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Background: The purpose of this study was to investigate the effects of halothane and sevoflurane on the magnitude of the increase in intracellular calcium with hypoxia in carotid body type I (glomus) cells. We wished to ascertain if the effects of these agents in single cells paralleled their known effects on the human hypoxic ventilatory response, where halothane depresses this response more than does sevoflurane.Methods: We studied single glomus cells from neonatal rat carotid bodies. Halothane and sevoflurane were administered in concentrations ranging from 0.18 to 1.45 and 0.1 to 2 minimum alveolar concentration (MAC), respectively (rat values). The intracellular calcium response to a approximately 90 s period of hypoxia was measured using indo-1 dye. We also assessed if these agents influenced the calcium response to exposure to potassium 100 mM.Results: Halothane depressed the increase in intracellular calcium with hypoxia more than did sevoflurane (P=0.036). Although halothane was depressive at all concentrations tested, sevoflurane depressed the calcium response only at 2 MAC. Both agents also depressed the calcium response to elevated extracellular potassium-halothane more so than sevoflurane (P=0.004).Conclusions: The actions of the agents in single cells reflect their known influence on human hypoxic ventilatory response, consistent with the notion that the cellular process underlies the whole-body effect. The responses to elevated extracellular potassium, which depolarizes the cell membrane, indicate that (in addition to molecular mechanisms previously proposed), voltage-activated calcium channels may also be involved in the anaesthetic effect. [ABSTRACT FROM AUTHOR]

The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia-inducible transcription factor (HIF), HIF-2α, as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2α, including isolated overexpression of HIF-2α but not HIF-1α, is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems.

Background and Aims: There is a lack of basic science data on the effect of dexmedetomidine on the hypoxic chemosensory reflex with both depression and stimulation suggested. The primary aim of this study was to assess if dexmedetomidine inhibited the cellular response to hypoxia in rat carotid body glomus cells, the cells of the organs mediating acute hypoxic ventilatory response (AHVR). Additionally, we used a small sample of mice to assess if there was any large influence of subsedative doses of dexmedetomidine on AHVR., Material and Methods: In the primary study, glomus cells isolated from neonatal rats were used to study the effect of 0.1 nM ( n = 9) and 1 nM ( n = 13) dexmedetomidine on hypoxia-elicited intracellular calcium [Ca 2% ]i influx using ratiometric fluorimetry. Secondarily, whole animal unrestrained plethysmography was used to study AHVR in a total of 8 age-matched C57BL6 mice, divided on successive days into two groups of four mice randomly assigned to receive sub-sedative doses of 5, 50, or 500 μg.kg -1 dexmedetomidine versus control in a crossover study design (total n = 12 exposures to drug with n = 12 controls)., Results: There was no effect of dexmedetomidine on the hypoxia-elicited increase in [Ca 2% ]i in glomus cells (a mean ± SEM increase of 95 ± 32 nM from baseline with control hypoxia, 124 ± 41 nM with 0.1 nM dexmedetomidine; P = 0.514). In intact mice, dexmedetomidine had no effect on baseline ventilation during air-breathing (4.01 ± 0.3 ml.g -1 .min -1 in control and 2.99 ± 0.5 ml.g -1 .min -1 with 500 μg.kg -1 dexmedetomidine, the highest dose; P = 0.081) or on AHVR (136 ± 19% increase from baseline in control, 152 ± 46% with 500 μg.kg -1 dexmedetomidine, the highest dose; P = 0.536)., Conclusion: Dexmedetomidine had no effect on the cellular responses to hypoxia. We conclude that it unlikely acts via inhibition of oxygen sensing at the glomus cell. The respiratory chemoreflex effects of this drug remain an open question. In our small sample of intact mice, hypoxic chemoreflex responses and basal breathing were preserved., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Anaesthesiology Clinical Pharmacology.)

Background: The degree to which different volatile anesthetics depress carotid body hypoxic response relates to their ability to activate TASK potassium channels. Most commonly, volatile anesthetic pairs act additively at their molecular targets. We examined whether this applied to carotid body TASK channels., Methods: We studied halothane and isoflurane effects on hypoxia-evoked rise in intracellular calcium (Ca2+i, using the indicator Indo-1) in isolated neonatal rat glomus cells, and TASK single-channel activity (patch clamping) in native glomus cells and HEK293 cell line cells transiently expressing TASK-1., Results: Halothane (5%) depressed glomus cell Ca2+i hypoxic response (mean ± SD, 94 ± 4% depression; P < 0.001 vs. control). Isoflurane (5%) had a less pronounced effect (53 ± 10% depression; P < 0.001 vs. halothane). A mix of 3% isoflurane/1.5% halothane depressed cell Ca2+i response (51 ± 17% depression) to a lesser degree than 1.5% halothane alone (79 ± 15%; P = 0.001), but similar to 3% isoflurane alone (44 ± 22%; P = 0.224), indicating subadditivity. Halothane and isoflurane increased glomus cell TASK-1/TASK-3 activity, but mixes had a lesser effect than that seen with halothane alone: 4% halothane/4% isoflurane yielded channel open probabilities 127 ± 55% above control, versus 226 ± 12% for 4% halothane alone (P = 0.009). Finally, in HEK293 cell line cells, progressively adding isoflurane (1.5 to 5%) to halothane (2.5%) reduced TASK-1 channel activity from 120 ± 38% above control, to 88 ± 48% (P = 0.034)., Conclusions: In all three experimental models, the effects of isoflurane and halothane combinations were quantitatively consistent with the modeling of weak and strong agonists competing at a common receptor on the TASK channel., (Copyright © 2020, the American Society of Anesthesiologists, Inc. All Rights Reserved.)

