Correlation Between Adenosine Triphosphate Levels, Dopamine Release and Electrical Activity in the Carotid Body: Support for the Metabolic Hypothesis of Chemoreception

Producción CientíficaAn unsolved issue for the arterial chemoreceptors is the mechanism by which hypoxia and other natural stimuli lead to an increase of activity in the carotid sinus nerve. According to the 'metabolic hypothesis', the hypoxic activation of the carotid body (CB) is mediated by a decrease of the ATP levels in the type I cells, which then release a neurotransmitter capable of exciting the sensory nerve endings. Using an in vitro preparation of cat CB, we report that ATP levels in the CB do in fact decrease when the organs are exposed to moderate, short lasting hypoxia (5 min 20% 02). Additionally, we found that decreases in ATP levels induced by 2-deoxyglucose (2 mM) or sodium cyanide (0.1 raM) are closely correlated with dopamine release from type I cells and electrical activity in the carotid sinus nerve elicited by these agents. The possible cause-effect relationship of these events is discusse

Effects of 2-Deoxy-D-Glucose on In Vitro Cat Carotid Body

Producción CientíficaThe process of chemosensory transduction in the arterial chemoreceptors is not well understood. According to the metabolic hypothesis of chemoreception, a decrease in arterial pO 2 will produce a decrease in the adenosine triphosphate (ATP) content in the chcmosensory 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 acetylcholinc, 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 raM. These concentrations of 2-deoxyglucose evoke a dose-dependent release of [3H]dopamine (synthesized from pH]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 Ca 2+ . These same concentrations of 2-deoxy-D-glucose significantly reduce the ATP content of the carotid bodies. The events postulated bv the metabolic hypothesis, i.e. decrease in ATP content, release of a putative neurotransminer and activation of the sensory nerve endings are found to occur simultaneously. A possible cause-effect relationship between these three events is discussed

The role of dihydropydine-sensitive Ca2 + channels in stimulus-evoked catecholamine release from chemoreceptor cells of the carotid body

Producción CientíficaAhatraet-The present study utilized an in vitro preparation of the rabbit carotid body, with tissue catecholamine stores labeled by incubation with ‘H-tyrosine. The goal was to characterize pharmacologically the vol~g~~n&nt Ca*+ channels present in the type I (glomus) cells of this arterial chemoreceptor organ, and to elucidate their role as pathways for Ca2+ entry. We found that release of ‘H-dopamine induced by high external potassium was over 95% dependent on external cakium concentration and that this release was 9&100% inhibited by the dihydropy~~ne antagonists, nisoldipine and nitrendipine, and was potentiated by the dihydropyridine agonist, BayK 8444. Therefore, any stimulus-induced, cakiumdependent release of 3H-dopamine that was inhibited by nisoldipine and potentiated by BayK 8644, was considered to be supported by Ca2+ entry into the cells via voltage-dependent Ca2+ channels. Significant differences were observed in the release of ‘H-dopamine induced by 75 vs 25mM K+. On prolonged stimulation, release induced by 75 mM K+ was large and transient, whilst that induced by 25 mM K+, although more moderate, was sustained. The release elicited by 75 mM K+ was inhibited approximately 90% by 1.5 mM Co2+ or 625 nM nisoldipine, while release by 25 mM K+ was completely blocked by 0.6 mM Co*+ or 125 nM nisoldipine. Low PO,-induced release of 3H-dopamine was 95% dependent on Ca*+, and was inhibited by nisoldipine (625 nM) in a manner inversely proportional to the intensity of hypoxic stimulation, i.e. 79% inhibition at a PO, of 49 Torr, and 20% inhibition at PO2 of 0 Torr. BayK 8644 potentiatcd the release induced by moderate hypoxic stimuli. Release elicited by high PCOJlow pH, or by Na+-propionate or dinitrophenol~n~ining solutions, was approximately 80% Ca’+-dependent, and the ~hyd~y~din~ failed to modify this release. It is concluded that type I mlls possess vol~~de~nd~t Ca ‘+ channels sensitive to the dihydropy~dines, which in agreement with previous el~trophysiolo~~l data should be defined as L-type Ca*+ channels. Calcium entry which supports the release of 3H-dopamine elicited by moderate hypoxia should occur mainly through these channels while the release induced by strong hypoxic stimuli will be SetNed by Ca2+ entry which occurs in part via voltage-dependent Ca2+ channels, and in part through an additional pathway, probably a Na+/Ca2+ exchanger. The insensitivity to dihydropyridines of the release of )H-dopamine induced by high 1DC02/low pH, Na+-propionate and dinitrophenol may indicate a complete loss of efficacy of the drugs to modulate Ca 2+ channels under these conditions or more likely, that other mechanisms are activated, probably the Na+-Ca’+ exchanger. Carotid body (CB) chemoreceptors are thought to be composite receptors in which the type I (glomus) cells detect changes in blood PO,, PCO, and pH and respond with the release of neurotransmitt~ to activate the closely apposed chemosensory nerve terminals.~** One such neurotransmitter that has received considerable attention in recent years and is known to be released by the type I cells is dopamine (DA). This biogenic amine has been shown to be released in proportion to both the intensity of stimulation and the resultant sensory discharge recorded from the carotid sinus nerve $To whom correspondence should be addressed. Abbr~~~~~~ CB, carotid body; CSN, carotid sinus nerve; DA, dopamine; DHMA, dihydrox~~delic acid, DOPAC, dihydroxyphenyl acetic acid; NE, norepinephrine. (CSN). This relationship between stimulu

