Lactate sensing mechanisms in arterial chemoreceptor cells

ARTICLE Lactate sensing mechanisms in arterial chemoreceptor cells Hortensia Torres-Torrelo 1,2,4,5, Patricia Ortega-Sáenz1,2,3,5, Lin Gao 1,2,3 & José López-Barneo 1,2,3✉ Classically considered a by-product of anaerobic metabolism, lactate is now viewed as a fundamental fuel for oxidative phosphorylation in mitochondria, and preferred over glucose by many tissues. Lactate is also a signaling molecule of increasing medical relevance. Lactate levels in the blood can increase in both normal and pathophysiological conditions (e.g., hypoxia, physical exercise, or sepsis), however the manner by which these changes are sensed and induce adaptive responses is unknown. Here we show that the carotid body (CB) is essential for lactate homeostasis and that CB glomus cells, the main oxygen sensing arterial chemoreceptors, are also lactate sensors. Lactate is transported into glomus cells, leading to a rapid increase in the cytosolic NADH/NAD+ratio. This in turn activates membrane cation channels, leading to cell depolarization, action potential firing, and Ca2+ influx. Lactate also decreases intracellular pH and increases mitochondrial reactive oxygen species production, which further activates glomus cells. Lactate and hypoxia, although sensed by separate mechanisms, share the same final signaling pathway and jointly activate glomus cells to potentiate compensatory cardiorespiratory reflexes. https://doi.org/10.1038/s41467-021-24444-7 OPEN 1Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain. 2Departamento de Fisiología Médica y Biofísica, Facultad de Medicina, Universidad de Sevilla, Seville, Spain. 3Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain. 4 Present address: Facultad de Medicina, Universidad San Pablo-CEU, CEU Universities, Madrid, Spain. 5 These authors contributed equally: Hortensia Torres-Torrelo, Patricia Ortega-Sáenz. ✉email: [email protected] NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications 1 1234567890():,; While lactate has classically been considered a waste product of anaerobic metabolism and a fatigue agent, this view has changed radically in recent years. It is now established that even under fully aerobic conditions, lactate is the final product of glycolysis in many cell types1,2. Lactate can be secreted, for example, by white skeletal muscle, erythrocytes or astrocytes and either consumed by neighboring cells (e.g., neurons)3or transported by the blood to cells in distant organs for energy production (e.g., in the heart) or gluconeogenesis (e.g., in the liver)4. In lactate-consuming cells, lactate is converted to pyruvate by lactate dehydrogenase in the cytosol or in the mitochondrial intermembrane space5–8. In addition to being a systemic metabolic substrate, preferred over glucose as a fuel in numerous tissues9, lactate is gaining relevance for its role as a signaling molecule. Lactate is co-transported with protons (H+) into cells by a broadly distributed family of reversible monocarboxylate transporters (MCTs)10. It can also bind to GPR81, a G-protein coupled putative lactate receptor expressed in adipose tissue and brain11,12. Lactate inhibits lipolysis13, favors angiogenesis through the induction of VEGF14 and elicits vasodilation15. Infused lactate increases circulating BDNF levels16, protects heart and ischemic neurons, promotes adult hippocampal neurogenesis17,18, and inhibits inflammation following organ injury19,20. Moreover, lactate’s interaction with histidine residues activates TREK1 K+channels21, while lactylation of histone lysines regulates gene expression22. Despite the vast amount of existing literature on lactate metabolism and signaling in numerous organs, the “where”and “how”blood lactate (lactatemia) is sensed to elicit adaptive responses in the organism are unknown. Acute hyperlactatemia occurs in response to physical exercise or exposure to systemic hypoxia4,7,23, however, the homeostatic role of these responses is not well understood. It has been suggested that lactate itself, apart from lactic acidosis, can induce ventilation24 and contribute to the initiation and maintenance of the hypoxic ventilatory response (HVR)25, possibly via an action on the carotid body (CB)26, the main acute O 2 -sensing organ in mammals27. It has also been reported that lactate activates the CB by means of binding to an atypical olfactory receptor (Olfr78) that is highly expressed in the CB’s chemoreceptor (or glomus) cells28. This latter proposal was disregarded because the findings could not be reproduced by others29–32. Nonetheless, in preliminary studies, we found that single Olfr78-deficient glomus cells are indeed directly activated by lactate31. Here we report that the CB is essential for lactate homeostasis and that glomus cells are physiologically relevant acute lactate sensors. Lactate is transported into glomus cells where it increases the cytosolic NADH/NAD+ ratio, decreases cytosolic pH, and stimulates mitochondrial