Oxygen regulation of breathing through an olfactory receptor activated by lactate

Abstract

Animals have evolved homeostatic responses to changes in oxygen availability that act on different timescales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long-term responses to low oxygen (hypoxia) has been established1, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor gene Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines2. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation3,4,5,6, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that, in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline.

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Figure 1: Olfr78 is expressed in carotid body glomus cells.
Figure 2: Ventilatory responses of Olfr78 null mutants to hypoxia and hypercapnia.
Figure 3: Olfr78 mediates carotid body oxygen sensing.
Figure 4: Lactate activates Olfr78 and carotid body sensory activity.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray and RNA sequencing data have been deposited in the Gene Expression Omnibus database under accession number GSE72166.

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Acknowledgements

We thank G. Fish for technical assistance; D. Riordan for assistance with RNA sequencing and analysis; L. He, B. Dinger, and S. Fidone for instruction on carotid body dissection; A. Gourine for instruction on carotid sinus nerve recordings; H. Matsunami for plasmids and advice about OR activity assays; A. Olson for assistance with two-photon imaging; R. Yu and K. Deisseroth for mouse strains; D. Cornfield and D. Bernstein for equipment; and P. Harbury and members of our laboratory for discussions and comments on the manuscript. This work was supported by Stanford University Dean’s Postdoctoral Fellowship, National Institutes of Health (NIH) K12 RFA-HL-07-004 Career Development Program, Helen Hay Whitney Postdoctoral Fellowship, and NIH Pediatric Research Loan Repayment Program (A.J.C.), Howard Hughes Medical Institute Gilliam Fellowship (F.E.O.), NIH MH065541 and Harold and Leila Y. Mathers Charitable Foundation (D.V.M.), NIH NS069375 (Stanford Neuroscience Microscopy Service), and the Howard Hughes Medical Institute (M.A.K.).

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Contributions

A.J.C. conducted all the experiments except for luciferase assays, which were performed with F.E.O., and animal surgeries for blood gases, which were performed by J.R. Electrophysiology experiments were performed by A.J.C. in the laboratory of D.V.M., who participated in design, conduct, and analysis of these experiments. A.J.C. and M.A.K. conceived the experiments, analysed the data, and wrote the manuscript.

Corresponding author

Correspondence to Mark A. Krasnow.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Model of oxygen sensing by the carotid body and the mitochondrion.

