Carotid Body Chemoreceptor Cells Research Articles

The Thirty-Fifth Anniversary of K+ Channels in O2 Sensing: What We Know and What We Don’t Know

On the thirty-fifth anniversary of the first description of O2-sensitive K+ channels in the carotid body chemoreceptors O2 sensing remains a salient issue in the literature. Whereas much has been learned about this subject, important questions such as the identity of the specific K+ channel subtype(s) responsible for O2 sensing by chemoreceptors and the mechanism(s) by which their activities are altered by hypoxia have not yet been definitively answered. O2 sensing is a fundamental biological process necessary for the acute and chronic responses to varying environmental O2 levels which allow organisms to adapt to hypoxia. Whereas chronic responses depend on the modulation of hypoxia-inducible transcription factors which determine the expression of numerous genes encoding enzymes, transporters and growth factors, acute responses rely mainly on the dynamic modulation of ion channels by hypoxia, causing adaptive changes in cell excitability, contractility and secretory activity in specialized tissues. The most widely studied oxygen-sensitive ion channels are potassium channels, but oxygen sensing by members of both the calcium and sodium channel families has also been demonstrated. Given the explosion of information on this topic, in this review, we will focus on the mechanisms of physiological oxygen chemotransduction by PO2-dependent K+ channels, with particular emphasis on their function in carotid body chemoreceptor cells (CBCC) and pulmonary artery smooth muscle cells (PASMC), highlighting areas of consensus and controversy within the field. We will first describe the most well-established concepts, those reproduced in multiple laboratories, and then discuss selected observations or questions that remain unresolved, and that limit our progress in this field.

Immunohistochemical distribution of Ca2+/calmodulin-dependent protein kinase II subunits in the rat carotid body

Carotid body (CB) activity stimulated by a lower partial oxygen pressure in rats is enhanced by exposure to chronic intermittent hypoxia. However, the mechanisms that modulate CB activity remain unclear. In the present study, the expression and distribution of one of the candidate molecules to modulate reactivity, Ca2+/calmodulin-dependent protein kinase II (CaMKII) were examined in the rat CB using reverse transcriptional polymerase chain reaction and immunofluorescence with isoform-specific antibodies. CaMKIIγ and CaMKIIδ were distributed in CB chemoreceptor cells, and exhibited intense immunoreactivity in dopamine β-hydroxylase-positive chemoreceptor cells. CaMKIIβ and CaMKIIγ were distributed in sensory nerve endings attached to chemoreceptor cells of the CB. In the petrosal ganglion, immunoreactivities for CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ were detected in the perinuclear region of ganglion cells. The present results indicate that CaMKIIγ and CaMKIIδ in chemoreceptor cells and CaMKIIβ and CaMKIIγ in sensory nerve endings enhanced reciprocal synaptic transmission, i.e., noradrenaline and ATP for cells to neurons and glutamate for neurons to cells.

TASK inhibition by mild acidosis increases Ca2+ oscillations to mediate pH sensing in rat carotid body chemoreceptor cells.

