The present study reveals three novel and mechanistically linked findings: (i) IH-evoked mitochondrial complex I inhibition requires ROS generation by Nox2 and S-glutathionylation of complex I subunits, (ii) inhibition of complex I increases mitochondrial ROS, and (iii) mitochondrial ROS contributes to sustained but not transient elevations in blood pressure in IH-treated rats.
We previously reported that IH activates Nox (26
) and inhibits mitochondrial complex I activity (27
) in the rat carotid body. However, carotid bodies being too small in size (weighing 50–80
μg) precluded analysis of potential interactions between Nox and complex I. PC12 cells respond similarly to IH with increased Nox and decreased mitochondrial complex I activities (38
) (), which prompted us to assess the interactions between Nox and mitochondrial complex I in this cell line. We further verified the data from PC12 cell cultures with brain stem tissue from intact mice or rats and found that IH increased Nox and decreased complex I activities in both preparations. However, Nox activity measured in brainstem tissue was lower than in PC12 cells. It is likely that Nox is expressed in certain population of neurons or a subset of glial cells, whereas PC12 cells are a homogenous population. The heterogeneity of brain stem tissue might account for the relatively lower complex I and Nox activities compared to PC12 cell cultures. Further, we also found similar changes in Nox and complex 1 activity in cerebral cortical tissue samples from IH-treated rats and mice, suggesting that these responses can be seen in other neural tissues as well.
The following observations demonstrate that activation of Nox, especially Nox2 by IH, mediates complex I inhibition: (a) two structurally distinct inhibitors of Nox prevented IH-evoked inhibition of complex I; (b) genetic silencing of Nox2 but not Nox 4 abrogated complex I inhibition in IH-treated cells, and (c) complex I inhibition by IH was absent in tissues from Nox2 KO mice. Consistent with previous studies (3
) we found that activation of Nox increases mitochondrial ROS. It has been proposed that mitochondrial permeability transition pore (mPTP) plays a critical role in mitochondrial redox signaling (6
). The possibility that IH-induced mitochondrial ROS might be due to changes in mPTP requires further investigation. The finding that Nox activation by IH causes mitochondrial complex I inhibition under IH is reminiscent of mitochondrial dysfunction by Nox activation by angiotensin (37
) and nitoglycerine (36
). Nox activation by IH was transient and reversible, whereas complex I inhibition was long lasting. It is likely that transient Nox activation by IH and the ensuing ROS triggers prolonged ROS generation by mitochondria via
inhibition of complex I activity. In other words, these data indicate that IH evokes ROS-induced ROS mechanism. Taken together these observations demonstrate a functional cross-talk between Nox and mitochondrial complex I activity under IH leading to mitochondrial dysfunction. Whether increased mitochondrial ROS impacts Nox activation as proposed recently (16
) also plays a role in IH remain to be investigated.
Our results provide further insight into how Nox activation by IH inhibits complex I. Yuan et al.
) reported that ROS generated by Nox leads to persistent elevation of baseline [Ca2+
in IH-treated PC12 cells. The following observations demonstrate that Ca2+
is critical for evoking complex I inhibition in IH-treated cells: (a) BAPTA-AM, a Ca2+
chelator or RR, an inhibitor of mitochondrial Ca2+
uniporter prevented IH-evoked complex I inhibition; (b) ionomycin, a Ca2+
ionophore inhibited complex I activity in control cells and this effect was prevented by RR; and (c) Ca2+
inhibited the complex I activity in control cells, and this effect was occluded in IH-treated cells. It can be argued that RR may inhibit Ca2+
release from the sarcoplasmic reticulum as evidenced in normal cardiomyocytes (4
). However, RR at the concentrations that prevented complex I inhibition in IH-treated cells had no impact on complex I activity in control PC12 cells. Taken together, these observations suggest that Ca2+
entry into mitochondria is critical for complex I inhibition in IH-treated cells.
How might Ca2+
inhibit the mitochondrial complex I activity? Previous studies identified two catalytically distinct forms of mitochondrial complex I, including an active A-form and the other deactivated D-form (8
and other divalent cations facilitate the transition of active-to-deactivated form of the complex I with altered substrate affinity (9
). IH decreased the substrate affinity of the complex I similar to that seen in control cells treated with Ca2+
, suggesting that IH causes Ca2+
-dependent conformational change in complex I similar to that reported with ischemia and re-perfusion in rat heart preparation (32
). Previous studies have shown that the deactivated, not the active, form of the complex I is susceptible to redox modifications (8
). Indeed IH caused redox modulation of modification of complex I as evidenced by increased S-glutathionylation of 75 and 50
kDa subunits both in cell cultures and in the brain tissues from IH-treated mice. The findings that IH-evoked S-glutathionylation can be blocked by a Ca2+
chelator as well as RR and mimicked by a Ca2+
ionophore further support the notion that IH-induced S-glutationylation involves a Ca2+
-dependent conformational change of complex I subunits.
-glutathionylation, in addition to conformational change of the complex I, also requires elevated GSSG levels (2
). Not only did IH elevate GSSG as evidenced by increased ratio of GSSG/GSH, but more importantly, addition of GSH (reduced form) abrogated complex I inhibition and restored the Vmax
of the reaction in IH-treated cells. These results demonstrate that S-glutathionylation is the critical signaling event responsible for IH-evoked complex I inhibition. The findings that IH-evoked S-glutathionylation could be prevented by a Nox inhibitor as well as by genetic silencing of Nox 2 in cell cultures and was absent in IH-treated Nox2 KO mice suggest that Nox 2 activation by IH and the resulting ROS mediate S-glutathionylation.
IH increased mitochondrial ROS, as evidenced by decreased mitochondrial aconitase activity, an in vivo
marker of ROS. This increase in mitochondrial ROS was due to complex I inhibition, because preventing complex I inhibition abrogated the changes in mitochondrial aconitase activity. What might be the physiological significance of increased mitochondrial ROS by IH? One of the hallmarks of IH is the elevated blood pressures [reviewed in (29
) and the present study]. Although ROS signaling has been implicated in IH-induced hypertension (28
), the relative importance of mitochondrial ROS has not been established. Our results showed that treatment with mito-tempol, a mitochondrial ROS scavenger, selectively abolished sustained but not the acute elevations of blood pressure in IH-treated rats, suggesting a role for mitochondrial ROS in IH-evoked hypertension. On the other hand, Nox inhibition by apocynin prevented both the transient as well as sustained elevations in blood pressures. Although cellular mechanisms by which mitochondrial ROS contribute to sustained elevations in blood pressure in IH-treated rats remain to be investigated, our results suggest a role for mitochondrial ROS generated by complex I inhibition resulting from Nox activation in mediating IH-induced long-lasting elevations of blood pressures.