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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. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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., 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.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer and there is great interest in applying inhibitors of HIF as anti-cancer 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 impact on 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 tumour growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatisation and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerisation 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 heterodimerisation with HIF-1β. Although PT2385 was well-tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.

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.

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.

Key points • TASK-like background potassium channels play a key role in the sensing of hypoxic, metabolic and acidic stimuli in arterial chemoreceptor cells. • In this study, we investigated the roles of TASK-1 and TASK-3 in forming these channels by using gene deletion in mice. • Deletion of Task-1 (Kcnk3) and/or Task-3 (Kcnk9) disrupted the main form of background K-channel activity. • In single knock-out mice, we observed, instead of the wild-type channel, the homomeric forms of TASK-1 in Task-3−/− and TASK-3 in Task-1−/−. • All forms of TASK were inhibited by hypoxia, cyanide and the uncoupler FCCP. • We conclude that the main form of background K-channel in chemoreceptor cells is a TASK-1/TASK-3 heterodimer and that both TASK-1 and TASK-3 subunits can couple to both oxygen and metabolic signalling pathways in these cells. Abstract 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.

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.

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 [Ca2+]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 [Ca2+]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.

I read with interest the publication by Lu et al. (2013) in which the authors claim to have found evidence for uncoupling and reciprocity in the responses of carotid body glomus cells to hypoxic and acidic stimuli. By measuring changes in intracellular calcium as an index of chemosensitivity, the authors argue that cells that are highly sensitive to hypoxia are less sensitive to acidosis and vice versa. The authors refer to this as a reciprocal sensitivity although it is not entirely clear if by this they mean that responses to acidosis and hypoxia are negatively correlated (i.e. Y=−aX+b) or inverse (Y=a/X+b). Whilst this is an intriguing hypothesis I do not believe that it is supported by the data. There are three main issues in particular to which I would draw your attention with regard to the analysis of the data presented. 1. The data set presented on Rat type-1 cells has not been subject to any statistical analysis that would support either the existence of distinct populations of cells (acid sensitive vs. hypoxia sensitive) or any correlation between acid sensitivity and oxygen sensitivity. Conclusions are presented based upon an apparently subjective analysis of the numbers of cells having responses greater or less than some arbitrarily chosen value. Moreover, casual inspection of the histogram in fig. 3E does not immediately suggest the presence of a bimodal distribution for either acid sensitivity or hypoxic sensitivity. 2. The data sets presented for mice (in fig. 6) have been manipulated in an extraordinary manner. Data have been divided into two groups based upon whether the [Ca2+]i response to cyanide (CN, used as a surrogate for hypoxia) is greater than the response to acidosis or not. The two groups are then plotted in different colours and subject to linear regression analysis separately with the regressions forced to pass through the origin. The only possible result from such an analysis is that the regression line fitted to the group of data selected such that CN>acid must have a gradient >1 and that for the group CN 0. The authors then argue that the (inevitable) differences in the gradients between the two groups ‘suggest two distinct cell populations’. They also argue that ‘the reciprocal nature of the responses was evident’ from this analysis despite the fact that it produced a positive gradient for both groups indicating a positive correlation between CN response and acid response (which is again inevitable as the regression is forced to go through 0 and all values for X and Y are positive). I believe that the segregation of the data into two groups in this manner introduces an undue bias that effectively invalidates any conclusions that might be drawn from this analysis. 3. Data sets appear underpowered. This is particularly true for one of the mouse genotypes (fig. 6C). Even in the rat data set however there are very few cells with ‘high’[Ca2+]i responses to acidosis and many of these may have come from just one cell cluster (that shown in fig. 3C). To test the hypothesis that there are distinct populations of cells within each carotid body it is also necessary to exclude variability in responses which may be due to differences between individuals (due to genetic background, age, environment or health). In summary I do not believe that any conclusions can be drawn from the analyses presented in this study regarding either correlation between acid sensitivity and oxygen sensitivity or the existence of discrete populations of glomus cells. The only conclusion which would appear to be supported empirically is that most glomus cells are sensitive in some degree to both stimuli (as has been previously reported).

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.

