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Hypoxia activates chemoreceptor cells of the carotid body (CB) promoting an increase in their normoxic release of neurotransmitters. Catecholamine (CA) release rate parallels the intensity of hypoxia. Coupling of hypoxia to CA release requires cell depolarization, produced by inhibition of O2-regulated K+ channels, and Ca2+ entering the cells via voltage-operated channels. In rat chemoreceptor cells hypoxia inhibits large-conductance, calcium-sensitive K channels (maxiK) and a two-pore domain weakly inward rectifying K+ channel (TWIK)-like acid-sensitive K+ channel (TASK)-like channel, but the significance of maxiK is controversial. A proposal envisions maxiK contributing to set the membrane potential (Em) and the hypoxic response, but the proposal is denied by authors finding that maxiK inhibition does not depolarize chemoreceptor cells or alters intracellular Ca2+ concentration or CA release in normoxia or hypoxia. We found that maxiK channel blockers (tetraethylammonium and iberiotoxin) did not modify CA release in rat chemoreceptor cells, in either normoxia or hypoxia, and iberiotoxin did not alter the Ca2+ transients elicited by hypoxia. On the contrary, both maxiK blockers increased the responses elicited by dinitrophenol, a stimulus we demonstrate does not affect maxiK channels in isolated patches of rat chemoreceptor cells. We conclude that in rat chemoreceptor cells maxiK channels do not contribute to the genesis of the Em, and that their full inhibition by hypoxia, preclude further inhibition by maxiK channel blockers. We suggest that full inhibition of this channel is required to generate the spiking behavior of the cells in acute hypoxia. [ABSTRACT FROM AUTHOR]

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 Ca2+ transients but a much higher catecholamine (CA) release response in intact CBs than intense acidic/hypercapnic stimuli (20% CO2, pH 6.6). Similarly, intense hypoxia ( ≈ 20 mmHg) produces smaller depolarizations and Ca2+ 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 ICa (∼60%; whole cell) and CA release (∼45%; intact CB) elicited by ionomycin and high K+. (2) Adenylate cyclase inhibition (SQ-22536; 80 μm) 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 × IC50) did not alter the hypoxic release response. (4) The Epac (exchange protein activated by cAMP) activator (8-pCPT-2′- O-Me-cAMP, 100 μm) reversed the effects of the cyclase inhibitor. (5) The Epac inhibitor brefeldin A (100 μm) inhibited (54%) hypoxic induced release. Our findings show for the first time that an Epac-mediated pathway mediates O2 sensing/transduction in chemoreceptor cells. [ABSTRACT FROM AUTHOR]

Agapito MT, Sanz-Alfayate G, Gomez-Niño A, Gonzalez C, Obeso A. General redox environment and carotid body chemoreceptor function. Am J Physiol Cell Physiol 296: C620-C63 I, 2009. First published January 14, 2009; doi:l0.l 152/ajpcell.00542.2008.-Carotid body (CB) chemoreceptor cells detect physiological levels of hypoxia and generate a hyperventilation, homeostatic in nature, aimed to minimize the deleterious effects of hypoxia. Intimate mechanisms involved in oxygen sensing in chemoreceptor cells remain largely unknown, but reactive oxygen species (ROS) had been proposed as mediators of this process. We have determined glutathione levels and calculated glutathione redox potential (EGSH; indicator of the general redox environment of cells) in rat diaphragms incubated in the presence of oxidizing agents of two types: nonpermeating and permeating through cell membranes; in the latter group, unspecific oxidants and inhibitors of ROS-disposing enzymes were used. Selected concentrations of oxidizing agents were tested for their ability to modify the normoxic and hypoxic activity of chemoreceptor cells measured in vitro as their rate of release of neurotransmitters. Results evidence variable relationships between E[subGSH] and the activity of chemoreceptor cells. The independence of chemoreceptor cell activity from the E[subGSH] would imply that the ability of the CB to play its homeostatic role is largely preserved in any pathological or toxicological contingency causing oxidative stress. Consistent with this suggestion, it was also found that CB-mediated hypoxic hyperventilation was not altered by treatment of intact animals with agents that markedly decreased the E[subGSH] in all tissues assayed. [ABSTRACT FROM AUTHOR]