production of reactive oxygen species (ROS), which in turn activate cation channels to induce membrane depolarization, Ca2+entry, and transmitter release. Lactate and hypoxia, although sensed by separate mechanisms, jointly activate glomus cells to potentiate adaptive cardiorespiratory reflexes. Results and discussion Carotid body dysfunction enhances hypoxia-induced lactatemia. The main systemic acute O 2 -sensing organ in mammals is the CB, which upon activation by hypoxia stimulates the central respiratory and autonomic centers to induce hyperventilation and sympathetic activation27. We tested whether the CB plays any role in lactate homeostasis by monitoring hypoxia-induced lactatemia in wild type mice (control) compared to mice insensitive to hypoxia due to deficient CB function. To this end, we used two mouse models previously studied in our laboratory: TH-HIF2α, with embryonic ablation of the gene Epas1 (coding HIF2α)in sympathoadrenal (tyrosine hydroxylase-positive) cells and exhibiting CB atrophy and strong inhibition of the HVR33 (Supplementary Fig. 1), and ERT2-HIF2α, with ubiquitous conditional ablation of Epas1 and showing normal CB development but selective abolition of responsiveness to hypoxia in adulthood34.In wild type mice, hypoxia, even if relatively mild, induced a graded and reversible increase in blood lactate within a few minutes that occurred in parallel with an increase in ventilation (Fig. 1a and c). In contrast, hypercapnia (5% CO 2 ), a stimulus known to activate CB cells27, had no effect on blood lactate but did evoke a ventilatory response stronger than that induced by hypoxia (Fig. 1b and c). Lactatemia elicited by breathing 10% O 2 , was much higher in CB-deficient mice (~30% and ~90% increase in TH-HIF2αand ERT2-HIF2αstrains, respectively) than in controls (Fig. 1d). These data indicate that although hypercapnia induces, as hypoxia, CB activation27 and hyperventilation it is not accompanied by lactatemia. CB activation during systemic hypoxia in normal mice reduces the intensity of hypoxemia33 and thereby blunts lactate release from tissues. Ca2+-dependent activation of carotid body glomus cells by lactate. We studied the effect of extracellular L-lactate (sodium salt) on mouse single glomus cells, the polymodal acute O 2 sensing neurosensory elements in the CB (Fig. 2a)27.Amperometric experiments showed that lactate induced a dose-dependent exocytotic catecholamine release from cells in CB slices (Fig. 2b and c). Cytosolic Ca2+measured in Fura 2-loaded dispersed glomus cells increased in response to high K+and hypoxia35.In~90%ofthese O 2 -sensitive cells, lactate also elicited an increase in cytosolic Ca2+ (Fig. 2d–f) which was abolished by extracellular cadmium (Cd2+), a non-selective blocker of high-threshold membrane Ca2+channels in glomus cells (Fig. 2g)35. Lactate-induced secretory activity was also blocked by Cd2+(Fig. 2h) and strongly inhibited by nifedipine (Fig. 2iandj),ablockerofL-typeCa 2+channels in rodent glomus cells36. Therefore, similar to hypoxia, lactate activates glomus cells in an external Ca2+-dependent manner. In agreement with these data in mouse CB, we recorded robust responses to lactate in dispersed rat glomus cells (Supplementary Fig. 2a) as well as in cells in rat CB slices (Supplementary Fig. 2b). It has recently been reported that membrane potential and conductance of rat glomus cells are unaffected by lactate, however, these experiments were performed with the whole-cell configuration of the patch-clamp technique and therefore intracellular dialysis may have altered the lactate signaling pathway37 (see below). Lactate is transported into glomus cells and leads to NADH accumulation and mitochondrial ROS production. In many cell types, lactate is co-transported with H+across the plasma membrane by monocarboxylate transporters (MCTs) and once inside the cell is converted to pyruvate (in the cytosol or organelles) by lactate dehydrogenase with production of NADH (see scheme in Fig. 3a). We studied by immunocytochemistry the expression in CB cells of MCT1,2,and4,themostrepresentativeMCTsinmammalian cells38.MCT2,ahigh-affinity (Km < 1 mM) lactate transporter expressed in neurons and other cell types38,39, was consistently detected in tyrosine hydroxylase (TH) positive glomus cells (Fig. 3b), however, this transporter was absent in glia-like, fibrillary acidic protein (GFAP) positive, CB type II cells (Fig. 3c; Supplementary Fig. 3). MCT2 was also expressed in neurons of the superior cervical ganglion (SCG) and in adrenal medulla (AM) chromaffin cells (Supplementary Fig. 4a and b), which are catecholaminergic (TH positive) cells that, as the CB glomus cells, derive from neural crest progenitors. Neither CB glomus cells (Fig. 3d) nor type II cells (Supplementary Fig. 5a and c) expressed MCT1, a ubiquitous lactate transporter with lower affinity (Km ~5 mM) than MCT238. