No description available.

<p> Reduced oxygen tension (i.e. hypoxia) regulates gene expression in various chromaffin cell types that synthesize catecholamines. In this study, the effect of chronic hypoxia on tyrosine hydroxylase (TH) mRNA and protein expression was investigated in the adrenomedullary chromaffin MAH cell line. RT-PCR results indicated that TH mRNA was expressed in MAH cells both during normoxia (20% 0 2) and hypoxia (5% 02). However, TH mRNA expression during chronic hypoxia was significantly higher than that during normoxia, increasing by approximately 2- fold after 16 hour exposure to chronic hypoxia. Western Blot analysis of the regulation of TH gene expression by chronic hypoxia indicated that TH protein initially decreased during 10 hr exposure to hypoxia and this was followed by a rapid increase in expression over the next 10 hr, and then by a slower increase (up to 1.3x initial control) after 72 hr exposure. Therefore, TH mRNA and protein levels were changed in MAH cells by hypoxia in a time-dependent manner. Surprisingly, cobalt treatment of MAH cells, expected to mimic the effects of chronic hypoxia, had little effect on TH gene expression. Interestingly, the decrease in TH expression protein after 10 hr exposure to hypoxia was prevented by nifedipine, an L-type calcium channel blocker. These results suggest that MAH cells represent a useful model system for examining hypoxia-induced gene regulation in an 02-sensitive cell line. Additionally, preliminary studies on HIF-1a expression in MAH cells showed that HIF-la mRNA was expressed and remained stable under both hypoxic and normoxic conditions. </p> / Thesis / Master of Science (MSc)

No description available.

Les cellules chromaffines des glandes surrénales sont des cellules neuroendocrines excitables impliquées dans la sécrétion de catécholamines. En réponse à un stress, ces hormones, parmi les premières à être libérées exercent de multiples actions sur leurs organes-cibles, contribuant à la réponse adaptive de l'organisme. Ainsi, élucider la physiopathologie du stress est un enjeu de santé publique et mieux connaître les mécanismes permettant au tissu médullosurrénalien d'optimiser la sécrétion de catécholamines aux besoins de l'organisme est un défi à relever.La sécrétion des catécholamines est liée à l'activité électrique des cellules chromaffines et élucider les mécanismes cellulaires qui en contrôlent l'excitabilité est d'intérêt. L'activité électrique de ces cellules est régulée par le nerf splanchnique ainsi que par des conductances ioniques intrinsèques. Dans ce contexte, les conductances opérant autour du potentiel de repos jouent un rôle majeur dans le déclenchement des potentiels d'action. C'est en particulier le cas du canal NALCN (sodium leak channel), récemment décrit comme régulant le potentiel de repos des neurones. C'est pourquoi nous avons orienté nos travaux vers la caractérisation du rôle de NALCN dans l'excitabilité des cellules chromaffines, dans des tranches de glandes surrénales de souris. L'enregistrement du potentiel de membrane révèle qu'environ 62% des cellules chromaffines présentent des potentiels d'action spontanés et que le profil de décharge suit un mode régulier ou un mode en bouffées. Des enregistrements plus longs révèlent qu'une même cellule présente alternativement ces 2 modes de décharge. Un changement de potentiel de quelques mV autour du potentiel de repos favorise un mode, indiquant que les courants ioniques actifs autour du potentiel de repos sont des composantes cruciales de l'excitabilité cellulaire. NALCN est-il un de ces courants?Pour commencer, nous avons observé, par hybridation in situ, la présence du transcrit codant NALCN dans les cellules chromaffines chez la souris (coll Dr. Ventéo, INM, Montpellier). Nous avons alors cherché à déterminer si NALCN est impliqué dans l'activité électrique des cellules chromaffines. Nous avons utilisé un protocole de diminution de la concentration extracellulaire de Na+, classiquement utilisé pour l'étude électrophysiologique de NALCN. La diminution du Na+ extracellulaire induit une hyperpolarisation et un arrêt des potentiels d'action. Cet effet n'est pas bloqué par la TTX. En potentiel imposé, la diminution du Na+ réduit le courant de maintien, elle n'est ni bloquée par la TTX ni par le Cs+. La courbe courant/potentiel du courant sensible à la réduction du Na+ révèle un courant linéaire entre -130 et -50 mV et un potentiel d'inversion en accord avec la contribution de plusieurs espèces ioniques. Ce courant présente une perméabilité majeure au Na+ vs K+. Ainsi, ces résultats décrivent une conductance ionique partageant des propriétés biophysiques et pharmacologiques similaires à celles de NALCN.Afin de poursuivre dans cette direction, nous avons initié des travaux ambitieux