Loss Of Nitric Oxide Production Research Articles

Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by β-blocker also restored NO formation by PAH cells, identifying one mechanism by which β-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.

Total polyphenol of Anemarrhena asphodeloides ameliorates advanced glycation end products‐induced endothelial dysfunction by regulation of AMP‐Kinase (知母多酚通过激活AMP‐激酶来缓解糖基化终末产物引起的内皮损伤)

AbstractObjectiveAnemarrhena asphodeloides Bunge is widely used in China for the treatment of diabetes and the polyphenol components are responsible for its anti‐diabetic action. This study aimed to investigate the effect of total polyphenol of Anemarrhena asphodeloides (TPAA) on endothelial dysfunction and to elucidate underlying mechanisms.MethodsWe stimulated endothelial cells with advanced glycation end products (AGEs) to establish the model of endothelial dysfunction in vitro and observed the effect of TPAA (10, 30, or 100 μg/mL) on AMP‐Kinase (AMPK) activation implicated in regulation of nitric oxide (NO) and endothelin‐1 (ET‐1) production. Meanwhile, nuclear factor‐κB (NF‐κB) activation, intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) and eNOS expression were investigated by western blot, fluorescence microscopy and real time‐quantitative PCR analysis, respectively.ResultsTotal polyphenol of Anemarrhena asphodeloides enhanced AMPK phosphorylation and promoted the basal NO production along with the inhibition of ET‐1 secretion in endothelial cells. TPAA inhibited NF‐κB activation by attenuating p65 phosphorylation and suppressed ROS production, well demonstrating its action in inhibition of ROS‐associated inflammation in the endothelium. Meanwhile, TPAA protected mitochondrial function and endothelial homeostasis against AGEs insult by restoring ΔΨm and mRNA expression of eNOS. AGEs stimulation inhibited AMPK activation and induced the loss of NO production together with increased secretion of ET‐1, but these changes were reversed by TPAA in a concentration‐dependent manner. Compound C, an AMPK inhibitor, attenuated the effects of TPAA mentioned above, indicating the involvement of AMPK.ConclusionsTotal polyphenol of Anemarrhena asphodeloides inhibited AGEs‐induced ROS‐associated inflammation and ameliorated endothelial dysfunction through beneficial regulation of AMPK activation.

Endothelial nitric oxide synthase S‐glutathionylation and Ser1177 phosphorylation modulate myocardial protection

Endothelial nitric oxide synthase (eNOS) plays an important role in cardiovascular function, myocardial injury and protection. The role of eNOS is well recognized in ischemic preconditioning (IPC); however, questions remain regarding how eNOS is regulated during ischemia‐reperfusion (I/R) and IPC. eNOS phosphorylation at Ser1177 enhances enzyme activation and nitric oxide (NO) production; however, S‐glutathionylation of eNOS uncouples the enzyme leading to loss of NO production and vascular dysfunction. We measure myocardial eNOS S‐glutathionylation and phosphorylation in the process of I/R injury and investigate the role of these modifications in cardioprotection. Isolated rat hearts were subjected to 30‐min of global ischemia and/or reperfusion with or without IPC stimuli. Ischemia induced a 4–5 fold increase in eNOS S‐glutathionylation that was completely reversed by â‐mercaptoethanol. Importantly, IPC reduced I/R‐induced eNOS S‐glutathionylation by 50%, decreased myocardial superoxide generation by >;80% and attenuated myocardial infarction by 75%. Levels of eNOS phosphorylation at Ser1177 decreased with I/R but were increased by IPC. Thus, IPC stimuli inhibit I/R‐induced eNOS S‐glutathionylation but increase Ser1177 phosphorylation to maintain eNOS coupling, activation and NO production that protect myocardium against I/R injury. (Support: NIH grants HL63744, HL65608 and HL38324).

