Ana Obeso

Oxygen and acid chemoreception in the carotid body chemoreceptors

The carotid bodies are arterial chemoreceptors that are sensitive to blood PO2, PCO2 and pH. They are the origin of reflexes that are crucial for maintaining PCO2 and pH in the internal milieu and for adjusting the O2 supply according to the metabolic needs of the organism in situations of increased demand, such as exercise and while breathing at decreased O2 partial pressures during ascent or when living at high altitude. Chemoreceptor cells of the carotid body transduce the blood-borne stimuli into a neurosecretory response that is dependent on external Ca2+. These cells have an O2-sensitive K+ current that is reversibly inhibited by low PO2. It is proposed that the depolarization produced by inhibition of this K+ current activates Ca2+ channels; Ca2+ influx and neurosecretion follow. The cells have also a potent Na(+)-Ca2+ antiporter that could be responsible for the intracellular Ca2+ rise required to trigger the release of neurotransmitters during high PCO2 or low pH stimulation.

Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia

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.

Molecular identification of Kvα subunits that contribute to the oxygen-sensitive K+ current of chemoreceptor cells of the rabbit carotid body

Rabbit carotid body (CB) chemoreceptor cells possess a fast‐inactivating K+ current that is specifically inhibited by hypoxia. We have studied the expression of Kvα 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.

Ionic mechanisms for the transduction of acidic stimuli in rabbit carotid body glomus cells.

1. The release of [3H]dopamine (DA) in response to inhibition of the Na+ pump or to intracellular acid load was studied in rabbit carotid bodies (CB) previously incubated with the precursor [3H]tyrosine. The ionic requirements of the release response and the involvement of specific ion transport systems were investigated. 2. Inhibition of the Na+ pump, by incubating the CB with ouabain or in K(+)‐free medium, evokes a DA release response which requires the presence of Na+ and Ca2+ in the medium and is insensitive to nisoldipine. This suggests that the response is triggered by entry of external Ca2+ through Na(+)‐Ca2+ exchange, a consequence of the increase in intracellular Na+ resulting from inhibition of the pump. 3. Incubation of the CB in medium equilibrated with 20% CO2 at pH 6.6, or in medium containing the protonophore dinitrophenol (DNP) or the weak acid propionate, elicits a DA release response which requires also the presence of Na+ and Ca2+ in the medium and is insensitive to dihydropyridines. 4. Ethylisopropylamiloride (EIPA), an inhibitor of the Na(+)‐H+ exchanger, markedly decreases the release response elicited by DNP or propionate in bicarbonate‐free medium, but has not any effect in bicarbonate‐buffered medium. In the latter condition, the EIPA‐insensitive release of DA is inhibited by reducing the HCO3‐ concentration in the medium to 2 mM or by removal of Cl‐, suggesting that in bicarbonate‐buffered medium a Na(+)‐dependent HCO3(‐)‐Cl‐ exchanger is involved in the release response. 5. It is concluded that the release of DA by the chemoreceptor cells in response to acidic stimulation is triggered by entry of external Ca2+ through Na(+)‐Ca2+ exchange. This exchange is promoted by the increase of intracellular Na+ that results from the operation of Na(+)‐coupled H(+)‐extruding mechanisms activated by the acid load.

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors.

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.

Chemoreception in the context of the general biology of ROS

Superoxide anion is the most important reactive oxygen species (ROS) primarily generated in cells. The main cellular constituents with capabilities to generate superoxide anion are NADPH oxidases and mitochondrial respiratory chain. The emphasis of our article is centered in critically examining hypotheses proposing that ROS generated by NADPH oxidase and mitochondria are key elements in O(2)-sensing and hypoxic responses generation in carotid body chemoreceptor cells. Available data indicate that chemoreceptor cells express a specific isoform of NADPH oxidase that is activated by hypoxia; generated ROS acting as negative modulators of the carotid body (CB) hypoxic responses. Literature is also consistent in supporting that poisoned respiratory chain can produce high amounts of ROS, making mitochondrial ROS potential triggers-modulators of the CB activation elicited by mitochondrial venoms. However, most data favour the notion that levels of hypoxia, capable of strongly activating chemoreceptor cells, would not increase the rate of ROS production in mitochondria, making mitochondrial ROS unlikely triggers of hypoxic responses in the CB. Finally, we review recent literature on heme oxygenases from two perspectives, as potential O(2)-sensors in chemoreceptor cells and as generators of bilirubin which is considered to be a ROS scavenger of major quantitative importance in mammalian cells.