Some age-related deficits in the ventilatory responses have been attributed to a decline in the functionality of the carotid body (CB) arterial chemoreceptors, but a systematic study of the CB function in ageing is lacking. In rats aged 3–24 months, we have performed quantitative morphometry on specific chemoreceptor tissue, assessed the function of chemoreceptor cells by measuring the content, synthesis and release of catecholamines (a chemoreceptor cell neurotransmitter) in normoxia and hypoxia, and determined the functional activity of the intact organ by measuring chemosensory activity in the carotid sinus nerve (CSN) in normoxia, hypoxia and hypercapnic acidosis. We found that with age CBs enlarge, but at the same time there is a concomitant decrease in the percentage of chemoreceptor tissue. CB content and turnover time for their catecholamines increase with age. Hypoxic stimulation of chemoreceptor cells elicits a smaller release of catecholamines in rats after 12 months of age, but a non-specific depolarizing stimulus elicits a comparable release at all ages. In parallel, there was a marked decrease in the responsiveness to hypoxia, but not to an acidic-hypercapnic stimulus, assessed as chemosensory activity in the CSN. We conclude that in aged mammals chemoreceptor cells become hypofunctional, leading to a decreased peripheral drive of ventilation. [ABSTRACT FROM AUTHOR]

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal ( IC50, 210 µm) and low intensity (PO2 ≈ 66 mm Hg/30 mm K+) stimulation-induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5′-( N-ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low-intensity hypoxia-induced release. 2- p-(2-Carboxyethyl)phenethyl-amino-5′- N-ethylcaboxamido-adenosine hydrochloride (CGS21680), 2-hexynyl-NECA (HE-NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8-cycle-1,3-dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8-(4-{[(4-cyanophenyl)carbamoylmethyl]-oxy}phenyl)-1,3-di(n-propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 × 10−9 m) and high (160 × 10−6 m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors. [ABSTRACT FROM AUTHOR]

Abstract: In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the “on-state” or the heme edge in the “off-state” but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (K d∼350 μM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded “on-state” forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded “off-state” forms. In particular, saturation with a regulatory ligand (the FeIII form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction. [Copyright &y& Elsevier]

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 P2X 2 and A 2A 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., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

The carotid bodies (CB) are peripheral chemoreceptors that sense changes in arterial blood O2, CO2, and pH levels. Hypoxia, hypercapnia, and acidosis activate the CB, which respond by increasing the action potential frequency in their sensory nerve, the carotid sinus nerve (CSN). CSN activity is integrated in the brain stem to induce a panoply of cardiorespiratory reflexes aimed, primarily, to normalize the altered blood gases, via hyperventilation, and to regulate blood pressure and cardiac performance, via sympathetic nervous system (SNS) activation. Besides its role in the cardiorespiratory control the CB has been proposed as a metabolic sensor implicated in the control of energy homeostasis and, more recently, in the regulation of whole body insulin sensitivity. Hypercaloric diets cause CB overactivation in rats, which seems to be at the origin of the development of insulin resistance and hypertension, core features of metabolic syndrome and type 2 diabetes. Consistent with this notion, CB sensory denervation prevents metabolic and hemodynamic alterations in hypercaloric feed animal. Obstructive sleep apnea (OSA) is another chronic disorder characterized by increased CB activity and intimately related with several metabolic and cardiovascular abnormalities. In this manuscript we review in a concise manner the putative pathways linking CB chemoreceptors deregulation with the pathogenesis of insulin resistance and arterial hypertension. Also, the link between chronic intermittent hypoxia (CIH) and insulin resistance is discussed. Then, a final section is devoted to debate strategies to reduce CB activity and its use for prevention and therapeutics of metabolic diseases with an emphasis on new exciting research in the modulation of bioelectronic signals, likely to be central in the future.