visant à éteindre l'expression du gène codant NALCN dans les cellules chromaffines, au travers d'une stratégie d'injection de virus in vivo. Une construction codant pour un shRNA dirigé contre NALCN, a été injectée dans la glande surrénale gauche. Les résultats sont très encourageants, montrant i) la présence, dans les glandes injectées, de cellules chromaffines transduites et ii) une diminution significative de l'expression de NALCN dans les glandes injectées avec le ShRNA-anti NALCN. Cette approche de transduction virale mérite d'être poursuivie.En conclusion, et même si les résultats actuels ne permettent pas d'affirmer avec certitude que NALCN contribue à l'excitabilité des cellules chromaffines, ce travail de thèse apporte néanmoins une contribution majeure à l'étude de l'excitabilité de ces cellules et ouvre des perspectives attractives quant au rôle de NALCN. / Adrenal chromaffin cells are excitable neuroendocrine cells involved in the secretion of catecholamines. Once delivered into the blood circulation, these hormones exert multiple actions, leading to physiological adjustments enabling the organism to cope with stress. Deciphering the physiology/pathology of stress is a major public health issue, especially in the field of the mechanisms that lead to optimal catecholamine secretion.The electrical activity of chromaffin cells critically shapes the catecholamine secretory pattern. Elucidating the mechanisms regulating the firing discharge is therefore of interest. In situ, chromaffin cell excitability is regulated by both the splanchnic nerve inputs and the intrinsic ion conductances expressed in cells. Regarding this, the conductances operating near the resting membrane potential are crucial in the cell competence to spontaneously fire. In particular, the background current flowing through the sodium leak channel NALCN has been recently reported to tune the resting potential of neuronal cells. This finding prompted us to investigate the possible contribution of NALCN to chromaffin cell excitability in mouse acute adrenal slices. The first part of my thesis was aimed at investigating chromaffin cell electrical firing pattern. Whole-cell recordings indicate that about 62% of mouse chromaffin cell spontaneously fire and exhibit two discharge patterns, a regular firing mode and a bursting mode. Long-lasting recordings of spontaneous electrical activity reveal that the two firing modes can occur in the same cells. When the membrane potential is challenged around the resting value, the firing pattern alternate between the two modes, indicating that currents operating around the resting membrane potential are key components in regulating cell excitability. Is NALN one of these currents?To answer this question, we first unveiled, by in situ hybridization, the presence of the transcript encoding NALCN in mouse chromaffin cells (coll with Dr. Ventéo, INM, Montpellier). Second, we performed electrophysiological experiments using protocols and pharmacological agents commonly used to study NALCN currents. Decreasing external NaCl leads to a robust membrane hyperpolarization, abrogating action potentials. This effect is not blocked by TTX. In voltage-clamp conditions, external Na+ reduction leads to a decrease in the holding current. This effect is not blocked by Cs+. Depolarizing voltage ramps unveil that the current blocked by lowering external Na+ blocks is linear between -130 and -50 mV, and displays a reversal potential arguing for a non-selective conductance. The ionic permeability is dominant for Na+ over K+. Collectively, our results describe a voltage-independent and non-selective cationic conductance operating near the resting potential of mouse chromaffin cells. Its electrophysiological and pharmacological properties recapitulate two NALCN attributes.In the third part, we developed an ambitious approach aiming at silencing NALCN expression specifically in chromaffin cells in vivo. Viral vectors encoding anti-NALCN shRNA under the control of the tyrosine hydroxylase promoter, as well as appropriate positive and negative viral constructs, were injected in the left gland. As promising results, transduced cells were detected in the injected glands only and a significant decrease in NALCN expression was observed in glands injected with the anti-NALCN shRNA. As such, the data collected from in vivo manipulation of NALCN expression are encouraging and this approach deserves to be pursued.This thesis describes a Na+-sensitive current operating near the resting membrane potential of mouse chromaffin cells, sharing biophysical and pharmacological properties with NALCN. Even though further experiments are needed to ascertain that NALCN supports this conductance, our work contributes to a better knowledge of chromaffin cell excitability.

