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Xiao Wang,1,* Ying Cao,2,* Jing Hu,3 Lian-Cheng Jia,4 Bo Li,3 Baoli Liu,5 Wei-Hai Yao,2 Xiao-Lu Pei,6 Wei Peng,2 Shuang Wang,2 Cun-Zhi Liu,1,7 Jian-Feng Tu,1,7 Zhi-Cheng Qu2 1School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 2Emergency Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 3Evidence Based Medicine Center, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 4Urinary Surgery, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 5Nephrology Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 6Nursing Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of China; 7International Acupuncture and Moxibustion Innovation Institute, Beijing University of Chinese Medicine, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhi-Cheng Qu, Emergency Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, 23 Meishuguanhou Street, Dongcheng District, Beijing, 100010, People’s Republic of China, Email qzhch0824@163.com Jian-Feng Tu, School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, No. 11, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100029, People’s Republic of China, Email tujianfeng1@126.comIntroduction: Analgesia is often delayed for suspected acute renal colic due to urinary calculi (ARCUC) even in emergency department. Acupuncture has a rapid onset and is effective for analgesia, however, evidence about early-acupuncture for suspected ARCUC is limited. This trial aims to evaluate the efficacy of early-intervention acupuncture compared with sham acupuncture on pain relief among emergency department suspected ARCUC patients.Methods and Analysis: A total of 84 eligible patients who are suspected diagnosed with ARCUC will be randomly allocated to the acupuncture group or the sham acupuncture group. Each patient will receive 1 session of acupuncture or sham acupuncture before diagnostic imaging. The primary outcome will be the response rate at 10 min after needle manipulation, defined as the proportion of patients whose Numeric Rating Scale (NRS) score decrease by at least 50% from baseline. Secondary outcomes will include pain intensity assessed by NRS, further analgesia requirement, revisit rate, surgical intervention rate, satisfaction evaluation, and adverse events. The final diagnosis rate determined by radiography will be recorded and reported. All patients who receive randomization will be included in the intention-to-treat analysis.Conclusion: This study’s findings are anticipated to evaluate the analgesic effect of early-intervention acupuncture for acute renal colic in emergency department, which could be useful for moving the timing of analgesia forward and aligning pain management for acute renal colic more with the guidelines.Trial Registration Number: ChiCTR2100049069 (https://www.chictr.org.cn/showproj.html?proj=125338).Keywords: acupuncture, complementary medicine, pain management, emergency medicine, renal colic

Xiao Wang,1 Ying Cao,2 Cun-Zhi Liu,1 Jian-Feng Tu,1 Zhi-Cheng Qu2 1School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 2Emergency Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Jian-Feng Tu, School of Acupuncture-Moxibustion and Tuina, Beijing University of Chinese Medicine, No. 11, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100029, People’s Republic of China, Email tujianfeng1@126.com Zhi-Cheng Qu, Emergency Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, 23 Meishuguanhou Street, Dongcheng District, Beijing, 100010, People’s Republic of China, Email qzhch0824@163.com

Background: The integration of acupuncture with intramuscular injection of diclofenac sodium can expedite the onset of analgesia in treating acute renal colic caused by urolithiasis. However, it remains unclear whether acupuncture can accelerate pain relief constantly until complete remission. This study aimed to explore the extent to which acupuncture can expedite the onset time of response or complete pain relief in treating acute renal colic, and the predictive value of patient characteristics for treatment efficacy., Methods: This secondary analysis utilized data from a prior randomized controlled trial. Eighty patients with acute renal colic were randomly assigned 1:1 to the acupuncture group or the sham acupuncture group. After intramuscular injection of diclofenac sodium, acupuncture or sham acupuncture was delivered to patients. The outcomes included time to response (at least a 50 % reduction in pain) and complete pain relief. Between-group comparison under the 2 events was estimated by Kaplan-Meier methodology. Subgroup analysis was performed utilizing the Cox proportional hazards model., Results: The median response time and complete pain relief time in the acupuncture group were lower than those in the sham acupuncture group (5 vs 30 min, Log Rank P < 0.001; 20 min vs not observed, Log Rank P < 0.001, respectively). Hazard Ratios (HRs) for response across all subgroups favored the acupuncture group. All HRs for complete pain relief favored acupuncture, expect large stone and moderate pain at baseline. No interaction was found in either event., Conclusion: Acupuncture can accelerate the response time and complete pain relief time for patients with acute renal colic, with the efficacy universally., Trial Registration: This study has been registered at Chinese Clinical Trial Registry: ChiCTR1900025202., (© 2024 Korea Institute of Oriental Medicine. Published by Elsevier B.V.)