Obstructive sleep apnea (OSA) consists of sleep-related repetitive obstructions of upper airways that generate episodes of recurrent or intermittent hypoxia (IH). OSA commonly generates cardiovascular and metabolic pathologies defining the obstructive sleep apnea syndrome (OSAS). Literature usually links OSA-associated pathologies to IH episodes that would cause an oxidative status and a carotid body-mediated sympathetic hyperactivity. Because cardiovascular and metabolic pathologies in obese patients and those with OSAS are analogous, we used models (24-wk-old Wistar rats) of IH (applied from weeks 22 to 24) and diet-induced obesity (O; animals fed a high-fat diet from weeks 12 to 24) to define the effect of each individual maneuver and their combination on the oxidative status and sympathetic tone of animals, and to quantify cardiovascular and metabolic parameters and their deviation from normality. We found that IH and O cause an oxidative status (increased lipid peroxides and diminished activities of superoxide dismutases), an inflammatory status (augmented C-reactive protein and nuclear factor kappa-B activation), and sympathetic hyperactivity (augmented plasma and renal artery catecholamine levels and synthesis rate); combined treatments worsened those alterations. IH and O augmented liver lipid content and plasma cholesterol, triglycerides, leptin, glycemia, insulin levels, and HOMA index, and caused hypertension; most of these parameters were aggravated when IH and O were combined. IH diminished ventilatory response to hypoxia, and hypercapnia and O created a restrictive ventilatory pattern; a combination of treatments led to restrictive hypoventilation. Data demonstrate that IH and O cause comparable metabolic and cardiovascular pathologies via misregulation of the redox status and sympathetic hyperactivity., (Copyright © 2014 the American Physiological Society.)

When de Castro entered the carotid body (CB) field, the organ was considered to be a small autonomic ganglion, a gland, a glomus or glomerulus, or a paraganglion. In his 1928 paper, de Castro concluded: "In sum, the Glomus caroticum is innervated by centripetal fibers, whose trophic centers are located in the sensory ganglia of the glossopharyngeal, and not by centrifugal [efferent] or secretomotor fibers as is the case for glands; these are precisely the facts which lead to suppose that the Glomus caroticum is a sensory organ." A few pages down, de Castro wrote: "The Glomus represents an organ with multiple receptors furnished with specialized receptor cells like those of other sensory organs [taste buds?]…As a plausible hypothesis we propose that the Glomus caroticum represents a sensory organ, at present the only one in its kind, dedicated to capture certain qualitative variations in the composition of blood, a function that, possibly by a reflex mechanism would have an effect on the functional activity of other organs… Therefore, the sensory fiber would not be directly stimulated by blood, but via the intermediation of the epithelial cells of the organ, which, as their structure suggests, possess a secretory function which would participate in the stimulation of the centripetal fibers." In our article we will recreate the experiments that allowed Fernando de Castro to reach this first conclusion. Also, we will scrutinize the natural endowments and the scientific knowledge that drove de Castro to make the triple hypotheses: the CB as chemoreceptor (variations in blood composition), as a secondary sensory receptor which functioning involves a chemical synapse, and as a center, origin of systemic reflexes. After a brief account of the systemic reflex effects resulting from the CB stimulation, we will complete our article with a general view of the cellular-molecular mechanisms currently thought to be involved in the functioning of this arterial chemoreceptor.

Obstructive sleep apnea syndrome (OSAS) is described as repetitive obstructions of the upper airways during sleep, causing concomitant episodes of systemic hypoxia and associated cardiovascular and metabolic pathologies. The mechanisms generating these pathologies are controversial. Because recurrent hypoxia is the element of inadequate respiration that leads to the pathology, experimental models of OSAS consist in the exposure of the animals to intermittent hypoxia (IH) by cycling O2 percentages in their habitats. A proposed mechanism linking the IH of OSAS to pathologies is the increased production of reactive oxygen species (ROS). However, it has been argued that many patients seem to lack oxidative stress and that, to augment ROS in IH animals, intense hypoxia, seldom encountered in patients, has to be applied. To solve the controversy, we have exposed rats to two intensities of IH (cycles of 10 or 5% O2, 40s, and then 21% O2, 80s; 8h/day, 15 days). We then measured reduced and oxidized glutathione and lipid peroxide levels, aconitase and fumarase activities, and ROS-disposal enzyme activity in liver, brain, and lung. Liver levels of nuclear NF-κB-p65 and plasma C-reactive protein (CRP), as well as lipid levels, were also assessed. Lowest hemoglobin saturations were 91.7 ± 0.8 and 73.5 ± 1.4%. IH caused tissue-specific oxidative stress related to hypoxic intensity. Nuclear NF-κB-p65 and lipid content in the liver and CRP in the plasma all increased with IH intensity, as did both plasma triglycerides and cholesterol. We conclude that IH, even of moderate intensity, causes oxidative stress probably related to the pathologies encountered in OSAS patients., (© 2013 Elsevier Inc. All rights reserved.)