No description available.

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca2+] in the vicinity of plasmalemmal Ca2+ channels due to Ca2+ influx, elicits exocytosis. This dissertation examines the effect on both spontaneous and elicited exocytosis of a rise in focal cytosolic [Ca2+] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca2+ syntillas. Ca2+ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (Scintilla, Latin for spark, found in nerve terminals, normally synaptic structures.) We have also observed Ca2+ syntillas in mouse adrenal chromaffin cells (ACCs). Here the effect of Ca2+syntillas on exocytosis is examined in ACCs, which are widely used as model cells for the study of neurosecretion. Elicited exocytosis employs two sources of Ca2+, one due to influx from the cell exterior through voltage-gated Ca2+ channels (VGCCs) and another due to release from intracellular stores. To eliminate complications arising from Ca2+ influx, the first part of this dissertation examines spontaneous exocytosis where influx is not activated. We report that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine after which syntillas were decreased. We conclude that Ca2+syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas. The second part of this dissertation examines the role of syntillas in elicited exocytosis whereby Ca2+ influx is activated by physiologically relevant levels of stimulation. Catecholamine and neuropeptide release from ACCs into the circulation is controlled by the sympathetic division of the Autonomic Nervous System. To ensure proper homeostasis tightly controlled exocytic mechanisms must exist both in resting conditions, where minimal output is desirable and under stress, where maximal, but not total release is necessary. It is thought that sympathetic discharge accomplishes this task by regulating the frequency of Ca2+ influx through VGCCs, which serves as a direct trigger for exocytosis. But our studies on spontaneous release in ACCs revealed the presence of Ca2+ syntillas, which had the opposite effect of inhibiting release. Therefore, assuming Ca2+-induced Ca2+ release (CICR) via RYRs due to Ca2+ influx through VGCCs, we are confronted with a contradiction. Sympathetic discharge should increase syntilla frequency and that in turn should decreaseexocytosis, a paradox. A simple “explanation” might be that the increase in syntillas would act as a brake to prevent an overly great exocytic release. But upon investigation of this question a different finding emerged. We examined the role of syntillas under varying levels of physiologic stimulation in ACCs using simulated action potentials (sAPs) designed to mimic native input at frequencies associated with stress, 15 Hz, and the basal sympathetic tone, 0.5 Hz. Surprisingly, we found that sAPs delivered at 15 Hz or 0.5 Hz were able to completely abolish Ca2+ syntillas within a time frame of two minutes. This was not expected. Further, a single sAP is all that was necessary to initiate suppression of syntillas. Syntillas remained inhibited after 0.5 Hz stimulation but were only temporarily suppressed (for 2 minutes) by 15 Hz stimulation, where global [Ca2+]i was raised to 1 – 2 μM. Thus we propose that CICR, if present in these cells, is overridden by other processes. Hence it appears that inhibition of syntillas by action potentials in ACCs is due to a new process which is the opposite of CICR. This process needs to be investigated, and that will be one of the very next steps in the future. Finally we conclude that syntilla suppression by action potentials is part of the mechanism for elicited exocytosis, resolving the paradox. In the last chapter speculation is discussed into the mechanisms by which physiologic input in the form of an action potential can inhibit Ca2+ syntillas and furthermore, how the Ca2+ syntilla can inhibit exocytic output.

