervention capable of aggravating medical conditions such as regional pneumonias, atelectases or general anaesthesia in adult life.

Potentiation of Hypoxic Pulmonary Vasoconstriction by Hydrogen Sulfide Precursors 3-Mercaptopyruvate and D-Cysteine Is Blocked by the Cystathionine γ Lyase Inhibitor Propargylglycine.

Although the gasotransmitter hydrogen sulfide (H(2)S) generally dilates systemic arteries in mammals, it causes constriction of pulmonary arteries. In isolated rat pulmonary arteries, we have shown that the H(2)S precursor cysteine enhances both hypoxic pulmonary vasoconstriction and tension development caused by the agonist prostaglandin F(2α) under normoxic conditions. These effects were blocked by propargylglycine (PAG), a blocker of the enzyme cystathionine γ lyase which metabolises cysteine to sulfide. In the present study, we evaluated whether 3-mercaptopyruvate (3-MP), a sulfide precursor which is thought to give rise to sulfide when it is metabolised by the enzyme mercaptopyruvate sulfurtransferase, also enhanced contraction. Application of 3-MP prior to hypoxic challenge caused a marked enhancement of HPV which was completely blocked by both L- and D,L-PAG (both 1 mM). Cumulative application of 3-1,000 µM 3-MP during an ongoing contraction to PGF(2α) under normoxic conditions also caused a marked increase in tension. Application of D-cysteine (1 mM) also enhanced HPV, and this effect was prevented by both the D-amino acid oxidase inhibitor sodium benzoate (500 µM) and 1 mM L-PAG.

Hypoxic pulmonary vasoconstriction in the absence of pretone: essential role for intracellular Ca2+ release.

Hypoxic pulmonary vasoconstriction (HPV) maintains blood oxygenation during acute hypoxia but contributes to pulmonary hypertension during chronic hypoxia. The mechanisms of HPV remain controversial, in part because HPV is usually studied in the presence of agonist-induced preconstriction ('pretone'). This potentiates HPV but may obscure and distort its underlying mechanisms. We therefore carried out an extensive assessment of proposed mechanisms contributing to HPV in isolated intrapulmonary arteries (IPAs) in the absence of pretone by using a conventional small vessel myograph. Hypoxia elicited a biphasic constriction consisting of a small transient (phase 1) superimposed upon a sustained (phase 2) component. Neither phase was affected by the L-type Ca2+ channel antagonists diltiazem (10 and 30 µm) or nifedipine (3 µm). Application of the store-operated Ca2+ entry (SOCE) blockers BTP2 (10 µm) or SKF96365 (50 µm) attenuated phase 2 but not phase 1, whereas a lengthy (30 min) incubation in Ca2+-free physiological saline solution similarly reduced phase 2 but abolished phase 1. No further effect of inhibition of HPV was observed if the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (30 µm) was also applied during the 30 min incubation in Ca2+-free physiological saline solution. Pretreatment with 10 µm ryanodine and 15 mm caffeine abolished both phases, whereas treatment with 100 µm ryanodine attenuated both phases. The two-pore channel blocker NED-19 (1 µm) and the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist BZ194 (200 µm) had no effect on either phase of HPV. The lysosomal Ca2+-depleting agent concanamycin (1 µm) enhanced HPV if applied during hypoxia, but had no effect on HPV during a subsequent hypoxic challenge. The cyclic ADP ribose antagonist 8-bromo-cyclic ADP ribose (30 µm) had no effect on either phase of HPV. Neither the Ca2+-sensing receptor (CaSR) blocker NPS2390 (0.1 and 10 µm) nor FK506 (10 µm), a drug which displaces FKBP12.6 from ryanodine receptor 2 (RyR2), had any effect on HPV. HPV was virtually abolished by the rho kinase blocker Y-27632 (1 µm) and attenuated by the protein kinase C inhibitor Gö6983 (3 µm). Hypoxia for 45 min caused a significant increase in the ratio of oxidised to reduced glutathione (GSSG/GSH). HPV was unaffected by the NADPH oxidase inhibitor VAS2870 (10 µm), whereas phase 2 was inhibited but phase 1 was unaffected by the antioxidants ebselen (100 µm) and TEMPOL (3 mm). We conclude that both phases of HPV in this model are mainly dependent on [Ca2+]i release from the sarcoplasmic reticulum. Neither phase of HPV requires voltage-gated Ca2+ entry, but SOCE contributes to phase 2. We can detect no requirement for cyclic ADP ribose, NAADP-dependent lysosomal Ca2+ release, activation of the CaSR, or displacement of FKBP12.6 from RyR2 for either phase of HPV. Sustained HPV is associated with an oxidising shift in the GSSG/GSH redox potential and is inhibited by the antioxidants ebselen and TEMPOL, consistent with the concept that it requires an oxidising shift in the cell redox state or the generation of reactive oxygen species.

