Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.
Nitric oxide (NO) is a gaseous signaling molecule and effector in various biological processes. In mammalian cells, NO is produced by a family of NO synthases (NOS). Three NOS isoforms have been identified as: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). In addition to NO, NOS also produces superoxide anion. This phenomenon is named NOS uncoupling as superoxide generation mainly occurs when NOS is not coupled with its cofactor or substrate. nNOS was first found to produce superoxide under L-arginine depletion condition. Further studies demonstrated that superoxide production is a general feature of all three NOS isoforms. In particular, superoxide generated from uncoupled eNOS has been found to play critical roles in the process of various cardiovascular diseases. Although NOS was first found to produce superoxide only when uncoupled with its cofactor or substrate, recent studies reveal that oxygen reduction to superoxide is an intrinsic process amid NO synthesis. Tetrahydrobiopterin plays a controlling role in preventing superoxide release from the eNOS oxygenase domain. Besides tetrahydrobiopterin, the regulation of eNOS uncoupling by the interactions with other proteins, protein phosphorylation, S-glutathionylation, and endogenous L-arginine derivatives, will be discussed in this review.
Inducible nitric oxide synthase (iNOS) plays important roles in cell injury and host defense. Our early study demonstrated that heat shock protein 90 (Hsp90) interacts with iNOS and this interaction enhances iNOS function. Recently, we reported that Hsp90 is also essential for iNOS gene transactivation. In the present study, we investigate the role of Hsp90 in controlling iNOS protein stability. In mouse macrophages, Hsp90 inhibition dissociated Hsp90 from iNOS and the latter subsequently formed aggregates. Aggregation deactivated iNOS. iNOS aggregates were cleared by the ubiquitin-proteasome system (UPS) inside cells. CHIP, an Hsp90-dependent E3 ligase, was previously implicated in iNOS turnover. However, CHIP knockdown had little effect on iNOS degradation in Hsp90-inhibited cells, indicating that other E3 ligases accounted for the clearance of iNOS aggregates. Further studies revealed that the SPRY domain-containing SOCS box protein 2 (SPSB2), an E3 ligase-recruiting protein, was essential for the ubiquitination of iNOS aggregates. SPSB2 knockdown or deleting the SPSB2-interacting domain on iNOS prevented the clearance of iNOS aggregates in Hsp90-inhibited cells. Thus, besides modulating iNOS function and gene transcription, Hsp90 is also essential for the protein stability of iNOS. Hsp90 blockade induces iNOS aggregation and SPSB2 is required for UPS degradation of iNOS aggregates.
Endothelial NO synthase (eNOS) function is critically modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (S1179, bovine)/1177 (S1177, human and rat) by Akt has emerged as a central mechanism of eNOS regulation under both physiological and pathological conditions. Endoplasmic reticulum (ER) stress is a fundamental unfolded protein response occurred in various diseases. Whether and how ER stress affects eNOS phosphorylation is unknown. To address this issue, we induced ER stress in bovine aortic endothelial cells (BAECs) with Brefeldin A (BFA, 5 μg/ml), a compound blocking protein transport from ER to Golgi apparatus. BFA time-dependently induced ER stress in BAECs as evidenced by the markedly increased expressions of ER chaperon Grp78. Parallel to the time course of ER stress, a progressive loss of eNOS S1179 phosphorylation was seen. ER stress-induced eNOS dephosphorylation was specific to S1179 because the phosphorylation status of eNOS T497 or S635 was unchanged. In cells exposed to BFA for 4 hr, eNOS S1179 phosphorylation was decreased more than 5 fold (17.9±0.1% of control,
P
<0.01, n=5). As a result, eNOS activity was diminished (from 3.32±0.28 to 0.85±0.08 pmol/mg/min,
P
<0.01, n=3). Further studies revealed that ER stress caused Akt T308 and S473 dephosphorylation leading to Akt deactivation. Besides BFA, the loss of eNOS and Akt phosphorylation was also measured in ER stress induced by depleting ER Ca
2+
content with
A23187
(2 μM) or perturbing ER oxidative environment with DTT (5 mM). To determine if these findings from cell culture occur in vivo, we monitored ER stress and eNOS S1177 phosphorylation in postischemic rat hearts. Indeed, severe ER stress and corresponding loss of eNOS S1177 phosphorylation and activity were detected in the infarcted areas of hearts after 1-hr coronary artery (LAD) occlusion followed by 24-hr reperfusion. Collectively, these results demonstrate that ER stress decreases eNOS S1179 phosphorylation and function via Akt deactivation. Ischemia/reperfusion cause ER stress, which, at least in part, accounts for the loss of eNOS S1179 phosphorylation and function in hearts. Thus, reducing ER stress may be an important approach to prevent eNOS dysfunction in postischemic hearts.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.