Objective: This study aimed to assess the correlation between coronal imbalance and lower-limb physiological parameters in degenerative scoliosis using the biplanar whole body imaging system (EOS)., Materials and Methods: A total of 101 successive EOS images were selected between January 2018 and December 2021. Of the selected images, 63 patients were in the degenerative scoliosis group (DSG) and 38 patients were in the control group (CG). Two independent observers performed measurements of the parameters and compared the two groups., Results: Among parameters examined, significant inter-group differences were found for coronal pelvic tilt angle (CPT), bilateral femoral length difference (ΔFL), and bilateral total lower limb length (ΔTL) difference. Additionally, the knee and ankle joints had more severe degeneration on the main curved side in patients with degenerative scoliosis. In the left curved group, 18 (42.86%) and 24 (57.1%) patients had more severe degeneration in the left knee and left ankle, respectively. In the right lateral bending group, 13 (61.9%) and 14 (66.7%) patients had more severe degeneration in the right knee and right ankle, respectively. Statistical differences were found in the degree of degeneration in both knee and ankle joints bilaterally., Conclusion: This study showed that biomechanical parameters of the lower limbs are affected in cases of degenerative scoliosis with altered coronal balance. The lower limb on the main curve side became shorter compared to its counterpart, and joint degeneration of the knee and ankle joints became more severe., Competing Interests: None., (AJTR Copyright © 2022.)

Importance: Renal colic is described as one of the worst types of pain, and effective analgesia in the shortest possible time is of paramount importance., Objectives: To examine whether acupuncture, as an adjunctive therapy to analgesics, could accelerate pain relief in patients with acute renal colic., Design, Setting, and Participants: This single-center, sham-controlled, randomized clinical trial was conducted in an emergency department in China between March 2020 and September 2020. Participants with acute renal colic (visual analog scale [VAS] score ≥4) due to urolithiasis were recruited. Data were analyzed from October 2020 to January 2022., Interventions: After diagnosis and randomization, all patients received 50 mg/2 mL of diclofenac sodium intramuscular injection immediately followed by 30-minute acupuncture or sham acupuncture., Main Outcomes and Measures: The primary outcome was the response rate at 10 minutes after needle manipulation, which was defined as the proportion of participants whose VAS score decreased by at least 50% from baseline. Secondary outcomes included response rates at 0, 5, 15, 20, 30, 45, and 60 minutes, rescue analgesia, and adverse events., Results: A total of 115 participants were screened and 80 participants (66 men [82.5%]; mean [SD] age, 45.8 [13.8] years) were enrolled, consisting of 40 per group. The response rates at 10 minutes were 77.5% (31 of 40) and 10.0% (4 of 40) in the acupuncture and sham acupuncture groups, respectively. The between-group differences were 67.5% (95% CI, 51.5% to 83.4%; P < .001). The response rates of acupuncture were also significantly higher than sham acupuncture at 0, 5, 15, 20 and 30 minutes, whereas no significant difference was detected at 45 and 60 minutes. However, there was no difference between the 2 groups in rescue analgesia rate (difference 2.5%; 95% CI -8.8% to 13.2%; P > .99). No adverse events occurred during the trial., Conclusions and Relevance: These findings suggest that acupuncture plus intramuscular injection of diclofenac is safe and provides fast and substantial pain relief for patients with renal colic compared with sham acupuncture in the emergency setting. However, no difference in rescue analgesia was found, possibly because of the ceiling effect caused by subsequent but robust analgesia of diclofenac. Acupuncture can be considered an optional adjunctive therapy in relieving acute renal colic., Trial Registration: Chinese Clinical Trial Registry: ChiCTR1900025202.