The influence of N-acetyl-L-cysteine on oxidative stress and nitric oxide synthesis in stimulated macrophages treated with a mustard gas analogue

BackgroundSulphur mustard gas, 2, 2'-dichlorodiethyl sulphide (HD), is a chemical warfare agent. Both mustard gas and its monofunctional analogue, 2-chloroethyl ethyl sulphide (CEES), are alkylating agents that react with and diminish cellular thiols and are highly toxic. Previously, we reported that lipopolysaccharide (LPS) significantly enhances the cytotoxicity of CEES in murine RAW 264.7 macrophages and that CEES transiently inhibits nitric oxide (NO) production via suppression of inducible NO synthase (iNOS) protein expression. NO generation is an important factor in wound healing. In this paper, we explored the hypotheses that LPS increases CEES toxicity by increasing oxidative stress and that treatment with N-acetyl-L-cysteine (NAC) would block LPS induced oxidative stress and protect against loss of NO production. NAC stimulates glutathione (GSH) synthesis and also acts directly as a free radical scavenger. The potential therapeutic use of the antibiotic, polymyxin B, was also evaluated since it binds to LPS and could thereby block the enhancement of CEES toxicity by LPS and also inhibit the secondary infections characteristic of HD/CEES wounds.ResultsWe found that 10 mM NAC, when administered simultaneously or prior to treatment with 500 μM CEES, increased the viability of LPS stimulated macrophages. Surprisingly, NAC failed to protect LPS stimulated macrophages from CEES induced loss of NO production. Macrophages treated with both LPS and CEES show increased oxidative stress parameters (cellular thiol depletion and increased protein carbonyl levels). NAC effectively protected RAW 264.7 cells simultaneously treated with CEES and LPS from GSH loss and oxidative stress. Polymyxin B was found to partially block nitric oxide production and diminish CEES toxicity in LPS-treated macrophages.ConclusionThe present study shows that oxidative stress is an important mechanism contributing to CEES toxicity in LPS stimulated macrophages and supports the notion that antioxidants could play a therapeutic role in preventing mustard gas toxicity. Although NAC reduced oxidative stress in LPS stimulated macrophages treated with CEES, it did not reverse CEES-induced loss of NO production. NAC and polymyxin B were found to help prevent CEES toxicity in LPS-treated macrophages.

Abstract 148: Superoxide Promotes Reversible Uncoupling Of Inducible Nitric Oxide Synthase, While Peroxynitrite Induces Irreversible And Complete Enzyme Inactivation

Inducible nitric oxide synthase (iNOS) is present in the post-ischemic heart, and plays an important role in the pathogenesis of injury and remodeling. Oxidants formed during reperfusion injury are known to alter NOS function. In this study we compare and contrast how two biologically relevant oxidants, peroxynitrite (ONOO − ) and superoxide (O 2 − ), alter iNOS function and define the mechanisms involved for each. Nitric oxide (NO) generation rate of purified iNOS was quantified by the methemoglobin formation, O 2 −. generation was measured using EPR spin-trapping, and the quaternary structure of iNOS by size-exclusion FPLC. Tetrahydrobiopterin (BH 4 )-replete iNOS (1 μM) was exposed to O 2 −. , and ONOO − in concentrations from 0.01 μM to 500μM and NO production was measured with and without addition of excess BH 4 . ONOO − (50μM) decreased NO production to 19% ± 0.17 and excess BH 4 only partially restored activity to 48% ± 3.4. O 2 −. (50 μM) reduced NO production to 42% ± 1.2 and the excess BH 4 completely restored activity to 100% ± 3.1. ONOO − exerted the highest decrease in NO production rate (96% ± 1.3 activity loss at 500 μM ONOO − ) and no activity was restored by excess BH 4 . In contrast, O 2 −. induced a significant decrease (90% ± 1.2 activity loss at 500 μM O 2 −. ), however excess BH 4 restored the iNOS activity back to 69% ± 5.6.O 2 −. exposure enhanced O 2 −. production from iNOS (up to 155% ± 0.72 at 50 μM O 2 −. ), while ONOO − at higher concentrations (>20μM) reduced O 2 −. production (10% ± 3.2 decrease at 50 μM ONOO − ). We also found that incubation with ONOO − and O 2 −. induced iNOS monomerization. In conclusion, both ONOO − and O 2 −. decrease iNOS NO production in a dose dependent manner. Adding excess BH 4 only partially restore the loss of NO production induced by ONOO − , but almost completely restore the loss of NO production induced by O 2 −.. Therefore, O 2 −. promotes the uncoupling of iNOS and elevates O 2 −. production by oxidizing the protein bound BH 4 with subsequent formation of NOS monomer in a reversible fashion. Conversely, ONOO − induces irreversible enzyme inactivation and decreases both NO and O 2 −. production. Although oxidation of BH 4 is involved in the observed ONOO − induced loss of iNOS activity, there are other causes, including oxidation of critical amino acid residues.