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1β, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1β. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

In humans the intravenous anaesthetic propofol depresses ventilatory responses to hypoxia and CO 2 . Animal studies suggest that this may in part be due to inhibition of synaptic transmission between chemoreceptor glomus cells of the carotid body and the afferent carotid sinus nerve. It is however unknown if propofol can also act directly on the glomus cell. Here we report that propofol can indeed inhibit intracellular Ca 2+ responses to hypoxia and hypercapnia in isolated rat glomus cells. Neither this propofol effect, nor the glomus cell response to hypoxia in the absence of propofol, were influenced by GABA receptor activation (using GABA, muscimol and baclofen) or inhibition (using bicuculline and 5-aminovaleric acid). Suggesting that these effects of propofol are not mediated through GABA receptors. Propofol inhibited calcium responses to nicotine in glomus cells but the nicotinic antagonists vecuronium and methyllycaconitine did not inhibit calcium responses to hypoxia. TASK channel activity was not altered by propofol. The glomus cell Ca 2+ response to depolarisation with 30 mM K + was however modestly inhibited by propofol. In summary we conclude that propofol does have a direct effect upon hypoxia signalling in isolated type-1 cells and that this may be partially due to its ability to inhibit voltage gated Ca 2+ v channels. We also note that propofol has the capacity to supress glomus cell excitation via nicotinic receptors and may therefore also interfere with paracrine/autocrine cholinergic signalling in the intact organ. The effects of propofol on chemoreceptor function are however clearly complex and require further investigation., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Sensing of hypoxia and acidosis in arterial chemoreceptors is thought to be mediated through the inhibition of TASK and possibly other (e.g., BK C a ) potassium channels which leads to membrane depolarization, voltage-gated Ca-entry, and neurosecretion. Here, we investigate the effects of pharmacological inhibitors on TASK channel activity and [Ca 2+ ] i -signaling in isolated neonatal rat type-1 cells. PK-THPP inhibited TASK channel activity in cell attached patches by up to 90% (at 400 nmol/L). A1899 inhibited TASK channel activity by 35% at 400 nmol/L. PK-THPP, A1899 and Ml 365 all evoked a rapid increase in type-1 cell [Ca 2+ ] i . These [Ca 2+ ] i responses were abolished in Ca 2+ -free solution and greatly attenuated by Ni 2+ (2 mM) suggesting that depolarization and voltage-gated Ca 2+ -entry mediated the rise in [Ca 2+ ] i. Doxapram (50 μmol/L), a respiratory stimulant, also inhibited type-1 cell TASK channel activity and increased [Ca 2+ ] i. . We also tested the effects of combined inhibition of BK C a and TASK channels. TEA (5 mmol/L) slightly increased [Ca 2+ ] i in the presence of PK-THPP and A1899. Paxilline (300 nM) and iberiotoxin (50 nmol/L) also slightly increased [Ca 2+ ] i in the presence of A1899 but not in the presence of PK-THPP. In general [Ca 2+ ] i responses to TASK inhibitors, alone or in combination with BK C a inhibitors, were smaller than the [Ca 2+ ] i responses evoked by hypoxia. These data confirm that TASK channel inhibition is capable of evoking membrane depolarization and robust voltage-gated Ca 2+ -entry but suggest that this, even with concomitant inhibition of BK C a channels, may be insufficient to account fully for the [Ca 2+ ] i -response to hypoxia., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Key Points: The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia. Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses. Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia. Inactivation of HIF-2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia. Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma-like carotid bodies. These changes, similar to those observed in hypoxia, are dependent on HIF-2α. Taken together, these findings demonstrate a key role for the PHD2-HIF-2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body., Abstract: The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests a rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialized chemoreceptive functions of that organ and, potentially, to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10% oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to (or from) that lineage. Inactivation of a specific HIF isoform, HIF-2α, in the Type I cells was associated with a greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multilineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF-2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF-2α) within Type I cells of the carotid body with respect to the oxygen sensing and adaptive functions of that organ., (© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Cardiovascular disease is the most prevalent age-related illness worldwide, causing approximately 15 million deaths every year. Hypertension is central in determining cardiovascular risk and is a strong predictive indicator of morbidity and mortality; however, there remains an unmet clinical need for disease-modifying and prophylactic interventions. Enhanced sympathetic activity is a well-established contributor to the pathophysiology of hypertension, however the cellular and molecular changes that increase sympathetic neurotransmission are not known. The aim of this study was to identify key changes in the transcriptome in normotensive and spontaneously hypertensive rats. We validated 15 of our top-scoring genes using qRT-PCR, and network and enrichment analyses suggest that glutamatergic signalling plays a key role in modulating Ca 2+ balance within these ganglia. Additionally, phosphodiesterase activity was found to be altered in stellates obtained from the hypertensive rat, suggesting that impaired cyclic nucleotide signalling may contribute to disturbed Ca 2+ homeostasis and sympathetic hyperactivity in hypertension. We have also confirmed the presence of these transcripts in human donor stellate samples, suggesting that key genes coupled to neurotransmission are conserved. The data described here may provide novel targets for future interventions aimed at treating sympathetic hyperactivity associated with cardiovascular disease and other dysautonomias.

