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Oxygen is such an essential element for life that multiple mechanisms have evolved to maintain oxygen homeostasis, including those which detect decreases in arterial O2 and generate adaptive responses to hypoxia. In mammals, oxygen sensing mechanisms are found in erythropoietin-producing cells, peripheral chemoreceptor cells (carotid and aortic bodies), pulmonary artery smooth muscle cells, pulmonary neuroepithelial cells, chromaffin cells, and some specific types of neurons., Programa Estratégico IBGM, Escalera de Excelencia, Ref. CCVC8485, Consejería de Educación, Junta de Castilla y León (Spain).

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., The present study was supported by grant reference BFU2015-70616-R from MINECOFEDER (Spain) and grant number VA106G18 (JCyL, Spain). Programa Estrategico IBGM, Escalera de Excelencia, ref. CCVC8485, Consejeria de Educacion, Junta de Castilla y León (Spain).

The molecular mechanisms underlying O

Guinea pigs (GP), originally from the Andes, have absence of hypoxia-driven carotid body (CB) reflex. Neonatal mammals have an immature CB chemo reflex and respond to hypoxia with metabolic changes arising from direct effects of hypoxia on adrenal medulla (AM). Our working hypothesis is that adult GP would mimic neonatal mammals. Plasma epinephrine (E) has an AM origin, while norepinephrine (NE) is mainly originated in sympathetic endings, implying that specific GP changes in plasma E/NE ratio, and in blood glucose and lactate levels during hypoxia would be observed. Experiments were performed on young adult GP and rats. Hypoxic ventilation (10% O

Resumen del trabajo presentado al European Respiratory Society (ERS) International Congress, celebrado en Paris (Francia) del 15 al 19 de septiembre de 2018., [Rationale & Aim]: Experimental evidences indicate a correlation among chronic intermittent hypoxia (CIH), increased carotid body (CB) activity, enhanced sympatho-respiratory coupling and arterial hypertension. The recurrent hypoxia/reoxygenation episodes in CIH models produce CB sensitization increasing secretory response and chemoreceptor input to the brainstem, exaggerating the sympathetic reflex. CIH effect in the guinea pig CB, with a hypofunctional CB and lack of ventilatory responses to hypoxia, has not been studied., [Methods]: Guinea pigs were exposed to CIH (21%O2-80s / 5%O2-40s 8h/day; 30 days) and CB secretory response, pletismographic parameters, systemic arterial pressure and sympathetic activity were measured and compared to control animals., [Results]: Ventilatory data showed that only intense hypoxia induced significant increase of minute ventilation in both groups of animals. CB response to hypoxia (catecholamine (CA) secretion or Ca2+i changes) were not observed in C or CIH animals. Plasma CA, heart rate and mean arterial blood pressure were significantly increased in CIH guinea pigs., [Conclusion]: CIH induced hypoxic activation of the sympathetic system non-dependent of CB chemoreceptors, promoting cardiovascular adjustments. Guinea pigs can be a model to study the CB-dependent and nondependent effects induced by CIH., Spanish Ministry of Economy and Competitiveness, MINECO/FEDER, UE (BFU2015-70616-R) and CIBERES, ISCiii (CB06/06/0050).