Severe levels of acidosis (pH < 6.8) have been shown to cause a sustained rise in cytosolic Ca2+ concentration in carotid body Type 1 (glomus) cells. To understand how physiologically relevant levels of acidosis regulate Ca2+ signaling in glomus cells, we studied the effects of small changes in extracellular pH (pHo) on the kinetics of Ca2+ oscillations. A decrease in pHo from 7.4 to 7.3 (designated mild) and 7.2 (designated moderate) acidosis produced significant increases in the frequency and amplitude of Ca2+ oscillations. These effects of acidosis on Ca2+ oscillations were not blocked by NS383 and amiloride [acid-sensing ion channel (ASIC) inhibitors]. Mild and moderate levels of acidosis, however, caused a small but significant inhibition of two-pore domain acid-sensing K+ channels (TASK) (TASK-1- and TASK-3-like channels) and depolarized the cell by 6-13 mV. Acidosis-induced increase in Ca2+ oscillations was inhibited by nifedipine (1 µM; L-type Cav inhibitor) and by TTA-P2 (20 µM; T-type Cav inhibitor). Mild inhibition of TASK activity by N-[(2,4-difluorophenyl)methyl]-2'-[[[2-(4methoxyphenyl)acetyl]amino]methyl][1,1'-biphenyl]-2-carboxamide (A1899) (0.3 µM) and 1-[1-[6-[[1,1'-biphenyl]-4-ylcarbonyl)-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine-4-yl]-4-piperidinyl]-1-butanon (PK-THPP) (0.1 µM) increased Ca2+ oscillation frequency to levels similar to those observed with mild-moderate acidosis. Mild acidosis (pHo 7.3) and mild hypoxia (∼5%O2) produced similar levels of changes in the kinetics of Ca2+ oscillations. Block of tetraethylammonium (TEA)-sensitive Kv channels did not affect acid-induced increase in Ca2+ oscillations. Our study shows that mild and moderate levels of acidosis increase the frequency and amplitude of Ca2+ oscillations primarily by inhibition of TASK without involving ASICs, and suggests a major role of TASK for signal transduction in response to a physiological change in pHo.

Carotid Body-Mediated Chemoreflex Function in Aging and the Role of Receptor-Interacting Protein Kinase.

Ventilatory impairment during aging has been linked to carotid body (CB) dysfunction. Anatomical/morphological studies evidenced CB degeneration and reductions in the number of CB chemoreceptor cells during aging. The mechanism(s) related to CB degeneration in aging remains elusive. Programmed cell death encompasses both apoptosis and necroptosis. Interestingly, necroptosis can be driven by molecular pathways related to low-grade inflammation, one hallmark of the aging process. Accordingly, we hypothesized that necrotic cell death dependent on receptor-interacting protein kinase-3 (RIPK3) may contribute, at least in part, to impair CB function during aging. Adult (3months) and aged (24months) wild type (WT) and RIPK3-/- mice were used to study chemoreflex function. Aging results in significant reductions in both the hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). Adult RIPK3-/- mice showed normal HVR and HCVR compared to adult WT mice. Remarkable, aged RIPK3-/- mice displayed no reductions in HVR nor in HCVR. Indeed, chemoreflex responses obtained in aged RIPK3-/- KO mice were undistinguishable from the ones obtained in adult WT mice. Lastly, we found high prevalence of breathing disorders during aging and this was absent in aged RIPK3-/- mice. Together our results support a role for RIPK3-mediated necroptosis in CB dysfunction during aging.

Neurochemical plasticity of the carotid body in hypertension.

The carotid body (CB), a main peripheral arterial chemoreceptor, has lately been implicated in the pathophysiology of various cardiovascular disorders. Emerging experimental evidence supports a causal relationship between CB dysfunction and augmented sympathetic outflow which is the common hallmark of human sympathetic-related diseases, including essential hypertension. To gain insight into the neurotransmitter profile of chemosensory cells in the hypertensive CB, we examined the expression and cellular localization of some classical neurotransmitters, neuropeptides, and gaseous signaling molecules as well as neurotrophic factors and their receptors in the CB of spontaneously hypertensive rats, a common animal model of hypertension. Our immunohistochemical experiments revealed an elevated catecholamine and serotonin content in the hypertensive CB compared to normotensive controls. GABA immunostaining was seen in some peripherally located glomus cells in the CB of SHR and it was significantly lower than in control animals. The density of substance P and vasoactive intestinal peptide-immunoreactive fibers was diminished whereas that of neuropeptide Y-immunostained nerve fibers was increased and that of calcitonin gene-related peptide-containing fibers remained almost unchanged in the hypertensive CB. We have further demonstrated that in the hypertensive state the production of nitric oxide is impaired and that the components of the neurotrophin signaling system display an abnormal enhanced expression. Our results provide immunohistochemical evidence that the altered transmitter phenotype of CB chemoreceptor cells and the elevated production of neurotrophic factors modulate the chemosensory processing in hypertensive animals which contributes to autonomic dysfunction and elicits sympathetic hyperactivity, consequently leading to elevated blood pressure.