Single or combinatorial administration of β-blockers is a mainstay treatment strategy for conditions caused by sympathetic overactivity. Conventional wisdom suggests that the main beneficial effect of β-blockers includes resensitization and restoration of β1-adrenergic signaling pathways in the myocardium, improvements in cardiomyocyte contractility, and reversal of ventricular sensitization. However, emerging evidence indicates that another beneficial effect of β-blockers in disease may reside in sympathetic neurons. We investigated whether β-adrenoceptors are present on postganglionic sympathetic neurons and facilitate neurotransmission in a feed-forward manner. Using a combination of immunocytochemistry, RNA sequencing, Förster resonance energy transfer, and intracellular Ca 2+ imaging, we demonstrate the presence of β-adrenoceptors on presynaptic sympathetic neurons in both human and rat stellate ganglia. In diseased neurons from the prehypertensive rat, there was enhanced β-adrenoceptor-mediated signaling predominantly via β 2 -adrenoceptor activation. Moreover, in human and rat neurons, we identified the presence of the epinephrine-synthesizing enzyme PNMT (phenylethanolamine-N-methyltransferase). Using high-pressure liquid chromatography with electrochemical detection, we measured greater epinephrine content and evoked release from the prehypertensive rat cardiac-stellate ganglia. We conclude that neurotransmitter switching resulting in enhanced epinephrine release, may provide presynaptic positive feedback on β-adrenoceptors to promote further release, that leads to greater postsynaptic excitability in disease, before increases in arterial blood pressure. Targeting neuronal β-adrenoceptor downstream signaling could provide therapeutic opportunity to minimize end-organ damage caused by sympathetic overactivity., (© 2018 The Authors.)

Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia-inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF-1α and HIF-2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen-treated Phd2(f/f);Rosa26(+/CreERT2) mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min(-1) g(-1) in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF-2α, but not HIF-1α. Furthermore, inducible inactivation of HIF-2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min(-1) g(-1) in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine-positive cells mm(-2) in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF-2α enzyme-substrate couple in modulating ventilatory sensitivity to hypoxia., (© 2015 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: It is difficult to design a system to reliably deliver volatile anaesthetics such as halothane or isoflurane to in vitro preparations such as tissues or cells cultures: the very volatility of the drugs means that they can rapidly dissipate from even carefully-prepared solutions. Furthermore, many experiments require the control of other gases (such as oxygen or carbon dioxide) which requires constant perfusion., New Method: We describe a constant perfusion system that is air-tight (i.e., allows the accurate administration of hypoxic or hypercapnic gas mixtures), in which volatile anaesthetic is delivered via calibrated vaporisers by constant bubbling into the perfusing solution (and continuously monitored for stability by infrared spectroscopy in the headspace above the solution)., Results: We have confirmed the accuracy (i.e., linear relationship of dissolved concentrations with vapour dial settings) and stability (i.e., over time) of the anaesthetic concentrations in solutions in samples taken from the bottles into which anaesthetic is bubbled, and from samples taken from the tissue perfusion bath, using gas chromatrography-mass spectrometry (GC-MS)., Conclusions: It is possible to deliver volatile anaesthetics in accurate concentrations to cell/tissue preparations whilst adjusting ambient air composition rapidly, stable over sustained time periods., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A functional role for the mitochondria in acute O2 sensing in the carotid body (CB) remains undetermined. Whilst total inhibition of mitochondrial activity causes intense CB stimulation, it is unclear whether this response can be moderated such that graded impairment of oxidative phosphorylation might be a mechanism that sets and modifies the O2 sensitivity of the whole organ. We assessed NADH autofluorescence and [Ca2+]i in freshly dissociated CB type I cells and sensory chemoafferent discharge frequency in an intact CB preparation, in the presence of varying concentrations of nitrite (NO2 −), a mitochondrial nitric oxide (NO) donor and a competitive inhibitor of mitochondrial complex IV. NO2 − increased CB type I cell NADH in a manner that was dose-dependent and rapidly reversible. Similar concentrations of NO2 − raised type I cell [Ca2+]i via L-type channels in a PO2-dependent manner and increased chemoafferent discharge frequency. Moderate inhibition of the CB mitochondria by NO2 − augmented chemoafferent discharge frequency during graded hypoxia, consistent with a heightened CB O2 sensitivity. Furthermore, NO2 − also exaggerated chemoafferent excitation during hypercapnia signifying an increase in CB CO2 sensitivity. These data show that NO2 − can moderate the hypoxia sensitivity of the CB and thus suggest that O2 sensitivity could be set and modified in this organ by interactions between NO and mitochondrial complex IV.

