Electron Transport Chain Inhibition May Act as the 02 Sensor in AMC

Several specific inhibitors of each of the four complexes of the ETC have been extensively characterized and can be utilized to investigate whether or not the 02-sensor is consistent with a mitochondrial location. Figure 3 (A and B) show that rotenone (a complex I inhibitor) can inhibit outward currents in neonatal AMC. It should be noted that hypoxia plus rotenone caused no further suppression of outward current, suggesting convergence of the two pathways (22). Figure 3C shows a typical response to hypoxia (~5 mmHg) of a spontaneously active AMC, recorded from a small cell cluster. Both hypoxia and rotenone induced membrane depolarizations that were not additive (Fig. 3C and D), and were associated with action potential broadening (Fig. 4). This suggests that hypoxia and block of electron transport at complex I utilize a common mechanism for ion channel inhibition, and tentatively localizes the 02 sensor of AMC to mitochondria. Interestingly, we did not observe any significant effect of 5 mM cyanide (a complex IV inhibitor) on neonatal AMC, with the exception of 1 of 5 cells tested where CN induced membrane hyperpolarization (Fig. 4) (22). This latter observation contrasts with that of Mojet et al. (21) who showed that CN raised intracellular Ca2+ and activated CA secretion from AMC, presumably mimicking hypoxia. One possibility to explain this discrepancy is that CN is acting at other 02-dependent cellular systems or that it activates secretion by a different pathway to hypoxia.

Figure 4. Hypoxia and rotenone prolong action potential duration in neonatal AMC. A: Hypoxia (Hyp) and rotenone (Rot) reversibly broadened action potentials recorded from spontaneously active neonatal chromaffin cells. For comparison purposes and to eliminate the effects of hypoxia-induced membrane depolarization, action potentials were adjusted to start at the same resting potential. "Rec", recovery/washout. B: Effects of hypoxia (n=90 spikes from 4 cells), rotenone (n=50 spikes from 3 cells), and cyanide (n=30 spikes from 2 cells) on action potential duration and spike frequency. Hypoxia and rotenone (5 pM), but not cyanide (2.5 mM), caused a slight increase in the rise time of the action potential CcriK), significantly prolonged the decay phase ("Way)and half width of the action potential (thalf). Hypoxia and rotenone, but not cyanide caused a slight but non-significant decrease in action potential frequency. Note that the effects of hypoxia were attenuated or abolished in the presence of rotenone, but not cyanide. All records were obtained using the nystatin perforated patch configuration of whole-cell recording in 1=0 mode.

Figure 4. Hypoxia and rotenone prolong action potential duration in neonatal AMC. A: Hypoxia (Hyp) and rotenone (Rot) reversibly broadened action potentials recorded from spontaneously active neonatal chromaffin cells. For comparison purposes and to eliminate the effects of hypoxia-induced membrane depolarization, action potentials were adjusted to start at the same resting potential. "Rec", recovery/washout. B: Effects of hypoxia (n=90 spikes from 4 cells), rotenone (n=50 spikes from 3 cells), and cyanide (n=30 spikes from 2 cells) on action potential duration and spike frequency. Hypoxia and rotenone (5 pM), but not cyanide (2.5 mM), caused a slight increase in the rise time of the action potential CcriK), significantly prolonged the decay phase ("Way)and half width of the action potential (thalf). Hypoxia and rotenone, but not cyanide caused a slight but non-significant decrease in action potential frequency. Note that the effects of hypoxia were attenuated or abolished in the presence of rotenone, but not cyanide. All records were obtained using the nystatin perforated patch configuration of whole-cell recording in 1=0 mode.

4.3. Reactive Oxygen Species May Function as the Second Messenger During Hypoxic Chemoreception

If hypoxia is sensed by the mitochondria, how is the signal transduced to plasmalemmal K+ channels? Our data suggest that hypoxia decreases ROS generation from the ETC at a site upstream of the classical 02 and CN binding site of cytochrome c oxidase (complex IV) and downstream of the rotenone binding site in complex I. We tested if alterations of ROS could account for K+ channel inhibition during hypoxia. Exogenous H202, an 02-signaling pathway second messenger, reversed the effect of hypoxia on K+ currents, and the ROS scavenger, N-acetylcysteine, mimicked and attenuated the hypoxic inhibition of K+ currents (Thompson and Nurse, unpublished data). Taken together, these data suggest that hypoxia decreases ROS generation by the ETC of AMC. A similar mechanism, involving inhibition of mitochondrial ROS generation has been proposed to explain the 02-sensitivity ofpulmonary myocytes (19).

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