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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.

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.)

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.

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.

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.

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.

The effects of hypercapnic acidosis and hypoxia on intracellular Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](i) rises. The percentage of cells responding was also dependent on the severity of the hypoxic stimulus. Raising CO(2) from 5 to 10 or 20% elicited a significant increase in [Ca(2+)](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.

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.

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)