Singlet molecular oxygen ( 1 O 2 ) has well-established roles in photosynthetic plants, bacteria and fungi 1-3 , but not in mammals. Chemically generated 1 O 2 oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine 4 , while enzymatic oxidation of tryptophan to N-formylkynurenine is catalyzed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1 5 . Under inflammatory conditions, this hemecontaining enzyme becomes expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure 6 . However, whether indoleamine 2,3-dioxygenase 1 forms 1 O 2 and whether this contributes to blood pressure control is unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of 1 O 2 . We observed that in the presence of hydrogen peroxide, the enzyme generates 1 O 2 and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophanderived hydroperoxide acts as a hitherto undiscovered signaling molecule in vivo, which induces arterial relaxation and decreases blood pressure dependent on cysteine residue 42 of protein kinase G1α. Our findings demonstrate a pathophysiological role for 1 O 2 in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions. Several small molecules, such as nitric oxide and hydrogen peroxide (H 2 O 2 ) regulate cellular signaling via interaction with proteins containing redox active metals and/or cysteine residues. Of these molecules, nitric oxide, formed from L-arginine by endothelial nitric oxide synthase, is an important regulator of vascular tone 7 . Sustained increases in nitric oxide synthesis by inducible nitric oxide synthase, as observed in pathological settings such as sepsis, are associated with profound hypotension 8 . Paradoxically, inhibitors of the nitric oxide pathway have generally failed to ameliorate severe septic shock 9,10 , suggesting involvement of additional mediators of hypotension.Based on functional similarity with the metabolism of L-arginine by nitric oxide synthase, we 3 reported previously that metabolism of L-tryptophan (Trp) to N-formylkynurenine (NFK) and kynurenine by endothelial indoleamine 2,3-dioxygenase 1 (IDO1) ( Fig. 1a) contributes to the regulation of vascular tone and blood pressure in inflammation 6 . We also showed that commercial kynurenine relaxed pre-constricted arteries, which suggested that kynurenine is an endotheliumderived relaxant factor 6 . Although others have since confirmed these findings 11,12 , we noticed that recently purchased kynurenine no longer caused arterial relaxation, and that HPLC-purified kynurenine and NFK also failed to relax naïve mouse arteries ( Fig. 1b). However, purified Trp relaxed pre-constricted mouse abdominal aortas that expressed IDO1, irrespective of whether IDO1 expression was...
Abstract-Dysregulated blood pressure control leading to hypertension is prevalent and is a risk factor for several common diseases. Fully understanding blood pressure regulation offers the possibility of developing rationale therapies to alleviate hypertension and associated disease risks. Although hydrogen sulfide (H 2 S) is a well-established endogenous vasodilator, the molecular basis of its blood-pressure lowering action is incompletely understood. H 2 S-dependent vasodilation and blood pressure lowering in vivo was mediated by it catalyzing formation of an activating interprotein disulfide within protein kinase G (PKG) Iα. However, this oxidative activation of PKG Iα is counterintuitive because H 2 S is a thiol-reducing molecule that breaks disulfides, and so it is not generally anticipated to induce their formation. This apparent paradox was explained by H 2 S in the presence of molecular oxygen or hydrogen peroxide rapidly converting to polysulfides, which have oxidant properties that in turn activate PKG by inducing the disulfide. These observations are relevant in vivo because transgenic knockin mice in which the cysteine 42 redox sensor within PKG has been systemically replaced with a redox-dead serine residue are resistant to H 2 S-induced blood pressure lowering. Thus, a primary mechanism by which the reductant molecule H 2 S lowers blood pressure is mediated somewhat paradoxically by the oxidative activation of PKG. (Hypertension. 2014;64:1344-1351.)
