Verónica A. Campanucci

A novel O2-sensing mechanism in rat glossopharyngeal neurones mediated by a halothane-inhibitable background K+ conductance

Modulation of K+ channels by hypoxia is a common O2‐sensing mechanism in specialised cells. More recently, acid‐sensitive TASK‐like background K+ channels, which play a key role in setting the resting membrane potential, have been implicated in O2‐sensing in certain cell types. Here, we report a novel O2 sensitivity mediated by a weakly pH‐sensitive background K+ conductance in nitric oxide synthase (NOS)‐positive neurones of the glossopharyngeal nerve (GPN). This conductance was insensitive to 30 mm TEA, 5 mm 4‐aminopyridine (4‐AP) and 200 μm Cd2+, but was reversibly inhibited by hypoxia (O2 tension (PO2) = 15 mmHg), 2–5 mm halothane, 10 mm barium and 1 mm quinidine. Notably, the presence of halothane occluded the inhibitory effect of hypoxia. Under current clamp, these agents depolarised GPN neurones. In contrast, arachidonic acid (5–10 μm) caused membrane hyperpolarisation and potentiation of the background K+ current. This pharmacological profile suggests the O2‐sensitive conductance in GPN neurones is mediated by a class of background K+ channels different from the TASK family; it appears more closely related to the THIK (tandem pore domain halothane‐inhibited K+) subfamily, or may represent a new member of the background K+ family. Since GPN neurones are thought to provide NO‐mediated efferent inhibition of the carotid body (CB), these channels may contribute to the regulation of breathing during hypoxia via negative feedback control of CB function, as well as to the inhibitory effect of volatile anaesthetics (e.g. halothane) on respiration.

Expression of Multiple P2X Receptors by Glossopharyngeal Neurons Projecting to Rat Carotid Body O2-Chemoreceptors: Role in Nitric Oxide-Mediated Efferent Inhibition

In mammals, ventilation is peripherally controlled by the carotid body (CB), which receives afferent innervation from the petrosal ganglion and efferent innervation from neurons located along the glossopharyngeal nerve (GPN). GPN neurons give rise to the “efferent inhibitory” pathway via a plexus of neuronal nitric oxide (NO) synthase-positive fibers, believed to be responsible for CB chemoreceptor inhibition via NO release. Although NO is elevated during natural CB stimulation by hypoxia, the underlying mechanisms are unclear. We hypothesized that ATP, released by rat CB chemoreceptors (type 1 cells) and/or red blood cells during hypoxia, may directly activate GPN neurons and contribute to NO-mediated inhibition. Using combined electrophysiological, molecular, and confocal immunofluorescence techniques, we detected the expression of multiple P2X receptors in GPN neurons. These receptors involve at least four different purinergic subunits: P2X2 [and the splice variant P2X2(b)], P2X3, P2X4, and P2X7. Using a novel coculture preparation of CB type I cell clusters and GPN neurons, we tested the role of P2X signaling on CB function. In cocultures, fast application of ATP, or its synthetic analog 2′,3′-O-(4 benzoylbenzoyl)-ATP, caused type I cell hyperpolarization that was prevented in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium. These data suggest that ATP released during hypoxic stress from CB chemoreceptors (and/or red blood cells) will cause GPN neuron depolarization mediated by multiple P2X receptors. Activation of this pathway will lead to calcium influx and efferent inhibition of CB chemoreceptors via NO synthesis and consequent release.

Mitochondrial Reactive Oxygen Species Inactivate Neuronal Nicotinic Acetylcholine Receptors and Induce Long-Term Depression of Fast Nicotinic Synaptic Transmission

Neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels implicated in a variety of cognitive, motor, and sensory behaviours, are targeted to compartments rich in mitochondria, particularly postsynaptic domains and presynaptic terminals, exposing these receptors to reactive oxygen species (ROS) generated by oxidative phosphorylation. In addition, these receptors can become exposed to ROS during the progression of certain neurodegenerative diseases. Because ROS are known to modify several membrane proteins, including some types of ion channels, it raises the question of whether elevations in cytosolic ROS alter the function of nAChRs. To address this, we elevated ROS in cultured sympathetic neurons, directly by perfusing neurons intracellularly with ROS, indirectly by blocking the mitochondrial electron transport chain, or noninvasively by transient NGF removal; we then simultaneously measured changes in cytosolic ROS levels and whole-cell ACh-evoked currents. In addition, we elevated cytosolic ROS in postganglionic neurons in intact ganglia and measured changes in nerve-evoked EPSPs. Our experiments indicate that mild elevations in cytosolic ROS, including that produced by transient interruption of NGF signaling, induce a use-dependent, long-lasting rundown of ACh-evoked currents on cultured sympathetic neurons and a long-lasting depression of fast nerve-evoked EPSPs. We show that these effects of cytosolic ROS are specific to nAChRs on neurons and do not cause rundown of ACh-evoked currents on muscle. Our results demonstrate that elevations in cytosolic ROS inactivate neuronal nAChRs in a use-dependent manner and suggest that mild oxidative stress impairs mechanisms mediated by cholinergic nicotinic signaling at neuronal–neuronal synapses.

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea.

Significance Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease caused by the failure to clear bacteria. Bacterial clearance depends on a layer of airway surface liquid (ASL) covering the airways, rich in antimicrobial compounds and mucins, that removes bacteria from the airway through mucociliary clearance. This study provides the first demonstration that inhalation of bacteria triggers CFTR-dependent ASL secretion. We suggest that this response to inhaled pathogens is an important but previously unknown part of the innate immune response that would be missing in CF patients, resulting in reduced bacterial killing and facilitating infection. Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation.

O2-sensing mechanisms in efferent neurons to the rat carotid body.

02-sensing by mammalian cells is a fundamental process that is important for adaptation to variable physiological situations. The mammalian carotid bodies (CB) are peripheral chemoreceptors which monitor blood levels of Po2, Pco2and pH (Gonzalezet al., 1994; Prabhakar, 2000; Lopez-Barneoet al., 2001). As a result they can initiate or modify respiratory reflexes in order to maintain P02homeostasis. Inhibition of K+channels appears to be an important step in CB chemotransduction during hypoxia (Peers, 1997; Lopez-Barneoet al., 2001). It is known that under certain conditions the responses of the CB to specific stimuli can be altered. Chemoreceptor inhibition is the mechanism by which the CB responses are reduced during chemical stimulation, leading to an inhibition of chemoreceptor discharge. Nitric oxide (NO) has been implicated as an important neurotransmitter in the efferent inhibition of rat CB chemoreceptors during hypoxia (Wanget al. 1993; 1994a & b; 1995a & b; Ho hleret al. 1994). An extensive plexus of NO synthase (NOS)-containing nerve fibers projects to the CB and is formed by sensory fibers from the petrosal ganglion and autonomic fibers from neurons located near the junction of the glossopharyngeal (GPN) and carotid sinus (CSN) nerves (WangeM/., 1993).

Purinergic stimulation of carotid body efferent glossopharyngeal neurones increases intracellular Ca2+ and nitric oxide production

•  What is the central question of this study? Nitric oxide is a strong modulator of carotid body (CB) chemoreceptor activity; however, the mechanisms leading to the production/release of NO during CB chemoexcitation remained largely unknown. •  What is the main finding and what is its importance? In the present study, we identified an important contribution of ATP and purinergic P2X receptor signalling in initiating the events leading to NO synthesis and release in efferent neurons to the CB. Acting via P2X receptors, ATP causes an increase in intracellular Ca2+ that triggers NO production in efferent neurones to the rat carotid body. In addition, ATP‐induced depolarization of these neurones activates voltage‐gated Ca2+ channels that further amplify the NO inhibitory signal.

