Patricia Ortega-Sáenz

Glia-like Stem Cells Sustain Physiologic Neurogenesis in the Adult Mammalian Carotid Body

Neurogenesis is known to occur in the specific niches of the adult mammalian brain, but whether germinal centers exist in the neural-crest-derived peripheral nervous system is unknown. We have discovered stem cells in the adult carotid body (CB), an oxygen-sensing organ of the sympathoadrenal lineage that grows in chronic hypoxemia. Production of new neuron-like CB glomus cells depends on a population of stem cells, which form multipotent and self-renewing colonies in vitro. Cell fate mapping experiments indicate that, unexpectedly, CB stem cells are the glia-like sustentacular cells and can be identified using glial markers. Remarkably, stem cell-derived glomus cells have the same complex chemosensory properties as mature in situ glomus cells. They are highly dopaminergic and produce glial cell line-derived neurotrophic factor. Thus, the mammalian CB is a neurogenic center with a recognizable physiological function in adult life. CB stem cells could be potentially useful for antiparkinsonian cell therapy.

Abnormal sympathoadrenal development and systemic hypotension in PHD3–/– mice.

ABSTRACT Cell culture studies have implicated the oxygen-sensitive hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 in the regulation of neuronal apoptosis. To better understand this function in vivo, we have created PHD3−/− mice and analyzed the neuronal phenotype. Reduced apoptosis in superior cervical ganglion (SCG) neurons cultured from PHD3−/− mice is associated with an increase in the number of cells in the SCG, as well as in the adrenal medulla and carotid body. Genetic analysis by intercrossing PHD3−/− mice with HIF-1a+/− and HIF-2a+/− mice demonstrated an interaction with HIF-2α but not HIF-1α, supporting the nonredundant involvement of a PHD3-HIF-2α pathway in the regulation of sympathoadrenal development. Despite the increased number of cells, the sympathoadrenal system appeared hypofunctional in PHD3−/− mice, with reduced target tissue innervation, adrenal medullary secretory capacity, sympathoadrenal responses, and systemic blood pressure. These observations suggest that the role of PHD3 in sympathoadrenal development extends beyond simple control of cell survival and organ mass, with functional PHD3 being required for proper anatomical and physiological integrity of the system. Perturbation of this interface between developmental and adaptive signaling by hypoxic, metabolic, or other stresses could have important effects on key sympathoadrenal functions, such as blood pressure regulation.

The Mitochondrial SDHD Gene Is Required for Early Embryogenesis, and Its Partial Deficiency Results in Persistent Carotid Body Glomus Cell Activation with Full Responsiveness to Hypoxia

ABSTRACT The SDHD gene encodes one of the two membrane-anchoring proteins of the succinate dehydrogenase (complex II) of the mitochondrial electron transport chain. This gene has recently been proposed to be involved in oxygen sensing because mutations that cause loss of its function produce hereditary familiar paraganglioma, a tumor of the carotid body (CB), the main arterial chemoreceptor that senses oxygen levels in the blood. Here, we report the generation of a SDHD knockout mouse, which to our knowledge is the first mammalian model lacking a protein of the electron transport chain. Homozygous SDHD −/− animals die at early embryonic stages. Heterozygous SDHD +/− mice show a general, noncompensated deficiency of succinate dehydrogenase activity without alterations in body weight or major physiological dysfunction. The responsiveness to hypoxia of CBs from SDHD +/− mice remains intact, although the loss of an SDHD allele results in abnormal enhancement of resting CB activity due to a decrease of K+ conductance and persistent Ca2+ influx into glomus cells. This CB overactivity is linked to a subtle glomus cell hypertrophy and hyperplasia. These observations indicate that constitutive activation of SDHD +/− glomus cells precedes CB tumor transformation. They also suggest that, contrary to previous beliefs, mitochondrial complex II is not directly involved in CB oxygen sensing.

Acute oxygen sensing in heme oxygenase-2 null mice.

Hemeoxygenase-2 (HO-2) is an antioxidant enzyme that can modulate recombinant maxi-K+ channels and has been proposed to be the acute O2 sensor in the carotid body (CB). We have tested the physiological contribution of this enzyme to O2 sensing using HO-2 null mice. HO-2 deficiency leads to a CB phenotype characterized by organ growth and alteration in the expression of stress-dependent genes, including the maxi-K+ channel α-subunit. However, sensitivity to hypoxia of CB is remarkably similar in HO-2 null animals and their control littermates. Moreover, the response to hypoxia in mouse and rat CB cells was maintained after blockade of maxi-K+ channels with iberiotoxin. Hypoxia responsiveness of the adrenal medulla (AM) (another acutely responding O2-sensitive organ) was also unaltered by HO-2 deficiency. Our data suggest that redox disregulation resulting from HO-2 deficiency affects maxi-K+ channel gene expression but it does not alter the intrinsic O2 sensitivity of CB or AM cells. Therefore, HO-2 is not a universally used acute O2 sensor.