Clinical and experimental evidence indicates a positive correlation between chronic intermittent hypoxia (CIH), increased carotid body (CB) chemosensitivity, enhanced sympatho-respiratory coupling and arterial hypertension and cardiovascular disease. Several groups have reported that both the afferent and efferent arms of the CB chemo-reflex are enhanced in CIH animal models through the oscillatory CB activation by recurrent hypoxia/reoxygenation episodes. Accordingly, CB ablation or denervation results in the reduction of these effects. To date, no studies have determined the effects of CIH treatment in chemo-reflex sensitization in guinea pig, a rodent with a hypofunctional CB and lacking ventilatory responses to hypoxia. We hypothesized that the lack of CB hypoxia response in guinea pig would suppress chemo-reflex sensitization and thereby would attenuate or eliminate respiratory, sympathetic and cardiovascular effects of CIH treatment. The main purpose of this study was to assess if guinea pig CB undergoes overactivation by CIH and to correlate CIH effects on CB chemoreceptors with cardiovascular and respiratory responses to hypoxia. We measured CB secretory activity, ventilatory parameters, systemic arterial pressure and sympathetic activity, basal and in response to acute hypoxia in two groups of animals: control and 30 days CIH exposed male guinea pigs. Our results indicated that CIH guinea pig CB lacks activity elicited by acute hypoxia measured as catecholamine (CA) secretory response or intracellular calcium transients. Plethysmography data showed that only severe hypoxia (7% O2) and hypercapnia (5% CO2) induced a significant increased ventilatory response in CIH animals, together with higher oxygen consumption. Therefore, CIH exposure blunted hyperventilation to hypoxia and hypercapnia normalized to oxygen consumption. Increase in plasma CA and superior cervical ganglion CA content was found, implying a CIH induced sympathetic hyperactivity. CIH promoted cardiovascular adjustments by increasing heart rate and mean arterial blood pressure without cardiac ventricle hypertrophy. In conclusion, CIH does not sensitize CB chemoreceptor response to hypoxia but promotes cardiovascular adjustments probably not mediated by the CB. Guinea pigs could represent an interesting model to elucidate the mechanisms that underlie the long-term effects of CIH exposure to provide evidence for the role of the CB mediating pathological effects in sleep apnea diseases., This research was supported by grants: MINECO/FEDER, UE BFU2015-70616R, and ISCiii CIBER CB06/06/0050., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Resumen del trabajo presentado al European Respiratory Society (ERS) International Congress, celebrado en Paris (Francia) del 15 al 19 de septiembre de 2018., [Rationale & Aim]: Chronic sustained hypoxia (CSH) initiates physiological adaptations to maintain oxygen levels but also has deleterious effects as pulmonary hypertension (PH). Chronic intermittent hypoxia (CIH), as found in obstructive sleep apnea, is associated with the development of systemic hypertension and cardiovascular pathologies. Our aim was to compare systemic and pulmonary hypertension and endothelial dysfunction development in rats exposed to CSH vs. CIH., [Methods]: We exposed male rats to sustained hypoxic atmosphere (10%O2; pO2≈70 mmHg, 14 days) or intermittent hypoxia (5%O2 40s, 21%O2 80s, 8h/day, 21 days). We measured pulmonary artery (PA) pressure by right heart catheterization, Fulton index, vasomotor responses in PA using small vessels myography and L- arginine-NO metabolism., [Results]: Systemic and pulmonary hypertension, besides left ventricular hypertrophy, occurs in response to CIH exposure in rats, preserving pulmonary artery endothelial integrity, but decreasing NO production and eNOS protein expression. Conversely, CSH exposure triggers CB mediated respiratory chemoreflex, PH and right ventricular hypertrophy, besides blunted pulmonary vascular relaxation to carbachol and reduced plasma L-arginine: asymmetric dimethyl arginine (ADMA) ratio., [Conclusion]: CSH and CIH point to different targets and different mechanisms to produce endothelial dysfunction and systemic and pulmonary hypertension in the rat., Spanish Ministry of Economy and Competitiveness, MINECO/FEDER, UE (BFU2015-70616-R); CIBER Enfermedades Respiratorias, ISCiii (CIBERES, CB06/06/0050).