Effects of cyanide and uncoupler on chemoreceptor activity and ATP content of the cat carotid body

In cat carotid bodies (c.b.s) incubated in vitro with [3H]tyrosine to label the stores of catecholamines, it was found that CN promotes dose- and Ca2+-dependent release of [3H]dopamine (DA) from c.b. tissues in parallel to the increased electrical activity recorded from the carotid sinus nerve (c.s.n.). Two different uncouplers, dinitrophenol (DNP) and carbonyl-cyanide-m-chlorophenyl-hydrazone (CCCP), both activate also in a dose-dependent fashion, release of DA and electrical activity in the c.s.n. However, while cyanide (CN) (10(-4) M) applied during 5 min reduced the adenosine triphosphate (ATP) content of the c.b. by 45%, DNP (2.5 x 10(-4) M) and CCCP (10(-6) M) applied for the same period of time did not modify the ATP levels of the organ. At the above concentrations, the 3 agents increased about 8-fold the electrical activity recorded from the c.s.n. Thus, contrary to the postulates of the metabolic hypotheses, our findings indicate that the decrease in the ATP content in the c.b. is not a prerequisite for the activation of the chemoreceptors. We propose alternative mechanisms to explain the chemostimulant action of the metabolic poisons.

CELLULAR MECHANISMS OF OXYGEN CHEMORECEPTION IN THE CAROTID BODY

The carotid bodies (CB) are arterial chemoreceptors that by sensing changes of arterial PO2, PCO2 and pH can initiate and modify ventilatory and cardiovascular reflexes in order to maintain PO2, PCO2 and pH within physiological levels. It is now generally accepted that the glomus or type I cells of the CB are the transducers of hypoxic stimuli, and relay chemosensory information to the brainstem via neurotransmitter release at synaptic contacts with afferent terminals of the carotid sinus nerve. This article reviews the mechanisms of the O2-sensing process at the cellular level. We consider first the transduction of the hypoxic stimulus, in which most of the experimental evidence currently favors a mechanism involving modulation of the electrical properties of type I cells. The last part of the article deals with the transmission of the stimulus between type I cells and afferent nerve terminals, and we present an overview on the issue of neurotransmission in the CB, summarizing the actions of the main neurotransmitters present in the organ.

Effects of high potassium on the release of [3H]dopamine from the cat carotid body in vitro.

Using an in vitro preparation of the cat carotid body, we have characterized the release of [3H]dopamine (DA) induced by high extracellular K+. Pulse superfusion (3 min) with high K+ Tyrode solution (20‐80 mM) evoked a concentration‐dependent release of [3H]DA from type I cells with a threshold at about 20 mM‐extracellular K+ and a plateau at about 60 mM‐extracellular K+. Equivalent low extracellular Na+ concentration ([Na+]o) solutions osmotically balanced with sucrose did not induce release. The high extracellular K+ concentration ([K+]o)‐evoked release of [3H]DA by type I cells was dependent on the presence of Ca2+ in the superfusion media. On prolonged (10‐14 min) superfusion with high K+ Tyrode solution, the [3H]DA release evoked by 60 mM‐extracellular K+ was transient, while that evoked by 30 mM‐extracellular K+ was sustained. In preparations superfused for 6 min with 60 mM‐extracellular K+ and zero extracellular Ca2+ concentration ([Ca2+]o) Tyrode solution, reintroduction of Ca2+ did not elicit a secretory response. Ba2+ was a potent secretagogue of [3H]DA in preparations superfused with normal and zero [Ca2+]o Tyrode solution. Additionally, Ba2+ was capable of eliciting a secretory response from type I cells in preparations previously exposed (6 min) to 60 mM‐extracellular K+, whether or not [Ca2+]o was present. With regards to stimulus‐secretion coupling, our results indicate that high [K+]o probably depolarizes type I cells. This effect would, in turn, activate voltage‐dependent Ca2+ channels, allowing the entrance of this ion to activate the neurosecretory response.

Effects of 2-deoxy-d-glucose on in vitro cat carotid body

The process of chemosensory transduction in the arterial chemoreceptors is not well understood. According to the metabolic hypothesis of chemoreception, a decrease in arterial pO2 will produce a decrease in the adenosine triphosphate (ATP) content in the chemosensory type I cells, leading to release of a neurotransmitter and increased sensory neural activity. There is a paucity of direct experimental support for this hypothesis, and in the present work, we have tested the postulates of the metabolic hypothesis in an in vitro preparation of cat carotid body using 2-deoxy-D-glucose as an ATP-depleting agent. This preparation, when superfused with Tyrode containing 5 mM Na+-pyruvate instead of glucose, responds normally to hypoxia, low pH and acetylcholine, and maintains normal ATP levels. Under these conditions, 2-deoxy-D-glucose is a chemostimulant, i.e. electrical activity in the carotid sinus nerve is increased, with a threshold concentration of 0.25 mM and a maximum response at about 2-4 mM. These concentrations of 2-deoxyglucose evoke a dose-dependent release of [3H]dopamine (synthesized from [3H]tyrosine) from the carotid bodies which parallels the electrical activity. The 2-deoxy-D-glucose-evoked release and electrical activity is dependent on the presence of extracellular Ca2+. These same concentrations of 2-deoxy-D-glucose significantly reduce the ATP content of the carotid bodies. The events postulated by the metabolic hypothesis, i.e. decrease in ATP content, release of a putative neurotransmitter and activation of the sensory nerve endings are found to occur simultaneously. A possible cause-effect relationship between these three events is discussed.