<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)