a, Anatomy and blood supply of the carotid body. The carotid body is located bilaterally at bifurcation of carotid artery (CA) in the neck. Its location can be variable as well as the source of its blood supply, which can come from branches of nearby internal and external carotid, occipital, pharyngeal arteries. Blood flows through fenestrated capillaries close to clusters of type I glomus cells and drains from carotid body into jugular vein (JV) on ventral side2. b, Cellular organization of carotid body. The carotid body is composed of several cell types, including type I glomus cells (red) that sense changes in blood oxygen and are organized in clusters, type II sustentacular cells (blue) that resemble neuroglia and surround glomus cell clusters, carotid sinus nerve (CSN) fibres that innervate glomus cells, and endothelial (E) and smooth muscle cells (not shown) that form the tortuous vasculature2. Panels a and b modified from Pardal, R., Ortega-Sáenz, P., Durán, R. & López-Barneo, J. Glia-like stem cells sustain physiologic neurogenesis in the adult mammalian carotid body, Cell 131, 364–377 (2007) (ref. 45) with permission from Elsevier. c, Oxygen-sensing respiratory circuit. The primary chemoreceptor for blood oxygen is the carotid body. A decrease in PaO2 of arterial blood from normoxia (100 mmHg) to hypoxia (< 80 mmHg) stimulates glomus cells to signal the carotid sinus nerve, a branch of glossopharyngeal nerve (GN) with cell bodies in petrosal ganglion (PG). Axons of the GN terminate in nucleus tractus solitarius (NTS) in brainstem, a site of many converging afferent inputs2. The signal from NTS is transmitted to the ventral respiratory group (VRG) that includes the pre-Bötzinger complex, a region essential for respiratory rhythm generation. From VRG, neurons project to premotor and motor neurons that innervate respiratory muscles, such as diaphragm and intercostal muscles46. In addition to carotid body, vagus nerve afferents can also contribute to respiratory behaviours under specialized conditions39. The vagus nerve innervates heart, lung, and oxygen-sensitive cells of the aortic body43. d, A current model of acute oxygen sensing by carotid body. A decrease in PaO2 in blood causes a decrease in O2 concentration inside carotid body glomus cells. This causes a decrease in activity of mitochondrial electron transport chain (ETC)47 and changes in other putative oxygen-sensing pathways, such as oxygen-sensitive K+ channels48,49, haem oxygenase50, AMP kinase51, and hydrogen sulphide signalling37. These changes are hypothesized to converge on oxygen-sensitive K+ channels, which close in hypoxia and depolarize the plasma membrane. Depolarization then opens voltage-gated Ca2+ channels, leading to an increase in intracellular calcium that stimulates transmitter release to carotid sinus nerve to increase breathing2. Mitochondria of carotid body cells are highly sensitive to hypoxia compared with other tissues, as assayed by imaging of mitochondrial membrane potential, NADH levels, and spectral properties23,52,53,54. Drugs and mutations that inhibit the ETC mimic the effect of hypoxia on carotid body activity and breathing23,55,56,57,58,59. e, Regulation of lactate production by oxygen. In normoxia, pyruvate produced by glycolysis is transported into mitochondria and efficiently used in Krebs cycle to supply electrons to ETC to produce ATP. In hypoxia, lack of oxygen to act as the final electron acceptor limits electron transport, causing pyruvate to build up and become converted to lactate5,6,27. The ETC poison cyanide inhibits the haem a3 subunit of cytochrome c oxidase to prevent transfer of electrons to oxygen, leading to lactate accumulation even in presence of adequate oxygen60. Cytosolic lactate accumulation results in transport of lactic acid (lactate and H+) out of the cell by monocarboxylate transporters6,61. In normoxia, lactate concentrations in blood, tissue, and tissue interstitium are 1–5 mM62,63.

Extended Data Figure 2 RNA sequencing and whole-genome microarrays detect Olfr78 transcripts enriched in the carotid body.

a, Histogram of frequency of genes for different levels of expression enrichment in carotid body relative to adrenal medulla by RNA sequencing. Values are log2(carotid body/adrenal medulla), with data binned for every log2 interval of 1.0 centred at integers. b, Plot of log2 values of RPKM in carotid body and adrenal medulla of all 1,126 OR genes annotated in RefSeq shown in alphanumerical order. The five OR genes expressed at RPKM >2 (dashed line) are indicated. Samples that had no transcripts are plotted at a value of −7.1, just below the smallest RPKM value for ORs. Data presented in Supplementary Table 1. c, Comparison of expression levels of >34,000 genes in adult mouse carotid body and adrenal medulla by whole-genome microarrays. Plot shows log2 of the ratio for carotid body relative to adrenal medulla of the fluorescence intensity values for the 45,000 probe sets. The three probe sets for Olfr78 transcripts are indicated (circles). Expression of Olfr78 was significantly different between carotid body and adrenal medulla for all three probe sets (P < 0.05 by ANOVA with control for false discovery rate). d, Histogram of the frequency of genes for different levels of expression enrichment in carotid body relative to adrenal medulla in microarray data. Values are log2(carotid body/adrenal medulla), with data binned for every log2 interval of 1.0 centred at integers. The three probe sets detecting Olfr78 mRNA (arrows) confirmed the RNA sequencing data (a, Fig. 1a, b and Extended Data Table 1) showing Olfr78 among the mRNAs most highly enriched in carotid body. Mouse carotid body Olfr78 expression is consistent with previous microarray data7,64. ad, Three cohorts of ten animals each. Data as mean. e, Genomic locus showing the large cluster of ~160 class I OR genes on chromosome 7, with region encoding MOR18 subfamily (Olfr78, Olfr558, and Olfr557) expanded below. We did not detect transcripts in either tissue for Olfr557, which lies adjacent to Olfr558 in the cluster, or for the intervening (Olfr33, Olfr559) and intronic (Olfr560) ORs. Clusters of genes encoding globins, Trims, and USP proteins are also found with this OR cluster. Large box, coding sequence; arrowhead, coding orientation; small box, non-coding exons.