Analysis of ATG4C function in vivo.

ABBREVIATIONS: ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).

Chronic Intermittent Hypoxia-Induced Dysmetabolism Is Associated with Hepatic Oxidative Stress, Mitochondrial Dysfunction and Inflammation.

The association between obstructive sleep apnea (OSA) and metabolic disorders is well-established; however, the underlying mechanisms that elucidate this relationship remain incompletely understood. Since the liver is a major organ in the maintenance of metabolic homeostasis, we hypothesize that liver dysfunction plays a crucial role in the pathogenesis of metabolic dysfunction associated with obstructive sleep apnea (OSA). Herein, we explored the underlying mechanisms of this association within the liver. Experiments were performed in male Wistar rats fed with a control or high fat (HF) diet (60% lipid-rich) for 12 weeks. Half of the groups were exposed to chronic intermittent hypoxia (CIH) (30 hypoxic (5% O2) cycles, 8 h/day) that mimics OSA, in the last 15 days. Insulin sensitivity and glucose tolerance were assessed. Liver samples were collected for evaluation of lipid deposition, insulin signaling, glucose homeostasis, hypoxia, oxidative stress, antioxidant defenses, mitochondrial biogenesis and inflammation. Both the CIH and HF diet induced dysmetabolism, a state not aggravated in animals submitted to HF plus CIH. CIH aggravates hepatic lipid deposition in obese animals. Hypoxia-inducible factors levels were altered by these stimuli. CIH decreased the levels of oxidative phosphorylation complexes in both groups and the levels of SOD-1. The HF diet reduced mitochondrial density and hepatic antioxidant capacity. The CIH and HF diet produced alterations in cysteine-related thiols and pro-inflammatory markers. The results obtained suggest that hepatic mitochondrial dysfunction and oxidative stress, leading to inflammation, may be significant factors contributing to the development of dysmetabolism associated with OSA.

Effects of cigarette smoke and chronic hypoxia on ventilation in guinea pigs. Clinical significance.

Ventilatory effects of chronic cigarette smoke (CS) alone or associated to chronic hypoxia (CH), as frequently occurs in chronic obstructive pulmonary disease (COPD), remain unknown. We have addressed this problem using whole-body plethysmography in guinea-pigs, common models to study harmful effects of CS on the respiratory system. Breathing frequencies (Bf) in control (2-5 months old) guinea pigs is 90-100 breaths/min, their tidal volume (TV) increased with age but lagged behind body weight gain and, as consequence, their minute volume (MV)/Kg decreased with age. MV did not change by acutely breathing 10% O(2) but doubled while breathing 5% CO(2) in air. Exposure to chronic sustained hypoxia (15 days, 12% O(2), CH) did not elicit ventilatory acclimatization nor adaptation. These findings confirm the unresponsiveness of the guinea pig CB to hypoxia. Exposure to CS (3 months) increased Bf and MV but association with CH blunted CS effects. We conclude that CS and CH association accelerates CS-induced respiratory system damage leading to a hypoventilation that can worsen the ongoing COPD process.

Maladaptive Pulmonary Vascular Responses to Chronic Sustained and Chronic Intermittent Hypoxia in Rat.