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 2NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021- 24444-7 | www.nature.com/naturecommunications However, TH positive CB cells expressed MCT4 (Fig. 3e), which was considered to be a low affinity (Km > 20 mM) transporter characteristic of lactate secreting cells38,40. A recent study indicates that MCT4 affinity for lactate (Km ~1 mM) is much higher than previously thought and that this transporter has a relevant affinity for pyruvate (Km ~4 mM). However, the higher affinity for lactate relative to pyruvate makes MCT4 suitable for a major role in lactate release from cells41. MCT4 appeared to be only slightly expressed in some type II cells (Supplementary Fig. 5b and d). The selective abundant expression of MCT2 in glomus cells suggest that they can easily uptake lactate from blood, a property compatible with their role as lactate sensors. MCT4 may also contribute to lactate uptake by glomus cells. This transporter, which is less sensitive to pyruvate than to lactate38,41, could also mediate lactate secretion from glomus cells in some circumstances, as for example in conditions of accelerated glycolysis secondary to mitochondrial dysfunction42. MCT4 might also be preferentially expressed in CB neuroblasts, which are immature TH-positive CB cells with the mitochondriabased acute O 2 -sensor still undeveloped43. Noteworthy, type II cells, which are glia-like supportive elements with quiescent stem cell function44,45, did not express MCT1 and seemed to have only low levels of MCT4 expression. In this regard, type II cells are different from astrocytes which release lactate to fuel and modulate the function of neighboring neurons3,39,46. In agreement with the immunocytochemical observations, we monitored lactate transport into glomus cells by measuring intracellular acidification using single-cell microfluorimetry (Fig. 3f and g). Control of the lactateinduced acidification signal was obtained by exposure of the cells to CO 2 ,whichstronglyacidifies the cytosol due to its conversion to carbonic acid by carbonic anhydrase present in CB glomus cells27. 0 10 20 min 21% PO2 ab 0 10 20 min 0.04% 5% PCO2 0.04% hypoxic ventilatory response 160 120 200 240 % O2 21 10 1 min 200 160 240 280 1 min 0.04 c d % CO2 10% or 15% 21% hypercapnic ventilatory response breaths/min breaths/min 15% O2 21% O2 10% O2 r21% O2 5% CO2 5.0 lactate sampling 5% lactate sampling 21% O 2 10% O 2 blood lactate (mM) 0 15 10 5 P = 0.0017 blood lactate (mM) 0 10 5 15 20 control TH-HIF2D ERT2-HIF2D control TH-HIF2D ERT2-HIF2D P = 0.0001 P < 0.0001 P = 0.0004 P < 0.0001 P < 0.0001 P < 0.0001 Fig. 1 Blunting of hypoxia-induced lactatemia by arterial chemoreceptors. a,bTop. Scheme of the experimental protocol followed to measure blood lactate (lactate sampling indicated by horizontal brown bars) under normoxic conditions (21% O 2 ) and during exposure to hypoxia (10% or 15% O 2 tension, pale blue) or hypercapnia (5% CO 2 , pale red). Bottom. Plethysmographic recordings showing the average increase in respiratory frequency (breaths/min) during exposure to hypoxia (blue dots; n=35 mice) or hypercapnia (red dots; n=27 mice). cScatter plots of blood lactate levels measured in the various experimental conditions. Values (mean ± SEM) are: normoxia (2.5 ± 0.1 mM, n=29 mice; gray), hypoxia 15% O 2 (3.6 ± 0.3 mM, n=17 mice; blue), hypoxia 10% O 2 (5.8 ± 0.5 mM, n=23 mice; blue), recovery in normoxia (r21% O 2 measured 5 min after returning to normoxia; 4.4 ± 0.3 mM, n= 14 mice; gray), and hypercapnia (2.2 ± 0.5 mM, n=4 mice; pale red). P-values calculated by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test are indicated. dScatter plots of blood lactate levels, during normoxia (gray) and hypoxia (blue), in control and mice with carotid body dysfunction due to embryonic or conditional adult deficiency of Hif2α.P-values calculated by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test are indicated. Mean ± SEM values are: TH-HIF2α(normoxia =3.2 ± 0.6 mM; hypoxia 10% O 2 =8.3 ± 1.2 mM, n=5 mice); ERT2-HIF2α (normoxia 2.9 ± 0.4 mM; hypoxia 10% O 2 =12.3 ± 1.7 mM, n=4 mice). Source data are provided as a Source data file. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 ARTICLE NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications 3 Exposure of dispersed glomus cells to lactate elicited a fast, highly reversible, and dose-dependent increase in intracellular NADH, as monitored by single-cell NAD(P)H autofluorescence34,42,47,48 (Fig. 4a and b). This signal was strongly inhibited by AR-C155858 (Fig. 4c and d), an inhibitor of MCT1 and MCT249. The lactate (10 mM)-dependent increase in NADH was abolished by the extracellular application of pyruvate (5 mM), a molecule also co-transported with H+by MCT1 and MCT2 but with a higher affinity (Km < 1 mM and <0.1 mM for MCT1 and MCT2, respectively) than lactate38,39. Application of a short pulse of pyruvate produced a marked decrease in the lactate-induced NADH signal to values below that even to basal levels, indicating that once inside the glomus cell, pyruvate, by consuming NADH, was