Extended Data Figure 3 Olfr78 and Olfr558 expression in tissues in the oxygen–sensing circuit.

Expression of Olfr78 reporter in heterozygous (a) and homozygous (bd) Olfr78-GFP-taulacZ reporter animals11. ac, Sections of carotid bifurcations stained for GFP (Olfr78 reporter; green), TH (red), and DAPI (nuclei; blue). a, Section of carotid body showing co-expression of reporter GFP and TH in glomus cells. Monoallelic expression would predict that only half of TH-positive cells express the reporter12. Arrowheads, clusters of glomus cells expressing both GFP and TH. b, c, Sections of the same carotid bifurcation. Panels on right show close-ups of boxed region (petrosal ganglion, PG). No GFP-positive cells were found in petrosal ganglion. TH-positive nerve fibres (arrowheads) and cell bodies were found in glossopharyngeal nerve (GN) and petrosal ganglion. Dashed circle indicates vagus nerve (VN). NG/JG, nodose/jugular ganglia. d, X-gal staining of a brain sagittal section. Reporter expression (blue) was restricted to olfactory bulb (arrowhead) in this section and complete brain serial sagittal sections. Anterior, right; dorsal, up. eh, Olfr558 expression in a knockout/reporter mouse in which the Olfr558 coding region is replaced with lacZ encoding β-galactosidase. e, Olfr558 reporter expression in blood vessels of carotid body and SCG by X-gal staining. Heterozygous Olfr558+/lacZ samples showed the same pattern of staining (data not shown). fh, Carotid body sections immunostained for β-galactosidase (Olfr558 reporter; green) and TH (red) with DAPI counterstain (blue) in f, and for β-galactosidase (green) and CD31 (red) in g or smooth muscle actin (red) in h. Scale bars, 100 μm (a, b right, c right, fh), 200 μm (b left, c left), 500 μm (e), and 2 mm (d).

Extended Data Figure 4 Tidal volume and minute ventilation of Olfr78−/− mutants exposed to hypoxia and hypercapnia.

Whole body plethysmography of unrestrained, unanaesthetized Olfr78+/+ control and Olfr78−/− mutant littermates (as in Fig. 2). a, b, Tidal volume (TV) and minute ventilation (MV) of animals exposed to hypoxia. Sample size n = 9 (+/+), 8 (−/−) animals. c, d, TV and MV of animals exposed to hypercapnia. Sample size n = 4 (+/+), 5 (−/−) animals. Data as mean ± s.e.m. *P < 0.05, ***P < 0.001 by paired t-test.

Extended Data Figure 5 Physiological responses of Olfr78/ mutants to hypoxia in vivo.

af, Arterial blood gas measurements of Olfr78+/+ control and Olfr78−/− mutant animals exposed to hypoxia. Values are PaO2 (a), PaCO2 (b), and pH (c) of blood collected from the right carotid artery of anaesthetized Olfr78+/+ control and Olfr78−/− mutant animals exposed to normoxia (21% O2) and hypoxia (10% O2) for 3 min. Oxygen saturation (SaO2, d), [HCO3] (e), and base excess of extracellular fluid (BEecf, f) calculated from PaO2 (a), PaCO2 (b), and pH (c) values. Sample size n = 4 (+/+, 21% O2), 5 (−/−, 21% O2), 4 (+/+, 10% O2), 6 (−/−, 10% O2) animals. g, Body temperature of unanaesthetized Olfr78+/+ control and Olfr78−/− mutant littermates in room air (21% O2) and exposed to hypoxia (10% O2) for indicated times. Sample size n = 4 (+/+), 6 (−/−) animals. hj, Metabolic values measured by indirect calorimetry of unanaesthetized Olfr78+/+ control and Olfr78−/− mutant littermates exposed to normoxia (21% O2) and hypoxia (10% O2) for 10 min. Sample size n = 4 (+/+), 6 (−/−) animals. Data as mean ± s.e.m. *P < 0.05 by unpaired t-test.