Hydroxycobalamin Reveals the Involvement of Hydrogen Sulfide in the Hypoxic Responses of Rat Carotid Body Chemoreceptor Cells.

Carotid 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.

Properties of Ca2+ oscillations in rat carotid body chemoreceptor cells

Properties of Ca²⁺ oscillations in rat carotid body chemoreceptor cells

High fat diet blunts the effects of leptin on ventilation and on carotid body activity.

Leptin plays a role in the control of breathing, acting mainly on central nervous system; however, leptin receptors have been recently shown to be expressed in the carotid body (CB), and this finding suggests a physiological role for leptin in the regulation of CB function. Leptin increases minute ventilation in both basal and hypoxic conditions in rats. It increases the frequency of carotid sinus nerve discharge in basal conditions, as well as the release of adenosine from the CB. However, in a metabolic syndrome animal model, the effects of leptin in ventilatory control, carotid sinus nerve activity and adenosine release by the CB are blunted. Although leptin may be involved in triggering CB overactivation in initial stages of obesity and dysmetabolism, resistance to leptin signalling and blunting of responses develops in metabolic syndrome animal models. Leptin plays a role in the control of breathing, acting mainly on central nervous system structures. Leptin receptors are expressed in the carotid body (CB) and this finding has been associated with a putative physiological role of leptin in the regulation of CB function. Since, the CBs are implicated in energy metabolism, here we tested the effects of different concentrations of leptin administration on ventilatory parameters and on carotid sinus nerve (CSN) activity in control and high-fat (HF) diet fed rats, in order to clarify the role of leptin in ventilation control in metabolic disease states. We also investigated the expression of leptin receptors and the neurotransmitters involved in leptin signalling in the CBs. We found that in non-disease conditions, leptin increases minute ventilation in both basal and hypoxic conditions. However, in the HF model, the effect of leptin in ventilatory control is blunted. We also observed that HF rats display an increased frequency of CSN discharge in basal conditions that is not altered by leptin, in contrast to what is observed in control animals. Leptin did not modify intracellular Ca2+ in CB chemoreceptor cells, but it produced an increase in the release of adenosine from the whole CB. We conclude that CBs represent an important target for leptin signalling, not only to coordinate peripheral ventilatory chemoreflexive drive, but probably also to modulate metabolic variables. We also concluded that leptin signalling is mediated by adenosine release and that HF diets blunt leptin responses in the CB, compromising ventilatory adaptation.

Topical Application of Connexin43 Hemichannel Blocker Reduces Carotid Body-Mediated Chemoreflex Drive in Rats.

The carotid body (CB) is the main arterial chemoreceptor involved in oxygen sensing. Upon hypoxic stimulation, CB chemoreceptor cells release neurotransmitters, which increase the frequency of action potentials in sensory nerve fibers of the carotid sinus nerve. The identity of the molecular entity responsible for oxygen sensing is still a matter of debate; however several ion channels have been shown to be involved in this process. Connexin-based ion channels are expressed in the CB; however a definitive role for these channels in mediating CB oxygen sensitivity has not been established. To address the role of these channels, we studied the effect of blockers of connexin-based ion channels on oxygen sensitivity of the CB. A connexin43 (Cx43) hemichannel blocking agent (CHBa) was applied topically to the CB and the CB-mediated hypoxic ventilatory response (FiO2 21, 15, 10 and 5%) was measured in adult male Sprague-Dawley rats (~250 g). In normoxic conditions, CHBa had no effect on tidal volume or respiratory rate, however Cx43 hemichannels inhibition by CHBa significantly impaired the CB-mediated chemoreflex response to hypoxia. CHBa reduced both the gain of the hypoxic ventilatory response (HVR) and the maximum HVR by ~25% and ~50%, respectively. Our results suggest that connexin43 hemichannels contribute to the CB chemoreflex response to hypoxia in rats. Our results suggest that CB connexin43 hemichannels may be pharmacological targets in disease conditions characterized by CB hyperactivity.