rapidly converted to lactate by lactate dehydrogenase (Fig. 4e and f). Treatment with α-ketobutyrate (α- KB), which was shown previously to rapidly decrease NADH autofluorescence in glomus cells due to its conversion to nonmetabolizable α-hydroxybutyrate50, also prevented the lactatedependent NADH rise in the cells (Supplementary Fig. 6a and b). In addition to decreasing NADH levels, pyruvate inhibited the secretory response to lactate in glomus cells (Fig. 4g). Even a small amount of extracellular pyruvate (100 μM), near the normal concentration in plasma51, was sufficient to partially inhibit lactate-induced secretion in glomus cells (Supplementary Fig. 6c and d). However, normal responses to high lactate were recorded in cells bathed by solutions containing physiological plasma values of pyruvate and lactate51 (Fig. 4h). Although in the presence of lactate a short exposure to pyruvate consistently inhibited the secretory activity in glomus cells, in most cases this inhibitory phase was followed by a late activation of the cells (Supplementary Fig. 6e). Indeed, although at high nonphysiological concentrations, pyruvate by itself (in the absence of lactate) induced a slow secretory activity in all cells tested probably due to intracellular acidification (Supplementary Fig. 6f). These results suggest that increases in extracellular lactate alter the extra and intracellular pyruvate/lactate equilibria, resulting in NADH accumulation (or an increase in the NADH/NAD+ratio), which participates in the regulation of Ca2+influx by lactate in 1 min gcytosolic calcium Cd 1 min Cd 0.1 lactate lactate 0.2 lactate Cd 1 min 340/380 ratio h lactate cadmium 2 pA calcium dependent secretory activity j lactate 1 min lactate nifedipine lactate 5 pA i lactate nifedipine lactate lactate 5 pA l actate 1 min calcium dependent secretory activity bsecretory activity 5 Lac 20 Lac15 Lac10 Lac 5 pA 1 min 0 2 4 6 8 secretion rate (pC/min) 520 1510 0 lactate (mM) glomus cell carbon fiber acd 40K lactate hypoxia cytosolic calcium 0.2 340/380 ratio hypoxia f 100 0 20 40 60 80 % of resp cells e Hx Lac 1 min 340/380 ratio 0 0.2 0.1 0.5 0.4 0.3 Hx Lac carotid artery glossopharyngeal nerve sinus nerve CB ICA ECA carotid sinus Fig. 2 Activation of carotid body cells by lactate. a Scheme of the carotid bifurcation representing the location of the carotid body (CB) and sensory innervation (ICA and ECA, internal and external carotid artery). b,cSecretory activity (catecholamine release measured with a polarized carbon fiber) induced by extracellular lactate in glomus cells in CB slices. Average values (mean ± SEM) in (c) are from: 5 Lac (n=5 cells/5 mice); 10 Lac (n=8 cells/8 mice); 15 Lac (6 cells/6 mice); 20 Lac (n=5 cells/5 mice). dIncrease in cytosolic Ca2+induced by K+(40 mM), hypoxia (O 2 tension ≈15 mm Hg, blue), and lactate (5 mM, gray) in dispersed single glomus cells. ePercentage of cells recorded that responded to hypoxia (blue) and lactate (5–10 mM; gray) (n=20 cells from 6 mice). fBox plots summarizing the amplitude of the responses to hypoxia and lactate (n=20 cells from 6 mice). Mean ± SEM values are: hypoxia: 0.19 ± 0.02; 5 mM lactate: 0.21 ± 0.02. In the boxplots the middle line is the median, the lower and upper hinges (IQR, interquartile range) correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR and the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR. gRepresentative recordings (n=5) of lactate (5 mM)-induced rise in cytosolic Ca2+and its inhibition by Cd2+.hRepresentative amperometric recording illustrating the reversible inhibition of lactate (10 mM)-induced secretory activity by extracellular Cd2+. Similar recordings were obtained in three separate experiments. i,jAmperometric recordings of the inhibitory effects of nifedipine (0.5 µM) on lactate (10 mM)-induced secretion applied before and after exposure to lactate (4 experiments in 3 mice). Source data are provided as a Source data file. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 4NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications these cells (see below). In addition, intracellular acidification produced by lactate transport may also contribute to activation of glomus cells. Together with NADH measurements we monitored the generation of mitochondrial ROS in glomus cells using a fluorescent genetic probe selectively directed to the mitochondrial matrix or intermembrane space (IMS)50. Lactate produced a dose-dependent reversible rise in matrix ROS (Fig. 4i, top), which likely reflected the increased fuel supply to the highly active glomus cell mitochondria50,52. Indeed, pyruvate at the same concentration also produced an increase in matrix ROS, which was in contrast to the decrease in matrix ROS measured in response to hypoxia (Fig. 4i, bottom and j)50. The increase in mitochondria ROS produced by lactate or pyruvate was also manifested at the IMS (Supplementary Fig. 6g and h). Lactate-induced depolarization and action potential firing is triggered by NADH-dependent