Pathophysiology of Contrast-Induced Nephropathy

Contrast-induced nephropathy (CIN) is the third leading cause of acute kidney injury in hospitalized patients and is associated with significant patient morbidity. The pathogenesis of CIN is complex and not fully understood, but iodinated contrast agents induce intense and prolonged vasoconstriction at the corticomedullary junction of the kidney. Moreover, high-osmolar dyes directly impair the autoregulatory capacity of the kidney through a loss of nitric oxide production. These effects, coupled with direct tubular toxicity of contrast media, lead to overt acute tubular necrosis and the syndrome of CIN.

The Role of β-Blockers as a Cornerstone of Cardiovascular Therapy

The beta-adrenergic blockade as a therapeutic approach first emerged in the 1950s. During the past five decades, the total number of indications that have been suggested, and the remarkable number approved by regulatory agencies, places beta-blockade far ahead of all competing treatments, not only in the cardiovascular area, but in all of therapeutics. Differentiation of beta-adrenergic blocking agents has been made on the basis of beta1 selectivity, duration of action, intrinsic sympathomimetic activity, lipophilicity, and whether or not the beta-adrenergic blocking action is accompanied by an alpha-adrenergic blocking action. With the development of nebivolol, a beta-adrenergic blocking agent that also activates nitric oxide synthase in blood vessels, comes a new therapeutic option. Endothelial dysfunction with loss of nitric oxide production is a common feature in many cardiovascular diseases. This fascinating class of drugs continues to provide us with new and important therapeutic opportunities.

Oxidant stress leads to impaired regulation of renal cortical oxygen consumption by nitric oxide in the aging kidney.

Structural and functional changes occur in the kidney with aging. Previous studies have suggested that loss of nitric oxide production contributes to these changes. The authors therefore explored regulation of renal cortical oxygen consumption, a nitric oxide mediated effect, in tissue from Fischer 344 rats at different ages (4, 13, and 23 mo) to characterize changes in renal nitric oxide production with age. Bradykinin, enalaprilat, and amlodipine significantly suppressed cortical oxygen consumption in 4-mo-old rats (bradykinin: -2.5 +/- 0.9% to -21 +/- 1.5%; enalaprilat: -0.7 +/- 0.5% to -26 +/- 1.2%; amlodipine: -1.3 +/- 0.9% to -18 +/- 1.2%; P < 0.05). Similar results were obtained in 13-mo-old animals. However, in 23-mo-old animals, the responses to bradykinin and enalaprilat were attenuated (bradykinin: 0 +/- 0% to -13 +/- 0.9%; enalaprilat: -0.3 +/- 0.3% to -17 +/- 2.1%; P < 0.05), whereas the response to an NO donor was unaffected, suggesting decreased bioavailability of NO. Addition of the superoxide radical scavenger tempol restored the ability of bradykinin, enalaprilat, and amlodipine to suppress oxygen consumption in tissue from 23-mo-old animals to levels seen in younger animals, suggesting NO destruction by superoxide as the reason for decreased NO availability. Apocynin, an inhibitor of NAD(P)H oxidase, similarly restored the ability of all three drugs to suppress oxygen consumption, suggesting NAD(P)H oxidase as the enzyme responsible for enhanced superoxide production in aging. Levels of eNOS protein, assessed by immunoblotting, did not change significantly with age. These results suggest that NO availability is decreased in the aging kidney and that this is due to scavenging of NO by superoxide produced by NAD(P)H oxidase. Oxidant stress, by depleting NO, may contribute to the structural and hemodynamic changes characteristic of the aging kidney.

Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction.

Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y-27632. Castration reduced the maximal erectile response (CCP/MAP) by 33%, and testosterone replacement restored the response (intact, 0.736 +/- 0.040; castrate, 0.492 +/- 0.022; testosterone, 0.681 +/- 0.073). Injection of Y-27632 increased CCP in all experimental groups; it also left shifted the voltage response curve and increased the maximal CCP/MAP response (intact, 0.753 +/- 0.091; castrate, 0.782 +/- 0.081; testosterone treated, 0.894 +/- 0.033). Y-27632 dose dependently relaxed phenylephrine-stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels after castration. Our data support the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction.