Inhibitors of mitochondrial function have been known to be stimulants of the carotid body for many decades. Recent experiments have demonstrated that in isolated neonatal rat type I cells both hypoxia and mitochondrial uncouplers inhibit background K+ currents. This leads to membrane depolarisation and voltage gated Ca2+ entry (Buckler, 1997; Buckler and Vaughan-Jones, 1998). Since these data are consistent with a role for mitochondria in oxygen sensing, we have determined whether other mitochondrial inhibitors mimic the effects of hypoxia on the type I cells.

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.

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use. [Ca2+]i was measured using the Ca(2+)-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L-type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non-selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (-40 to -60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)

The postulated mechanisms for hypoxic and acidic chemotransduction by type I cells that we have described here are summarized in the diagrams of Fig. 4. Most if not all of these require more complete evaluation and, as we have described, there are obvious points of contention that need to be resolved. Nevertheless, it is apparent that studies of isolated type I cell preparations carried out over the last six years have provided significant advancements in our understanding of chemotransduction in the type I cell. Only when the functioning of these cells has been fully described can we hope to understand the mechanisms underlying the responses of the intact organ to chemostimuli.

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.

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.

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.

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.

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 μM) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl) tetrahydropryazine-1(2H)-carbox-amide (BCTC; 1 μM). 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. Electronic supplementary material The online version of this article (doi:10.1007/s00424-009-0715-6) contains supplementary material, which is available to authorized users.

background K-current leads to type-1 cell membrane depolarisation (Buckler, 1997), voltage-gated calcium entry and thus excitation of the carotid body. The mechanisms by which hypoxia modulates these K channels is still unknown, however the effects of hypoxia are mimicked by a wide range of inhibitors of oxidative phosphorylation. Uncouplers, electron transport inhibitors (e.g. cyanide, rotenone & myxothiazol) and inhibitors of ATP synthase (oligomycin) are all potent stimulants of the carotid body (Anichkov & Belen`kii, 1963; Gonzalez et al., 1994) and potent inhibitors of background K-current (Wyatt & Buckler, 2004). Moreover background K-current sensitivity to hypoxia is lost in the presence of metabolic inhibitors (Wyatt & Buckler, 2004) suggesting that ATP synthesis may be a prerequisite for the expression of oxygen sensitivity. Further support for the idea that background K-channel activity may be dependent upon cellular ATP levels comes from the observation that following patch excision (from the cell attached to the inside out configuration) there is an abrupt rundown of background K-channel activity which can be partially reversed by the addition of mM levels of ATP to the intracellular solution (Williams & Buckler, 2004). In this study we have further investigated the action of ATP on background K-channels in order to evaluate the potential role for ATP in modulating channel activity and to identify general mechanisms by which ATP might act.

Although the carotid body is the principal arterial pH receptor, little is known about its ionic mechanisms for pH-detection. A prime candidate for the H+ chemo sensory element is the type I (glomus) cell, also believed to be the site of O2-chemoreception. This cell contains a rich variety of neuroactive compounds, most notably catecholamines, which are secreted in response to extracellular acid/base challenges and to hypoxia (Fidone & Gonzalez, 1986). In this way, local endings of the carotid sinus nerve are believed to be elicited. Indirect evidence has suggested that, in order to get chemoreception of extracellular acidosis/alkalosis there must first be a change of intracellular pH in the type I cell (Hanson, Nye & Torrance, 1981; Rigual, Lopez-Lopez Gonzalez, 1991). Until recently, however, there was no accurate way of directly measuring pH in this cell. Consequently, it was not known if pH, was regulated by acid equivalent efflux and influx mechanisms as it is in many other cells (Roos & Boron, 1981; Thomas, 1984 & Vaughan-Jones, 1988). In the present work we have therefore used the intracellular fluorescent probe SNARF-1 to examine intracellular pH and its regulation in type I cells isolated enzymically from the neonatal rat carotid body.

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.

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+]iby 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+]ihomeostasis 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.