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity

Producción CientíficaLowoxygen sensing in chemoreceptor cells involves the inhibition of specific plasma membrane K+ channels, suggesting that mitochondria-derived reactive oxygen species (ROS) link hypoxia to K+ channel inhibition, subsequent cell depolarization and activation of neurotransmitter release.We have used several mitochondrial poisons, alone and in combination with the antioxidant N-acetylcysteine (NAC), and quantify their capacity to alter GSH/GSSG levels and glutathione redox potential (EGSH) in rat diaphragm. Selected concentrations of mitochondrial poisons with or without NAC were tested for their capacity to activate neurotransmitter release in chemoreceptor cells and to alter ATP levels in intact rat carotid body (CB).We found that rotenone (1 M), antimycin A (0.2 g/ml) and sodium azide (5mM) decreased EGSH; NAC restored EGSH to control values. At those concentrations mitochondrial poisons activated neurotransmitter release from CB chemoreceptor cells and decreased CB ATP levels, NAC being ineffective to modify these responses. Additional experiments with 3-nitroprionate (5 mM), lower concentrations of rotenone and dinitrophenol revealed variable relationships between EGSH and chemoreceptor cell neurotransmitter release responses and ATP levels. These findings indicate a lack of correlation between mitochondrialgenerated modifications of EGSH and chemoreceptor cells activity. This lack of correlation renders unlikely that alteration of mitochondrial production of ROS is the physiological pathway chemoreceptor cells use to signal hypoxia

Intracellular Ca2 + deposits and catecholamine secretion by chemoreceptor cells of the rabbit carotid body

Producción CientíficaThe pívotal role of íntracellular free [Ca2+] fluctuatíons in the control of cellular functíons such as contraction and secretíon, íncludíng the release of neurotransmítters, was recognized many decades ago (see Rubín, 1982). More recently, the list of cellular functíons tríggered or modulated by the levels of Ca2+¡ has grown enormously. Addítional functíons regulated by [Ca2+)¡ include neuronal excítabílity, synaptic plastícíty, gene ex­ pressíon, cellular metabolísm, cell dívísíon and dífferentíatíon, and programmed cell dead (Míller, 1991; Clapham, 1995). Parallelíng the growth in this líst of Ca2+-controlled func­ tíons, a multíplicity of cellular mechanísms aimed at maintaining resting free [Ca2+)¡ in the range of l 00 nM for most cells has been described, allowing increases in Ca2+¡ levels that are specific in their magnitude, time course and spatial distributíon, accordíng to the cell function activated (Toescu, 1995). Since Ca2+ cannot be metabolized, cells regulate theír cytoplasmic levels of free Ca2+ through numerous bínding proteíns and influx and efflux mechanisms (Fíg 1). Ca2+ ínflux to cell cytoplasm from the extracellular milieu occurs vía voltage or receptor operated channels or vía yet ill-defined capacítatíve pathways; the Na+/Ca 2+ exchanger can also produce in sorne círcumstances net ínflux of Ca2+ (Míller, 1991; Clapham, 1995). Ca2+ ef­ flux to the extracellular space occurs against electrochemical gradíents, and thereby the pumpíng out of Ca2+ is directly (Caz+ pump) or indirectly (Na+/Ca2+) coupled to the hy­ drolysis of ATP