Chronic sustained hypoxia (CSH), as found in individuals living at a high altitude or in patients suffering respiratory disorders, initiates physiological adaptations such as carotid body stimulation to maintain oxygen levels, but has deleterious effects such as pulmonary hypertension (PH). Obstructive sleep apnea (OSA), a respiratory disorder of increasing prevalence, is characterized by a situation of chronic intermittent hypoxia (CIH). OSA is associated with the development of systemic hypertension and cardiovascular pathologies, due to carotid body and sympathetic overactivation. There is growing evidence that CIH can also compromise the pulmonary circulation, causing pulmonary hypertension in OSA patients and animal models. The aim of this work was to compare hemodynamics, vascular contractility, and L-arginine-NO metabolism in two models of PH in rats, associated with CSH and CIH exposure. We demonstrate that whereas CSH and CIH cause several common effects such as an increased hematocrit, weight loss, and an increase in pulmonary artery pressure (PAP), compared to CIH, CSH seems to have more of an effect on the pulmonary circulation, whereas the effects of CIH are apparently more targeted on the systemic circulation. The results suggest that the endothelial dysfunction evident in pulmonary arteries with both hypoxia protocols are not due to an increase in methylated arginines in these arteries, although an increase in plasma SDMA could contribute to the apparent loss of basal NO-dependent vasodilation and, therefore, the increase in PAP that results from CIH.

Chronic Intermittent Hypoxia Induces Early-Stage Metabolic Dysfunction Independently of Adipose Tissue Deregulation.

Several studies demonstrated a link between obstructive sleep apnea (OSA) and the development of insulin resistance. However, the main event triggering insulin resistance in OSA remains to be clarified. Herein, we investigated the effect of mild and severe chronic intermittent hypoxia (CIH) on whole-body metabolic deregulation and visceral adipose tissue dysfunction. Moreover, we studied the contribution of obesity to CIH-induced dysmetabolic states. Experiments were performed in male Wistar rats submitted to a control and high-fat (HF) diet. Two CIH protocols were tested: A mild CIH paradigm (5/6 hypoxic (5% O2) cycles/h, 10.5 h/day) during 35 days and a severe CIH paradigm (30 hypoxic (5% O2) cycles, 8 h/day) during 15 days. Fasting glycemia, insulinemia, insulin sensitivity, weight, and fat mass were assessed. Adipose tissue hypoxia, inflammation, angiogenesis, oxidative stress, and metabolism were investigated. Mild and severe CIH increased insulin levels and induced whole-body insulin resistance in control animals, effects not associated with weight gain. In control animals, CIH did not modify adipocytes perimeter as well as adipose tissue hypoxia, angiogenesis, inflammation or oxidative stress. In HF animals, severe CIH attenuated the increase in adipocytes perimeter, adipose tissue hypoxia, angiogenesis, and dysmetabolism. In conclusion, adipose tissue dysfunction is not the main trigger for initial dysmetabolism in CIH. CIH in an early stage might have a protective role against the deleterious effects of HF diet on adipose tissue metabolism.

Type 2 diabetes progression differently affects endothelial function and vascular contractility in the aorta and the pulmonary artery.

Type 2 diabetes (T2D) is associated with cardiovascular and pulmonary disease. How T2D affects pulmonary endothelial function is not well characterized. We investigated the effects of T2D progression on contractility machinery and endothelial function in the pulmonary and systemic circulation and the mechanisms promoting the dysfunction, using pulmonary artery (PA) and aorta. A high-fat (HF, 3 weeks 60% lipid-rich diet) and a high-fat/high-sucrose (HFHSu, combined 60% lipid-rich diet and 35% sucrose during 25 weeks) groups were used as prediabetes and T2D rat models. We found that T2D progression differently affects endothelial function and vascular contractility in the aorta and PA, with the contractile machinery being altered in the PA and aorta in prediabetes and T2D animals; and endothelial function being affected in both models in the aorta but only affected in the PA of T2D animals, meaning that PA is more resistant than aorta to endothelial dysfunction. Additionally, PA and systemic endothelial dysfunction in diabetic rats were associated with alterations in the nitrergic system and inflammatory pathways. PA dysfunction in T2D involves endothelial wall mineralization. The understanding of the mechanisms behind PA dysfunction in T2D can lead to significant advances in both preventative and therapeutic treatments of pulmonary disease-associated diabetes.