Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, March 2000. / Includes bibliographical references. Also available on the Internet.

Thesis (doctoral)--Lund University, 1997. / Added t.p. with thesis statement inserted.

<p> Asphyxia (hypoxia, hypercapnia, and acidosis) sensing by neonatal adrenal chromaffin cells (AMC) plays a key role in the adaptation of the neonate to extrauterine environment. During birth, exposure of the neonate to episodes of acute asphyxia results in secretion of catecholamines from AMC, independent of neural inputs. This catecholamine (CAT) secretion promotes re-absorption of fluid and surfactant production in the lung, improved cardiac conductance, and assists in neonatal arousal. The asphyxia-sensing responses in AMC are lost postnatally during a time course that parallels splanchnic innervation. </p> </p> <p> The primary goal of this thesis was to uncover the mechanisms of asphyxia sensing in neonatal rat AMC, using primary and immortalized chromaffin cells, and to elucidate the potential role of innervation in the postnatal loss of these mechanisms. The experimental approaches relied on patch clamp techniques to record ionic currents and membrane potential, carbon fiber amperometry to monitor CAT secretion, chemiluminescence to measure reactive oxygen species (ROS) production and ATP secretion, and molecular techniques to examine protein and gene expression. </p> <p> In primary neonatal, but not juvenile AMC, hypoxia (PO₂ = 15 mmHg) caused a suppression of outward K⁺ current, membrane depolarization, and increased secretion. These effects were associated with a decrease in mitochondrial ROS production, were reversed by exogenous H₂O₂, and where mimicked by antioxidants. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occulded the effects of hypoxia. The immortalized chromaffin cell line (MAH cells) behaved similarly, and became hypoxia-insensitive when depleted of functional mitochondria (ρ0 MAH). Both neonatal AMC and immortalized MAH cells also responded to increased CO₂ (hypercapnia) with K⁺ current inhibition, membrane depolarized, increased intracellular Ca²⁺, and CAT secretion. However, these responses were independent of functional mitochondria and were associated with the expression of carbonic anhydrase II (CAII). </p> <p> Since the splanchnic nerve supplies both cholinergic and opioid peptidergic innervation to AMC, I tested the hypothesis that postsynaptic activation of the corresponding receptors might underlie the postnatal loss of asphyxia-sensing. First, to determine whether nicotinic acetylcholine receptor stimulation was involved, pregnant rats were exposed to nicotine (or saline) during gestation and AMC were examined in the offspring, soon after birth. Control AMC isolated from pups born to saline-treated dams displayed typical responses to hypoxia and hypercapnia, including inhibition of outward K⁺ current, membrane depolarization, increased cytosolic calcium, and CAT secretion. In contrast, AMC from pups born to nicotine-treated dams showed a dramatic loss of hypoxic sensitivity, though hypercapnic sensitivity and the expression of CO₂ markers (i.e. carbonic anhydrase I and II) appeared normal. This effect of chronic nicotine could be reproduced in cultured chromaffin cells in vitro and was mediated via activation of α7-nicotinic acetylcholine receptors (nAChR), leading to upregulation of ATP-dependent (K_ATP) K⁺ channels, which open during hypoxia and cause membrane hyperpolarization. The upregulation of K_ATP channels involved expression ofthe hypoxia inducible transcription factor (HIF 2α). Second, to determine whether opioid receptor stimulation was also involved, cultured chromaffin cells were chronically exposed to mu, delta, or kappa opioid agonists in vitro. Treatment with both mu and delta, but not kappa, agonists resulted in the loss of both hypoxia and hypercapnia sensing and a decrease in CAII expression. </p> <p> Taken together, these studies suggest that neonatal chromaffin cells sense hypoxia via a reduction in ROS generation located at or upstream of mitochondrial complex I. Chronic stimulation of nicotinic α7 ACh receptors leads to a selective loss of hypoxia sensing and this appears to be mediated via a HIF 2α-dependent upregulation of K_ATP channels. These channels open during hypoxia causing membrane hyperpolarization and reduced excitability. The result of this upregulation of K_ATP channels results in cells deficient in the ability to respond to hypoxia. </p> / Thesis / Doctor of Philosophy (PhD)