Purines and Carotid Body: New Roles in Pathological Conditions

It is known that adenosine and adenosine-5′-triphosphate (ATP) are excitatory mediators involved in carotid body (CB) hypoxic signaling. The CBs are peripheral chemoreceptors classically defined by O2, CO2, and pH sensors. When hypoxia activates the CB, it induces the release of neurotransmitters from chemoreceptor cells leading to an increase in the action potentials frequency at the carotid sinus nerve (CSN). This increase in the firing frequency of the CSN is integrated in the brainstem to induce cardiorespiratory compensatory responses. In the last decade several pathologies, as, hypertension, diabetes, obstructive sleep apnea and heart failure have been associated with CB overactivation. In the first section of the present manuscript we review in a concise manner fundamental aspects of purine metabolism. The second section is devoted to the role of purines on the hypoxic response of the CB, providing the state-of-the art for the presence of adenosine and ATP receptors in the CB; for the role of purines at presynaptic level in CB chemoreceptor cells, as well as, its metabolism and regulation; at postsynaptic level in the CSN activity; and on the ventilatory responses to hypoxia. Recently, we have showed that adenosine is involved in CB hypersensitization during chronic intermittent hypoxia (CIH), which mimics obstructive sleep apnea, since caffeine, a non-selective adenosine receptor antagonist that inhibits A2A and A2B adenosine receptors, decreased CSN chemosensory activity in animals subjected to CIH. Apart from this involvement of adenosine in CB sensitization in sleep apnea, it was recently found that P2X3 ATP receptor in the CB contributes to increased chemoreflex hypersensitivity and hypertension in spontaneously hypertension rats. Therefore the last section of this manuscript is devoted to review the recent findings on the role of purines in CB-mediated pathologies as hypertension, diabetes and sleep apnea emphasizing the potential clinical importance of modulating purines levels and action to treat pathologies associated with CB dysfunction.

Endocytosis: another pathway in receptor-Gq-TASK signalling.

TASKs (TASK-1: K2P3.1 and TASK-3: K2P9.1) are expressed in many tissues, but are particularly highly expressed in cerebellar granule neurons, brainstem motor neurons, carotid body chemoreceptor cells and cells of the adrenal gland in the rat. This article is protected by copyright. All rights reserved

Adenosine and dopamine oppositely modulate a hyperpolarization-activated current Ih in chemosensory neurons of the rat carotid body in co-culture.