The carotid body is a peripheral arterial chemoreceptor which senses oxygen, carbon dioxide and arterial pH. In response to hypoxia, hypercapnia or acidosis it evokes an increase in neural discharge within afferent fibres of the carotid sinus nerve which project to cardiovascular and respiratory control centres in the brain stem. This results in increased ventilation, stimulation of the sympathetic nervous system and modulation of blood flow and cardiac output (Daly, 1997). The primary sensory element within the carotid body is the type-1 or glomus cell (Fidone & Gonzalez, 1986; Gonzalez et al. 1994). Type-1 cells express a background potassium channel (KB channel) with biophysical and pharmacological properties similar to those of the TASK group of tandem-p-domain K+ channels, including weak rectification (similar to that predicted by the Goldman–Hodgkin–Katz constant field equation); inhibition by acidosis, quinidine and bupivacaine; activation by halothane; and insensitivity to TEA and 4-AP (Buckler et al. 2000). These channels are active over a wide range of membrane potentials and are the predominant K+ conductance at the resting membrane potential (Buckler, 1997; Williams & Buckler, 2004). As a consequence modulation of these channels has a marked influence upon the type-1 cell. Their inhibition by either hypoxia or acidosis, the two main physiological stimuli of arterial chemoreceptors, causes destabilization of the resting membrane potential resulting in a depolarizing receptor potential which then initiates electrical activity and voltage-gated calcium entry (Buckler & Vaughan-Jones, 1994a, 1994b; Buckler, 1997; Buckler et al. 2000). Inhibition of other K+ channels, particularly the large-conductance Ca2+-activated K+ channels by hypoxia and other chemostimuli (Peers, 1990; Peers & O'Donnell, 1990; Peers & Green, 1991) may facilitate some aspects of this electrical signalling process although their precise role is still unclear (Peers & Wyatt, 2007). This calcium signal in turn leads to neurosecretion from the type-1 cell (Montoro et al. 1996) and consequently excitation of chemoreceptor afferents (through release of ATP and acetylcholine and the concomitant activation of postsynaptic P2X and nicotinic receptors; Zhang et al. 2000; Rong et al. 2003). Modulation of type-1 cell background K+ channels is therefore thought to be a pivotal event in the chemotransduction process for both hypoxia and acidic stimuli. Similar chemosensory roles have also recently been suggested for TASK-like potassium channels in mediating responses to hypoxia in pulmonary vascular smooth muscle (Olschewski et al. 2006) and in mediating the excitatory actions of acidosis in some putative central chemoreceptive neurons (Bayliss et al. 2001; Washburn et al. 2002; Washburn et al. 2003). In addition to being able to sense hypoxia and acidosis we have also shown that the background K+ current of type-1 cells is sensitive to inhibitors of mitochondrial energy metabolism (Wyatt & Buckler, 2004). Metabolic inhibition results in a rapid decline in background K+ current, membrane depolarization, voltage-gated Ca2+ entry (Buckler & Vaughan-Jones, 1998; Wyatt & Buckler, 2004), and neurosecretion (Ortega Saenz et al. 2003) just as for physiological stimuli. This observation provides a part explanation for the long established phenomenon that the carotid body is rapidly and powerfully excited by numerous inhibitors of oxidative phosphorylation (Heymans et al. 1931; Shen & Hauss, 1939; Anichkov & Belen'kii, 1963; Mulligan & Lahiri, 1981; Mulligan et al. 1981; Obeso et al. 1989) (see also Fidone & Gonzalez, 1986; Gonzalez et al. 1994 and refs therein). Moreover the ability of these channels to respond to hypoxic stimuli is ablated when mitochondrial function is inhibited suggesting a strong link between energy metabolism and oxygen sensing (Wyatt & Buckler, 2004). Indeed it may be that this organ primarily responds to metabolic status rather than oxygen per se since it can also be excited by inhibitors of glycolysis (Obeso et al. 1986) and, in some preparations, by hypoglycaemia (Pardal & Lopez Barneo, 2002). In this respect it is of interest to note that another endogenous TASK-like potassium channel has recently been implicated in mediating glucose sensing in orexin neurons (Burdakov et al. 2006). The capacity to sense some aspect of metabolic status could therefore be another emerging role for endogenous TASK-like K+ channels. The nature of the link between metabolism and background K+ channel activity has not yet been established. We have, however, noted in previous studies that background K+ channel activity in excised membrane patches can be enhanced by millimolar levels of ATP (Williams & Buckler, 2004). Here we investigate the potential for channel modulation by cytosolic nucleotides in greater detail. We find that, in excised inside-out patches, type-1 cell background K+ channels are indeed strongly modulated by variation in MgATP at levels within the physiological range (with a K1/2 in the low millimolar range). We also describe sensitivity to a number of other Mg-nucleotides, including GTP, UTP, AMP-PCP and ATP-γ-S, which suggests a mechanism that may involve some form of magnesium–nucleotide sensor rather than an enzymatic process. These data are consistent with a direct link between metabolism and cell excitability mediated through changes in cytosolic, or submembrane, ATP levels.