Carotid body oxygen sensing

The carotid body (CB) is a neural crest-derived organ whose major function is to sense changes in arterial oxygen tension to elicit hyperventilation in hypoxia. The CB is composed of clusters of neuron-like glomus, or type-I, cells enveloped by glia-like sustentacular, or type-II, cells. Responsiveness of CB to acute hypoxia relies on the inhibition of O2-sensitive K+ channels in glomus cells, which leads to cell depolarisation, Ca2+ entry and release of transmitters that activate afferent nerve fibres. Although this model of O2 sensing is generally accepted, the molecular mechanisms underlying K+ channel modulation by O2 tension are unknown. Among the putative hypoxia-sensing mechanisms there are: the production of oxygen radicals, either in mitochondria or reduced nicotinamide adenine dinucleotide phosphate oxidases; metabolic mitochondrial inhibition and decrease of intracellular ATP; disruption of the prolylhydroxylase/hypoxia inducible factor pathway; or decrease of carbon monoxide production by haemoxygenase-2. In chronic hypoxia, the CB grows with increasing glomus cell number. The current authors have identified, in the CB, neural stem cells, which can differentiate into glomus cells. Cell fate experiments suggest that the CB progenitors are the glia-like sustentacular cells. The CB appears to be involved in the pathophysiology of several prevalent human diseases.

Rotenone selectively occludes sensitivity to hypoxia in rat carotid body glomus cells

Carotid body glomus cells release transmitters in response to hypoxia due to the increase of excitability resulting from inhibition of O2 ‐regulated K+ channels. However, the mechanisms involved in the detection of changes of O2 tension are unknown. We have studied the interaction between glomus cell O2 sensitivity and inhibition of the mitochondrial electron transport chain (ETC) in a carotid body thin slice preparation in which catecholamine release from intact single glomus cells can be monitored by amperometry. Inhibition of the mitochondrial ETC at proximal and distal complexes induces external Ca2+‐dependent catecholamine secretion. At saturating concentration of the ETC inhibitors, the cellular response to hypoxia is maintained. However, rotenone, a complex I blocker, selectively occludes the responsiveness to hypoxia of glomus cells in a dose‐dependent manner. The effect of rotenone is mimicked by 1‐methyl‐4‐phenylpyridinium ion (MPP+), an agent that binds to the same site as rotenone, but not by complex I inhibitors acting on different sites. In addition, the effect of rotenone is not prevented by incubation of the cells with succinate, a substrate of complex II. These data strongly suggest that sensitivity to hypoxia of carotid body glomus cells is not linked in a simple way to mitochondrial electron flow and that a rotenone (and MPP+)‐sensitive molecule critically participates in acute oxygen sensing in the carotid body.

Carotid body chemosensory responses in mice deficient of TASK channels

Background K+ channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in TASK1−/− or double TASK1/3−/− mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in TASK1−/− cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either TASK1−/− or double TASK1/3−/− cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-K+ channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background K+ current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for O2/CO2 sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of TASK1/3−/− glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-K+ channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function.

Mechanisms of Low-Glucose Sensitivity in Carotid Body Glomus Cells

OBJECTIVE—Glucose sensing is essential for the adaptive counterregulatory responses to hypoglycemia. We investigated the mechanisms underlying carotid body (CB) glomus cells activation by low glucose. RESEARCH DESIGN/METHODS AND RESULTS—Removal of extracellular glucose elicited a cell secretory response, abolished by blockade of plasma membrane Ca2+ channels, and a reversible increase in cytosolic Ca2+ concentration. These data indicated that glucopenia induces transmembrane Ca2+ influx and transmitter secretion. In patch-clamped glomus cells, exposure to low glucose resulted in inhibition of macroscopic outward K+ currents and in the generation of a depolarizing receptor potential (DRP). The DRP was abolished upon removal of extracellular Na+. The membrane-permeable 1-oleoyl-2-acetyl-sn-glycerol induced inward currents of similar characteristics as the current triggered by glucose deficiency. The functional and pharmacological analyses suggest that low glucose activates background cationic Na+-permeant channels, possibly of the transient receptor potential C subtype. Rotenone, a drug that occludes glomus cell sensitivity to hypoxia, did not abolish responsiveness to low glucose. The association of Glut2 and glucokinase, characteristic of some high glucose–sensing cells, did not seem to be needed for low glucose detection. CONCLUSIONS—Altogether, these data support the view that the CB is a multimodal chemoreceptor with a physiological role in glucose homeostasis.

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.

Mechanisms of acute oxygen sensing by the carotid body: lessons from genetically modified animals.

We have studied carotid body (CB) glomus cell sensitivity to changes in O(2) tension in three different genetically engineered animals models using thin CB slices and monitoring the secretory response to hypoxia by amperometry. Glomus cells from partially HIF-1alpha deficient mice exhibited a normal sensitivity to hypoxia. Animals with complete deletion of the small membrane anchoring subunit of succinate dehydrogenase (SDHD) died during embryonic life but heterozygous SDHD +/- mice showed a normal CB response to low O(2) tension. SDHD +/- mice had, however, a clear CB phenotype characterized by a decrease of K(+) current amplitude, an increase of basal catecholamine release from glomus cells, and a slight organ growth. The lack of hemeoxygenase-2 (HO-2), a ubiquitous powerful antioxidant enzyme, produces a notable CB phenotype, characterized by hypertrophy and alteration in the level of CB expression of some stress-dependent genes (including down-regulation of the maxi-K(+) channel alpha-subunit). Nevertheless, in HO-2 deficient mice the exquisite intrinsic O(2) responsiveness of CB glomus cells remains unaltered. Therefore, HO-2 is not absolutely necessary for acute CB O(2) sensing. Although the nature of the CB acute O(2) sensor(s) is yet unknown, studies similar to those summarized here serve to test the existing hypothesis and help to distinguish between those that need to be explored further and those that definitively lack experimental support.