Adenosine and dopamine (DA) are neuromodulators in the carotid body (CB) chemoafferent pathway, but their mechanisms of action are incompletely understood. Using functional co-cultures of rat CB chemoreceptor (type I) cells and sensory petrosal neurons (PNs), we show that adenosine enhanced a hyperpolarization-activated cation current Ih in chemosensory PNs via A2a receptors, whereas DA had the opposite effect via D2 receptors. Adenosine caused a depolarizing shift in the Ih activation curve and increased firing frequency, whereas DA caused a hyperpolarizing shift in the curve and decreased firing frequency. Acute hypoxia and isohydric hypercapnia depolarized type I cells concomitant with increased excitation of adjacent PNs; the A2a receptor blocker SCH58261 inhibited both type I and PN responses during hypoxia, but only the PN response during isohydric hypercapnia. We propose that adenosine and DA control firing frequency in chemosensory PNs via their opposing actions on Ih . Adenosine and dopamine (DA) act as neurotransmitters or neuromodulators at the carotid body (CB) chemosensory synapse, but their mechanisms of action are not fully understood. Using a functional co-culture model of rat CB chemoreceptor (type I) cell clusters and juxtaposed afferent petrosal neurons (PNs), we tested the hypothesis that adenosine and DA act postsynaptically to modulate a hyperpolarization-activated, cyclic nucleotide-gated (HCN) cation current (Ih ). In whole-cell recordings from hypoxia-responsive PNs, cAMP mimetics enhanced Ih whereas the HCN blocker ZD7288 (2μm) reversibly inhibited Ih . Adenosine caused a potentiation of Ih (EC50 ∼ 35nm) that was sensitive to the A2a blocker SCH58261 (5nm), and an ∼16mV depolarizing shift in V½ for voltage dependence of Ih activation. By contrast, DA (10μm) caused an inhibition of Ih that was sensitive to the D2 blocker sulpiride (1-10μm), and an ∼11mV hyperpolarizing shift in V½ . Sulpiride potentiated Ih in neurons adjacent to, but not distant from, type I cell clusters. DA also decreased PN action potential frequency whereas adenosine had the opposite effect. During simultaneous paired recordings, SCH58261 inhibited both the presynaptic hypoxia-induced receptor potential in type I cells and the postsynaptic PN response. By contrast, SCH58261 inhibited only the postsynaptic PN response induced by isohydric hypercapnia. Confocal immunofluorescence confirmed the localization of HCN4 subunits in tyrosine hydroxylase-positive chemoafferent neurons in tissue sections of rat petrosal ganglia. These data suggest that adenosine and DA, acting through A2a and D2 receptors respectively, regulate PN excitability via their opposing actions on Ih .

Guinea Pig Oxygen-Sensing and Carotid Body Functional Properties.

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

Evidence that 5-HT stimulates intracellular Ca2+ signalling and activates pannexin-1 currents in type II cells of the rat carotid body.

5-HT is a neuromodulator released from carotid body (CB) chemoreceptor (type I) cells and facilitates the sensory discharge following chronic intermittent hypoxia (CIH). In the present study, we show that, in addition to type I cells, adjacent glial-like type II cells express functional, ketanserin-sensitive 5-HT2 receptors, and their stimulation increases cytoplasmic Ca2+ derived from intracellular stores. In type II cells, 5-HT activated a ketanserin-sensitive inward current (I5-HT ) that was similar to that (IUTP ) activated by the P2Y2R agonist, UTP. As previously shown for IUTP , I5-HT was inhibited by BAPTA-AM and carbenoxolone (5μm), a putative blocker of ATP-permeable pannexin (Panx)-1 channels; IUTP was reversibly inhibited by the specific Panx-1 mimetic peptide channel blocker, 10 Panx peptide. Paracrine stimulation of type II cells by 5-HT, leading to ATP release via Panx-1 channels, may contribute to CB excitability, especially in pathophysiological conditions associated with CIH (e.g. obstructive sleep apnoea). Carotid body (CB) chemoreceptor (type I) cells can synthesize and release 5-HT and increased autocrine-paracrine 5-HT2 receptor signalling contributes to sensory long-term facilitation during chronic intermittent hypoxia (CIH). However, recent studies suggest that adjacent glial-like type II cells can respond to CB paracrine signals by elevating intracellular calcium (Δ[Ca2+ ]i ) and activating carbenoxolone-sensitive, ATP-permeable, pannexin (Panx)-1-like channels. In the present study, using dissociated rat CB cultures, we found that 5-HT induced Δ[Ca2+ ]i responses in a subpopulation of type I cells, as well as in most (∼67%) type II cells identified by their sensitivity to the P2Y2 receptor agonist, UTP. The 5-HT-induced Ca2+ response in type II cells was dose-dependent (EC50 ∼183nm) and largely inhibited by the 5-HT2A receptor blocker, ketanserin (1μm), and also arose mainly from intracellular stores. 5-HT also activated an inward current (I5-HT ) in type II cells (EC50 ∼200nm) that was reversibly inhibited by ketanserin (1-10nm), the Ca2+ chelator BAPTA-AM (5μm), and low concentrations of carbenoxolone (5μm), a putative Panx-1 channel blocker. I5-HT reversed direction at approximately -11mV and was indistinguishable from the UTP-activated current (IUTP ). Consistent with a role for Panx-1 channels, IUTP was reversibly inhibited by the specific Panx-1 mimetic peptide blocker 10 Panx (100μm), although not by its scrambled control peptide (sc Panx). Because ATP is an excitatory CB neurotransmitter, it is possible that the contribution of enhanced 5-HT signalling to the increased sensory discharge during CIH may occur, in part, by a boosting of ATP release from type II cells via Panx-1 channels.

