Using redox-sensitive fluorescent probes to record real-time reactive oxygen species production in cells from mouse carotid body slices.

Reactive oxygen species (ROS) are important signaling molecules for physiologic processes such as acute response to hypoxia. However, reliable real-time ROS measurement in cells has been a long-standing methodological challenge. Here, we present a protocol to record acute changes in ROS production in sensory cells from mouse carotid body (CB) slices using redox-sensitive green fluorescent protein probes and microfluorimetry. This protocol provides sensitive and reproducible quantification of ROS during acute hypoxia in different subcellular compartments of CB glomus cells. For complete details on the use and execution of this protocol, please refer to Fernández-Agüera et al. (2015) and Arias-Mayenco et al. (2018).

Acute O2 Sensing: Role of Coenzyme QH2/Q Ratio and Mitochondrial ROS Compartmentalization.

Acute O2 sensing by peripheral chemoreceptors is essential for mammalian homeostasis. Carotid body glomus cells contain O2-sensitive ion channels, which trigger fast adaptive cardiorespiratory reflexes in response to hypoxia. O2-sensitive cells have unique metabolic characteristics that favor the hypoxic generation of mitochondrial complex I (MCI) signaling molecules, NADH and reactive oxygen species (ROS), which modulate membrane ion channels. We show that responsiveness to hypoxia progressively disappears after inducible deletion of the Ndufs2 gene, which encodes the 49 kDa subunit forming the coenzyme Q binding site in MCI, even in the presence of MCII substrates and chemical NAD+ regeneration. We also show contrasting effects of physiological hypoxia on mitochondrial ROS production (increased in the intermembrane space and decreased in the matrix) and a marked effect of succinate dehydrogenase activity on acute O2 sensing. Our results suggest that acute responsiveness to hypoxia depends on coenzyme QH2/Q ratio-controlled ROS production in MCI.

Monitoring Functional Responses to Hypoxia in Single Carotid Body Cells.

The carotid body is the main arterial chemoreceptor in mammals that mediates the cardiorespiratory reflexes activated by acute hypoxia. Here we describe the protocols followed in our laboratory to study responsiveness to hypoxia of single, enzymatically dispersed, glomus cells monitored by microfluorimetry and the patch-clamp technique.

Gene expression analyses reveal metabolic specifications in acute O2 -sensing chemoreceptor cells.

KEY POINTS: Glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM) are essential for reflex cardiorespiratory adaptation to hypoxia. However, the mechanisms whereby these cells detect changes in O2 tension are poorly understood. The metabolic properties of acute O2 -sensing cells have been investigated by comparing the transcriptomes of CB and AM cells, which are O2 -sensitive, with superior cervical ganglion neurons, which are practically O2 -insensitive. In O2 -sensitive cells, we found a characteristic prolyl hydroxylase 3 down-regulation and hypoxia inducible factor 2α up-regulation, as well as overexpression of genes coding for three atypical mitochondrial electron transport subunits and pyruvate carboxylase, an enzyme that replenishes tricarboxylic acid cycle intermediates. In agreement with this observation, the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. The responsiveness of peripheral chemoreceptor cells to acute hypoxia depends on a 'signature metabolic profile'. ABSTRACT: Acute O2 sensing is a fundamental property of cells in the peripheral chemoreceptors, e.g. glomus cells in the carotid body (CB) and chromaffin cells in the adrenal medulla (AM), and is necessary for adaptation to hypoxia. These cells contain O2 -sensitive ion channels, which mediate membrane depolarization and transmitter release upon exposure to hypoxia. However, the mechanisms underlying the detection of changes in O2 tension by cells are still poorly understood. Recently, we suggested that CB glomus cells have specific metabolic features that favour the accumulation of reduced quinone and the production of mitochondrial NADH and reactive oxygen species during hypoxia. These signals alter membrane ion channel activity. To investigate the metabolic profile characteristic of acute O2 -sensing cells, we used adult mice to compare the transcriptomes of three cell types derived from common sympathoadrenal progenitors, but exhibiting variable responsiveness to acute hypoxia: CB and AM cells, which are O2 -sensitive (glomus cells > chromaffin cells), and superior cervical ganglion neurons, which are practically O2 -insensitive. In the O2 -sensitive cells, we found a characteristic mRNA expression pattern of prolyl hydroxylase 3/hypoxia inducible factor 2α and up-regulation of several genes, in particular three atypical mitochondrial electron transport subunits and some ion channels. In addition, we found that pyruvate carboxylase, an enzyme fundamental to tricarboxylic acid cycle anaplerosis, is overexpressed in CB glomus cells. We also observed that the inhibition of succinate dehydrogenase impairs CB acute O2 sensing. Our data suggest that responsiveness to acute hypoxia depends on a 'signature metabolic profile' in chemoreceptor cells.