Increased sympathetic activity is a well-known pathophysiological mechanism in insulin resistance (IR) and hypertension (HT). The carotid bodies (CB) are peripheral chemoreceptors that classically respond to hypoxia by increasing chemosensory activity in the carotid sinus nerve (CSN), causing hyperventilation and activation of the sympathoadrenal system. Besides its role in the control of ventilation, the CB has been proposed as a glucose sensor implicated in the control of energy homeostasis. However, to date no studies have anticipated its role in the development of IR. Herein, we propose that CB overstimulation is involved in the etiology of IR and HT, core metabolic and hemodynamic disturbances of highly prevalent diseases like the metabolic syndrome, type 2 diabetes, and obstructive sleep apnoea. We demonstrate that CB activity is increased in IR animal models and that CSN resection prevents CB overactivation and diet-induced IR and HT. Moreover, we show that insulin triggers CB, highlighting a new role for hyperinsulinemia as a stimulus for CB overactivation. We propose that CB is implicated in the pathogenesis of metabolic and hemodynamic disturbances through sympathoadrenal overactivation and may represent a novel therapeutic target in these diseases.

Sustained hypoxia produces a carotid body (CB) sensitization, known as acclimatization, which leads to an increase in carotid sinus nerve (CSN) activity and ensuing hyperventilation greater than expected from the prevailing partial pressure of oxygen. Whether sustained hypoxia is physiological (high altitude) or pathological (lung disease), acclimatization has a homeostatic implication because it tends to minimize hypoxia. Caffeine, the most commonly ingested psychoactive drug and a nonselective adenosine receptor antagonist, alters CB function and ventilatory responses when administered acutely. Our aim was to investigate the effect of chronic caffeine intake on CB function and acclimatization using four groups of rats: normoxic, caffeine-treated normoxic, chronically hypoxic (12% O₂, 15 days), and caffeine-treated chronically hypoxic rats. Caffeine was administered in drinking water (1 mg/ml). Caffeine ameliorated ventilatory responses to acute hypoxia in normoxic animals without altering the output of the CB (CSN neural activity). Caffeine-treated chronically hypoxic rats exhibited a decrease in the CSN response to acute hypoxia tests but maintained ventilation compared with chronically hypoxic animals. The findings related to CSN neural activity combined with the ventilatory responses indicate that caffeine alters central integration of the CB input to increase the gain of the chemoreflex and that caffeine abolishes CB acclimatization. The putative mechanisms involved in sensitization and its loss were investigated: expression of adenosine receptors in CB (A(2B)) was down-regulated and that in petrosal ganglion (A(2A)) was up-regulated in caffeine-treated chronically hypoxic rats; both adenosine and dopamine release from CB chemoreceptor cells was increased in chronic hypoxia and in caffeine-treated chronic hypoxia groups.

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.

Neural plasticity is defined as a persistent change in the morphology and/or function based on prior experiences. Plasticity is well evident when the triggering experience occurs early in life, but in the case of respiratory control plasticity, it also can be triggered in adult life. We have combined a 10 days postnatal hypoxic (PH) (0-10 days of age;11% O(2)) and a 15 days intermittent hypoxia (IH) exposures in the adulthood (90-105 days of age; 5% O(2), 40 s/20% O(2), 80 s; 8 h/day) to test if early PH interacts with IH of the adulthood to generate detrimental plastic changes. After recording of ventilatory parameters, the brains were studied immunocytochemically for localization of the organization pattern of non-phosphorylated subunit of neurofilament H (NFH) and tyrosine hydroxylase (TH) expression in the nucleus tractus solitarius (Sol) and caudal (CVL) and rostral ventrolateral reticular (RVL) nuclei, areas related to central cardio-respiratory regulation. In comparison to control, PH male rats (but not females) at 1 month of age hyperventilated at rest, in response to moderate hypoxia (12% O(2)) and 5% CO(2), the effect being due to increased tidal volume. At 3.5 months sex differences in ventilation disappeared and it was indistinguishable between control and PH. IH tended to decrease ventilation in both control (C) and PH animals. PH augmented PENH values in air and in hypoxic conditions when compared with C group. IH in both groups, tended to decrease the PENH value, being statistically different in PH+IH. Results also show an increment of disorganization of NFH-positive labeled structures at the level of Sol and CVL/RVL nuclei in PH, IH and HP+HI groups. PH rats showed differences in the number of TH-positive neurons at the level of CVL/RVL nuclei, which was increased in the PH and PH+IH groups with respect to C one. In conclusion, PH alters the central morpho-physiological organization and the catecholaminergic components of cardio-respiratory nuclei, whose effects were enhanced after a period of IH in the adulthood.