No description available.

Les cellules neuroendocrines sécrètent hormones et neuropeptides dans la circulation sanguine par un processus d’exocytose régulée par le calcium. Engendrant un apport de membrane important à la surface cellulaire, l'exocytose doit être suivie par un processus d’endocytose compensatrice afin de maintenir l’homéostasie cellulaire et assurer le recyclage des composés vésiculaires. La connaissance précise des mécanismes moléculaires qui régulent la sécrétion neuroendocrine est primordiale. En effet, de nombreux cancers neuroendocrines sont associés à un dysfonctionnement de la sécrétion hormonale. Bien que connus des cliniciens, les mécanismes cellulaires et moléculaires qui induisent de telles perturbations de sécrétion restent à ce jour inexplorés. Les travaux réalisés au laboratoire démontrent que la sécrétion hormonale dans les cellules neuroendocrines est contrôlée par RhoA, Rac1 et Cdc42, trois GTPases de la famille Rho. Néanmoins, les voies moléculaires contrôlant le cycle d’activation-inactivation de ces GTPases Rho lors de l’exocytose sont peu connues. Mon projet de thèse s’est articulé autour de deux axes principaux. Un premier objectif fut d’étudier, au cours de la sécrétion neuroendocrine, l’implication potentielle de l’oligophrénine-1, une protéine GAP capable d'inactiver certaines GTPases Rho. En utilisant les cellules chromaffines de la glande surrénale comme modèle cellulaire, j'ai découvert un double rôle de la protéine oligophrénine-1. En effet, en inactivant RhoA, l’oligophrénine-1 permettrait la mise en place du pore de fusion nécessaire à l'exocytose tandis que par le biais de son domaine BAR, elle contrôle l'endocytose compensatrice. Mon second objectif fut d’appréhender les bases cellulaires et moléculaires à l’origine du dysfonctionnement de la sécrétion dans les cancers neuroendocrines en utilisant les phéochromocytomes humains comme modèle expérimental. En combinant des analyses ampérométriques et protéomiques sur des tumeurs humaines, j'ai pu montrer que l'hypersécrétion catécholaminergique des phéochromocytomes est bien la conséquence d’une augmentation de l’activité sécrétrice. J'ai également pu identifier des acteurs protéiques, dont plusieurs modulateurs des GTPases de type Rho, qui pourraient être impliqués dans ces défauts de sécrétion. L’ensemble de mon projet de thèse m’a permis de mieux comprendre les mécanismes de régulation des GTPases Rho au cours de la sécrétion mais également de proposer les premières bases moléculaires et cellulaires abordant les perturbations de la sécrétion dans les tumeurs neuroendocrines. / Neuroendocrine cells release hormones and neuropeptides in the bloodstream through a calcium regulated exocytosis process. This process leads to an important increase of membrane at the cell surface which needs to be compensated through endocytosis in order to maintain cell homeostasis as well as the recycling of vesicular compounds. Many neuroendocrine cancers have been associated with a dysfunction in hormone secretion, urging toward a need to understand the molecular mechanisms which regulate neuroendocrine secretion. Even though these perturbations are known at a clinical level, the underlying cellular and molecular mechanism remains to be unraveled. Previous works in the team demonstrate that hormone secretion is regulated by RhoA, Rac1 and Cdc42, which are all GTPases of the Rho family. Nevertheless the molecular pathways which control the activation and inactivation cycle of these RhoGTPases during exocytosis remain unknown. My PhD project covered essentially two aspects. My first goal was to characterize the potential role of oligophrenin-1, a GAP protein which can inactivate Rho-GTPases, in neuroendocrine secretion. By using adrenal gland chromaffin cells as a cellular model, I have discovered a dual role for oligophrenin-1. Indeed, through RhoA inactivation, oligophrenin-1 allows the set up of a fusion pore, which is necessary for exocytosis, while controlling through the BAR domain the compensatory endocytosis. My second goal was to characterize the molecular and cellular basis which leads to a secretion dysfunction in neuroendocrine cancers by using human pheochromocytomas as an experimental model. By combining amperometric and proteomic analyses on human tumors, I was able to demonstrate that pheochromocytoma catecholaminergic secretion was indeed due to an increase in secretion activity. I could also identify major actors, including several Rho GTPase modulators, which could be implied in these secretion defects. All together, these approaches gave me a better understanding of RhoGTPase regulation during secretion but also an insight into the molecular and cellular basis of impaired secretion in neuroendocrine tumors.

