Sruti Shiva

Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species.

The functional role of mitochondria in cell physiology has previously centered around metabolism, with oxidative phosphorylation playing a pivotal role. Recently, however, this perspective has changed significantly with the realization that mitochondria are active participants in signal transduction pathways, not simply the passive recipients of injunctions from the rest of the cell. In this review the emerging role of the mitochondrion in cell signaling is discussed in the context of cytochrome c release, hydrogen peroxide formation from the respiratory chain, and the nitric oxide-cytochrome c oxidase signaling pathway.

Mitochondria, nitric oxide, and cardiovascular dysfunction ☆

Cardiovascular diseases encompass a wide spectrum of abnormalities with diverse etiologies. The molecular mechanisms underlying these disorders include a variety of responses such as changes in nitric oxide- (NO) dependent cell signaling and increased apoptosis. An interesting aspect that has received little or no attention is the role mitochondria may play in the vascular changes that occur in both atherosclerosis and hypertension. With the changing perspective of the organelle from simply a role in metabolism to a contributor to signal transduction pathways, the role of mitochondria in cells with relatively low energy demands such as the endothelium has become important to understand. In this context, the definition of the NO-cytochrome c oxidase signaling pathway and the influence this has on cytochrome c release is particularly important in understanding apoptotic mechanisms involving the mitochondrion. This review examines the role of compromised mitochondrial function in a variety of vascular pathologies and the modulation of these effects by NO. The interaction of NO with the various mitochondrial respiratory complexes and the role NO plays in modulating mitochondrial-mediated apoptosis in these systems will be discussed.

The role of iNOS in alcohol‐dependent hepatotoxicity and mitochondrial dysfunction in mice

Nitric oxide (NO) is now known to control both mitochondrial respiration and organelle biogenesis. Under conditions of ethanol‐dependent hepatic dysfunction, steatosis is increased, and this is associated with increased expression of inducible nitric oxide synthase (iNOS). We have previously shown that after chronic exposure to ethanol, the sensitivity of mitochondrial respiration to inhibition by NO is enhanced, and we have proposed that this contributes to ethanol‐dependent hypoxia. This study examines the role of iNOS in controlling the NO‐dependent modification of mitochondrial function. Mitochondria were isolated from the livers of both wild‐type (WT) and iNOS knockout (iNOS−/−) mice that were fed an isocaloric ethanol‐containing diet for a period of 5 weeks. All animals that consumed ethanol showed some evidence of fatty liver; however, this was to a lesser extent in the iNOS−/− mice compared to controls. At this early stage in ethanol‐dependent hepatic dysfunction, infiltration of inflammatory cells and the formation of nitrated proteins was also decreased in response to ethanol feeding in the iNOS−/− animals. Mitochondria isolated from wild‐type ethanol‐fed mice showed a significant decrease in respiratory control ratio and an increased sensitivity to NO‐dependent inhibition of respiration relative to their pair‐fed controls. In contrast, liver mitochondria isolated from iNOS−/− mice fed ethanol showed no change in the sensitivity to NO‐dependent inhibition of respiration. In conclusion, the hepatic response to chronic alcohol‐dependent cytotoxicity involves a change in mitochondrial function dependent on the induction of iNOS. (HEPATOLOGY 2004;40:565–573.)

Nitric Oxide and cGMP-dependent Protein Kinase Regulation of Glucose-mediated Thrombospondin 1-dependent Transforming Growth Factor-β Activation in Mesangial Cells

Excessive transforming growth factor-β (TGF-β) activity in hyperglycemia contributes to the development of diabetic nephropathy. Glucose stimulation of TGF-β activity and matrix synthesis are dependent on autocrine thrombospondin 1 (TSP1) to convert latent TGF-β to its biologically active form. The mechanisms by which glucose regulates TSP1 are not known. High glucose inhibits nitric oxide (NO) bioavailability and decreased NO increases TGF-β activity and extracellular matrix accumulation. Yet, the impact of NO signaling on TSP1 activation of TGF-β is unknown. We tested the role of NO signaling in the regulation of TSP1 expression and TSP1-dependent TGF-β activity in rat mesangial cells exposed to high glucose. On exposure to 30 mmglucose, NO accumulation in the conditioned media and intracellular cGMP levels were significantly decreased. The addition of an NO donor prevented the glucose-dependent increase in TSP1 mRNA, protein, and TGF-β bioactivity. The effects of the NO donor were blocked by ODQ (a soluble guanylate cyclase inhibitor) or Rp-8-pCPT-cGMPS (an inhibitor of cGMP-dependent protein kinase). These effects of high glucose were also reversed by the nitric-oxide synthase cofactor tetrahyrobiopterin (BH4). These results show that high glucose mediates increases in TSP1 expression and TSP1-dependent TGF-β bioactivity through down-modulation of NO-cGMP-dependent protein kinase signaling.

Mechanisms of the interaction of nitroxyl with mitochondria

It is now thought that NO* (nitric oxide) and its redox congeners may play a role in the physiological regulation of mitochondrial function. The inhibition of cytochrome c oxidase by NO* is characterized as being reversible and oxygen dependent. In contrast, peroxynitrite, the product of the reaction of NO* with superoxide, irreversibly inhibits several of the respiratory complexes. However, little is known about the effects of HNO (nitroxyl) on mitochondrial function. This is especially important, since HNO has been shown to be more cytotoxic than NO*, may potentially be generated in vivo, and elicits biological responses with some of the characteristics of NO and peroxynitrite. In the present study, we present evidence that isolated mitochondria, in the absence or presence of substrate, convert HNO into NO* by a process that is dependent on mitochondrial concentration as well as the concentration of the HNO donor Angelis salt. In addition, HNO is able to inhibit mitochondrial respiration through the inhibition of complexes I and II, most probably via modification of specific cysteine residues in the proteins. Using a proteomics approach, extensive modification of mitochondrial protein thiols was demonstrated. From these data it is evident that HNO interacts with mitochondria through mechanisms distinct from those of either NO* or peroxynitrite, including the generation of NO*, the modification of thiols and the inhibition of complexes I and II.

Induction of the permeability transition and cytochrome c release by 15-deoxy-Δ12,14-prostaglandin J2 in mitochondria

The electrophilic lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is known to allow adaptation to oxidative stress in cells at low concentrations and apoptosis at high levels. The mechanisms leading to adaptation involve the covalent modification of regulatory proteins, such as Keap1, and augmentation of antioxidant defences in the cell. The targets leading to apoptosis are less well defined, but mitochondria have been indirectly implicated in the mechanisms of cell death mediated by electrophilic lipids. To determine the potential of electrophilic cyclopentenones to induce pro-apoptotic effects in the mitochondrion, we used isolated liver mitochondria and demonstrated that 15d-PGJ2 promotes Ca2+-induced mitochondrial swelling and cytochrome c release. The mechanisms involved are consistent with direct modification of protein thiols in the mitochondrion, rather than secondary formation of reactive oxygen species or lipid peroxidation. Using proteomic analysis in combination with biotinylated 15d-PGJ2, we were able to identify 17 potential targets of the electrophile-responsive proteome in isolated liver mitochondria. Taken together, these results suggest that electrophilic lipid oxidation products can target a sub-proteome in mitochondria, and this in turn results in the transduction of the electrophilic stimulus to the cell through cytochrome c release.