De récentes études ont montré dans les cellules chromaffines que la libération des granules de sécrétion est temporellement et spatialement couplée au processus d’endocytose. Nous avons proposé l’hypothèse que la membrane du granule préserve son intégrité au sein de la membrane plasmique durant l’exocytose avant d’être internalisée ainsi avec ses composants. Cependant, les mécanismes moléculaires de ce processus d’endocytose compensatrice sont encore inconnus. Ainsi, mon projet de thèse vise a répondre à la question suivante : Quels sont les différents mécanismes déclenchant et régulant l’exocytose et l’endocytose compensatrice? Les propriétés physiques des lipides jouent des rôles fondamentaux dans le trafic membranaire. Ils servent de système d’échafaudage pour maintenir la machinerie spécifique à des endroits précis de la membrane plasmique. Par exemple, la formation de microdomaines de gangliosides et de PIP2 au niveau des sites d’exocytose ou encore le mélange de lipides au sein de la bicouche lipidique représentent des processus attractifs pour permettre cette fonction au cours des événements d’exo-endocytose dans les cellules neuroendocrines. De plus, en raison de leur implication importante dans les processus d’exo-endocytose ou dans le remodelage des lipides, l’annexine A2, la synaptotagmine 1, l’oligophrénine1 et la scramblase 1 doivent être considérées comme des signaux potentiels pour le déclenchement de l’endocytose de la membrane granulaire. Au cours de mon doctorat, je me suis intéressée à étudier comment l’exocytose et l’endocytose compensatrice sont régulées par la scramblase1 et l’oligophrénine1 dans les cellules chromaffines de la glande surrénale. / Recent studies in neuroendocrine chromaffin cells have suggested that the secretory granule release is temporally and spatially coupled to a compensatory endocytic process. Hence, we hypothesized that the secretory granule membrane would preserve its integrity within the plasma membrane after exocytosis before being retrieved as such along with its components. However, the underlying molecular mechanisms of this compensatory endocytic process are largely unknown today. Therefore my thesis project is aiming to address the following specific question: What are the different mechanisms triggering and regulating exocytosis and the compensatory endocytosis? Physical properties of lipids play fundamental roles in membrane trafficking. They act as a scaffolding system to maintain specific machinery at restricted site of the plasma membrane. For example, the formation of ganglioside- and PIP2-enriched microdomains at the exocytic sites or the phospholipid scrambling across the bilayer plasma membrane, represent attractive processes to fulfill this function during exo- endocytosis events in neuroendocrine cells. Moreover, in view to their important implication in exo-endocytotic processes or lipid remodeling, annexin-A2, synaptotagmin- 1, oligophrenin-1 and phospholipid scramblase-1 have to be considered as potential signal-triggers of the granule endocytosis. During my PhD, I focused in investigating how exocytosis and compensatory endocytosis are regulated by PLSCR-1 and OPHN1 in adrenal chrommaffin cells.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on January 30, 2008) Vita. Includes bibliographical references.