Background: Acute renal colic caused by urinary calculi (ARCUC) has a considerable impact on the quality of life. Acupuncture might be a potential treatment option. However, the evidence is limited. We will conduct this trial to evaluate the efficacy and safety of acupuncture as adjunctive treatment to diclofenac for ARCUC., Methods/design: A total of 80 eligible patients who are diagnosed with urinary stone renal colic will be randomly allocated to the acupuncture group or the sham acupuncture group. Each patient will receive 1 session of acupuncture or sham acupuncture. The primary outcome will be the response rate of patients achieving a reduction of > 50% on visual analog score (VAS) from baseline to 10 min after treatment. Secondary outcomes will include the VAS, remedial analgesia, re-visit and admission rate, blinding assessment, credibility and expectancy, and adverse event. All patients who receive randomization will be included in the intent-to-treat analysis., Discussion: The finding of this trial will provide evidence on the efficacy and safety of acupuncture for the treatment of ARCUC. The results of this study will be published in peer-reviewed journals., Trial Registration: ClinicalTrials.gov ChiCTR 1900025202 . Registered on August 16, 2019., (© 2021. The Author(s).)

Objective: MiR-551b has been reported to display tumor-suppressive and oncogenic potential in several cancers, but there has been no study on the roles of miR-551b in colorectal cancer (CRC). In this work, we aimed to explore the potential functions and mechanisms of miR-551b in the modulation of the CRC progression., Patients and Methods: The expressions of miR-551b were examined in 122 pairs of CRC cancer tissues and adjacent non-tumor samples by Real Time-Polymerase Chain Reaction (RT-PCR). The clinical significance of miR-551b in CRC patients was explored using a Chi-square test, Kaplan-Meier assays, and multivariate analysis. MiR-551b mimics and inhibitors were used to establish miR-551b upregulation and downregulation models in CRC cells to examine the functions of miR-551b on cells proliferation, migration, invasion, and apoptosis. Dual-luciferase reporter assays were conducted for the validation of the possible modulation of a putative target of miR-551b., Results: We showed that miR-551b was significantly down-regulated in CRC tissues and cell lines. It was observed that miR-551b expressions were correlated with lymph nodes metastasis, TNM stage, and poor prognosis. Multivariate analysis identifies low level of miR-551b expressions as an independent predictor for a shorter overall survival. The functional assessment suggested that forced miR-551b expression distinctly suppressed CRC cells growth, invasion, and migration, while the suppression of miR-551b displayed the opposite trend. Mechanistic studies confirmed that PTP4A3 was identified as a direct target of miR-551b in CRC. Interesting observations revealed that the cells capacities were higher in miR-551b +PTP4A3 group, when compared with those in miR-551b group, indicating that up-regulation of PTP4A3 rescued the repressive functions of miR-551b overexpression on CRC cells growth and migration., Conclusions: The findings of our study first showed that miR-551b, a potential tumor suppressor, may be used as a promising prognostic predictor and therapeutic target for CRC.

Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species., (Copyright © 2016 the American Physiological Society.)

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 µM and complete inhibition at 50 µM. Loading cells with 100 µM ascorbate also decreased the basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25%, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 µM L-NAME (but not D-NAME) as well as by 30 µM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema.

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8-1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 H. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25-50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism., (© 2015 International Union of Biochemistry and Molecular Biology.)

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 min and an apparent transport Km of 21 μM, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion., (Copyright © 2015 Elsevier Inc. All rights reserved.)

High glucose concentrations due to diabetes increase apoptosis of vascular pericytes, impairing vascular regulation and weakening vessels, especially in brain and retina. We sought to determine whether vitamin C, or ascorbic acid, could prevent such high glucose-induced increases in pericyte apoptosis. Culture of human microvascular brain pericytes at 25 mM compared to 5mM glucose increased apoptosis measured as the appearance of cleaved caspase 3. Loading the cells with ascorbate during culture decreased apoptosis, both at 5 and 25 mM glucose. High glucose-induced apoptosis was due largely to activation of the receptor for advanced glycation end products (RAGE), since it was prevented by specific RAGE inhibition. Culture of pericytes for 24h with RAGE agonists also increased apoptosis, which was completely prevented by inclusion of 100 μM ascorbate. Ascorbate also prevented RAGE agonist-induced apoptosis measured as annexin V binding in human retinal pericytes, a cell type with relevance to diabetic retinopathy. RAGE agonists decreased intracellular ascorbate and GSH in brain pericytes. Despite this evidence of increased oxidative stress, ascorbate prevention of RAGE-induced apoptosis was not mimicked by several antioxidants. These results show that ascorbate prevents pericyte apoptosis due RAGE activation. Although RAGE activation decreases intracellular ascorbate and GSH, the prevention of apoptosis by ascorbate may involve effects beyond its function as an antioxidant., (Copyright © 2014 Elsevier Inc. All rights reserved.)