cation channels. Because lactate induction of secretory activity in glomus cells was dependent on Ca2+influx, we hypothesized that lactate might elicit membrane depolarization and, in this way, give rise to the opening of voltage-gated Ca2+channels. Experiments on patch-clamped (perforated patch configuration) dispersed glomus cells showed that lactate (10 mM) elicited a reversible depolarization of 9.5 ± 1mV (n=17 cells) from a relatively low spontaneous basal resting potential (RP ≈−30 to −40 mV) (Fig. 5a and b). When current clamped cells were hyperpolarized to ~ −70 mV to deinactivate voltage-gated ion channels, lactate evoked a larger (31 ± 4 mV; n=16) depolarization accompanied by the firing of action potentials (Fig. 5b and c). Because glomus cells have a Na+-permeant standing conductance at rest53,54, replacement of extracellular Na+with N-methy-D-glucamine (NMDG, a large membrane-impermeant cation) resulted in cell hyperpolarization and abolition of the response to lactate (Fig. 5d). As shown above for the recordings of NADH autofluorescence and glomus cell secretion, application of a fast pulse of pyruvate rapidly and reversibly inhibited the cell depolarization and action potential firing induced by lactate (Fig. 5e). Finally, the electrophysiological effects of lactate (Fig. 5f) and lactate-induced secretory activity (Fig. 5g) were abolished by 2-APB, a non-selective blocker of some transient receptor potential (TRP) and other cationic channels in CB55. Taken together, these data suggest that lactateinduced increase in the cytosolic NADH/NAD+ratio leads to a cation channel-dependent depolarization of glomus cells with subsequent opening of Ca2+channels, Ca2+influx, and transmitter release. SCG neurons and AM chromaffin cells showed lactate-induced increases in NAD(P)H autofluorescence (Supplementary Fig. 7a, c, e), which is compatible with the expression of MCT2 in these cells (see Supplementary Fig. 4). However, ab H+ H+ lactate lactate MCT pyruvate NADH NAD+ LDH extracellular cytosol plasma membrane intracellular pH 490/440 ratio 0.25 0.5 f lactatelactate CO 2 g 1 min MCT2 / TH / DAPI MCT2 / GFAP / DAPI MCT1 / TH / DAPI MCT4 / TH / DAPI c d e 10 µm MCT2 MCT2 MCT1 MCT4 TH TH TH GFAP intracellular acidification 490/440 ratio 0.5 1.0 Lac 1.5 0.0 CO 2 P = 0.0019 Fig. 3 Expression of lactate transporters in glomus cells. a Scheme of proton-coupled lactate co-transport across the plasma membrane and conversion of lactate to pyruvate by lactate dehydrogenase (LDH). MCT (monocarboxylate transporter). b,cImmunocytochemical demonstration of MCT2 expression in TH-positive CB glomus cells and absence of MCT2 expression in GFAP-positive type II cells. dLack of expression of MCT1 in TH-positive CB glomus cells. eExpression of MCT4 in TH-positive CB glomus cells. Photographs in (b–e) are representative examples of similar data obtained in n=4 mice. Scale bar in (b, left) is the same for all panels. fRepresentative microfluorimetric recording of intracellular glomus cell acidification induced by lactate (Lac, 10 mM) and hypercapnia (~12% CO 2 ). gBox plots representing the distribution of the amplitude of pH signals induced by the two stimuli (n=16 cells from 2 mice). Mean ± SEM values are: 10 mM lactate (gray): 0.36 ± 0.6; 12% CO 2 (red):0.83 ± 0.13. The boxplots indicate median (middle line), 25th, 75th percentile (box), and largest and smallest values extending no further than 1.5× interquartile range (whiskers). Data beyond the end of the whiskers (outlying points) are plotted individually (gray). The P-value calculated by two tails, paired ttest are indicated. Source data are provided as a Source data file. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 ARTICLE NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications 5 patch clamped dispersed SCG neurons that were reversibly depolarized by high extracellular K+were unaltered, or even slightly hyperpolarized, by lactate (Supplementary Fig. 7b, d). On the other hand, hypercapnia induced catecholamine release from adult AM chromaffin cells56, however, these cells appeared to be practically insensitive to lactate (Supplementary Fig. 7f). These observations further support a selective role as lactate sensors for CB glomus cells. The differential sensitivity of glomus cells to lactate, respecting SCG neurons or AM chromaffin cells, may be a consequence of the different expression of membrane ion channels. However, it could be also due to the fact that neither SCG neurons nor AM chromaffin cells are able to generate a relevant mitochondrial ROS signal in response to lactate. Pyridine nucleotides are promiscuous signaling molecules that can regulate, directly or indirectly, a large variety of ion channel families57, most of which are represented in glomus cells27,30,52,58 and therefore could mediate lactate-dependent depolarization. A good candidate forming part of this response is the TRPC5 channel because the mRNA coding this protein is highly abundant in glomus cells30 and in