Extended Data Figure 6 Carotid body chemosensory responses assayed by carotid sinus nerve activity.

a, b, Raw discharge frequency (extracellular recording) of carotid sinus nerves from Olfr78+/+ control and Olfr78−/− mutant animals at time 0 (a) and 9 min (b) after the change in gas bubbling the perfusion buffer from 95% O2/5% CO2 to 95% N2/5% CO2. c, d, Carotid sinus nerve activity of an Olfr78+/− nerve 9 min after the change in gas to 95% N2/5% CO2 (c) and 2 min later after addition of 7.5 μM TTX while still bubbling 95% N2/5% CO2 (d). Scored action potentials are marked by filled circles. eh, Time course of carotid sinus nerve activity in the Olfr78 genotypes indicated, scored using Spike2 software (e, g) or by hand (f, h) and showing mean ± s.e.m. (e, f) or individual (g, h) values. The residual responses of Olfr78−/− nerves to hypoxia were more apparent when scored by hand. Sample size n = 6 (3 +/+, 3 +/−), 5 (−/−) animals. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t-test. Olfr78+/+ and Olfr78+/− recordings were not significantly different from each other at any time point, except for time = 11 min, by unpaired t-test (P > 0.05). i, j, Time course of raw discharge of carotid sinus nerves from Olfr78+/+ control and Olfr78−/− mutant animals in response to acetate (30mM, 5 min), propionate (30 mM, 5 min), and lactate (30 mM, 5 and 10 min), and pH 7.0 (5 min) scored using Spike2 software (i) or by hand (j). Recovery times were 15 min between acetate, propionate, and lactate, and at least 30 min between lactate and pH 7.0. To minimize the contribution of endogenous hypoxic signals, the superperfusion buffer in the chamber was maintained at hyperoxic conditions (PO2 = 625 mmHg). Sample size n = 5 (+/+), 5 (−/−) animals. Data as mean ± s.e.m. *P < 0.05, **P < 0.01 by unpaired t-test.

Extended Data Figure 7 Lactate activates Olfr78 expressed in HEK293T cells and increases acutely in blood in hypoxia in vivo.

a, b, HEK293T cells transfected with empty vector pCI (a) or pCI-Rho-Olfr78 (b) and RTP1S (OR transport protein) and cytoplasmic GFP (co-transfection marker) plasmids. Transfected cells were stained before fixation to detect Rho-tagged Olfr78 (anti-Rho; red) on the cell surface. GFP (transfection marker, green); DAPI (nuclei, blue). Bar, 100 μm. c, Quantitation of cells expressing GFP and Rho as percentage of DAPI-positive cells in fields shown in a and b. Sample size n = 164 (pCI), 108 (pCI-Rho-Olfr78) cells. Data as percentage ± standard error of percentage. d, Dose–response curves for propionate, acetate, and chloride compared with lactate in activation of Olfr78 in transfected HEK293T cells as in Fig. 4a. Sample size n = 8 (propionate), 12 (acetate), 4 (chloride), and 12 (lactate) wells. Data as mean ± s.e.m. By analysis of variance (ANOVA), all chemicals except chloride (P = 0.309) showed significant difference (P < 0.001). e, Dose–response curves as in c except cells were transfected with empty vector (pCI). ANOVA showed no significant difference (P > 0.05) for any chemical. f, EC50, 95% confidence interval of EC50, and relative maximal activation values from fitted curves in c. ND, not determined owing to lack of curve fitting to data. g, Structures of the short-chain fatty acids. h, Lactate concentrations in blood collected from tail artery of restrained, unanaesthetized Olfr78+/+ control and Olfr78−/− mutant littermates exposed to hypoxia (10% O2) for 4–5 min. Values for animals in normoxia (21% O2) are likely to be an overestimate of baseline concentrations owing to greater restraint required to immobilize animals in normoxia41. Sample size n = 5 (+/+), 6 (−/−) animals. Data as mean ± s.e.m. *P < 0.05 by paired t-test.