Nitric oxide regulates the apoptotic pathway in explanted failing human hearts

Cardiomyocyte apoptosis causes ventricular remodeling in heart failure. Caspases are intracellular enzymes that mediate apoptosis. We investigated nitric oxide (NO) mediated regulation of caspase activity in failing human myocardium to determine if loss of NO production in heart failure contributes to apoptosis. Methods: Ventricular myocardium was obtained from 10 explanted failing human hearts at heart transplantation. 5 patients (pts) were on chronic angiotensin-converting enzyme inhibitors (ACEI) prior to transplantation. Caspase-3 activity was induced using recombinant caspases 8 and 9 in cytosolic extracts of myocardium and measured colorimetrically. Effect of following on caspase activity was measured-exogenous NO donor, S-nitroso-N-acetyl penicillamine (SNAP, 10-5M, 10-4M), endogenous NO inhibitor, nitro-L-arginine methyl ester (L-NAME, 10-4M), and ACEI (enalapril, 10-4M). Results: (i) Caspase 8 and 9 induced caspase-3 activity in myocardial cell lysates (5.44+/-0.6 pmol/10 mcg/min). Exogenous NO donor, SNAP, caused a direct, dose-dependent decrease in induced caspase 3 activity downstream of caspases 8 and 9 (0.52+/-0.5 pmol/10mcg/min at highest dose, p<0.01) (ii) Caspase-3 activity was lower in the 5 pts on chronic ACEI therapy and increased significantly with addition of L-NAME, indicating that ACEIs may decrease caspase activity through increased NO production. This was confirmed by the ability of exogenously added enalapril to reduce myocardial caspase-3 activity (2.48+/-0.4 pmol/10mcg/min). (iii) Caspase activity was significantly higher in nonischemic (9.86+/-1) versus ischemic hearts (5.14+/-0.6) (p<0.05) including caspase-9 induced caspase-3 activity suggesting upregulation of the mitochondrial pathway of apoptosis. Conclusion: NO regulates apoptosis by inhibiting downstream caspase-3 activity in explanted failing human hearts and can potentially rescue a cell from apoptosis even after activation of the caspase cascade. This may have implications for use of NO agonists to inhibit apoptotis in heart failure.

Effects of sustained flow reduction on postnatal intestinal circulation.

Studies were conducted to determine the effect of mechanically induced sustained flow reduction on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine. In vitro gut loops were perfused under controlled-pressure conditions from an oxygenated blood reservoir at age-appropriate perfusion pressures; pressure was rapidly reduced to a level that lowered flow rate to approximately 50% of its baseline value, and pressure was then kept at that level for 2 h. In 3-day-old intestine, vascular resistance (Ri) increased by 20% immediately after pressure and flow reduction but then stabilized for 3-4 min; thereafter, flow began to decrease despite maintenance of perfusion pressure, so that Ri increased an additional 15% by 30 min after flow reduction. Flow continued to diminish over the next 90 min, though at much slower rate. Intestine from 35-day-old swine demonstrated an immediate increase in Ri after pressure and flow reduction, but thereafter Ri increased very little. The protocol was repeated within in vitro gut loops perfused under controlled-flow conditions, and within autoperfused, innervated gut loops developed in vivo and similar observations were made in both preparations. In 3-day-old intestine, pretreatment with the L-arginine analog Nomega-monomethyl-L-arginine (10(-4) M) had no effect on the immediate rise in resistance occurring in the first 1 min but substantially attenuated the subsequent slow, progressive rise noted thereafter. Pretreatment with the angiotensin 1A receptor antagonist losartan (2 x 10(-6) M) had no effect on hemodynamic changes during the first 60 min after mechanical perfusion pressure reduction but attenuated the very slight increase in resistance noted during the final 60 min of the protocol. The postnatal intestinal circulation demonstrates progressive vasoconstriction when its flow rate is mechanically reduced in a sustained manner, and this effect is age specific, occurring in 3- but not 35-day-old swine. These changes in gut vascular resistance may be consequent to loss of nitric oxide production and/or local production of angiotensin.