Arterial chemoreceptors play a vital role in cardiorespiratory control by providing the brain with information regarding blood oxygen, carbon dioxide, and pH. The main chemoreceptor, the carotid body, is composed of sensory (type 1) cells which respond to hypoxia or acidosis with a depolarising receptor potential which in turn activates voltage-gated calcium entry, neurosecretion and excitation of adjacent afferent nerves. The receptor potential is generated by inhibition of Twik-related acid-sensitive K(+) channel 1 and 3 (TASK1/TASK3) heterodimeric channels which normally maintain the cells' resting membrane potential. These channels are thought to be directly inhibited by acidosis. Oxygen sensitivity, however, probably derives from a metabolic signalling pathway. The carotid body, isolated type 1 cells, and all forms of TASK channel found in the type 1 cell, are highly sensitive to inhibitors of mitochondrial metabolism. Moreover, type1 cell TASK channels are activated by millimolar levels of MgATP. In addition to their role in the transduction of chemostimuli, type 1 cell TASK channels have also been implicated in the modulation of chemoreceptor function by a number of neurocrine/paracrine signalling molecules including adenosine, GABA, and serotonin. They may also be instrumental in mediating the depression of the acute hypoxic ventilatory response that occurs with some general anaesthetics. Modulation of TASK channel activity is therefore a key mechanism by which the excitability of chemoreceptors can be controlled. This is not only of physiological importance but may also offer a therapeutic strategy for the treatment of cardiorespiratory disorders that are associated with chemoreceptor dysfunction.

The identity of the oxygen sensor in arterial chemoreceptors has been the subject of much speculation. One of the oldest hypotheses is that oxygen is sensed through oxidative phosphorylation. There is a wealth of data demonstrating that arterial chemoreceptors are excited by inhibitors of oxidative phosphorylation. These compounds mimic the effects of hypoxia inhibiting TASK1/3 potassium channels causing membrane depolarisation calcium influx and neurosecretion. The TASK channels of Type-I cells are also sensitive to cytosolic MgATP. The existence of a metabolic signalling pathway in Type-1 cells is thus established; the contentious issue is whether this pathway is also used for acute oxygen sensing. The main criticism is that because cytochrome oxidase has a high affinity for oxygen (P50 ≈ 0.2 mmHg) mitochondrial metabolism should be insensitive to physiological hypoxia. This argument is however predicated on the assumption that chemoreceptor mitochondria are analogous to those of other tissues. We have however obtained new evidence to support the hypothesis that type-1 cell mitochondria are not like those of other cells in that they have an unusually low affinity for oxygen (Mills E, Jobsis FF, J Neurophysiol 35(4):405-428, 1972; Duchen MR, Biscoe TJ, J Physiol 450:13-31, 1992a). Our data confirm that mitochondrial membrane potential, NADH, electron transport and cytochrome oxidase activity in the Type-1 cell are all highly sensitive to hypoxia. These observations not only provide exceptionally strong support for the metabolic hypothesis but also reveal an unknown side of mitochondrial behaviour.

The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate PO2 led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ∼33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The hypoxia-inducible factor (HIF) family of transcription factors coordinates diverse cellular and systemic responses to hypoxia. Chuvash polycythemia (CP) is an autosomal recessive disorder in humans in which there is impaired oxygen-dependent degradation of HIF, resulting in long-term systemic elevation of HIF levels at normal oxygen tensions. CP patients demonstrate the characteristic features of ventilatory acclimatization to hypoxia, namely, an elevated baseline ventilation and enhanced acute hypoxic ventilatory response (AHVR). We investigated the ventilatory and carotid-body phenotype of a mouse model of CP, using whole-body plethysmography, immunohistochemistry, and electron microscopy. In keeping with studies in humans, CP mice had elevated ventilation in euoxia and a significantly exaggerated AHVR when exposed to 10% oxygen, with or without the addition of 3% carbon dioxide. Carotid-body immunohistochemistry demonstrated marked hyperplasia of the oxygen-sensing type I cells, and the cells themselves appeared enlarged with more prominent nuclei. This hypertrophy was confirmed by electron microscopy, which also revealed that the type I cells contained an increased number of mitochondria, enlarged dense-cored vesicles, and markedly expanded rough endoplasmic reticulum. The morphological and ultrastructural changes seen in the CP mouse carotid body are strikingly similar to those observed in animals exposed to chronic hypoxia. Our study demonstrates that the HIF pathway plays a major role, not only in regulating both euoxic ventilatory control and the sensitivity of the response to hypoxia, but also in determining the morphology of the carotid body.

In rat arterial chemoreceptors, background potassium channels play an important role in maintaining resting membrane potential and promoting depolarization and excitation in response to hypoxia or acidosis. It has been suggested that these channels are a heterodimer of TASK-1 and TASK-3 based on their similarity to heterologously expressed TASK-1/3 fusion proteins. In this study, we sought to confirm the identity of these channels through germline ablation of Task-1 (Kcnk3) and Task-3 (Kcnk9) in mice. Background K-channels were abundant in carotid body type-1 cells from wild-type mice and comparable to those previously described in rat type-1 cells with a main conductance state of 33 pS. This channel was absent from both Task-1(-/-) and Task-3(-/-) cells. In its place we observed a larger (38 pS) K(+)-channel in Task-1(-/-) cells and a smaller (18 pS) K(+)-channel in Task-3(-/-) cells. None of these channels were observed in Task-1(-/-)/Task-3(-/-) double knock-out mice. We therefore conclude that the predominant background K-channel in wild-type mice is a TASK-1/TASK-3 heterodimer, whereas that in Task-1(-/-) mice is TASK-3 and, conversely, that in Task-3(-/-) mice is TASK-1. All three forms of TASK channel in type-1 cells were inhibited by hypoxia, cyanide and the uncoupler FCCP, but the greatest sensitivity was seen in TASK-1 and TASK-1/TASK-3 channels. In summary, the background K-channel in type-1 cells is predominantly a TASK-1/TASK-3 heterodimer. Although both TASK-1 and TASK-3 are able to couple to the oxygen and metabolism sensing pathways present in type-1 cells, channels containing TASK-1 appear to be more sensitive.