Soluble epoxide hydrolase (sEH) is inhibited by electrophilic lipids by their adduction to Cys521 proximal to its catalytic center. This inhibition prevents hydrolysis of the enzymes' epoxyeicosatrienoic acid (EET) substrates, so they accumulate inducing vasodilation to lower blood pressure (BP). We generated a Cys521Ser sEH redoxdead knockin (KI) mouse model that was resistant to this mode of inhibition. The electrophilic lipid 10-nitro-oleic acid (NO 2 -OA) inhibited hydrolase activity and also lowered BP in an angiotensin II-induced hypertension model in wild-type (WT) but not KI mice. Furthermore, EET/dihydroxy-epoxyeicosatrienoic acid isomer ratios were elevated in plasma from WT but not KI mice following NO 2 -OA treatment, consistent with the redox-dead mutant being resistant to inhibition by lipid electrophiles. sEH was inhibited in WT mice fed linoleic acid and nitrite, key constituents of the Mediterranean diet that elevates electrophilic nitro fatty acid levels, whereas KIs were unaffected. These observations reveal that lipid electrophiles such as NO 2 -OA mediate antihypertensive signaling actions by inhibiting sEH and suggest a mechanism accounting for protection from hypertension afforded by the Mediterranean diet.thiol | cardiovascular
Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodelling during stress. Here we show that mitochondrial pyruvate carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as in the myocardium of mice induced to fail by angiotensin II or through transverse aortic constriction. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-[ 13 C 2 ]glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse aortic constriction protected against cardiac hypertrophy and failure. Collectively, our findings demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.Healthy myocardial mitochondria primarily utilize oxidative phosphorylation to generate adenosine triphosphate (ATP), which is required to meet the heart's energy-demanding function as a blood pump. In healthy myocardium with sufficient oxygen supply, oxidation of fatty acids provides approximately 60-90% of the myocardial acetyl-coenzyme A that contributes to ATP generation, with 10-40% arising from pyruvate oxidation 1,2 . However, the stressed human heart changes its fuel preference 3,4 , switching from fatty acids to glucose as a favoured carbon source 5,6 . Consistent with this observation, several studies with animal models of pressure overload-induced hypertrophy have shown reduced fatty acid oxidation rates with enhanced glucose uptake and glycolysis, accompanied by a compensatory anaplerosis to maintain the tricarboxylic acid cycle flux in the pathological heart [7][8][9] . Interestingly, this enhanced glycolysis and carbon influx via anaplerosis did not augment ATP production, consistent with an 'uncoupling' between glycolysis and glucose oxidation during pathological hypertrophy 7,10,11 . Furthermore, instead of pyruvate predominantly being oxidized in the mitochondria, it is metabolized by alternative pathways, including reductive fermentation to lactate, despite sufficient oxygen availability 12,13 . This is reminiscent of the Warburg effect, whereby many cancer cells increase glucose uptake and convert it to lactate via the reduction of pyruvate despite oxygen availability.Since the identification of the mitochondrial pyruvate carrier (MPC) in 2012 (refs. 14,15 ...
A complete elucidation of blood pressure homeostasis is important because its dysregulation commonly results in hypertension, increasing the risk of kidney injury, myocardial infarction, heart failure, and stroke. Three principal pathways control vasodilation and blood pressure lowering, including nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). EDHF is largely absent in conduit vessels, but in resistance vessels, which are the principal regulators of blood pressure, it is a prevalent and perhaps the predominant mechanism controlling vasodilation. [1][2][3][4] NO formation is stimulated by shear stress and circulating factors such as bradykinin, acetylcholine, and adenosine. The ability of NO to stimulate vessel relaxation is extensively characterized and involves its interaction with the heme center of guanylate cyclase, stimulating the catalytic ability of the enzyme to convert guanosine-5´-triphosphate to the second messenger cGMP. cGMP transduces many of the biological effects of NO by directly binding to and stimulating the activity of cGMP-dependent protein kinase, also known as protein kinase G (PKG). PKG activation induces substrate phosphorylation in vascular smooth muscle cells, resulting in blood vessel vasodilation by decreasing intracellular Ca 2+ and myofilament Ca 2+ sensitivity, thereby attenuating myosin actin crossbridge cycling.In addition to the classic NO-cGMP pathway, PKG can also be activated by an oxidation mechanism during which the homodimer complex forms an interprotein disulfide. 