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.

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.

Key points Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation.Oxygen‐sensitive signals are transduced in animal cells by post‐translational hydroxylation of transcription factors termed hypoxia‐inducible factors (HIFs).Mice heterozygous for the principal ‘oxygen‐sensing’ HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia.To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF‐1α and HIF‐2α.Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF‐2α.Inducible inactivation of HIF‐2α, but not HIF‐1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation.These findings demonstrate a key role for PHD2 and HIF‐2α in ventilatory control and carotid body biology. Abstract 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 Phd2f/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, Key points Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation.Oxygen‐sensitive signals are transduced in animal cells by post‐translational hydroxylation of transcription factors termed hypoxia‐inducible factors (HIFs).Mice heterozygous for the principal ‘oxygen‐sensing’ HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia.To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF‐1α and HIF‐2α.Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF‐2α.Inducible inactivation of HIF‐2α, but not HIF‐1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation.These findings demonstrate a key role for PHD2 and HIF‐2α in ventilatory control and carotid body biology.

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.

TREK-1 is a member of the two-pore domain potassium (K2P) channel family that is mechano-, heat, pH, voltage and lipid sensitive. It is highly expressed in the central nervous system and probably encodes one of the previously described arachidonic acid-activated K+ channels. Polyunsaturated fatty acids and lysophospholipids protect the brain against global ischaemia. Since both lipids are openers of TREK-1, it has been suggested that this K2P channel is directly involved in neuroprotection. Recently, however, this view has been challenged by a report claiming that TREK-1 and its activation by arachidonic acid is inhibited by hypoxia. In the present study, we demonstrate that the bubbling of saline with gases results in the loss of arachidonic acid from solution. Using experimental conditions which obviate this experimental artefact we demonstrate that TREK-1 is resistant to hypoxia and is strongly activated by arachidonic acid even at low PO2 (< 4 Torr). Furthermore, hypoxia fails to affect basal as well as 2,4,6-trinitrophenol- and acid-stimulated TREK-1 currents. These data are supportive for a possible role of TREK-1 in ischaemic neuroprotection and in cell signalling via arachidonic acid.

Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN−) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca2+, which was inhibited on removal of extracellular Ca2+. Under current clamp conditions rotenone and CN− caused a rapid membrane depolarization and elevation of [Ca2+]i. Voltage clamping cells to −70 mV substantially attenuated this rise in [Ca2+]i. Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K+ currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K+ current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN− or rotenone or the mitochondrial uncoupler p-trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14–16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 μM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.

The effects of hypercapnic acidosis and hypoxia on intracellular Ca2+concentration ([Ca2+]i) were determined with Indo 1 in enzymatically isolated single type I cells from neonatal rat carotid bodies. Type I cells responded to graded hypoxic stimuli with graded [Ca2+]i rises. The percentage of cells responding was also dependent on the severity of the hypoxic stimulus. Raising CO2 from 5 to 10 or 20% elicited a significant increase in [Ca2+]i in the same cells as those that responded to hypoxia. Thus both stimuli can be sensed by each individual cell. When combinations of hypoxic and acidic stimuli were given simultaneously, the responses were invariably greater than the response to either stimulus given alone. Indeed, in most cases, the response to hypercapnia was slightly potentiated by hypoxia. These data provide the first evidence that the classic synergy between hypoxic and hypercapnic stimuli observed in the intact carotid body may, in part, be an inherent property of the type I cell.