Mammals have developed different mechanisms to maintain oxygen supply to cells in response to hypoxia. One of those mechanisms, the carotid body (CB) chemoreceptors, is able to detect physiological hypoxia and generate homeostatic reflex responses, mainly ventilatory and cardiovascular. It has been reported that guinea pigs, originally from the Andes, have a reduced ventilatory response to hypoxia compared to other mammals, implying that CB are not completely functional, which has been related to genetically/epigenetically determined poor hypoxia-driven CB reflex. This study was performed to check the guinea pig CB response to hypoxia compared to the well-known rat hypoxic response. These experiments have explored ventilatory parameters breathing different gases mixtures, cardiovascular responses to acute hypoxia, in vitro CB response to hypoxia and other stimuli and isolated guinea pig chemoreceptor cells properties. Our findings show that guinea pigs are hypotensive and have lower arterial pO2 than rats, probably related to a low sympathetic tone and high hemoglobin affinity. Those characteristics could represent a higher tolerance to hypoxic environment than other rodents. We also find that although CB are hypo-functional not showing chronic hypoxia sensitization, a small percentage of isolated carotid body chemoreceptor cells contain tyrosine hydroxylase enzyme and voltage-dependent K+ currents and therefore can be depolarized. However hypoxia does not modify intracellular Ca2+ levels or catecholamine secretion. Guinea pigs are able to hyperventilate only in response to intense acute hypoxic stimulus, but hypercapnic response is similar to rats. Whether other brain areas are also activated by hypoxia in guinea pigs remains to be studied., This study was supported by grants BFU2015-70616R, SAF 2014-55399 and SAF 2016-77222-R (MINECO-FEDER), and CIBER CB06/06/0050 (ISCIII)., We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI)

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.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.

The views presented in this article are the fruit of reflections and discussion with my colleagues at Valladolid and with the members of the Sleep Apnea Hypopnea Syndrome Group of the CIBERES (Spain). We have assembled the article in three sections. In the first one we provide a mechanistic description of obstructive sleep apnea (OSA) and all of its components, including the repetitive episodes of upper airways (UA) obstruction and accompanying hypoxic hypoxia, the respiratory efforts to fight and overcome the obstruction, and the sleep fragmentation due to the hypoxia-triggered arousal reactions, all events occurring during sleep hours with frequencies that might reach up40-50 episodes/sleep hour. When OSA is accompanied by some of the elements of a big cohort of associated pathologies (vascular, metabolic, and neuropsychiatric) it conforms the obstructive sleep apnea syndrome (OSAS). The high frequency of OSAS in adults (35 years old) and the costs in every regard of the treatment makes the syndrome a primary importance socio-sanitary problem. In the second section, we describe the experimental models of OSAS, basically the episodic repetitive hypoxic model described by Fletcher and coworkers in 1992, today named in short intermittent hypoxia (IH). From these lines, we want to call for some kind of consensus among researchers to lessen the dispersion of IH protocols. Finally, in the last section we intend to share our optimism with all ISAC members. The optimism is based on the recognition that carotid body (CB) chemoreceptors are critical elements of one of the main pathophysiologic loops in the genesis of OSAS. Therefore, we believe that all of us, as ISAC members, are well qualified to contribute in multidisciplinary research teams with well defined translational interests.

Serotonin or 5-HT is a biogenic amine present in the carotid body (CB) of several species as evidenced in many immunocytochemical studies and in a few biochemical measurements. Early literature on 5-HT actions in the CB in all studied species has lead to the conclusion that it does not participate in the setting of conducted action potentials in the sensory nerve of the CB. However, during the last 10 years very important roles in the cellular physiology of the CB have been proposed for this biogenic amine. These roles include a primary role in setting the excitability of chemoreceptor cells via an autocrine or paracrine action, and thereby, the conducted activity in the carotid sinus nerve, and a critical role in the genesis of long term sensory facilitation observed in CBs of animals exposed to intermittent hypoxia. These facts, along with important discrepancies in the endogenous levels of 5-HT in the CB prompted present study conducted in rat CBs. We measured CB endogenous 5-HT content by HPLC with electrochemical detection and found levels of 5-HT in the range of 15-22 pmole/mg tissue in control and chronically hypoxic animals either sustained or intermittent, with no significant differences among them. 5-HT and the 5-HT2A antagonist ketanserin dose-dependently activated chemoreceptor cells as assessed by their capacity to release catecholamines from freshly isolated CB. In preliminary experiments we have observed that intense hypoxia and high extracellular K(+) promote a small release of 5-HT from CB which is not dependent on the presence of extracellular Ca(2+). Further studies are needed to firmly establish the dynamics of 5-HT in the CB of the rat.