Sensory neurons are able to detect tissue ischaemia and both transmit information to the brainstem as well as release local vasoactive mediators. Their ability to sense tissue ischaemia is assumed to be primarily mediated through proton sensing ion channels, lack of oxygen however may also affect sensory neuron function. In this study we investigated the effects of anoxia on isolated capsaicin sensitive neurons from rat nodose ganglion. Acute anoxia triggered a reversible increase in [Ca2+]i that was mainly due to Ca2+-efflux from FCCP sensitive stores and from caffeine and CPA sensitive ER stores. Prolonged anoxia resulted in complete depletion of ER Ca2+-stores. Mitochondria were partially depolarised by acute anoxia but mitochondrial Ca2+-uptake/buffering during voltage-gated Ca2+-influx was unaffected. The process of Ca2+-release from mitochondria and cytosolic Ca2+-clearance following Ca2+ influx was however significantly slowed. Anoxia was also found to inhibit SERCA activity and, to a lesser extent, PMCA activity. Hence, anoxia has multiple influences on [Ca2+]i homeostasis in vagal afferent neurons, including depression of ATP-driven Ca2+-pumps, modulation of the kinetics of mitochondrial Ca2+ buffering/release and Ca2+-release from, and depletion of, internal Ca2+-stores. These effects are likely to influence sensory neuronal function during ischaemia., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. A common criticism of this hypothesis is that mitochondrial function is insensitive to physiological levels of hypoxia. Here we investigate the effects of hypoxia (down to 0.5% O2) on mitochondrial function in neonatal rat type-1 cells. The oxygen sensitivity of mitochondrial [NADH] was assessed by monitoring autofluorescence and increased in hypoxia with a P50 of 15 mm Hg (1 mm Hg = 133.3 Pa) in normal Tyrode or 46 mm Hg in Ca(2+)-free Tyrode. Hypoxia also depolarised mitochondrial membrane potential (m, measured using rhodamine 123) with a P50 of 3.1, 3.3 and 2.8 mm Hg in normal Tyrode, Ca(2+)-free Tyrode and Tyrode containing the Ca(2+) channel antagonist Ni(2+), respectively. In the presence of oligomycin and low carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 75 nm) m is maintained by electron transport working against an artificial proton leak. Under these conditions hypoxia depolarised m/inhibited electron transport with a P50 of 5.4 mm Hg. The effects of hypoxia upon cytochrome oxidase activity were investigated using rotenone, myxothiazol, antimycin A, oligomycin, ascorbate and the electron donor tetramethyl-p-phenylenediamine. Under these conditions m is maintained by complex IV activity alone. Hypoxia inhibited cytochrome oxidase activity (depolarised m) with a P50 of 2.6 mm Hg. In contrast hypoxia had little or no effect upon NADH (P50 = 0.3 mm Hg), electron transport or cytochrome oxidase activity in sympathetic neurons. In summary, type-1 cell mitochondria display extraordinary oxygen sensitivity commensurate with a role in oxygen sensing. The reasons for this highly unusual behaviour are as yet unexplained.

Oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen-sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min(-1) g(-1) in controls; P < 0.01). HVR was similarly exaggerated in PHD2(+/-) animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min(-1) g(-1); P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2(+/-) animals versus 0.0015 ± 0.00019 mm(3) in controls; P < 0.01). In contrast, HVR in PHD1(-/-) and PHD3(-/-) mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.

Hypertension is associated with the early onset of cardiac sympathetic hyperresponsiveness and enhanced intracellular Ca(2+) concentration [Ca(2+)](i) in sympathetic neurons from both prehypertensive and hypertensive, spontaneously hypertensive rats (SHRs). Oxidative stress is a hallmark of hypertension, therefore, we tested the hypothesis that the inhibitory action of the nitric oxide-cGMP pathway on [Ca(2+)](i) transients is impaired in cardiac sympathetic neurons from the SHR. Stellate ganglia were isolated from young prehypertensive SHRs and age-matched normotensive Wistar-Kyoto rats. [Ca(2+)](i) was measured by ratiometric fluorescence imaging. Neurons from the prehypertensive SHR ganglia had a significantly higher depolarization evoked [Ca(2+)](i) transient that was also associated with decreased expression of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylate cyclase and cGMP when compared with the Wistar-Kyoto rat ganglia. Soluble guanylate cyclase inhibition or nNOS inhibition increased [Ca(2+)](i) in the Wistar-Kyoto rats but had no effect in SHR neurons. A nitric oxide donor decreased [Ca(2+)](i) in both sets of neurons, although this was markedly less in the SHR. A novel noradrenergic cell specific vector (Ad.PRSx8-nNOS/Cherry) or its control vector (Ad.PRSx8-Cherry) was expressed in sympathetic neurons. In the SHR, Ad.PRSx8-nNOS/Cherry-treated neurons had a significantly reduced peak [Ca(2+)](i) transient that was associated with increased tissue levels of nNOS protein and cGMP concentration compared with gene transfer of Ad.PRSx8-Cherry alone. nNOS inhibition significantly increased [Ca(2+)](i) after Ad.PRSx8-nNOS/Cherry expression. We conclude that artificial upregulation of stellate sympathetic nNOS via targeted gene transfer can directly attenuate intracellular Ca(2+) and may provide a novel method for decreasing enhanced cardiac sympathetic neurotransmission.