1. Mitochondrial uncouplers are potent stimulants of the carotid body. We have therefore investigated their effects upon isolated type I cells. Both 2,4-dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) caused an increase in [Ca2+]i which was largely inhibited by removal of extracellular Ca2+ or Na+, or by the addition of 2 mM Ni2+. Methoxyverapamil (D600) also partially inhibited the [Ca2+]i response. 2. In perforated-patch recordings, the rise in [Ca2+]i coincided with membrane depolarization and was greatly reduced by voltage clamping the cell to -70 mV. Uncouplers also inhibited a background K+ current and induced a small inward current. 3. Uncouplers reduced pHi by 0.1 unit. Alkaline media diminished this acidification but had no effect on the [Ca2+]i response. 4. FCCP and DNP also depolarized type I cell mitochondria. The onset of mitochondrial depolarization preceded changes in cell membrane conductance by 3-4 s. 5. We conclude that uncouplers excite the carotid body by inhibiting a background K+ conductance and inducing a small inward current, both of which lead to membrane depolarization and voltage-gated Ca2+ entry. These effects are unlikely to be caused by cell acidification. The inhibition of background K+ current may be related to the uncoupling of oxidative phosphorylation.

1. The effects of cholinergic agonists upon intracellular free Ca2+ levels ([Ca2+]i) have been studied in enzymically isolated rat carotid body single type I cells, using indo-1. 2. Acetylcholine (ACh) dose-dependently increased [Ca2+]i in 55% of cells studied (EC50 = 13 microM). These [Ca2+]i rises were partially inhibited by atropine or mecamylamine. 3. Specific nicotinic and muscarinic agonists also elevated [Ca2+]i in a dose-dependent manner (nicotine, EC50 = 15 microM; methacholine, EC50 = 20 microM). 4. While the majority of the ACh-sensitive cells responded to both classes of cholinergic agonist, 29% responded exclusively to nicotinic stimulation and 9% responded exclusively to muscarinic stimulation. 5. In the presence of nicotinic agonists, Ca2+i responses were transient. In the presence of muscarinic agonists, Ca2+i responses consisted of an initial rise, which then declined to a lower plateau level. 6. Nicotinic responses were rapidly abolished in Ca(2+)-free medium, suggesting that they are dependent on Ca2+ influx. 7. The plateau component of the muscarinic-activated response was also abolished in Ca(2+)-free conditions. The rapid initial [Ca2+]i rise, however, could still be evoked after several minutes in Ca(2+)-free medium. Muscarine also increased Mn2+ quenching of intracellular fura-2 fluorescence. These data suggest that the full muscarinic response depends on both Ca2+ release from intracellular stores and Ca2+o influx. 8. The results indicate that, in rat carotid body type I cells, both nicotinic and muscarinic acetylcholine receptors increase [Ca2+]i, but achieve this via different mechanisms. ACh may therefore play a role in carotid body function by modulating Ca2+i in the chemosensory type I cells.

Hypertension is associated with cardiac noradrenergic hyperactivity, although it is not clear whether this precedes or follows the development of hypertension itself. We hypothesized that Ca 2+ homeostasis in postganglionic sympathetic neurons is impaired in spontaneously hypertensive rats (SHRs) and may occur before the development of hypertension. The depolarization-induced rise in intracellular free calcium concentration ([Ca 2+ ] i ; measured using fura-2-acetoxymethyl ester) was significantly larger in cultured sympathetic neurons from prehypertensive SHRs than in age matched normotensive Wistar-Kyoto rats. The decay of the [Ca 2+ ] i transient was also faster in SHRs. The endoplasmic reticulum Ca 2+ content and caffeine-induced [Ca 2+ ] i amplitude were significantly greater in the young SHRs. Lower protein levels of phospholamban and more copies of ryanodine receptor mRNA were also observed in the young SHRs. Depleting the endoplasmic reticulum Ca 2+ store did not alter the difference of the evoked [Ca 2+ ] i transient and decay time between young SHRs and Wistar-Kyoto rats. However, removing mitochondrial Ca 2+ buffering abolished these differences. A lower mitochondrial membrane potential was also observed in young SHR sympathetic neurons. This resulted in impaired mitochondrial Ca 2+ uptake and release, which might partly be responsible for the increased [Ca 2+ ] i transient and faster decay in SHR sympathetic neurons. This Ca 2+ phenotype seen in early development in cardiac stellate and superior cervical ganglion neurons may contribute to the sympathetic hyperresponsiveness that precedes the onset of hypertension.

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 (p0.001). 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% (p0.001). 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, p0.001). 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.

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.

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.

