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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

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...

Oxidative post-translational modifications of proteins resulting from events that increase cellular oxidant levels play important roles in physiological and pathophysiological processes. Evaluation of alterations to protein redox states is increasingly common place because of methodological advances that have enabled detection, quantification and identification of such changes in cells and tissues. This mini-review provides a synopsis of biochemical methods that can be utilized to monitor the array of different oxidative and electrophilic modifications that can occur to protein thiols and can be important in the regulatory or maladaptive impact oxidants can have on biological systems. Several of the methods discussed are valuable for monitoring the redox state of established redox sensing proteins such as Keap1.

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...

Pregnant women who are obese or whose gestational weight gain is above recommended limits are at increased risk of obstetric complications, 1 but there may also be longer term consequences for the health of the child.2 A high maternal prepregnancy body mass index and excessive gestational weight gain have been frequently independently associated with a heightened risk of offspring metabolic dysfunction and obesity 3 and there is increasing evidence to suggest that cardiovascular function may also be compromised. 4 Studies of maternal calorie-rich diets and obesity in rodents, sheep, and nonhuman primates have provided unequivocal evidence for persistent and adverse influences on the offspring. 5 We and others have repeatedly observed cardiovascular dysfunction secondary to maternal obesity in animal models. [6][7][8] Previously, we observed that juvenile offspring of obese rats have aberrant autonomic control of blood pressure (BP) resulting in hypertension, which occurs before the development of increased adiposity and persists into adulthood, 7 and that these animals demonstrate hyperphagia and leptin resistance associated with a functional, structural, and cell-signaling deficit in leptin-sensitive processes in the arcuate nucleus and paraventricular nucleus of the hypothalamus. 9 The cardiovascular response to exogenous leptin, which is mediated through hypothalamic sympathetic efferent activity, 10,11 was, conversely, enhanced. 7 This differential response to leptin, sometimes termed selective leptin resistance, had been observed in rodents, but previously only in association with chronic obesity. 12 We also reported that the offspring of obese dams (OffOb) exhibit an exaggerated and prolonged physiological postnatal serum leptin surge, 9 known to play a critical role in the development of the normal rodent hypothalamus. 13 We hypothesized that neonatal exposure to supranormal leptin concentrations during this important window of hypothalamic plasticity may play a causal role in offspring cardiovascular Abstract-The prevalence of obesity among pregnant women is increasing. Evidence from human cohort studies and experimental animals suggests that offspring cardiovascular and metabolic function is compromised through early life exposure to maternal obesity. Previously, we reported that juvenile offspring of obese rats develop sympathetically mediated hypertension associated with neonatal hyperleptinemia. We have now addressed the hypothesis that neonatal exposure to raised leptin in the immediate postnatal period plays a causal role. Pups from lean Sprague-Dawley rats were treated either with leptin (3 mg/kg IP) or with saline twice daily from postnatal day 9 to 15 to mimic the exaggerated postnatal leptin surge observed in offspring of obese dams. Cardiovascular function was assessed by radiotelemetry at 30 days, and 2 and 12 months. In juvenile (30 days) leptin-treated rats, hearts were heavier and night-time (active period) systolic blood pressure was raised (mm Hg; mean±SEM: male leptin-treated, 132±1 v...

Background-Although nitroglycerin has remained in clinical use since 1879, the mechanism by which it relaxes blood vessels to lower blood pressure remains incompletely understood. Nitroglycerin undergoes metabolism that generates several reaction products, including oxidants, and this bioactivation process is essential for vasodilation. Protein kinase G (PKG) mediates classic nitric oxide-dependent vasorelaxation, but the 1␣ isoform is also independently activated by oxidation that involves interprotein disulfide formation within this homodimeric protein complex. We hypothesized that nitroglycerin-induced vasodilation is mediated by disulfide activation of PKG1␣. Methods and Results-Treating smooth muscle cells or isolated blood vessels with nitroglycerin caused PKG1␣ disulfide dimerization. PKG1␣ disulfide formation was increased in wild-type mouse aortas by in vivo nitroglycerin treatment, but this oxidation was lost as tolerance developed. To establish whether kinase oxidation underlies nitroglycerin-induced vasodilation in vivo, we used a Cys42Ser PKG1␣ knock-in mouse that cannot transduce oxidant signals because it does not contain the vital redox-sensing thiol. This redox-dead knock-in mouse was substantively deficient in hypotensive response to nitroglycerin compared with wild-type littermates as measured in vivo by radiotelemetry. Resistance blood vessels from knock-ins were markedly less sensitive to nitroglycerin-induced vasodilation (EC 50 ϭ39.2Ϯ10.7 mol/L) than wild-types (EC 50 ϭ12.1Ϯ2.9 mol/L). Furthermore, after Ϸ24 hours of treatment, wild-type controls stopped vasodilating to nitroglycerin, and the vascular sensitivity to nitroglycerin was decreased, whereas this tolerance phenomenon, which routinely hampers the management of hypertensive patients, was absent in knock-ins. Conclusions-PKG1␣ disulfide formation is a significant mediator of nitroglycerin-induced vasodilation, and tolerance to nitroglycerin is associated with loss of kinase oxidation. (Circulation. 2012;126:287-295.)