It has been proposed that endogenous H(2)S mediates oxygen sensing in chemoreceptors; this study investigates the mechanisms by which H(2)S excites carotid body type 1 cells. H(2)S caused a rapid reversible increase in intracellular calcium with EC(50) ≈ 6 μM. This [Ca(2+)](i) response was abolished in Ca-free Tyrode. In perforated patch current clamp recordings, H(2)S depolarised type 1 cells from -59 to -35 mV; this was accompanied by a robust increase in [Ca(2+)](i). Voltage clamping at the resting membrane potential abolished the H(2)S-induced rise in [Ca(2+)](i). H(2)S inhibited background K(+) current in whole cell perforated patch and reduced background K(+) channel activity in cell-attached patch recordings. It is concluded that H(2)S excites type 1 cells through the inhibition of background (TASK) potassium channels leading to membrane depolarisation and voltage-gated Ca(2+) entry. These effects mimic those of hypoxia. H(2)S also inhibited mitochondrial function over a similar concentration range as assessed by NADH autofluorescence and measurement of intracellular magnesium (an index of decline in MgATP). Cyanide inhibited background K channels to a similar extent to H(2)S and prevented H(2)S exerting any further influence over channel activity. These data indicate that the effects of H(2)S on background K channels are a consequence of inhibition of oxidative phosphorylation. Whilst this does not preclude a role for endogenous H(2)S in oxygen sensing via the inhibition of cytochrome oxidase, the levels of H(2)S required raise questions as to the viability of such a mechanism.

In humans the ventilatory response to sustained isocapnic hypoxia is biphasic: after an initial rapid rise there follows a steady decline of the next 20-30 min termed hypoxic ventilatory decline (HVD). It is not known whether this secondary phase resides in a reducing activity of the peripheral or the central chemoreflex. We wished to assess if the Ca(2+) transient that occurs in glomus cells in response to hypoxia exhibits a form of HVD with sustained hypoxia that parallels the human ventilatory response, or if it exhibits a different response. Glomus cells enzymatically isolated from rat pups were exposed to 10 min sustained hypoxia (5% CO(2) in N(2)), asphyxia (20% CO(2) in N(2)), hypercapnia (20% CO(2) in air), 100 mM K+ and 2 mM Ba(2+). Intracellular Ca(2+) transients [Ca(2+)]i were measured using indo-1 dye. Hypoxia elicited rapid increase in [Ca(2+)]i followed by a gradual persistent decline over 10 min to 50% of the peak value. Asphyxia also elicited a biphasic response, with the acute response twice as great as that for hypoxia and the subsequent decline also twice as large occurring over a similar time course. Hypercapnia- and hyperkalaemia-evoked [Ca(2+)]i responses displayed a more rapid initial decline (within 2- min) but then stabilised. Exposure to Ba(2+) evoked characteristic spiking activity in the [Ca(2+)]i signal. Although the glomus cell shows some adaptation of response to a variety of stimuli, its response to hypoxia is characterized by a biphasic response with continued secondary decline in [Ca(2+)]i in a manner akin to HVD.

The purpose of this study was to ascertain if effects of halothane and sevoflurane (0.18-1.45 MAC) on the magnitude of the rise in intracellular calcium ([Ca(2+)]i with approximately 90s hypoxia (measured using indo-1 dye) in rat pup carotid body type I glomus cells. paralleled their known effects on the human hypoxic ventilatory response, where halothane is more depressive. We also assessed these agents' effect on [Ca(2+)]i response to 100 mM K(+). Halothane depressed the [Ca(2+])i transient in hypoxia more than sevoflurane (p = 0.036). Both agents also depressed the [Ca(2+)]i response to K+ - halothane more than sevoflurane (p = 0.004). These actions reflect their known influence on human hypoxic ventilatory response, consistent with the notion that the cellular process underlies the whole-body effect. The responses to K(+), which depolarises the cell membrane, indicates that in addition to a putative effect on K(+) channels, voltage-activated Ca(2+) channels may also be involved in the anaesthetic effect.

We recently reported that volatile anaesthetics directly depress the isolated glomus cell response to hypoxia, halothane more so than sevoflurane, in a manner mimicking the action of these agents on the human hypoxic ventilatory response. We wished to extend these investigations to action of another agent (isoflurane), and we planned to examine the effects of this agent and halothane on background K(+) channels. In an isolated rat pup glomus cell preparation intracellular calcium [Ca(2+)]i (measured using indo-1 dye), halothane and isoflurane (0.45-2.73 MAC) depressed the Ca(2+) transient response to hypoxia (p = 0.028), halothane more than isoflurane (p < 0.001). Evaluating the effects of halothane, isoflurane (both 2.5 MAC) and hypoxia on the open probability of background TASK-like K(+) channels in cell attached patch recordings, halothane in euoxia strongly increased channel activity (2 fold) but isoflurane only increased activity by 50% (p < 0.001). In the presence of hypoxia halothane also increased channel activity (3 fold) while isoflurane again only had weak effects (p = 0.004). Thus there were marked differences between these agents on K(+) channel activity, comparable to their effects on the hypoxia induced Ca(2+) transient. When glomus cells were exposed to a depolarising stimulus using 100 mM K(+), both halothane and isoflurane modestly reduced the magnitude of the resulting Ca(2+) transient (by 44% and 10% respectively, p < 0.001). We conclude that the effect of volatile anaesthetics on the glomus cell response to hypoxia is mediated at least in part by their effect on background K(+) channels, and that this plausibly explains their whole-body effect. An additional effect on voltage-gated Ca(2+) is also possible.

