Mitochondria as Oxygen Sensors

The mitochondrion's role as the predominant site for 02 consumption and ATP synthesis makes it an obvious candidate site for an 02 sensor. The finding that inhibition of the mitochondrial electron transport chain (ETC) mimics hypoxia further supports this hypothesis (4). Indeed ETC inhibitors cause pulmonary vasoconstriction, systemic vasodilation and carotid body activation, a set of responses elicited by few other stimuli save hypoxia (4).

Carotid Body Oxygen Sensing Mech

Figure 4: Mitochondria regulate K+ channel function through the production of diffusible reducing equivalents and AOS in response to hypoxia. Hypoxia inhibits the proximal ETC (pETC), resulting in decreased production of AOS (02'~ and H202). The loss of tonic normoxic AOS reduces and inhibits Kv channels, depolarizing the PASMC and opening voltage-gated, L-type Ca2+ channels, causing [Ca2+]j to rise and constriction to occur. PDH, pyruvate dehydogenase.

Figure 4: Mitochondria regulate K+ channel function through the production of diffusible reducing equivalents and AOS in response to hypoxia. Hypoxia inhibits the proximal ETC (pETC), resulting in decreased production of AOS (02'~ and H202). The loss of tonic normoxic AOS reduces and inhibits Kv channels, depolarizing the PASMC and opening voltage-gated, L-type Ca2+ channels, causing [Ca2+]j to rise and constriction to occur. PDH, pyruvate dehydogenase.

In 1981, Rounds and McMurtry (103) reported that certain inhibitors of ETC and oxidative phosphorylation (including azide, cyanide, antimycin A, and rotenone) mimicked the HPV in isolated blood-perfused lungs. Furthermore, the same mitochondrial inhibitors which block cytochrome c oxidase (cyanide or azide), stimulated hypoxia-mediated activation ofthe carotid body. These early studies suggested that the basis for this mimicry of HPV was a decrease in ATP production due to inhibition of oxidative phosphorylation. In support of this "energy hypothesis" inhibition of glycolysis also had similar effects as hypoxia or ETC inhibitors (79, 115). However, it appears that it is the mitochondrion's ability to alter cellular redox state and produce diffusible redox mediators, rather than its well-established role in producing ATP, that underlies its role as an 02 sensor. Subsequent studies of HPV (induced by moderate hypoxia in perfused lungs) showed no association between HPV and depletion of ATP and adenylate charge (23). Although anoxia (P02 <10 mmHg) does cause ATP depletion, this results in pulmonary vasodilation, rather than constriction, in part by the activation of KATPchannels (129). In the isolated rat lung, it has been reported that ATP and ATP/ADP ratios are preserved after exposure to an alveolar Po2 of

7 mmHg, or to C0/02 ratios of 10/1 for up to 1 hr. Using 31P-NMR, Buescher et al. showed no change in energy status in whole lungs during hypoxic ventilation when compared to its control normoxic counterpart (23). Moreover, other metabolic markers such as pH, phosphocreatinine, and phosphate are also conserved in PAs exposed to hypoxia (55). Thus, the theory that a change in energy status acts as an 02 sensing mechanism seems unlikely. Teleologically, it would seem undesirable to wait until energy had been depleted before optimizing ventilation-perfusion matching (which could provide more 02). Furthermore, the lungs consume little 02 and, unlike in the heart, monitoring lung ATP levels provides little insight into the organ's function.

Another school of thought in 02-sensing is that the mitochondria are involved in 02 sensing through the production of AOS. In 1986, our laboratory proposed a link between mitochondrial AOS production, cellular redox status, K+ conductance, membrane potential, and HPV (7). Subsequently, we found that inhibitors of complexes I and III, but not complex IV, cause pulmonary vasoconstriction, inhibit K+ current (IK), and prevent further HPV. Furthermore, inhibitors of complex I and III also cause systemic vasodilatation and do not inhibit IK in systemic arterial SMCs, once again mimicking hypoxia (72). It is noteworthy that one of the putative mediators of HPV mimic hypoxia's opposing effects on the pulmonary and systemic vasculature (72). Further studies have shown that mitochondria generate superoxide anion (02'~) at complex I and III that is dismutated by mitochondrial specific manganese superoxide dismutase (MnSOD), generating the diffusible signal molecule hydrogen peroxide (H202). Several other groups have recently suggested that complex III may be more important than complex I (53, 125), although our group continues to find a role for both complexes in both the PA (72) and ductus arteriosus (74). The question remains as to how mitochondria are able to accomplish this task.

The 02 sensing function of PASMC mitochondria is tied to the redox cascade within the ETC. Electrons from reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), are transferred down a redox gradient from potential of -0.35 for NADH/NAD+ to +0.82 for 02/H20 (Fig. 5) (4). The terminal electron transfer to forms water. Four multicomponent megacomplexes accomplish the transfer of electrons. With each electron transferred down the ETC, a hydrogen ion is translocated across the inner mitochondrial membrane creating the very negative mitochondrial membrane potential (AvPm). It is the potential energy from this proton gradient that is utilized for ATP synthesis. Thus there is a link between electron donors, electron flux and production of AOS. It appears that complexes I and III are particularly important in 02 sensing mechanisms because it is at these sites that AOS are generated (10, 19). The peroxides can then diffuse to the plasma membrane whilst it may be that superoxide anion can move through diisothiocyano-2,2 disulfonic acid stilbene (DIDS)- and voltage-sensitive anion channels, validating mitochondria as sources of cytosolic superoxide anion (39).

i'Îr I Mala te, a-ketoghitarate(FAD) risocitratc, pyruvate (FAD)

y Acetyl CoA

NADH ^ I FAIJH

Complex I Rote twite

Complex IÏ

TTFA

Cyt c Fe>7Fe1+

Cyt!>

|

t

ATP

i Cyte,

Figure 5: Sites of AOS generation in the mitochondrial ETC. Electrons flow down the ETC driven by a difference in the redox potential between -0.4 V in complex I and +0.8 V in complex IV. As the electrons flow towards their final target, 02, they release electrochemical energy, which is used to synthesize ATP. Most of the "drop" in redox potential occurs in complexes I and III, sites that are responsible for most of the mitochondrial AOS production and ATP synthesis. Rotenone blocks complex I; thenoyltrifluoroacetone (TTFA) blocks complex II. Both agents inhibit electron entry to ubiquinone. Antimycin and cyanide are blockers of complexes III and IV, respectively.

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