5 The disulfide forms in the N-terminus of PKG1α, which is held together by a leucine zipper, with structural studies confirming that Cys42 on each chain closely aligns to explain the susceptibility to oxidation. Oxidation to the disulfide state is sufficient in itself to enable PKG catalytic activity. Classic activation increases PKG V max , whereas disulfide activation increases the kinase affinity for substrate. H 2 O 2 or related oxidants contribute to EDHF-dependent vasodilation of resistance vessel. [6][7][8][9][10][11] This is at least, in part, attributed to EDHF-induced oxidation of PKG1α. PKG oxidation contributes to basal blood pressure as transgenic "redox-dead" Cys42Ser PKG1α knock-in mice Abstract-Protein kinase G (PKG) is activated by nitric oxide (NO)-induced cGMP binding or alternatively by oxidantinduced interprotein disulfide formation. We found preactivation with cGMP attenuated PKG oxidation. 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) blockade of cGMP production increased disulfide PKG to 13±2% and 29±4% of total in aorta and mesenteries, respectively. This was potentially anomalous, because we observed 2.7-fold higher NO levels in aorta than mesenteries; consequently, we had anticipated that ODQ would induce more disulfide in the conduit vessel. ODQ also constricted aorta, whereas it had no effect on mesenteries. Thus, mesenteries, but not aorta, can compensate for loss of NO-cGMP by recruiting disulfide activation of PKG. Mechanistically, t...
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Sepsis is a common life-threatening clinical syndrome involving complications as a result of severe infection. A cardinal feature of sepsis is inflammation that results in oxidative stress. Sepsis in wildtype mice induced oxidative activation of cGMP-dependent protein kinase 1 alpha (PKG Iα), which increased blood vessel dilation and permeability, and also lowered cardiac output. These responses are typical features of sepsis and their combined effect is a lowering of blood pressure. This hypotension, a hallmark of sepsis, resulted in underperfusion of end organs, resulting in their damage. A central role for PKG Iα oxidative activation in injury is supported by oxidation-resistant Cys42Ser PKG Iα knock-in mice being markedly protected from these clinical indices of injury during sepsis. We conclude that oxidative activation of PKG Iα is a key mediator of hypotension and consequential organ injury during sepsis.redox | cardiovascular function | endotoxin S epsis, a prevalent medical condition caused by severe infection with systemic inflammation, causes substantial morbidity and mortality (1). Prognosis is poor with 85% survival in uncomplicated sepsis, falling to 20% in those with multiorgan failure (2). The cost of acute care is enormous (3), but survivors often suffer long-term cognitive impairment generating a chronic health care burden (4). Sepsis is characterized by systemic inflammation (5), decreased peripheral vascular resistance (1), microvascular leak (6), and decreased cardiac output (1). The combined effect of these alterations is low blood pressure (hypotension), a major clinical feature of sepsis (1). This hypotension results in underperfusion of end organs that leads to their functional failure and too often patient death (1).Oxidative stress is a hallmark of sepsis, consistent with the inflammatory respiratory burst by neutrophils generating high levels of oxidants (5). However, multiple oxidant-generating systems, including nicotinamide adenine dinucleotide phosphate oxidase, uncoupled nitric oxide synthase (NOS) (7), lysozyme-c (8), and mitochondria (9) are activated during sepsis. Consistent with this, the levels of superoxide and hydroxyl radicals, hydrogen peroxide, peroxynitrite, nitrogen dioxide (7), nitroxyl (10), and nitrosothiols (11) can increase during sepsis. Because oxidants can activate cGMP-dependent protein kinase 1 alpha (PKG Iα) to lower blood pressure (12-14), we hypothesized this process underlies sepsis-induced hypotension and consequential organ injury. Because PKG couples to enhance endothelial permeability (6, 15), oxidative activation would also account for the enhanced microvascular leak that would further exacerbate the hypotension. PKG is also negatively inotropic (13, 16); therefore, oxidative activation might also explain the attenuated cardiac output characteristic of sepsis, further exacerbating the hypotension. Results and DiscussionTo investigate the hypothesis that PKG Iα oxidation mediates septic injury, we used a "redox-dead" Cys42Ser PKG Iα knock-in (KI) mo...
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