Les récepteurs Erythropoietin-producing hepatocyte (EPH) constituent la plus grande famille de récepteurs à activité tyrosine kinase transmembranaires. Leur activité kinase peut être induite par leurs ligands, les éphrines (EFN). Une fois activés, ces récepteurs sont impliqués dans la régulation de la fonction cellulaire par transduction antérograde ou rétrograde du signal EPH-EFN. Au cours de la dernière décennie, nos études ont démontré que les EPH / EFN jouent un rôle important dans la régulation de la pression artérielle par la modulation de la contractilité des cellules musculaires lisses vasculaires (VSMC). EPHB6, EFNB1 et EFNB3 ont un effet négatif sur la contractilité des VSMC et la pression artérielle, tandis que EPHB4 et EFNB2 montrent un effet positif. La famille EPH / EFN est donc un nouveau système yin et yang qui ajuste finement l'homéostasie de la pression artérielle. Nous avons également constaté que les catécholamines urinaires de 24 h sont réduites chez les souris mâles EPHB6 knockout (KO), suggérant que l’EPHB6 régule la pression artérielle non seulement via les VSMC mais aussi par la sécrétion de catécholamine (CAT). La régulation de CAT par l’EPHB6 dépend de la testostérone car (1) les niveaux réduits de CAT ne sont pas observés chez les souris femelles EPHB6 KO ; et (2) la castration chez les souris mâles EPHB6 KO ramène la CAT à des niveaux normaux. Durant ma thèse, nous avons étudié le mécanisme impliqué dans la régulation de la sécrétion et de la synthèse des catécholamines chez les cellules chromaffines des glandes surrénales (AGCC) par la voie de signalisation de l’EPHB6. En ex vivo, la teneur totale en épinéphrine et la sécrétion d'épinéphrine déclenchée par l'acétylcholine (ACh) sont toutes deux réduites dans les glandes surrénales venant des souris KO mâles mais pas dans celles venant des femelles ou de mâles castrés. Ensuite, nous avons observé une diminution de l’afflux de Ca2+ dépendant de l'ACh dans les AGCC venant des souris mâles EPHB6 KO, ce qui découle de l'effet non-génomique de la testostérone. En appliquant le patch clamping de cellules entières sur les AGCC, nous avons démontré que la diminution d’afflux de Ca2+ dans ces cellules est causée par l’augmentation des courants de potassium à grande conductance activé par le calcium (BK). En utilisant l'enregistrement ampérométrique, nous avons constaté que la sécrétion de CAT par les AGCC est compromise en l'absence d'EPHB6. Nous avons également observé une diminution du désassemblage de la F-actine corticale dans les AGCC venant de souris mâles KO associée à une diminution de l'exocytose des vésicules contenant es catécholamines. Ces deux phénomènes n’ont pas été observés chez les femelles KO ni chez les mâles castrés. Des études complémentaires ont montré que le désassemblage défectueux de la F-actine dans les AGCC est régulé par la signalisation inverse de l'EPHB6 à l'EFNB1 via deux voies de signalisations différentes : la voie du membre A de la famille des homologues Ras (RHOA) et la voie de la tyrosine kinase proto-oncogène de la famille Src (FYN) / proto-oncogène c-ABL / la calponine monooxygénase associée aux microtubules et le domaine LIM contenant 1 (MICAL-1). En outre, nous avons observé que la diminution de la teneur totale en épinéphrine dans la glande surrénale venant des souris mâles KO est causée par une expression altérée de la tyrosine hydroxylase (TH), qui est l’enzyme limitant la vitesse dans la biosynthèse des CAT. L'effet non génomique de la testostérone a également participé dans ce processus. Nous avons révélé que la signalisation inverse d'EPHB6 à EFNB1 contribue à la surexpression de TH dans les AGCC par l’augmentation de son niveau de transcription. La voie en aval de cette signalisation inverse implique la petite famille Rac GTPase 1 (RAC1) / MAP kinase kinase 7 (MKK7) / c-Jun N-terminal kinase (JNK) / proto-oncogène c-Jun / activator protein 1 (AP1) / réponse de croissance précoce 1 (EGR1). Ces travaux démontrent pour la première fois un rôle spécifique de la famille EPH / EFN dans la régulation de la biologie médullaire de la glande surrénale. La signalisation rétrograde d’EPHB6 via EFNB1 régule la synthèse et la sécrétion des catécholamines de concert avec la testostérone dans les AGCC. / Erythropoietin-producing hepatocyte (Eph) receptors are the largest family of cell surface transmembrane receptor tyrosine kinases. Their kinase activity can be activated by their ligands, ephrins (EFNs), and involved in cell function regulation through either EPH-EFN forward or reverse signaling transduction. In the last decade, we have revealed the previously unknown function of EPHs/EFNs in the regulation of blood pressure by modulating the contractility of vascular smooth muscle cells (VSMCs). EPHB6, EFNB1, and EFNB3 have a negative effect on the VSMCs contractility and blood pressure, while EPHB4 and EFNB2 show a positive effect instead. Thus, EPH/EFN family is a novel yin and yang system that finely tunes blood pressure homeostasis. EPHB6 also targets cells responsible for catecholamine (CAT) secretion in addition to the VSMCs, since we found that the 24-h urine catecholamines are reduced in male EPHB6 knockout (KO) mice. This phenotype in EPHB6 KO mice is testosterone-dependent because the reduced CAT levels are not observed in female KO mice; castration in KO male mice reverts the CAT levels to a normal range. In this research, we investigated the mechanism for the regulation of catecholamine secretion and synthesis in adrenal gland chromaffin cells (AGCCs) by EPHB6 signaling. In ex vivo, the total content of epinephrine and the acetylcholine (ACh)-triggered epinephrine secretion were both reduced in the adrenal gland from KO male but not female or castrated mice. Then, we found a reduced ACh-dependent Ca2+ influx in AGCCs from male EPHB6 KO mice, and this effect depended on the non-genomic effect of testosterone. The results of whole-cell patch clamping on AGCCs indicated that the enhanced large-conductance calcium-activated potassium (BK) currents were responsible for the reduced Ca2+ influx in these cells. Using amperometry recording, we found that CAT secretion by AGCCs was compromised in the absence of EPHB6. The cortical F-actin disassembly in AGCCs from KO male but not female or castrated mice was reduced, accompanied by decreased catecholamine vesicle exocytosis. Further study showed such defective F-actin disassembly in AGCCs was regulated by the reverse signaling from EPHB6 to EFNB1 via the Ras homolog family member A (RHOA) and proto-oncogene Src family tyrosine kinase (FYN)/proto-oncogene c-ABL/microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways. Further, we observed that the reduced total content of epinephrine in the adrenal gland from male KO mice was caused by impaired expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in CAT biosynthesis. The non-genomic effect of testosterone was also involved in this process. We revealed that the reverse signaling from EPHB6 to EFNB1 contributed to the up-regulation of TH expression in AGCCs by enhancing its transcription. The downstream pathway of this reverse signaling involved Rac family small GTPase 1 (RAC1)/MAP kinase kinase 7 (MKK7)/c-Jun N-terminal kinase (JNK)/ proto-oncogene c-Jun/activator protein 1 (AP1)/early growth response 1 (EGR1). The present research, for the first time, revealed the specific role of the EPH/EFN family on the regulation of the adrenal gland medullary biology. The EPHB6 reverse signaling through EFNB1 in concert with testosterone regulates the catecholamine synthesis and secretion in AGCCs.