A number of tandem P-domain K(+)- channels (K(2)P) generate background K(+)-currents similar to those found in enteroreceptors that sense a diverse range of physiological stimuli including blood pH, carbon dioxide, oxygen, potassium and glucose. This review presents an overview of the properties of both cloned K(2)P tandem-P-domain K-channels and the endogenous chemosensitive background K-currents found in central chemoreceptors, peripheral chemoreceptors, the adrenal gland and the hypothalamus. Although the identity of many of these endogenous channels has yet to be confirmed they show striking similarities to a number of K(2)P channels especially those of the TASK subgroup. Moreover these channels seem often (albeit not exclusively) to be involved in pH and nutrient/metabolic sensing.

Ischaemia excites sensory neurones (generating pain) and promotes calcitonin gene-related peptide release from nerve endings. Acidosis is thought to play a key role in mediating excitation via the activation of proton-sensitive cation channels. In this study, we investigated the effects of acidosis upon Ca2+ signalling in sensory neurones from rat dorsal root ganglia. Both hypercapnic (pHo 6.8) and metabolic-hypercapnic (pHo 6.2) acidosis caused a biphasic increase in cytosolic calcium concentration ([Ca2+] i ). This comprised a brief Ca2+ transient (half-time approximately 30 s) caused by Ca2+ influx followed by a sustained rise in [Ca2+] i due to Ca2+ release from caffeine and cyclopiazonic acid-sensitive internal stores. Acid-evoked Ca2+ influx was unaffected by voltage-gated Ca2+-channel inhibition with nickel and acid sensing ion channel (ASIC) inhibition with amiloride but was blocked by inhibition of transient receptor potential vanilloid receptors (TRPV1) with (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide (AMG 9810; 1 microM) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropryazine-1(2H)-carbox-amide (BCTC; 1 microM). Combining acidosis with anoxia and aglycaemia increased the amplitude of both phases of Ca2+ elevation and prolonged the Ca2+ transient. The Ca2+ transient evoked by combined acidosis, aglycaemia and anoxia was also substantially blocked by AMG 9810 and BCTC and, to a lesser extent, by amiloride. In summary, the principle mechanisms mediating increase in [Ca2+] i in response to acidosis are a brief Ca2+ influx through TRPV1 followed by sustained Ca2+ release from internal stores. These effects are potentiated by anoxia and aglycaemia, conditions also prevalent in ischaemia. The effects of anoxia and aglycaemia are suggested to be largely due to the inhibition of Ca2+-clearance mechanisms and possible increase in the role of ASICs.

Nociceptive neurons play an important role in ischemia by sensing and transmitting information to the CNS and by secreting peptides and nitric oxide, which can have local effects. While these responses are probably primarily mediated by acid sensing channels, other events occurring in ischemia may also influence neuron function. In this study, we have investigated the effects of anoxia and anoxic aglycemia on Ca2+ regulation in sensory neurons from rat dorsal root ganglia. Anoxia increased [Ca2+]i by evoking Ca2+ release from two distinct internal stores one sensitive to carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and one sensitive to caffeine, cyclopiazonic acid (CPA), and ryanodine [assumed to be the endoplasmic reticulum (ER)]. Anoxia also promoted progressive decline in ER Ca2+ content. Despite partially depolarizing mitochondria, anoxia had relatively little effect on mitochondrial Ca2+ uptake when neurons were depolarized but substantially delayed mitochondrial Ca2+ release and subsequent Ca2+ clearance from the cytosol on repolarization. Anoxia also reduced both sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity and Ca2+ extrusion [probably via plasma membrane Ca2+-ATPase (PMCA)]. Thus anoxia has multiple effects on [Ca2+]i homeostasis in sensory neurons involving internal stores, mitochondrial buffering, and Ca2+ pumps. Under conditions of anoxic aglycemia, there was a biphasic and more profound elevation of [Ca2+]i, which was associated with complete ER Ca2+ store emptying and progressive, and eventually complete, inhibition of Ca2+ clearance by PMCA and SERCA. These data clearly show that loss of oxygen, and exhaustion of glycolytic substrates, can profoundly affect many aspects of cell Ca2+ regulation, and this may play an important role in modulating neuronal responses to ischemia.