RAGE mediates the inactivation of nAChRs in sympathetic neurons under high glucose conditions

Autonomic dysfunction is a serious complication of diabetes and can lead to cardiovascular abnormalities and premature death. It was recently proposed that autonomic dysfunction is triggered by oxidation‐mediated inactivation of neuronal nicotinic acetylcholine receptors (nAChRs), impairing synaptic transmission in sympathetic ganglia and resulting in autonomic failure. We investigated whether the receptor for advanced glycation end products (RAGE) and its role in the generation of reactive oxygen species (ROS) could be contributing to the events that initiate sympathetic malfunction under high glucose conditions. Using biochemical, live imaging and electrophysiological tools we demonstrated that exposure of sympathetic neurons to high glucose increases RAGE expression and oxidative markers, and that incubation with RAGE ligands (e.g. AGEs, S100 and HMGB1) mimics both ROS elevation and nAChR inactivation. In contrast, co‐treatment with either antioxidants or an anti‐RAGE IgG prevented the inactivation of nAChRs. Lastly, a role for RAGE in this context was corroborated by the lack of sensitivity of sympathetic neurons from RAGE knock‐out mice to high glucose. These data define a pivotal role for RAGE in initiating the events associated with exposure of sympathetic neurons to high glucose, and strongly support RAGE signaling as a potential therapeutic target in the autonomic complications associated with diabetes.

Cystic fibrosis swine fail to secrete airway surface liquid in response to inhalation of pathogens

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel, which can result in chronic lung disease. The sequence of events leading to lung disease is not fully understood but recent data show that the critical pathogenic event is the loss of the ability to clear bacteria due to abnormal airway surface liquid secretion (ASL). However, whether the inhalation of bacteria triggers ASL secretion and whether this is abnormal in cystic fibrosis has never been tested. Here we show, using a novel synchrotron-based in vivo imaging technique, that wild-type pigs display both a basal and a Toll-like receptor-mediated ASL secretory response to the inhalation of cystic fibrosis relevant bacteria. Both mechanisms fail in CFTR−/− swine, suggesting that cystic fibrosis airways do not respond to inhaled pathogens, thus favoring infection and inflammation that may eventually lead to tissue remodeling and respiratory disease.Cystic fibrosis is caused by mutations in the CFTR chloride channel, leading to reduced airway surface liquid secretion. Here the authors show that exposure to bacteria triggers secretion in wild-type but not in pig models of cystic fibrosis, suggesting an impaired response to pathogens contributes to infection.

RAGE-dependent potentiation of TRPV1 currents in sensory neurons exposed to high glucose

Diabetes mellitus is associated with sensory abnormalities, including exacerbated responses to painful (hyperalgesia) or non-painful (allodynia) stimuli. These abnormalities are symptoms of diabetic peripheral neuropathy (DPN), which is the most common complication that affects approximately 50% of diabetic patients. Yet, the underlying mechanisms linking hyperglycemia and symptoms of DPN remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) channel plays a central role in such sensory abnormalities and shows elevated expression levels in animal models of diabetes. Here, we investigated the function of TRPV1 channels in sensory neurons cultured from the dorsal root ganglion (DRG) of neonatal mice, under control (5mM) and high glucose (25mM) conditions. After maintaining DRG neurons in high glucose for 1 week, we observed a significant increase in capsaicin (CAP)-evoked currents and CAP-evoked depolarizations, independent of TRPV1 channel expression. These functional changes were largely dependent on the expression of the receptor for Advanced Glycation End-products (RAGE), calcium influx, cytoplasmic ROS accumulation, PKC, and Src kinase activity. Like cultured neurons from neonates, mature neurons from adult mice also displayed a similar potentiation of CAP-evoked currents in the high glucose condition. Taken together, our data demonstrate that under the diabetic condition, DRG neurons are directly affected by elevated levels of glucose, independent of vascular or glial signals, and dependent on RAGE expression. These early cellular and molecular changes to sensory neurons in vitro are potential mechanisms that might contribute to sensory abnormalities that can occur in the very early stages of diabetes.

Autonomic Nervous System: Carotid Body and Chemoception

Mammalian carotid bodies are small, bilateral organs that detect chemical signals (e.g., O2, CO2, pH, and glucose) in blood and initiate reflexes so as to maintain homeostasis. They are richly vascularized and contain clusters of endocrine-like receptor cells, linked by chemical and electrical synapses. These cells respond to chemical signals by releasing neurotransmitters, including ATP, that excite apposed sensory (afferent) nerve endings. Afferent nerve activity is modulated locally by released autocrine–paracrine mediators and by efferent autonomic nerves that release nitric oxide. This activity ultimately influences brain stem neurons that control a variety of vital respiratory, cardiovascular, and neuroendocrine functions.