The mammalian adrenal gland (or suprarenal gland) is a small organ located on the superior aspect of the kidney. The central region of the gland, the medulla, consists of chromaffin cells, which release catecholamines into the blood during periods of stress. This is best known as the 'fight or flight' response and is regulated, in the adult animal, by neuronal signals from the cholinergic sympathetic fibres of the splanchnic nerve. Interestingly, in some mammals, such as rat and human, sympathetic innervation is immature at birth, yet the chromaffin cells can still secrete catecholamines in response to physiological stessors, e.g. hypoxia. Increased plasma catecholamines is thought to provide a vital protective role for the neonatal animal during, and following birth. This is mediated in part by promoting lung fluid absorption, surfactant secretion, heart rate stabilization, and brown fat mobilization. The observation that, in the neonate, catecholamines are secreted in the absence of functional sympathetic innervation suggests that the chromaffin cells possess other mechanisms for directly 'sensing' a fall in blood O2 tension (hypoxia). The primary goal of this thesis was to uncover the mechanisms of oxygen-sensing in developing chromaffin cells from the rat and mouse, using primary short-term cell cultures of chromaffin cells. The experimental approaches relied on patch clamp techniques to record ionic currents and membrane potential, carbon fibre electrochemistry to record catecholamine secretion from cell clusters, and fluorescent indicators to measure reactive oxygen species generation. Hypoxic chemosensitivity was found in embryonic and neonatal, but not juvenile chromaffin cells from both the rat and mouse. Exposure to hypoxia or anoxia caused a reversible suppression of whole-cell current, which was comprised of the differential modulation of three K+ currents: (1) suppression of a large-conductance Ca2+-dependent K+ current; (2) suppression of a delayed rectifier K+ current; and (3) activation of an ATP-sensitive K+ current. Hypoxia also induced membrane depolarization that was not initiated by any of these three voltage-dependent K+ currents. Additionally, hypoxia broadened action potentials in chromaffin cells that showed spontaneous activity, and this was mediated by a prolongation of the time course of membrane repolarization. All of these factors likely contribute to catecholamine secretion by enhancing the influx of Ca2+ through depolarization-activated L-type Ca2+ channels. Two sets of experiments were designed to identify the oxygen sensor in neonatal chromaffin cells. First, cells from transgenic mice, deficient in the gp91^phox component of the putative O2-sensor protein, NADPH oxidase, responded to hypoxia in the same way as wild type cell, indicating that NADPH oxidase is not primarily responsible for oxygen sensitivity in these cells. Second, inhibitors of the proximal electron transport chain (e.g. rotenone and antimycin A) mimicked and attenuated the hypoxic response, while inhibitors of the distal electron transport chain (cyanide) and uncouplers of oxidative phosphorylation (2,4-dinitrophenol) had no effect. Furthermore, reactive oxygen species production, primarily H2O2, decreased during exposure to hypoxia or inhibitors of the proximal electron transport chain, revealing a potential mitochondrial mechanism for 'sensing' of the hypoxic stimulus. Reduced oxygen availability to the electron transport chain is proposed to cause a fall in cellular reactive oxygen species (ROS), principally H2O2. This fall in ROS signals closure of Ca2+-dependent and Ca2+-independent K+ channels, which causes broadening action potentials and increases Ca2+ influx. The latter is further enhanced by the hypoxia-induced membrane depolarization, which in turn increases the probability of cell firing. The rise in intracellular Ca2+ then acts as the signal for catecholamine release from the chromaffin cells. / Thesis / Doctor of Philosophy (PhD)