We tested the hypothesis that long-term caffeine intake prevents the development of insulin resistance and hypertension in two pathological animal models: the high-fat (HF) and the high-sucrose (HSu) diet rat. We used six groups of animals: control; caffeine-treated (Caff; 1 g/l in drinking water during 15 d); HF; caffeine-treated HF (HFCaff); HSu; caffeine-treated HSu (HSuCaff). Insulin sensitivity was assessed using the insulin tolerance test. Blood pressure, weight gain, visceral fat, hepatic glutathione, plasma caffeine, insulin and NO, and serum NEFA and catecholamines were measured. Caffeine reversed insulin resistance and hypertension induced by both the HF and HSu diets. In the HF-fed animals caffeine treatment restored fasting insulin levels to control values and reversed increased weight gain and visceral fat mass. In the HSu group, caffeine reversed fasting hyperglycaemia and restored NEFA to control values. There were no changes either in plasma NO or in hepatic glutathione levels. In contrast, caffeine totally prevented the increase in serum catecholamines induced by HF and HSu diets. To test the hypothesis that inhibition of the sympathetic nervous system prevents the development of diet-induced insulin resistance we administered carvedilol, an antagonist of β1, β2 and also α1 adrenoceptors, to HF and HSu rats. Carvedilol treatment fully prevented diet-induced insulin resistance and hypertension, mimicking the effect of caffeine. We concluded that long-term caffeine intake prevented the development of insulin resistance and hypertension in HF and HSu models and that this effect was related to a decrease in circulating catecholamines.

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.

Caffeine is the most commonly psychoactive drug, an habitual drink in high altitude sporting, and when acutely taken, it causes profound alterations in carotid body (CB) function and ventilation via adenosine receptors antagonism. In the present work we have investigated the effects of chronic caffeine ingestion in catecholamine (CA) dynamics in the carotid body of control and chronic hypoxic rats. Four groups of animals were used: normoxic (N), caffeine-treated normoxic (1 mg/mL in drinking water 15 days; CafN), chronic hypoxic (CH, 12%O(2), 15 days) and chronically hypoxic-caffeine-treated (CafH).. Caffeine intake in controls rats did not modify CA content, synthesizing, and releasing responses, and the expression of tyrosine hydroxylase. CH increased dopamine content, synthesis, and basal and acute hypoxia-induced release; chronic caffeine ingestion augmented CH effects. Findings indicate that chronic caffeine ingestion in normoxic rats did not modify dopamine dynamics at the CB, but increases dopaminergic system during chronic hypoxia.

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.

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 up >40-50 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.

Carotid bodies (CBs) are secondary sensory receptors in which the sensing elements, chemoreceptor cells, are activated by decreases in arterial PO(2) (hypoxic hypoxia). Upon activation, chemoreceptor cells (also known as Type I and glomus cells) increase their rate of release of neurotransmitters that drive the sensory activity in the carotid sinus nerve (CSN) which ends in the brain stem where reflex responses are coordinated. When challenged with hypoxic hypoxia, the physiopathologically most relevant stimulus to the CBs, they are activated and initiate ventilatory and cardiocirculatory reflexes. Reflex increase in minute volume ventilation promotes CO(2) removal from alveoli and a decrease in alveolar PCO(2) ensues. Reduced alveolar PCO(2) makes possible alveolar and arterial PO(2) to increase minimizing the intensity of hypoxia. The ventilatory effect, in conjunction the cardiocirculatory components of the CB chemoreflex, tend to maintain an adequate supply of oxygen to the tissues. The CB has been the focus of attention since the discovery of its nature as a sensory organ by de Castro (1928) and the discovery of its function as the origin of ventilatory reflexes by Heymans' group (1930). A great deal of effort has been focused on the study of the mechanisms involved in O(2) detection. This review is devoted to this topic, mechanisms of oxygen sensing. Starting from a summary of the main theories evolving through the years, we will emphasize the nature and significance of the findings obtained with veratridine and tetrodotoxin (TTX) in the genesis of current models of O(2)-sensing.

