Mitochondrial Reactive Oxygen Species are Required and Sufficient to Elicit Hypoxic Pulmonary Vasoconstriction

HPV occurs mainly in the small distal pulmonary arteries. Recent studies have indicated that acute hypoxia selectively inhibits voltage-gated K+ channels (Kv) (3). This results in membrane depolarization, opening of L-type voltage-dependent Ca2+channels, and elevation of [Ca2+]j. An increase in [Ca2+], causes calmodulin-mediated activation of myosin light chain kinase, actin-myosin interaction, and contraction. Voltage-gated Ca2+ channels provide a major influx pathway for the increase in [Ca2"1"],. An alternative model proposes that the initial release of Ca2+occurs from sarcoplasmic reticulum (SR) (10, 25). In this model, hypoxia causes the release of Ca2+ from intracellular stores, causing an inhibition of Kv channels, membrane depolarization, and opening of L-type voltage-gated

Ca2+ channels. Clearly, an elevation in [Ca2+]i; irrespective of the source, is responsible for the contraction. How decreases in oxygen tension are detected

and coupled to an increase in Ca is not fully understood.

Figure 2. Hypoxia increases the generation of mitochondrial superoxide at the ubisemiquinone site. Subsequently, the superoxide escapes mitochondria into the cytosol via anion channels. In the cytosol, superoxide is converted to hydrogen peroxide, which is sufficient and required for the hypoxia-induced [Ca2+]j increases in PA myocytes

Figure 2. Hypoxia increases the generation of mitochondrial superoxide at the ubisemiquinone site. Subsequently, the superoxide escapes mitochondria into the cytosol via anion channels. In the cytosol, superoxide is converted to hydrogen peroxide, which is sufficient and required for the hypoxia-induced [Ca2+]j increases in PA myocytes

We have proposed that mitochondria serve as oxygen sensors by increasing superoxide production within complex III of the electron transport chain during hypoxia in PASMCs (34, 35). The increase levels in mitochondrial superoxide production results in efflux of this anion into the cytosol through anion channels localized on both inner and outer mitochondrial membranes. Subsequently, the superoxide is converted to hydrogen peroxide which is sufficient and required to elevate [Ca2+], (Fig. 2). Primary evidence for this model comes from parallel experiments in isolated perfused lungs and PASMCs from rats. Pulmonary vasoconstriction in isolated lungs or PASMC contraction during hypoxia is blocked by myxothiazol, diphenyleneiodonium (DPI), or rotenone. All three mitochondrial inhibitors block electron transport chain upstream from the ubisemiquinone site of superoxide production. None of these pharmacological electron transport agents affect the vasoconstriction induced by U46619 under normoxia. DPI, rotenone, and myxothiazol also attenuate the increase in in response to hypoxia without affecting the rise in due to angiotensin II during normoxia. Antimycin A, which inhibits mitochondrial electron transport chain downstream from the ubisemiquinone site of superoxide production, maintains HPV and has no effect on hypoxia-induced calcium signaling. The mitochondrial anion channel blocker DIDS abolishes pulmonary vasoconstriction in isolated lungs and contraction in PASMCs during hypoxia without affecting the response to U46619. Ebselen, a synthetic glutathione peroxidase, abolishes pulmonary vasoconstriction in isolated lungs and contraction in PASMCs during hypoxia without affecting the response toU46619. H202 is sufficientto invoke contraction in PASMCs and to increase in calcium during normoxia. Adenovirus transfection of PASMCs with catalase prevents the increase in calcium during hypoxia or in H202 during normoxia, without affecting the increase in calcium due to angiotensin II during normoxia. The p° PASMCs retained the contractile response to U46619 but failed to respond to hypoxia. These results suggest that HPV in isolated lungs, as well as the increase in calcium and subsequent PASMC contraction during hypoxia, occur independently of mitochondrial ATP production and are triggered by increased release of superoxide from the ubisemiquinone site within the mitochondrial electron transport chain.

Our findings that mitochondria function as the 02 sensor upstream of the increase in calcium in triggering HPV has been corroborated by Ward and colleagues (20). They found that rotenone and myxothiazol attenuated hypoxia-induced increases in and subsequent vasoconstriction. By contrast, inhibition of complex IV with cyanide augmented the hypoxic response but had no effect on [Ca2+];. Interestingly, in pulmonary arteries that had been treated with rotenone, Ward and colleagues were able to restore HPV by using succinate to shuttle electrons into complex III via complex II, effectively bypassing the rotenone inhibition of complex I. However our observations have been challenged by Archer and colleagues who have reported a decrease in mitochondrial ROS generation. According to their hypothesis, hypoxia reduces mitochondrial electron transport thereby changing the ratio of redox couples (i.e., GSSG/GSH and NAD7NADH) toward a more reduced state resulting in a decrease in ROS generation. This leads to an inhibition of Kv channels, membrane depolarization, Ca2+ influx via L-type Ca2+ channels, increased [Ca2+]i, and vasoconstriction (23) (see Chapter 9). Thus the major controversy in 02 sensing in PASMC lies in whether ROS levels increase or decrease during hypoxia.

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