Purinergic signalling mediates bidirectional crosstalk between chemoreceptor typeI and glial-like typeII cells of the rat carotid body.

Carotid body chemoreceptors are organized in clusters containing receptor type I and contiguous glial-like type II cells. While type I cells depolarize and release ATP during chemostimulation, the role of type II cells which express purinergic P2Y2 receptors (P2Y2Rs) and ATP-permeable pannexin-1 (Panx-1) channels, is unclear. Here, we show that in isolated rat chemoreceptor clusters, type I cell depolarization induced by hypoxia, hypercapnia, or high K(+) caused delayed intracellular Ca(2+) elevations (Δ[Ca(2+)]i) in nearby type II cells that were inhibited by the P2Y2R blocker suramin, or by the nucleoside hydrolase apyrase. Likewise, stimulation of P2Y2Rs on type II cells caused a delayed, secondary Δ[Ca(2+)]i in nearby type I cells that was inhibited by blockers of Panx-1 channels, adenosine A2A receptors and 5'-ectonucleotidase. We propose that reciprocal crosstalk between type I and type II cells contributes to sensory processing in the carotid body via purinergic signalling pathways. The mammalian carotid body (CB) is excited by blood-borne stimuli including hypoxia and acid hypercapnia, leading to respiratory and cardiovascular reflex responses. This chemosensory organ consists of innervated clusters of receptor type I cells, ensheathed by processes of adjacent glial-like type II cells. ATP is a major excitatory neurotransmitter released from type I cells and type II cells express purinergic P2Y2 receptors (P2Y2Rs), the activation of which leads to the opening of ATP-permeable, pannexin-1 (Panx-1) channels. While these properties support crosstalk between type I and type II cells during chemotransduction, direct evidence is lacking. To address this, we first exposed isolated rat chemoreceptor clusters to acute hypoxia, isohydric hypercapnia, or the depolarizing stimulus high K(+), and monitored intracellular [Ca(2+)] using Fura-2. As expected, these stimuli induced intracellular [Ca(2+)] elevations (Δ[Ca(2+)]i) in type I cells. Interestingly, however, there was often a delayed, secondary Δ[Ca(2+)]i in nearby type II cells that was reversibly inhibited by the P2Y2R antagonist suramin, or by the nucleoside hydrolase apyrase. By contrast, type II cell stimulation with the P2Y2R agonist uridine-5'-triphosphate (100 μm) often led to a delayed, secondary Δ[Ca(2+)]i response in nearby type I cells that was reversibly inhibited by the Panx-1 blocker carbenoxolone (5 μm). This Δ[Ca(2+)]i response was also strongly inhibited by blockers of either the adenosine A2A receptor (SCH 58261) or of the 5'-ectonucleotidase (AOPCP), suggesting it was due to adenosine arising from breakdown of ATP released through Panx-1 channels. Collectively, these data strongly suggest that purinergic signalling mechanisms mediate crosstalk between CB chemoreceptor and glial cells during chemotransduction.

Hypoxic pulmonary vasoconstriction, carotid body function and erythropoietin production in adult rats perinatally exposed to hyperoxia.