Hydroxycobalamin reveals the involvement of hydrogen sulfide in the hypoxic responses of rat carotid body chemoreceptor cells

Producción CientíficaCarotid body (CB) chemoreceptor cells sense arterial blood PO2, generating a neurosecretory response proportional to the intensity of hypoxia. Hydrogen sulfide (H2S) is a physiological gaseous messenger that is proposed to act as an oxygen sensor in CBs, although this concept remains controversial. In the present study we have used the H2S scavenger and vitamin B12 analog hydroxycobalamin (Cbl) as a new tool to investigate the involvement of endogenous H2S in CB oxygen sensing. We observed that the slow-release sulfide donor GYY4137 elicited catecholamine release from isolated whole carotid bodies, and that Cbl prevented this response. Cbl also abolished the rise in [Ca2+]i evoked by 50 µM NaHS in enzymatically dispersed CB glomus cells. Moreover, Cbl markedly inhibited the catecholamine release and [Ca2+]i rise caused by hypoxia in isolated CBs and dispersed glomus cells, respectively, whereas it did not alter these responses when they were evoked by high [K+]e. The L-type Ca2+ channel blocker nifedipine slightly inhibited the rise in CB chemoreceptor cells [Ca2+]i elicited by sulfide, whilst causing a somewhat larger attenuation of the hypoxia-induced Ca2+ signal. We conclude that Cbl is a useful and specific tool for studying the function of H2S in cells. Based on its effects on the CB chemoreceptor cells we propose that endogenous H2S is an amplifier of the hypoxic transduction cascade which acts mainly by stimulating non-L-type Ca2+ channels.Ministerio de Economía, Industria y Competitividad - Fondo Europeo de Desarrollo Regional (project BFU2015-70616-R)Junta de Castilla y León (project VA106G18)Centro de Investigación Biomédica en Red de Enfermedades Respiratorias . Instituto de Salud Carlos III (project CIBER CB06/06/0050

Evidence for two types of nicotinic receptors in the cat carotid body chemoreceptor cells

Producción CientíficaCurrent concepts on the location and functional significance of nicotinic receptors in the carotid body rest on a-bungarotoxin binding and autoradiographic studies. Using an in vitro preparation of the cat carotid body whose catecholamine deposits have been labeled by prior incubation with the tritiated natural precursor w3Hxtyrosine, we have found that nicotine induces release of w3Hxcatecholamines in a dose-dependent manner IC50s9.81 mM.. We also found that mecamylamine 50 mM. completely abolished the nicotine-induced release, while a-bungarotoxin 100 nM; f20 times its binding Kd. only reduced the release by 56%. These findings indicate that chemoreceptor cells, and perhaps other carotid body structures, contain nicotinic receptors that are not sensitive to a-bungarotoxin and force a revision of the current concepts on cholinergic mechanisms in the carotid body chemoreception

Significance of ROS in oxygen sensing in cell systems with sensitivy to ohysiological 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

Ca2 + Dynamics in chemoreceptor cells: an overview

Producción CientíficaThe carotid body (CB) was defined as a sensory organ by De Castro in 1928. Two years later, Heymanns and coworkers demostrated that the organ was sensitive to alterations in blood gases and pH, in such a way that a decrease in blood P02 or pH or an increase in blood PC02 produced activation of the CB and, reflexely, hyperventilation. De Castro postulated that glomus cells were the sensor structures and that they should release sorn substance to transmit the stimulus to the sensory nerve endings (De Castro, 1928). De Castro's point of view, was widely accepted, and therefore the CB was considered a secondary sensory receptor. As a consequence, the principal aims of many workers in the chemoreception field have been to define the nature of the sensing mechanims ( sensory transduction process ) and to identify the substances released by chern cells

The role of NADPH oxidase in carotid body arterial chemoreceptors

Producción CientíficaO2-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca2+ entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O2-sensitivity of these cells currently focus on the coupling between local PO2 and the open-closed state of K+-channels. Amongst multiple competing hypotheses is the notion that K+-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing PO2. In O2-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that EM and K+-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O2 facilitate cell repolarization via specific subsets of K+-channels