Oxygen-sensing cells release neurotransmitters, including serotonin, in response to hypoxia in the blood or surrounding air/water. This stimulates the glossopharyngeal and/or vagal nerves, triggering increased ventilation via the respiratory reflex. In the adult, they are located in the carotid body (glomus cells) and lung epithelia (pulmonary neuroendocrine cells) of amniotes, and in the epithelia of the gills and orobranchial cavity (‘neuroepithelial cells’) of anamniotes. Despite their physiological importance, little is known about the molecular mechanisms of their development, while the evolutionary relationships between the various oxygen-sensing cell types are unknown. The chromaffin cells of the mammalian adrenal medulla are hypoxia-sensitive transiently during neonatal life. Both carotid body glomus cells and adrenal chromaffin cells arise from the neural crest and require the transcription factors Phox2b and Ascl1 for their development. Given these similarities, I aimed to test the hypothesis that the same molecular mechanisms underlie their development. Expression analysis of 13 sympathoadrenal pathway genes throughout chicken carotid body development revealed striking similarities with adrenal chromaffin cell development. Analysis of mouse mutants showed that the transcription factors Hand2, Sox4 and Sox11 are required for carotid body development. In addition, loss of the receptor tyrosine kinase Ret or the transcription factor AP-2β, which significantly affects sympathetic ganglion but not adrenal chromaffin cell development, has no effect on the carotid body. Adrenal chromaffin cells differentiate from neurons that migrate into the adrenal gland from ‘primary’ sympathetic ganglia at the dorsal aorta. Carotid body glomus cells were previously proposed to arise from neuronal “émigrés” from neighbouring ganglia: the superior cervical ganglion in mammals and the nodose ganglion in the chick. However, nodose neurons are considered to be nodose placode-derived. Using electroporation and grafting in the chick, I confirmed that the nodose placode does not contribute to the carotid body, identified a small population of autonomic neural crest-derived neurons in the nodose ganglion, and confirmed the existence of “bridges” of neurons between the nodose ganglion and the carotid body. My data suggest that, like adrenal chromaffin cells, carotid body glomus cells differentiate from autonomic neural crest-derived neurons in nearby ganglia, which migrate into the carotid body primordium and down-regulate neuronal markers. The proposed evolutionary relationship between the carotid body glomus cells and the serotonin-positive neuroepithelial cells of anamniote gills has never been tested. Using vital dye labelling, neural fold grafts, genetic lineage-tracing in zebrafish and analysis of zebrafish mutants lacking all neural crest cells, I found that serotonin-positive cells in the gills and orobranchial epithelia of lamprey (jawless fish), zebrafish (ray-finned bony fish) and frog (anamniote tetrapod) are not neural crest-derived, and hence are not homologous to carotid body glomus cells. Genetic lineage-tracing in mouse and neural fold grafts in chick also confirmed that serotonin-positive neuroendocrine cells in the lung are not neural crest- derived, hence must have an endodermal origin (since the lungs are out-pocketings of the gut). My results suggest that the neuroepithelial cells of anamniotes are not related to carotid body glomus cells, but rather are homologous to the oxygen-sensing cells of the lung. Consistent with this hypothesis, I found that many genes expressed during carotid body development are not expressed by the epithelia of either chick lungs or lamprey gills. Taken together, my data suggest that as air-breathing evolved, gut endoderm- derived cells that originally responded to hypoxia in water were maintained in the lungs to monitor oxygen levels in air, while a population of neural crest-derived chromaffin cells near the pharyngeal arch arteries was recruited to monitor oxygen levels in blood.

No description available.

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactivity (CART-LI) was studied in the rat spinal cord, sympathetic ganglia and adrenal glands by immunohistochemical methods, utilizing a polyclonal antiserum raised against the CART peptide fragment 55-102. CART-LI was detected in nerve fibers and in basket-like terminals surrounding many postganglionic neurons of the superior cervical ganglion (SCG), stellate, paravertebral and prevertebral ganglia. Postganglionic neurons exhibited low or non-detectable levels of CART-LI. Surgical sectioning of the cervical sympathetic trunk for 6-7 days resulted in a nearly complete loss of CART-LI fibers and terminals in the SCG. In the adrenal gland, CART-LI nerve fibers formed a plexus underneath the capsule, some of which bifurcated and made a sharp turn toward the adrenal medulla, where clusters of chromaffin cells were intensely labeled. The detection of CART-LI in sympathetic ganglia and adrenal glands extends the previous observation of the presence of CART-LI in sympathetic preganglionic neurons and further supports the notion that CART peptide(s) may function as a signaling molecule in the sympatho-adrenal axis.

No description available.

No description available.