Adult animals that have been perinatally exposed to oxygen-rich atmospheres (hyperoxia), recalling those used for oxygen therapy in infants, exhibit a loss of hypoxic pulmonary vasoconstriction, whereas vasoconstriction elicited by depolarizing agents is maintained. Loss of pulmonary hypoxic vasoconstriction is not linked to alterations in oxygen-sensitive K(+) currents in pulmonary artery smooth muscle cells. Loss of hypoxic vasoconstriction is associated with early postnatal oxidative damage and corrected by an antioxidant diet. Perinatal hyperoxia damages carotid body chemoreceptor cell function and the antioxidant diet does not reverse it. The hypoxia-elicited increase in erythropoietin plasma levels is not affected by perinatal hyperoxia. The potential clinical significance of the findings in clinical situations such as pneumonia, chronic obstructive pulmonary disease or general anaesthesia is considered. Adult mammalians possess three cell systems that are activated by acute bodily hypoxia: pulmonary artery smooth muscle cells (PASMC), carotid body chemoreceptor cells (CBCC) and erythropoietin (EPO)-producing cells. In rats, chronic perinatal hyperoxia causes permanent carotid body (CB) atrophy and functional alterations of surviving CBCC. There are no studies on PASMC or EPO-producing cells. Our aim is to define possible long-lasting functional changes in PASMC or EPO-producing cells (measured as EPO plasma levels) and, further, to analyse CBCC functional alterations. We used 3- to 4-month-old rats born and reared in a normal atmosphere or exposed to perinatal hyperoxia (55-60% O2 for the last 5-6days of pregnancy and 4weeks after birth). Perinatal hyperoxia causes an almost complete loss of hypoxic pulmonary vasoconstriction (HPV), which was correlated with lung oxidative status in early postnatal life and prevented by antioxidant supplementation in the diet. O2 -sensitivity of K(+) currents in the PASMC of hyperoxic animals is normal, indicating that their inhibition is not sufficient to trigger HPV. Perinatal hyperoxia also abrogated responses elicited by hypoxia on catecholamine and cAMP metabolism in the CB. An increase in EPO plasma levels elicited by hypoxia was identical in hyperoxic and control animals, implying a normal functioning of EPO-producing cells. The loss of HPV observed in adult rats and caused by perinatal hyperoxia, comparable to oxygen therapy in premature infants, might represent a previously unrecognized complication of such a medical intervention capable of aggravating medical conditions such as regional pneumonias, atelectases or general anaesthesia in adult life.

Hydrogen sulfide and hypoxia-induced changes in TASK (K2P3/9) activity and intracellular Ca2+ concentration in rat carotid body glomus cells

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that hydrogen sulfide (H2S) may serve as an oxygen sensor/signal in the carotid body during acute hypoxia. To further test such a role for H2S, we studied the effects of H2S on the activity of TASK channel and [Ca2+]i, which are considered important for mediating the glomus cell response to hypoxia. Like hypoxia, NaHS (a H2S donor) inhibited TASK activity and elevated [Ca2+]i. To inhibit the production of H2S, glomus cells were incubated (3h) with inhibitors of cystathionine-β-synthase and cystathionine-γ-lyase (dl-propargylglycine, aminooxyacetic acid, β-cyano-l-alanine; 0.3mM). SF7 fluorescence was used to assess the level of H2S production. The inhibitors blocked L-cysteine- and hypoxia-induced elevation of SF7 fluorescence intensity. In cells treated with the inhibitors, hypoxia produced an inhibition of TASK activity and a rise in [Ca2+]i, similar in magnitude to those observed in control cells. L-cysteine produced no effect on TASK activity or [Ca2+]i and did not affect hypoxia-induced inhibition of TASK and elevation of [Ca2+]i. These findings suggest that under normal conditions, H2S is not a major signal in hypoxia-induced modulation of TASK channels and [Ca2+]i in isolated glomus cells.

