Mechanisms of oxygen-chemosensitivity in adrenal medullary chromaffin cells from the developing rat and mouse

Abstract

<p>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 gp91phox component of the putative O 2 -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 H2 O2 , decreased during exposure to hypoxia or inhibitors of the proximal electron transport chain, revealing a potential mitochondrial mechanism for 'sensing' of the hypoxic stimulus. (Abstract shortened by UMI.)</p>