a comparative microarray analysis we showed that TRPC5 mRNA is more highly expressed in the CB than in SCG or AM cells52. To test this possibility, we performed experiments on CB glomus cells from TRPC5 ROS-matrix 5 Lac10 Lac 10 Lac i 400/484 ratio 0.005 400/484 ratio 0.005 20 arb. units 1min 10 Lac10 Lac10 Lac AR-C155858 25 arb. units 1 min 10 Lac 10 Lac 5 Lac 10 Lac 2.5 Lac 50 arb. units 1 min 0 20 40 80 60 Δ NADH (arb. units) 2.5 20105 lactate (mM) abc g 10 Lac 2.5 Lac 5 Pyr ef 10 Lac 10 Pyr hypoxia 2 min 2 min 20 lactate 10 pyruvate 1 min 2 pA 10 Lac 0.1 pyruvate + 1.4 lactate 1 min 5 pA 5 Lac hypoxia h -100 0.3 AR-C control 1 AR-C 0 100 200 300 Δ NADH (arb. units) d 40 20 -20 -40 Lac -60 Pyr Δ NADH (arb. units) 0 400/484 ratio 0.000 0.005 5 Lac 10 Pyr 10 Lac 0.010 0.015 20 Lac secretory response in slices intracellular NADH j P = 0.001 P < 0.0001 P < 0.0001 Fig. 4 Lactate-induced intracellular NADH and ROS signals. a,bIncrease in NAD(P)H autofluorescence (ΔNADH) induced by lactate (Lac in mM, gray) in dispersed glomus cells. Average values (mean ± SEM) in (b) are from: 2.5 Lac (n=21 cells/6 mice); 5 Lac (n=27 cells/8 mice); 10 Lac (28 cells/7 mice); 20 Lac (n=25 cells/4 mice). cReversible blockade of a lactate (10 mM, gray)-induced rise in intracellular NADH (ΔNADH) by MCT inhibition with AR-C155858 (0.3 μM, light blue). dBox plots representing the distribution of lactate-induced NADH signals under control conditions (gray) and with two different doses (0.3 and 1 μM; light blue) of AR-C155858 (n=22 cells from 4 mice). Statistical non-parametric tests were used. Calculated mean ± SEM values are: 10 mM lactate: 87.9 ± 11.7; 0.3 μM of AR-C155858: 28.8 ± 6.4; 1 μM of AR-C155858: 3.2 ± 8.5. Pvalues calculated with Mann–Whitney test are indicated. eInhibition of lactate (10 mM, gray)-induced NADH signal by the application of a short pulse of pyruvate (Pyr, 5 mM, light brown). fBox plots representing the distribution of changes in NADH signals (ΔNADH) induced by lactate (10–20 mM, gray) and following exposure to pyruvate (5–10 mM, light brown) (n=10 cells from 5 mice). Calculated mean ± SEM values are: 10–20 mM lactate: 22.8 ± 4.0; 5–10 mM pyruvate: −32.3 ± 6.1. The boxplots in (d,f) indicate median (middle line), 25th, 75th percentile (box), and largest and smallest values extending no further than 1.5× interquartile range (whiskers). Data beyond the end of the whiskers (outliers) are plotted individually (gray). The Pvalue calculated with Mann–Whitney test is indicated. gRepresentative amperometric recordings illustrating the inhibition of lactate (20 mM, gray)-induced secretion in a glomus cell by pyruvate (10 mM, light brown) (similar results were obtained in n=7 cells from 5 mice). hRepresentative amperometric recordings illustrating the secretory response to high (5 and 10 mM) lactate (gray) and hypoxia (≈15 mm Hg; pale blue) of glomus cells maintained in physiological resting levels of pyruvate/lactate concentrations (0.1 and 1.4 mM, respectively). iReversible and dose-dependent increase in mitochondrial matrix ROS induced by lactate (top and bottom; gray) and pyruvate (bottom; light brown) at the indicated concentrations (mM). Note that hypoxia (O 2 tension ≈15 mm Hg, pale blue) induces a decrease in matrix ROS. jAverage (mean ± SEM) increases in matrix ROS induced by lactate or pyruvate (in mM). Data are from: 2.5 Lac (n=23 cells/3 mice); 5 Lac (n=7 cells/2 mice); 10 Lac (n=30 cells/3 mice); 20 Lac (n=11 cells/2 mice). Arbitrary units (arb. units). Source data are provided as a Source data file. ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 6NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications knockout mice, which exhibited normal responses to hypoxia and lactate (Supplementary Fig. 8a–c). We also studied CB glomus cells from mice lacking TRPC6 channels, another member of the canonical sub-family of TRP channels59, which is expressed in glomus cells58 and has been suggested to be essential for hypoxic pulmonary vasoconstriction60. Our data indicated that sensitivity to lactate was not significantly affected in either TRPC6-deficient glomus cells or in cells obtained from Trpc5/Trpc6 double knockout mice (Supplementary Fig. 8d–g). In addition to TRPC5, mRNAs coding for subunits of TRPC3 and TRPM7 channels, as well as a beta subunit of non-voltage sensitive epithelial Na+ channels have been reported to be abundant in glomus cells30. Nonetheless, pharmacological experiments with several specific blockers (Pry3, FTY720, and amiloride for TRPC3, TRPM7, and epithelial Na+channels, respectively)61–63 showed that these channel types are not essential for lactate sensing by glomus cells (Supplementary Fig. 9a–f). These results suggest that the intracellular NADH and ROS produced by lactate are promiscuous signals that probably redundantly modulate several types of cationic channels in glomus cells. Lactate- and O 2 -sensing are mediated by separate mechanisms. It is well established that activation of CB glomus cells by