Selective accumulation of biotin in arterial chemoreceptors: requirement for carotid body exocytotic dopamine secretion.

KEY POINTS: Biotin, a vitamin whose main role is as a coenzyme for carboxylases, accumulates at unusually large amounts within cells of the carotid body (CB). In biotin-deficient rats biotin rapidly disappears from the blood; however, it remains at relatively high levels in CB glomus cells. The CB contains high levels of mRNA for SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Animals with biotin deficiency exhibit pronounced metabolic lactic acidosis. Remarkably, glomus cells from these animals have normal electrical and neurochemical properties. However, they show a marked decrease in the size of quantal dopaminergic secretory events. Inhibitors of the vesicular monoamine transporter 2 (VMAT2) mimic the effect of biotin deficiency. In biotin-deficient animals, VMAT2 protein expression decreases in parallel with biotin depletion in CB cells. These data suggest that dopamine transport and/or storage in small secretory granules in glomus cells depend on biotin. ABSTRACT: Biotin is a water-soluble vitamin required for the function of carboxylases as well as for the regulation of gene expression. Here, we report that biotin accumulates in unusually large amounts in cells of arterial chemoreceptors, carotid body (CB) and adrenal medulla (AM). We show in a biotin-deficient rat model that the vitamin rapidly disappears from the blood and other tissues (including the AM), while remaining at relatively high levels in the CB. We have also observed that, in comparison with other peripheral neural tissues, CB cells contain high levels of SLC5a6, a biotin transporter, and SLC19a3, a thiamine transporter regulated by biotin. Biotin-deficient rats show a syndrome characterized by marked weight loss, metabolic lactic acidosis, aciduria and accelerated breathing with normal responsiveness to hypoxia. Remarkably, CB cells from biotin-deficient animals have normal electrophysiological and neurochemical (ATP levels and catecholamine synthesis) properties; however, they exhibit a marked decrease in the size of quantal catecholaminergic secretory events, which is not seen in AM cells. A similar differential secretory dysfunction is observed in CB cells treated with tetrabenazine, a selective inhibitor of the vesicular monoamine transporter 2 (VMAT2). VMAT2 is highly expressed in glomus cells (in comparison with VMAT1), and in biotin-deficient animals VMAT2 protein expression decreases in parallel with the decrease of biotin accumulated in CB cells. These data suggest that biotin has an essential role in the homeostasis of dopaminergic transmission modulating the transport and/or storage of transmitters within small secretory granules in glomus cells.

Oxygen Sensing by Arterial Chemoreceptors Depends on Mitochondrial Complex I Signaling.

O2 sensing is essential for mammalian homeostasis. Peripheral chemoreceptors such as the carotid body (CB) contain cells with O2-sensitive K(+) channels, which are inhibited by hypoxia to trigger fast adaptive cardiorespiratory reflexes. How variations of O2 tension (PO2) are detected and the mechanisms whereby these changes are conveyed to membrane ion channels have remained elusive. We have studied acute O2 sensing in conditional knockout mice lacking mitochondrial complex I (MCI) genes. We inactivated Ndufs2, which encodes a protein that participates in ubiquinone binding. Ndufs2-null mice lose the hyperventilatory response to hypoxia, although they respond to hypercapnia. Ndufs2-deficient CB cells have normal functions and ATP content but are insensitive to changes in PO2. Our data suggest that chemoreceptor cells have a specialized succinate-dependent metabolism that induces an MCI state during hypoxia, characterized by the production of reactive oxygen species and accumulation of reduced pyridine nucleotides, which signal neighboring K(+) channels.

Cellular properties and chemosensory responses of the human carotid body.

The carotid body (CB) is the major peripheral arterial chemoreceptor in mammals that mediates the acute hyperventilatory response to hypoxia. The CB grows in response to sustained hypoxia and also participates in acclimatisation to chronic hypoxaemia. Knowledge of CB physiology at the cellular level has increased considerably in recent times thanks to studies performed on lower mammals, and rodents in particular. However, the functional characteristics of human CB cells remain practically unknown. Herein, we use tissue slices or enzymatically dispersed cells to determine the characteristics of human CB cells. The adult human CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin-positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF). We found that GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. Moreover, glomus cells exhibited voltage-dependent Na(+), Ca(2+) and K(+) currents that were qualitatively similar to those reported in lower mammals. These cells responded to hypoxia with an external Ca(2+)-dependent increase of cytosolic Ca(2+) and quantal catecholamine secretion, as reported for other mammalian species. Interestingly, human glomus cells are also responsive to hypoglycaemia and together these two stimuli can potentiate each other's effects. The chemosensory responses of glomus cells are also preserved at an advanced age. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.