1. Intracellular pH (pHi) was recorded ratiometrically in isolated guinea-pig ventricular myocytes using the pH-sensitive fluoroprobe, carboxy-SNARF-1 (carboxy-seminaphthorhodafluor). 2. Following an intracellular acid load (10 mM NH4 Cl removal), pHi recovery in HEPES-buffered Tyrode solution was inhibited by 1.5 mM amiloride (Na(+)-H+ antiport blocker). In the presence of amiloride, switching from HEPES buffer to HCO3-/CO2 (pHo of both solutions = 7.4) stimulated a pHi recovery towards more alkaline levels. 3. Amiloride-resistant, HCO(3-)-dependent pHi recovery was inhibited by removal of external Na+ (replaced by N-methyl-D-glucamine), whereas removal of external Cl- (replaced by glucuronate, leading to depletion of internal Cl-), removal of external K+, or decreasing external Ca2+ by approximately tenfold had no inhibitory effect. These results suggest that the amiloride-resistant recovery is due to a Na(+)-HCO3- cotransport into the cell. 4. The stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 500 microM) slowed Na(+)-HCO(3-)-dependent pHi recovery. 5. Intracellular pH increased in Cl(-)-free solution and this increase still occurred in Na(+)-free solution indicating that it is not caused via Na(+)-HCO3- symport and is more likely to be due to Cl- efflux in exchange for HCO3- influx on a sarcolemmal Cl(-)-HCO3- exchanger. The lack of any significant pHi recovery from intracellular acidosis in Na(+)-free solution suggests that this exchanger does not contribute to acid-equivalent extrusion. 6. Possible voltage sensitivity and electrogenicity of the co-transport were examined by using the whole-cell patch clamp technique in combination with SNARF-1 recordings of pHi. Stepping the holding potential from -110 to -40 mV did not affect amiloride-resistant pHi recovery from acidosis. Moreover, following an intracellular acid load, the activation of Na(+)-HCO3- co-influx (by switching from HEPES to HCO3-/CO2 buffer) produced no detectable outward current (outward current would be expected if the coupling of HCO3- with Na+ were > 1.0). 7. Intracellular intrinsic buffering power (beta i) was assessed as a function of pHi (beta i computed from the decrease of pHi following reduction of extracellular NH4 Cl in amiloride-containing solution). beta i in the ventricular myocyte increases roughly linearly with a decrease in pHi according the following equation: beta i = -28(pHi) +222.6. 8. Comparison of acid-equivalent efflux via Na(+)-HCO3- symport and Na(+)-H+ antiport showed that, following an intracellular acidosis, the symport accounts for about 40% of total acid efflux, the other 60% being carried by the antiport.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

We report the use of a new pH-sensitive dualemission fluoroprobe, carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1) for ratiometric recording of intracellular pH (pHi) in small isolated cells. The method is illustrated with pHi measurement in single type-1 cells (cell diameter ∼10 μm) isolated from the carotid body of the neonatal rat. Carboxy-SNARF-1 is loaded using bath application of the acetoxymethyl ester. When excited at 540 nm, the fluoroprobe gives strong, inversely related emission signals at 590 nm and 640 nm. Stable ratiometric recordings of pHi can be achieved from a single cell (pHi 8.5-6.5) for up to 50 min. Photobleaching of the probe is minimised by illuminating at relatively low light intensity (50 W xenon lamp with 0.2% transmission neutral density filter). The probe can be calibrated in situ using the nigericin technique and this is in good quantitative agreement with the independent null-point technique (extracellular weak acid/weak base application) of Eisner et al. (1989). This fluoroprobe offers certain advantages over the other commonly used probe for pHi 2′,7′-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF): (i) because of its two strong pH-sensitive peak emissions, SNARF displays a good signal-to-noise ratio for ratiometric recording at low light intensities; (ii) unlike BCECF, the dual emisson of SNARF requires no sequential mechanical switching of excitation filters, thus simplifying the epifluorescence set-up; (iii) because carboxy-SNARF-1 emission signals are at the yellow/red end of the visible spectrum, fluorescent drugs like amiloride, ethyl-isopropyl-amibride (EIPA), 4,4′-diisothiocyanostilbene 2,2′-disulphonic acid (DIDS) and cinnamate analogues do not interfere with the pHi recording, even when used at high concentrations.

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.