Carotid body chemoreceptor cells in response to hypoxic and hypercapnic stimulus increase their resting rate of release of neurotransmitters and their action potential frequency in the carotid sinus sensory nerve. When chemoreceptor activity is assessed at the level of the carotid sinus nerve and on ventilation, there exists an interaction between hypoxic and hypercapnic stimulus so that the response to both stimuli combined is additive or more than additive, over a wide range of stimulation. It is not clear if this interaction occurs at chemoreceptor cell or directly acting on the sensory nerve. In the present work we demonstrate for the first time the existence of a positive interaction between hypoxic and hypercapnic-acidotic stimuli at the level of both, membrane potential depolarization and neurotransmitter release in rat and rabbit carotid body. Inhibition of adenylate cyclase (SQ-22536) abolished the positive interaction between stimuli and the Epac (exchange proteins activated by cAMP) activator 8-pCPT-2'-O-Me-cAMP reversed the effect of adenylate cyclase inhibition. These results suggest that this interaction between the two natural stimuli is mediated by cAMP via an Epac-dependent pathway, at least at the level of neurotransmitter release.

Carotid body chemoreceptor cells in response to hypoxic and hypercapnic stimulus increase their resting rate of release of neurotransmitters and their action potential frequency in the carotid sinus sensory nerve. When chemoreceptor activity is assessed at the level of the carotid sinus nerve and on ventilation, there exists an interaction between hypoxic and hypercapnic stimulus so that the response to both stimuli combined is additive or more than additive, over a wide range of stimulation. It is not clear if this interaction occurs at chemoreceptor cell or directly acting on the sensory nerve. In the present work we demonstrate for the first time the existence of a positive interaction between hypoxic and hypercapnic-acidotic stimuli at the level of both, membrane potential depolarization and neurotransmitter release in rat and rabbit carotid body. Inhibition of adenylate cyclase (SQ-22536) abolished the positive interaction between stimuli and the Epac (exchange proteins activated by cAMP) activator 8-pCPT-2'-O-Me-cAMP reversed the effect of adenylate cyclase inhibition. These results suggest that this interaction between the two natural stimuli is mediated by cAMP via an Epac-dependent pathway, at least at the level of neurotransmitter release., This work was supported by grants BFU2007-61848 (DGICYT), CIBER CB06/06/0050 (FISS-ICIII).

Chemoreceptor cells of the carotid bodies (CB) are activated by hypoxia and acidosis, responding with an increase in their rate of neurotransmitter release, which in turn increases the electrical activity in the carotid sinus nerve and evokes a homeostatic hyperventilation. Studies in isolated chemoreceptor cells have shown that moderate hypoxias ( 46 mmHg) produces smaller depolarisations and comparable Ca(2+) transients but a much higher catecholamine (CA) release response in intact CBs than intense acidic/hypercapnic stimuli (20% CO(2), pH 6.6). Similarly, intense hypoxia ( 20 mmHg) produces smaller depolarizations and Ca(2+) transients in isolated chemoreceptor cells but a higher CA release response in intact CBs than a pure depolarizing stimulus (30-35 mm external K(+)). Studying the mechanisms responsible for these differences we have found the following. (1) Acidic hypercapnia inhibited I(Ca) (60%; whole cell) and CA release (45%; intact CB) elicited by ionomycin and high K(+). (2) Adenylate cyclase inhibition (SQ-22536; 80 microm) inhibited the hypoxic release response (50%) and did not affect acidic/hypercapnic release, evidencing that the high gain of hypoxia to elicit neurotransmitter release is cAMP dependent. (3) The last effect was independent of PKA activation, as three kinase inhibitors (H-89, KT 5720 and Rp-cAMP; 10 x IC(50)) did not alter the hypoxic release response. (4) The Epac (exchange protein activated by cAMP) activator (8-pCPT-2-O-Me-cAMP, 100 microm) reversed the effects of the cyclase inhibitor. (5) The Epac inhibitor brefeldin A (100 microm) inhibited (54%) hypoxic induced release. Our findings show for the first time that an Epac-mediated pathway mediates O(2) sensing/transduction in chemoreceptor cells.