hypoxia is a cell autonomous process that can be elicited in the absence of lactate or any other endocrine or paracrine signal27. Recently, it was shown that hypoxia-induced glomus cell transmitter release depends on both NADH, shuttled from the mitochondria to the cytosol, and ROS that are primarily generated at the mitochondrial IMS (probably at mitochondrial complexes—MC-I and III), which rapidly inhibit nearby membrane K+channels to induce depolarization and Ca2+influx34,42,50. During more protracted exposures to hypoxia, cationic channels, activated by intracellular Ca2+or cytosolic diffusion of the signaling molecules, may also -40 -60 -20 -80 -10 pA 1 min current clamp in isolated glomus cells -40 -35 -30 -20 -25 -45 lactate membrane potential (mV) -40 -60 -20 -80 1 min Pyr Pyr 1 min -40 -60 -20 NMDG 1 min abc ed membrane potential (mV) membrane potential (mV) membrane potential (mV) 1 min -40 -60 -20 -80 2-APB f membrane potential (mV) lactate lactate lactate lactate lactate lactate lactate lactatelactate lactate lactate lactatelactate 2 pA 2APB 1 min lactate lactate lactate g depolarization (mV) RP 40 20 0 60 - 70 mV secretory response in slices Fig. 5 Depolarization and action potential firing induced by lactate in glomus cells. a–cRepresentative examples of lactate (10 mM)-induced reversible depolarization in a current clamped cell (perforated patch) maintained at the spontaneous resting potential (RP ≈30-40 mV). cIn cells hyperpolarized to ~ −70 mV by current injection (red line and arrow) lactate (10 mM) induced larger depolarizations and the firing of action potentials. bBox plots representing the amplitudes of lactate (10 mM)-induced depolarizations in the two conditions. Mean ± SEM values were: 9.5 ± 1 mV (n=17 cells from 16 mice) at RP; 31.2 ± 4 mV (n=16 cells from 10 mice) at ~ −70 mV. The boxplot represents the median (middle line), 25th, 75th percentile (box), and largest and smallest values extending no further than 1.5× interquartile range (whiskers). Source data are provided as a Source data file. dCell hyperpolarization and abolition of the lactate (10 mM)-induced depolarization after replacement of extracellular Na+with N-methyl-D-glucamine (NMDG). Similar results were obtained in all cells tested (7 cells from 4 mice). eInhibition of lactate (10 mM)-induced depolarization and action potential firing by application of a short pulse of pyruvate (Pyr, 5 mM). Representative example of 8 cells recorded from 5 mice. Red line indicates current injection through the recording electrode to maintain a resting potential of ~ −70 mV. fInhibition of lactate (10 mM)-induced depolarization and action potential firing by 2-APB (10 μM), a non-selective cation channel blocker. Representative example of 3 cells recorded from 3 mice. Red line indicates current injection through the recording electrode to maintain a resting potential of approximately −70 mV. Note that action potential amplitude is truncated due to the relatively long sampling interval (4 ms). gReversible inhibition of the lactate (20 mM)-induced secretory response by 2-APB (10 μM). Representative example of 4 experiments in 3 mice. NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 ARTICLE NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications 7 secondarily contribute to this response64. Here we have shown that lactate activation of glomus cells depends on similar signals (NADH and ROS), although NADH is produced in the cytosol as a consequence of imported lactate and its rapid conversion to pyruvate, and ROS are primarily generated at the mitochondrial matrix. To further determine whether hypoxia and lactate share similar sensing/signaling mechanisms, we tested the effect of rotenone, a blocker of MCI that induces glomus cell secretion and occludes any further effect of hypoxia65. We observed that rotenone does not prevent the powerful glomus cell activation by lactate (Fig. 6a). Moreover, while responsiveness to hypoxia was practically abolished in mice with ablation of the Ndufs2 gene, which codes a 49 KDa protein essential for MCI assembly and function42,50, activation of the Ndufs2-deficient and O 2 -insensi- tive glomus cells by lactate (NADH accumulation and catecholamine release) remained unaltered (Fig. 6b–e). Moreover, CB cells from mice deficient of Hif2α, which have a normal MCI but strong inhibition of responsiveness to acute hypoxia due to down-regulation of atypical MCIV mitochondrial subunits34, also showed robust responses to lactate (Fig. 6f). These data indicate that lactate and oxygen sensing are mediated by separate mechanisms, although they share compartmentalized signals (rise of cytosolic NADH/NAD+ratio and ROS) that induce depolarization, Ca2+influx, and glomus cell transmitter release (Fig. 6g). In support of this notion, we found that between 65 and 90% of glomus cells that responded to hypoxia also responded to lactate (Fig. 7a–c; see Fig. 2d–f). Moreover, both the NADH signal and the secretory response to hypoxia were strongly