High glucose concentrations due to diabetes increase leakage of plasma constituents across the endothelial permeability barrier. We sought to determine whether vitamin C, or ascorbic acid (ascorbate), could reverse such high glucose-induced increases in endothelial barrier permeability. Human umbilical vein endothelial cells and two brain endothelial cell lines cultured at 25 mM glucose showed increases in endothelial barrier permeability to radiolabeled inulin compared to cells cultured at 5mM glucose. Acute loading of the cells for 30-60 min with ascorbate before the permeability assay prevented the high glucose-induced increase in permeability and decreased basal permeability at 5mM glucose. High glucose-induced barrier leakage was mediated largely by activation of the receptor for advanced glycation end products (RAGE), since it was prevented by RAGE blockade and mimicked by RAGE ligands. Intracellular ascorbate completely prevented RAGE ligand-induced increases in barrier permeability. The high glucose-induced increase in endothelial barrier permeability was also acutely decreased by several cell-penetrant antioxidants, suggesting that at least part of the ascorbate effect could be due to its ability to act as an antioxidant., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ascorbic acid enhances synthesis of norepinephrine from dopamine in adrenal chromaffin cells by serving as a co-factor for chromaffin granule dopamine β-hydroxylase (DβH). However, there is controversy regarding in situ kinetics of the ascorbate effect in chromaffin cells, as well as whether they apply to neuronal cells. In this study we evaluated the stimulation of norepinephrine synthesis from dopamine in cultured SH-SY5Y neuroblastoma cells. These cells contained neither ascorbate nor norepinephrine in culture, but when provided with dopamine, they generated intracellular norepinephrine at rates that were stimulated several-fold by intracellular ascorbate. Ascorbate-induced increases in norepinephrine synthesis in dopamine-treated cells were linear over 60 min, despite saturation of intracellular ascorbate. Norepinephrine accumulation after 60 min of incubation with 100 μM dopamine was half-maximal at intracellular ascorbate concentrations of 0.2-0.5 mM, which fits well with the literature K(m) for ascorbate of DβH using dopamine as a substrate. Moreover, these ascorbate concentrations were generated by initial extracellular ascorbate concentrations of less than 25 μM due to concentrative accumulation by the ascorbate transporter. Treatment with 100 μM dopamine acutely increased cellular superoxide generation, which was prevented by ascorbate loading, but associated with a decrease in intracellular ascorbate when the latter was present at concentrations under 1 mM. These results show that ascorbate promptly enhances norepinephrine synthesis from dopamine by neuronal cells that it does so at physiologic intracellular concentrations in accord with the kinetics of DβH, and that it both protects cells from superoxide and by providing electrons to DβH., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Ascorbic acid is well known to acutely stimulate norepinephrine synthesis in neurosecretory cells, but it has also been shown over several days to increase tyrosine hydroxylase mRNA and norepinephrine synthesis in cultured neurons. Since tyrosine hydroxylase is the rate-limiting step in catecholamine synthesis, an effect of ascorbate to increase tyrosine hydroxylase protein could contribute to its ability to increase or sustain catecholamine synthesis. Therefore, we evaluated whether tyrosine hydroxylase protein expression and function is increased in SH-SY5Y neuroblastoma cells by physiologically relevant intracellular ascorbate concentrations. SH-SY5Y neuroblastoma cells did not contain ascorbate and had only very low levels of norepinephrine in culture with L-tyrosine, the substrate for tyrosine hydroxylase. However, treatment of cells with ascorbate for 6 h or more markedly increased norepinephrine synthesis, such that intracellular ascorbate and norepinephrine increased in parallel with half maximal intracellular concentrations of about 1 mM ascorbate and 150 μM norepinephrine. This increase was enhanced by supplementing tetrahydrobiopterin, but was not mimicked by several antioxidants or by catalase or superoxide dismutase. Tyrosine hydroxylase protein expression increased at intracellular ascorbate concentrations above 1.5 mM. This contributed to norepinephrine generation, which was decreased 50-60% by inhibition of protein synthesis with cycloheximide at high intracellular ascorbate. These results suggest that expected physiologic neuronal ascorbate concentrations enhance norepinephrine synthesis both by maintaining tetrahydrobiopterin and increasing tyrosine hydroxylase expression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with N(ω)-nitro-l-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Oxidative stress acutely increases the permeability of the vascular endothelium to large molecules that would not otherwise cross the barrier. Ascorbic acid is an antioxidant that tightens the endothelial permeability barrier, so we tested whether it might also prevent the increase in endothelial permeability due to cellular oxidative stress. Treatment of EA.hy926 endothelial cells cultured on filter inserts with H(2) O(2) , menadione, and buthionine sulfoximine increased endothelial permeability to radiolabeled inulin. Short-term ascorbate loading of the cells to what are likely physiologic concentrations of the vitamin by treating them with dehydroascorbate prevented the increase in endothelial permeability due to these agents. The nonphysiologic antioxidants dithiothreitol and tempol also prevented increases in endothelial barrier permeability induced by the agents. These results suggest that oxidative stress induced directly by oxidants or indirectly by glutathione depletion impairs endothelial barrier function and that intracellular ascorbate may serve to prevent this effect., (Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.)