Expression of iNOS, eNOS, and peroxynitrite-modified proteins in experimental anti-myeloperoxidase associated crescentic glomerulonephritis

Nitric oxide radicals are recognized as important mediators in various physiological and pathophysiological processes. During inflammation, increased amounts of nitric oxide (NO) are produced, but it is unclear whether NO radicals are either protective or harmful. To obtain more insight into the role of NO in glomerular inflammation, we studied the temporal expression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) in conjunction with platelet aggregation, inflammatory cell influx, superoxide anion production cells, and nitrotyrosine formation in an experimental model of anti-myeloperoxidase (MPO) associated necrotizing crescentic glomerulonephritis (NCGN). Brown Norway rats were immunized with MPO in complete Freund's adjuvant (CFA) or CFA alone. After two weeks, the left kidney was perfused with a neutrophil lysosomal extract and H2O2. Rats were sacrificed at 24 hours, four days, and 10 days after perfusion. Kidney sections were stained by immunohistochemistry for eNOS, iNOS, platelets, nitrotyrosines, polymorphonuclear cells (PMN), monocytes, and T-cells. Superoxide anion producing cells were identified by enzyme cytochemistry using diaminobenzidine. Strong staining for eNOS was found in glomerular capillaries and interstitial tubular capillaries and larger vessels from non-perfused kidneys. At 24 hours after perfusion, glomerular and interstitial eNOS staining was greatly reduced, which was associated with massive platelet aggregation. At later time points, eNOS expression was absent in severely damaged glomeruli. Inducible NOS expression was found at all time points in infiltrating inflammatory cells, which by double labeling studies were identified as PMNs and monocytes. The peak in iNOS expression was observed at four days after perfusion but declined thereafter. Superoxide anion and nitrotyrosine generating cells were also found at all time points, but were most abundantly present at four days after perfusion, coinciding with the peak in iNOS expression. Double labeling experiments revealed that most nitrotyrosine generating cells also produced superoxide anions and expressed iNOS. In conclusion, these studies suggest that during the course of anti-MPO associated NCGN, loss of NO production by eNOS in conjunction with NO radical production by iNOS contribute to tissue injury. This is compatible with a protective role for eNOS contrasting with the possibly harmful effects of iNOS in anti-MPO associated NCGN.

953-34 Pulmonary Vascular Endothelium is Capable of Producing Nitric Oxide After Cardiopulmonary Bypass and Circulatory Arrest

Cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) can lead to clinically significant pulmonary dysfunction and increased pulmonary vascular resistance (PVR). Endothelial production of nitric oxide (NO), a vasodilator, is an important mediator of pulmonary vascular tone. Endothelial injury during CPB and DHCA with loss of NO production may result in elevated PVR postoperatively. To assess the importance of NO production before and after CPB and DHCA, twelve 4–6 week old piglets were instrumented with pulmonary artery (PA) and left atrial Millar micromanometers and a PA ultrasonic flow probe. NO synthase was blocked with a 5 mg/kg bolus of N w -Nitro-L-Arginine Methyl Ester (L-NAME) into the main PA either before (Control, n = 6) or after CPB and DHCA (DHCA, n = 6). The DHCA animals were placed on CPB, cooled to 18°C, arrested for 60 minutes, rewarmed to 37°C, and weaned from CPB. The L-NAME bolus was given 15 minutes after weaning from CPB in this group. PVR and input impedance Il in ) were measured immediately before and 10 minutes after L-NAME in both groups. The results are expressed as means ± SEM. Minutes PVR (dyne sec cm -5 ) Z in (dyne sec cm -5 ) Post-L-NAME Control Post-DHCA Control Post-DHCA 0 526 ± 53 1993 ± 399 † 1006 ± 52 2543 ± 436 † 10 1456 ± 136 * 3578 ± 668 * 2100 ± 69 * 4323 ± 687 * † p &lt; 0.006 vs Control by unpaired two-tailed t-test. * p &lt; 0.01 vs Time 0 by paired twotailed t-test 1) PVR and Z in are significantly increased by CPB and DHCA. 2) Blockade of NO synthase increases PVR and Z in to a similar degree preand post-DHCA, demonstrating that the pulmonary endothelium produces NO after CPB and DHCA. 3) Since the endothelium continues to produce NO following CPB and DHCA, other mechanisms, such as increased endothelial production of vasoconstrictors or interstitial edema, may be important mediators of post-CPB pulmonary dysfunction.