Extended Data Figure 8 Calcium imaging of responses of carotid body glomus cells to chemosensory stimuli.

a, Carotid body of a Th-Cre; ROSA-tdTomato adult immunostained for the Cre-dependent reporter tdTomato (red) and TH (green) to show glomus cells31,32,34 and counterstained with DAPI (nuclei, blue). tdTomato labelled glomus cells. be, Tissue preparations for calcium imaging of carotid bodies from TH-Cre; ROSA-GCaMP3 animals that express the calcium indicator GCaMP3 selectively in glomus cells31,32,33. b, Differential interference contrast image of whole mount carotid bifurcation with GCaMP3 fluorescence pseudocoloured green. c, High-magnification, two-photon image of boxed region in b. d, Differential interference contrast image of carotid body tissue slice with GCaMP3 fluorescence pseudocoloured green. e, Two-photon image of carotid body slice in d. Inset shows glomus cell marked by asterisk at higher magnification. GCaMP3 fluorescence was seen in cytoplasm and excluded from nucleus of glomus cells. Bars, 100 μm (a), 200 μm (b), 50 μm (ce). fi, Time course of calcium responses of individual glomus cells to hypoxia, lactate, and cyanide. Whole mount carotid bodies were exposed sequentially to hypoxia (40–50 mmHg), lactate (30 mM), and cyanide (2 mM). Interval between data points is ~2 min, the time required to acquire a stack of images through the carotid body, excluding the 2 min ramp times between stimuli. All glomus cells analysed (n = 42 cells) responded strongly to cyanide. Fluorescence traces shown are the 29 individual glomus cells that responded to both hypoxia and lactate, arranged in order of decreasing initial fluorescence intensity. The other 13 glomus cells responded either to hypoxia (9 cells) or to lactate (4 cells). Multiple data points for buffer or stimuli were averaged to generate the data presented in Fig. 4c. Background colours match bar colours in Fig. 4d.

Extended Data Table 1 Top 150 genes highly expressed in carotid body versus adrenal medulla by RNA sequencing
Extended Data Table 2 Expression of genes associated with olfactory neurons

Supplementary information

Supplementary Table 1 - Olfactory receptors expressed in carotid body and adrenal medulla by RNA-seq

*Carotid body (CB) and adrenal medulla (AM) values are reads per kilobase per million (RPKM). Olfactory receptors genes with no aligned reads in both CB and AM (1,029 of 1,123 genes) not included. P < 0.05 and P < 0.01 between CB and AM by paired t test. (PDF 318 kb)

Locomotory behavior of an Olfr78-/- mutant in normoxia and hypoxia

An adult Olfr78-/- mouse was placed in a plethysmography chamber in normoxia (21% O2), followed by a 1-minute ramp to hypoxia (12% O2). Note the reduction in locomotory activity that began during the ramp and persisted in hypoxia (12% O2). After 5 minutes in hypoxia (12% O2), the gas was shifted back to normoxia (21% O2), and the animal resumed normal locomotor activity. Similar behavior in normoxia and hypoxia was observed for wild-type Olfr78+/+ littermates (data not shown). (MP4 4308 kb)

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Chang, A., Ortega, F., Riegler, J. et al. Oxygen regulation of breathing through an olfactory receptor activated by lactate. Nature 527, 240–244 (2015). https://doi.org/10.1038/nature15721

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