potentiated by simultaneous application of extracellular lactate (Fig. 7d–g). In sum, we show here that the carotid arterial chemoreceptors play a major role in lactate homeostasis, a physiological process of broad functional and medical relevance. CB chemoreceptor cells TH-NDUFS2 CO2 10 lactate 40K 40K hypoxia 100 0 20 40 60 80 hypoxia CO2 % of responding cells lactate 10 Lac hypoxia 25 arb. units 5 pA ab Δ NADH Control (wild type) 1 μM rotenone 1 pA 20 lactate 1 min c 1 min 1 min de TH-NDUFS2 2 pA 1 min 10 lactate hypoxia f g Ca2+ Glomus cell K+ channels H+ H+ Lac lactate NADH NAD+ LDH Ca2+ channels Vm Vm N: NADH R: ROS Pyr cation channels O2 hypoxia and lactate signaling pathways 50 150 100 0 hypoxia lactate Δ NADH (arb. units) 10 Lac N R 20 lactate ERT2- HIF2α MCT P = 0.0009 Fig. 6 Separate lactate- and O 2 -sensing mechanisms in glomus cells. a Amperometric recording illustrating the secretory response to lactate (20 mM, gray) in the presence of rotenone (1 μM). Similar recordings were obtained in 7 cells from 3 mice. b,cNADH signals induced by lactate (Lac, 10 mM; gray) in a hypoxia (Hx, O 2 tension ≈15 mm Hg; pale blue)-insensitive glomus cell dispersed from a Ndufs2-deficient CB. Box plots representing the distribution of changes in NADH signals (ΔNADH) in 22 cells from 3 mice exposed to the two stimuli. In these experiments, all cells responded to lactate (10 mM) but only 40% of the cells showed some responsiveness to hypoxia (O 2 tension ≈15 mm Hg). Mean ± SEM values are: 10 mM lactate: 52.5 ± 4.3; hypoxia: 28.2 ± 3.2. Pvalue calculated by two tails, unpaired ttest is indicated. The boxplot represents the median (middle line), 25th, 75th percentile (box), and largest and smallest values extending no further than 1.5 × interquartile range (whiskers). Source data are provided as a Source data file. d,eRepresentative amperometric recordings from an Ndufs2-deficient CB slice showing that cells insensitive to hypoxia (pale blue) are activated by lactate (10 mM, gray), hypercapnia (10% CO 2 , pale red) and K+(40 mM)-induced depolarization. Similar experiments (n=6) were performed in CBs from 3 mice. None of the cells from these mice showed a secretory response to hypoxia. fSecretory responses to lactate (gray) in a hypoxia-insensitive (pale blue) glomus cell deficient of the Epas1 (coding Hif2α) gene. Similar experiments (n=2) were performed in CBs from two mice. gSchematic representation of the major compartmentalized steps in acute oxygen and lactate sensing by carotid body glomus cells. The decrease of oxygen (hypoxia) is detected by mitochondria, giving rise to the production of NADH (N) and reactive oxygen species (R) which inhibit nearby membrane K+channels to induce depolarization. On the other hand, lactate is transported into the cells by MCTs and rapidly converted to pyruvate with the production of NADH, which activates membrane cation channels to produce cell depolarization (an effect that may be facilitated by intracellular acidification). Pyruvate can also increase the production of reactive oxygen species at the mitochondria and in this manner contribute to the activation of glomus cells. Both hypoxia and lactate increase cytosolic [Ca2+] and induce transmitter release. MCT monocarboxylate transporter, LDH lactate dehydrogenase, ΔVm membrane depolarization. Arbitrary units (arb. units). ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 8NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications are activated by systemic hypoxia and thereby reduce the intensity of hypoxia-induced lactatemia. In addition, glomus cells are lactate sensors because they efficiently take up and metabolize lactate to increase the cytosolic NADH/NAD+ratio and mitochondrial ROS production, which modulate the activity of membrane ion channels. Both lactate and hypoxia, although sensed by separate mechanisms, converge on producing cell depolarization and opening of Ca2+channels (see Fig. 6g) and, therefore, have additive effects on glomus cells. The potentiation of CB activation by lactate during hypoxia facilitates a rapid reflex ventilatory compensation of hypoxemia and, secondarily, blunts peripheral lactate release (Fig. 7h). This adaptive response could minimize potential deleterious effects due to lactate release (e.g., metabolic acidosis) or excessive lactate uptake by cells. CB ab de c 1 min lactate 100 Hx 0 20 40 60 80 % responsive cells 5-10 Lac 1 pA Lac HxLac Hx Δ NADH 10 arb. units lactate 1min 1 pA hypoxia hypoxia lactate + hypoxia fg hlactate potentiation of the hypoxic response hyperventilation carotid body lactate release from tissues hypoxia hypoxia 50 20 10 0 Lac + Hx Hx Δ NADH (arb. units) 30 40 1 min Lac Hx 0 10 7.5 secretion rate (pC/min) 2.5 5 15 Hx 0 10 5 secretion rate (pC/min) Lac + Hx Hx P = 0.0353 P = 0.0009 P = 0.0044 NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24444-7 ARTICLE NATURE COMMUNICATIONS | (2021) 12:4166 | https://doi.org/10.1038/s41467-021-24444-7 | www.nature.com/naturecommunications 9