Sensory neurons can detect ischemia and transmit pain from various organs. Whereas the primary stimulus in ischemia is assumed to be acidosis, little is known about how the inevitable metabolic challenge influences neuron function. In this study we have investigated the effects of anoxia, aglycemia, and acidosis upon intracellular Mg(2+) concentration [Mg(2+)](i) and intracellular pH (pH(i)) in isolated sensory neurons. Anoxia, anoxic aglycemia, and acidosis all caused a rise in [Mg(2+)](i) and a fall in pH(i). The rise in [Mg(2+)](i) in response to acidosis appears to be due to H(+) competing for intracellular Mg(2+) binding sites. The effects of anoxia and aglycemia were mimicked by metabolic inhibition and, in a dorsal root ganglia (DRG)-derived cell line, the rise in [Mg(2+)](i) during metabolic blockade was closely correlated with fall in intracellular ATP concentration ([ATP](i)). Increase in [Mg(2+)](i) during anoxia and aglycemia were therefore assumed to be due to MgATP hydrolysis. Even brief periods of anoxia (<3 min) resulted in rapid internal acidosis and a rise in [Mg(2+)](i) equivalent to a decline in MgATP levels of 15-20%. With more prolonged anoxia (20 min) MgATP depletion is estimated to be around 40%. With anoxic aglycemia, the [Mg(2+)](i) rise occurs in two phases: the first beginning almost immediately and the second after an 8- to 10-min delay. Within 20 min of anoxic aglycemia [Mg(2+)](i) was comparable to that observed following complete metabolic inhibition (dinitrophenol + 2-deoxyglucose, DNP + 2-DOG) indicating a near total loss of MgATP. The consequences of these events therefore need to be considered in the context of sensory neuron function in ischemia.

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO-cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca(2+) influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca(2+) influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca(2+) transients by 44%. nNOS specific inhibition by N-[(4S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]-N'-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca(2+) transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.

The carotid body's physiological role is to sense arterial oxygen, CO(2) and pH. It is however, also powerfully excited by inhibitors of oxidative phosphorylation. This latter observation is the cornerstone of the mitochondrial hypothesis which proposes that oxygen is sensed through changes in energy metabolism. All of these stimuli act in a similar manner, i.e. by inhibiting a background TASK-like potassium channel (K(B)) they induce membrane depolarization and thus neurosecretion. In this study we have evaluated the role of ATP in modulating K(B) channels. We find that K(B) channels are strongly activated by MgATP (but not ATP(4)(-)) within the physiological range (K(1/2) = 2.3 mm). This effect was mimicked by other Mg-nucleotides including GTP, UTP, AMP-PCP and ATP-gamma-S, but not by PP(i) or AMP, suggesting that channel activity is regulated by a Mg-nucleotide sensor. Channel activation by MgATP was not antagonized by either 1 mm AMP or 500 microm ADP. Thus MgATP is probably the principal nucleotide regulating channel activity in the intact cell. We therefore investigated the effects of metabolic inhibition upon both [Mg(2+)](i), as an index of MgATP depletion, and channel activity in cell-attached patches. The extent of increase in [Mg(2+)](i) (and thus MgATP depletion) in response to inhibition of oxidative phosphorylation were consistent with a decline in [MgATP](i) playing a prominent role in mediating inhibition of K(B) channel activity, and the response of arterial chemoreceptors to metabolic compromise.

Chemosensing by type-1 cells of the carotid body involves a series of events which culminate in the calcium-dependent secretion of neurotransmitter substances which then excite afferent nerves. This response is mediated via membrane depolarisation and voltage-gated calcium entry. Studies utilising isolated cells indicates that the membrane depolarisation in response to hypoxia, and acidosis, appears to be primarily mediated via the inhibition of a background K(+)-current. The pharmacological and biophysical characteristics of these channels suggest that they are probably closely related to the TASK subfamily of tandem-P-domain K(+)-channels. Indeed they show greatest similarity to TASK-1 and -3. In addition to being sensitive to hypoxia and acidosis, the background K(+)-channels of the type-1 cell are also remarkably sensitive to inhibition of mitochondrial energy metabolism. Metabolic poisons are known potent stimulants of the carotid body and cause membrane depolarisation of type-1 cells. In the presence of metabolic inhibitors hypoxic sensitivity is lost suggesting that oxygen sensing may itself be mediated via depression of mitochondrial energy production. Thus these TASK-like background channels play a central role in mediating the chemotransduction of several different stimuli within the type-1 cell. The mechanisms by which metabolic/oxygen sensitivity might be conferred upon these channels are briefly discussed.

The carotid body plays an important role in initiating protective responses to hypoxemia. The primary oxygen sensing cells are the glomus or type 1 cells. Hypoxia evokes the secretion of neurotransmitters from these cells which then excite afferent nerves. This response is mediated via membrane depolarization and voltage-gated Ca2+ entry. Studies from this laboratory have revealed that membrane depolarization in response to hypoxia is primarily the result of inhibition of background K+ channels which show strong similarities to the acid sensitive tandem-P-domain K+ channels TASK-1 and TASK-3. The background K+ channels of type-1 cells are also very sensitive to inhibition of mitochondrial energy metabolism and, in excised patches, appear to be directly activated by ATP. Thus these TASK-like background channels would appear to confer the ability to sense changes in oxygen levels, pH and metabolism upon the type 1 cell. The key issue of whether the effects of hypoxia are mediated through changes in metabolism remains unanswered but the effects of inhibition of mitochondrial energy metabolism and of hypoxia upon background K+ channels is mutually exclusive suggesting that there is a close link between metabolism and oxygen sensing in the type 1 cell.