Background: Samples used for genotyping and transcription studies are obtained and conserved in very specific conditions. The possibility to use autopsy tissue samples, which contain nucleic acids of very poor quality, would open new possibilities for genetic studies., Methods: We have used liver tissue samples from autopsy cases to (i) determine its quality; (ii) study gene expression of 13 genes involved in different cell processes, before and after cDNA pre-amplification (quantitative reverse transcriptase polymerase chain reaction); and (iii) analyze the presence of 2 common polymorphisms of relevance for illness (ACE I/D genotype by PCR amplification, and TNF-α promoter gene polymorphism, by DNA sequencing)., Results: Samples were grouped according to different buffered formalin fixation times (group 1, <15 days; group 2, 60-90 days; group 3, 150-180 days; group 4, 240-270 days). Nucleic acids showed a time-dependent degradation. The expression of 13 genes could be studied in all cases from groups 1 and 2, only 7 from group 3 and none from group 4. cDNA preamplification allowed the study of all genes in all samples. DNA genotyping for ACE and TNF-α promoter region was possible in all cases., Conclusions: We conclude that nucleic acids extracted from autopsy specimens after prolonged periods of time in formalin were of sufficient quality to study gene expression and genotyping using currently available methodology and cDNA pre-amplification.

The characterization of the molecular mechanisms involved in low-oxygen chemotransduction has been an active field of research since the first description of an oxygen-sensitive K(+) channel in rabbit carotid body (CB) chemoreceptor cells. As a result, a large number of components of the transduction cascade, from O(2) sensors to O(2)-sensitive ion channels, have been found. Although the endpoints of the process are analogous, the heterogeneity of the elements involved in the different chemoreceptor tissues precludes a unifying theory of hypoxic signaling, and it has been a source of controversy. However, when these molecular constituents of the hypoxic cascade are brought back to their physiological context, it becomes clear that the diversity of mechanisms is necessary to build up an integrated cellular response that demands the concerted action of several O(2) sensors and several effectors.

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.

We have previously demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A2B receptors. Here, we have tested the hypothesis that the control is exerted via an interaction between adenosine A2B and dopamine D2 receptors present in chemoreceptor cells. Experiments were performed in vitro in CB from 3 months rats. The effect of A2B adenosine and D2 dopamine agonists and antagonists applied alone or in combination were studied on basal (20%O2) and hypoxia (10%O2)-evoked release of CA and cAMP content of CB. We have found that adenosine A2 agonists and D2 antagonists dose-dependently increased basal and evoked release CA from the CB while A2 antagonists and D2 agonists had an inhibitory action. The existence of A2B-D2 receptor interaction was established because the inhibitory action of A2 antagonists was abolished by D2 antagonists, and the stimulatory action of A2 agonists was abolished by D2 agonists. Further, A2 agonists increased and D2 agonist decreased cAMP content in the CB; their co-application eliminated the response. The present results provide direct pharmacological evidence that an antagonistic interaction between A2B adenosine and D2 dopamine receptors exist in rat CB and would explain the dopamine-adenosine interactions on ventilation previously observed.

We have assessed the expression, molecular identification and functional role of Na+ channels (Na(v)) in carotid bodies (CB) obtained from normoxic and chronically hypoxic adult rats. Veratridine evoked release of catecholamines (CA) from an in vitro preparation of intact CBs obtained from normoxic animals, the response being Ca2+ and Na+-dependent and sensitive to tetrodotoxin (TTX). TTX inhibited by 25-50% the CA release response evoked by graded hypoxia. Immunoblot assays demonstrated the presence of Na(v)alpha-subunit (c. 220 kDa) in crude homogenates from rat CBs, being evident an up-regulation (60%) of this protein in the CBs obtained from chronically hypoxic rats (10% O2; 7 days). This up-regulation was accompanied by an enhanced TTX-sensitive release response to veratridine, and by an enhanced ventilatory response to acute hypoxic stimuli. RT-PCR studies demonstrated the expression of mRNA for Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.6 and Na(v)1.7 isoforms. At least three isoforms, Na(v)1.1, Na(v)1.3 and Na(v)1.6 co-localized with tyrosine hydroxylase in all chemoreceptor cells. RT-PCR and immunocytochemistry indicated that Na(v)1.1 isoform was up-regulated by chronic hypoxia in chemoreceptor cells. We conclude that Na(v) up-regulation represents an adaptive mechanism to increase chemoreceptor sensitivity during acclimatization to sustained hypoxia as evidenced by enhanced ventilatory responses to acute hypoxic tests.