Hydrogen Sulfide and Hypoxia‐Induced Changes in K2P3/9 (TASK) current and [Ca2+]i in Rat Carotid Body Glomus Cells

Acute hypoxia depolarizes carotid body chemoreceptor (glomus) cells and elevates intracellular Ca2+ concentration ([Ca2+]i). Recent studies suggest that hydrogen sulfide (H2S) may serve as an oxygen sensor/signal in the carotid body during acute hypoxia. To further test such a role for H2S, we studied the effects of NaHS (a H2S donor) on the activity of TASK channel and intracellular [Ca2+]i, which are considered crucial for mediating the hypoxic response. Like hypoxia, NaHS inhibited TASK activity and elevated [Ca2+]i. To block the production of H2S, glomus cells were incubated (3 hr) with DL‐propargylglycine, aminooxyacetic acid, beta‐cyano‐L‐alanine (0.3 mM) that inhibit cystathionine‐beta‐synthase and cystathionine‐gamma‐lyase. Cell H2S production induced by L‐cysteine and hypoxia was strongly reduced by the inhibitors, as assessed using SF‐7 fluorescence. In cells treated with inhibitors, the hypoxia‐induced reduction of TASK activity and elevation of [Ca2+]i were similar in magnitude to those observed in untreated cells. In control or inhibitor‐treated cells, BK channel was not open at rest. These findings suggest that H2S does not mediate the hypoxia‐induced changes in TASK activity and [Ca2+]i in rat glomus cells.

Coexpression of Galanin and Nestin in the Chemoreceptor Cells of the Human Carotid Body.

The carotid body is a highly specialized chemoreceptive organ of neural crest origin whose role is to detect changes in arterial oxygen content. The sensory units are the chemoreceptor cells, which are neuronal-like cells, surrounded by sustentacular or glial-like cells. It is suggested that the carotid body contains self-renewing multipotent stem cells, which are putatively represented by glial-like sustentacular cells. The mechanisms of renewal of neuronal-like cells are unclear. Recently, we have demonstrated the expression of galanin, a peptide promoting neurogenesis, in chemoreceptor cells in the human CB. Thus, in the present study we seek to determine whether galanin expression in chemoreceptor cells could be matched with that of nestin, a peptide that is a marker of multipotent neural stem cells, or rather with the glial fibrillary acidic protein (GFAP), a marker for glial cells. The latter would underscore the pluasibly essential role of sustentacular cells in the self-renewal capability of chemorecetors. We found that galanin expression is matched with nestin in chemoreceptor cells of the human carotid body, but not with that of GFAP. Thus, galanin expression in chemoreceptor cells could provide a signal for neurogenesis and chemoreceptor cell differentiation in the carotid body.

Role of K₂p channels in stimulus-secretion coupling.

Two-pore domain K(+) (K2P) channels are involved in a variety of physiological processes by virtue of their high basal activity and sensitivity to various biological stimuli. One of these processes is secretion of hormones and transmitters in response to stimuli such as hypoxia, acidosis, and receptor agonists. The rise in intracellular [Ca(2+)] ([Ca(2+)]i) that is critical for the secretory event can be achieved by several mechanisms: (a) inhibition of resting (background) K(+) channels, (b) activation of Na(+)/Ca(2+)-permeable channels, and (c) release of Ca(2+) from intracellular stores. Here, we discuss the role of TASK and TREK in stimulus-secretion mechanisms in carotid body chemoreceptor cells and adrenal medullary/cortical cells. Studies show that stimuli such as hypoxia and acidosis cause cell depolarization and transmitter/hormone secretion by inhibition of TASK or TREK. Subsequent elevation of [Ca(2+)]i produced by opening of voltage-dependent Ca(2+) channels then activates a Na(+)-permeable cation channel, presumably to help sustain the depolarization and [Ca(2+)]i. Agonists such as angiotensin II may elevate [Ca(2+)]i via multiple mechanisms involving both inhibition of TASK/TREK and Ca(2+) release from internal stores to cause aldosterone secretion. Thus, inhibition of resting (background) K(+) channels and subsequent activation of voltage-gated Ca(2+) channels and Na(+)-permeable non-selective cation channels may be a common ionic mechanism that lead to hormone and transmitter secretion.