José López-Barneo

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.

Absolute requirement of GDNF for adult catecholaminergic neuron survival

GDNF is a potent neurotrophic factor that protects catecholaminergic neurons from toxic damage and induces fiber outgrowth. However, the actual role of endogenous GDNF in the normal adult brain is unknown, even though GDNF-based therapies are considered promising for neurodegenerative disorders. We have generated a conditional GDNF-null mouse to suppress GDNF expression in adulthood, hence avoiding the developmental compensatory modifications masking its true physiologic action. After Gdnf ablation, mice showed a progressive hypokinesia and a selective decrease of brain tyrosine hydroxylase (Th) mRNA, accompanied by pronounced catecholaminergic cell death, affecting most notably the locus coeruleus, which practically disappears; the substantia nigra; and the ventral tegmental area. These data unequivocally demonstrate that GDNF is indispensable for adult catecholaminergic neuron survival and also show that, under physiologic conditions, downregulation of a single trophic factor can produce massive neuronal death.

Low glucose–sensing cells in the carotid body

Decreased plasma glucose concentration elicits a complex neuroendocrine response that prevents or rapidly corrects hypoglycemia as required to preserve brain function; however, where and how low glucose is sensed is unknown. Here we show that low glucose increases secretion from glomus cells in the carotid bodies, sensory organs whose stimulation by hypoxia produces sympathetic activation, by a process that depends on extracellular Ca2+ influx and is paralleled by inhibition of voltage-gated K+ channels. We propose a new glucose-sensing role for the carotid body glomus cell that serves to integrate information about blood glucose and O2 levels and to activate counterregulatory responses.

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.

Single K+ channels in membrane patches of arterial chemoreceptor cells are modulated by O2 tension.

Type I cells of the carotid body are known to participate in the detection of O2 tension in arterial blood but the primary chemotransduction mechanisms are not well understood. Here we report the existence in excised membrane patches of type I cells of a single K+ channel type modulated by changes in PO2. Open probability of the O2-sensitive K+ channel reversibly decreased by at least 50% on exposure to hypoxia but single-channel conductance (approximately 20 pS) was unaltered. In the range between 70 and 150 mmHg (1 mmHg = 133 Pa) the decrease of single-channel open probability was proportional to the PO2 measured in the vicinity of the membrane patch. The inhibition of K+ channel activity by low PO2 was independent of the presence of non-hydrolyzable guanine triphosphate analogues at the internal face of the membrane. The results indicate that the O2 sensor of type I cells is in the plasma membrane and suggest that environmental O2 interacts directly with the K+ channels.

Oxygen-sensing by ion channels and the regulation of cellular functions

From bacteria to mammals, ambient O2 tension influences such diverse cellular functions as gene expression, secretion, contraction and the patterns of electrical activity. Some of the effects of O2 are attributed to its interaction with various classes of voltage-dependent ion channels. In glomus cells of the carotid body, the differential properties of O2-sensitive K+ and Ca2+ channels help us to understand the basic features of O2 chemoreception. Modifications of ion-channel activity in response to changes in the partial pressure of O2 are also involved in the adjustments of vascular tone to hypoxia as well as in the response of chemoreceptors in pulmonary airways. Direct O2-sensing by ion channels might also help to explain the alterations of brain function by low O2 tension. The O2-sensitivity of ion-channel activity appears to be a broadly distributed phenomenon contributing to a wide variety of cellular responses to hypoxia.

Cellular and Functional Recovery of Parkinsonian Rats after Intrastriatal Transplantation of Carotid Body Cell Aggregates

We have tested the suitability of chromaffin-like carotid body glomus cells for dopamine cell replacement in Parkinsonian rats. Intrastriatal grafting of cell aggregates resulted in almost optimal abolishment of motor asymmetries and deficits of sensorimotor orientation. Recovery of transplanted animals was apparent 10 days after surgery and progressed throughout the 3 months of the study. The behavioral effects were correlated with the long survival of glomus cells in the host brain. In host tissue, glomus cells were organized into glomerulus-like structures and retained the ability to secrete dopamine. Several weeks after transplantation, dopaminergic fibers emerged from the graft, reinnervating the striatal gray matter. The special durability of grafted glomus cells in the conditions of brain parenchyma could be related to their sensitivity to hypoxia, which is known to induce cell growth, excitability, and dopamine synthesis. This work should stimulate research on the clinical applicability of carotid body autotransplants in Parkinsons disease.

Low glucose|[ndash]|sensing cells in the carotid body

Decreased plasma glucose concentration elicits a complex neuroendocrine response that prevents or rapidly corrects hypoglycemia as required to preserve brain function; however, where and how low glucose is sensed is unknown. Here we show that low glucose increases secretion from glomus cells in the carotid bodies, sensory organs whose stimulation by hypoxia produces sympathetic activation, by a process that depends on extracellular Ca2+ influx and is paralleled by inhibition of voltage-gated K+ channels. We propose a new glucose-sensing role for the carotid body glomus cell that serves to integrate information about blood glucose and O2 levels and to activate counterregulatory responses.

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.

Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.

1. Calcium currents were recorded from smooth muscle cells dispersed from conduit and resistance rabbit pulmonary arteries. We tested the hypothesis that Ca2+ channel activity was regulated by environmental O2 tension. 2. Conduit (proximal) and resistance (distal) myocytes differ in their Ca2+ channel density and responses to low PO2. Ca2+ current density in distal myocytes (20.7 +/‐ 7.4 pA pF‐1, n = 10) is almost twice the value in proximal myocytes (12.6 +/‐ 5.5 pA pF‐1, n = 39). In proximal myocytes, the predominant response to reductions in PO2 is inhibition of the calcium current (n = 12) at membrane potentials below 0 mV, whereas potentiation of current amplitude is observed in distal myocytes (n = 24). 3. Hypoxia also produces opposite shifts in the conductance‐voltage relationships along the voltage axis. The average displacements induced by low PO2 are +5.05 +/‐ 2.98 mV (n = 5) in proximal myocytes and ‐6.06 +/‐ 2.45 (n = 10) in distal myocytes. 4. These findings demonstrate longitudinal differences in Ca2+ channel density and O2 sensitivity in myocytes along the pulmonary arterial tree. These results may help to understand the differential reactivity to hypoxia of the pulmonary vasculature: vasodilatation in conduit arteries and vasoconstriction in resistance vessels.