Two current hypotheses of O2 sensing in the carotid body (CB) chemoreceptors suggest participation of oxygen reactive (ROS) species, but they are mechanistically opposed. One postulates that hypoxia decreases ROS levels; the other that hypoxia increases them. Yet, both propose that the ensuing alteration in the cellular redox environment is the key signal triggering hypoxic chemoreception. Since the glutathione redox pair is the main cellular buffer for ROS and the main determinant of the general redox environment of the cells, a way to test whether ROS participate in chemoreception is to determine glutathione levels and to correlate them with the activity of CB chemoreceptor cells. We found that hypoxia does not alter the glutathione reduction potential but that it activates chemoreceptor cell neurosecretion. Incubation of tissues with reduced glutathione increases the glutathione-reducing potential but does not activate chemoreceptor cells in normoxia nor does it modify hypoxic activation. Like reduced glutathione, N-acetylcysteine promoted a general reducing environment in the cells without alteration of chemoreceptor cell activity. N-(mercaptopropionyl)-glycine, like the two previous agents, increases the reduction potential of glutathione. In contrast, the compound activated chemoreceptor cells in normoxia, promoting a dose- and Ca(2+)-dependent neurosecretion and a potentiation of the hypoxic responses. The existence of multiple relationships between glutathione reduction potential in the cells and their activity indicates that the general cellular redox environment is not a factor determining chemoreceptor cell activation. It cannot be excluded that the local redox environments of restricted microdomain(s) in the cells with specific regulating mechanisms are important signals for chemoreceptor cell activity.

Reactive oxygen species (ROS) are oxygen-containing molecular entities which are more potent and effective oxidizing agents than is molecular oxygen itself. With the exception of phagocytic cells, where ROS play an important physiological role in defense reactions, ROS have classically been considered undesirable byproducts of cell metabolism, existing several cellular mechanisms aimed to dispose them. Recently, however, ROS have been considered important intracellular signaling molecules, which may act as mediators or second messengers in many cell functions. This is the proposed role for ROS in oxygen sensing in systems, such as carotid body chemoreceptor cells, pulmonary artery smooth muscle cells, and erythropoietin-producing cells. These unique cells comprise essential parts of homeostatic loops directed to maintain oxygen levels in multicellular organisms in situations of hypoxia. The present article examines the possible significance of ROS in these three cell systems, and proposes a set of criteria that ROS should satisfy for their consideration as mediators in hypoxic transduction cascades. In none of the three cell types do ROS satisfy these criteria, and thus it appears that alternative mechanisms are responsible for the transduction cascades linking hypoxia to the release of neurotransmitters in chemoreceptor cells, contraction in pulmonary artery smooth muscle cells and erythropoietin secretion in erythropoietin producing cells., (Copyright 2002 Elsevier Science B.V.)

Rabbit carotid body (CB) chemoreceptor cells possess a fast-inactivating K+ current that is specifically inhibited by hypoxia. We have studied the expression of Kvalpha subunits, which might be responsible for this current. RT-PCR experiments identified the expression of Kv1.4, Kv3.4, Kv4.1 and Kv4.3 mRNAs in the rabbit CB. There was no expression of Kv3.3 or Kv4.2 transcripts. Immunocytochemistry with antibodies to tyrosine hydroxylase (anti-TH) and to specific Kv subunits revealed the expression of Kv3.4 and Kv4.3 in chemoreceptor cells, while Kv1.4 was only found in nerve fibres. Kv4.1 mRNA was also found in chemoreceptor cells following in situ hybridization combined with anti-TH antibody labelling. Kv4.1 and Kv4.3 appeared to be present in all chemoreceptor cells, but Kv3.4 was only expressed in a population of them. Electrophysiological experiments applying specific toxins or antibodies demonstrated that both Kv3.4 and Kv4.3 participate in the oxygen-sensitive K+ current of chemoreceptor cells. However, toxin application experiments confirmed a larger contribution of members of the Kv4 subfamily. [Ca2+]i measurements under hypoxic conditions and immunocytochemistry experiments in dispersed CB cells demonstrated the expression of Kv3.4 and Kv4.3 in oxygen-sensitive cells; the presence of Kv3.4 in the chemoreceptor cell membrane was not required for the response to low PO2. In summary, three Kv subunits (Kv3.4, Kv4.1 and Kv4.3) may be involved in the fast-inactivating outward K+ current of rabbit CB chemoreceptor cells. The homogeneous distribution of the Kv4 subunits in chemoreceptor cells, along with their electrophysiological properties, suggest that Kv4.1, Kv4.3, or their heteromultimers, are the molecular correlate of the oxygen-sensitive K+ channel.