Abstract Mildly oxidized low density lipoprotein (mLDL) acutely increases the permeability of the vascular endothelium to molecules that would not otherwise cross the barrier. This study has shown that ascorbic acid tightens the permeability barrier in the endothelial barrier in cells, so this work tested whether it might prevent the increase in endothelial permeability due to mLDL. Treatment of EA.hy926 endothelial cells with mLDL decreased intracellular GSH and activated the cells to further oxidize the mLDL. mLDL also increased endothelial permeability over 2 h to both inulin and ascorbate in cells cultured on semi-permeable filters. This effect was blocked by microtubule and microfilament inhibitors, but not by chelation of intracellular calcium. Intracellular ascorbate both prevented and reversed the mLDL-induced increase in endothelial permeability, an effect mimicked by other cell-penetrant antioxidants. These results suggest a role for endothelial cell ascorbate in ameliorating an important facet of endothelial dysfunction caused by mLDL.

Endothelial dysfunction is an early manifestation of atherosclerosis caused in part by oxidized LDL (oxLDL). Since vitamin C, or ascorbic acid, prevents several aspects of endothelial dysfunction, the effects of oxLDL on oxidative stress and regulation of the ascorbate transporter, SVCT2, were studied in cultured EA.hy926 endothelial cells. Cells cultured for 18 h with 0.2 mg/ml oxLDL showed increased lipid peroxidation that was prevented by a single addition of 0.25 mM ascorbate at the beginning of the incubation. This protection caused a decrease in intracellular ascorbate, but no change in the cell content of GSH. In the absence of ascorbate, oxLDL increased SVCT2 protein and function during 18 h in culture. Although culture of the cells with ascorbate did not affect SVCT2 protein expression, the oxLDL-induced increase in SVCT2 protein expression was prevented by ascorbate. These results suggest that up-regulation of endothelial cell SVCT2 expression and function may help to maintain intracellular ascorbate during oxLDL-induced oxidative stress, and that ascorbate in turn can prevent this effect.

Ascorbic acid improves endothelial barrier function by decreasing the permeability of endothelial cells cultured on semi-porous membrane filters. This decrease was not due to enhanced collagen synthesis and was mimicked by the collagen synthesis inhibitor ethyl-3,4-dihydroxybenzoic acid (EDHB). Since EDHB is known to chelate intracellular free iron, the effects of two membrane-permeant iron chelators were tested on endothelial permeability. Both 2,2'-dipyridyl and desferrioxamine decreased trans-endothelial permeability in a concentration-dependent manner. Increasing intracellular iron with a chelate of 8-hydroxyquinoline and ferric iron prevented effects of both EDHB and intracellular ascorbate. That EDHB and ascorbate did in fact chelate intracellular iron was supported by finding that they both decreased the cellular fluorescence quenching of the iron-sensitive dye Phen green SK. These results show that chelation of intracellular iron decreases endothelial barrier permeability and implicate this mechanism in the ability of EDHB and possibly intracellular ascorbate to tighten the endothelial barrier., (2010 Elsevier Inc. All rights reserved.)

To determine how ascorbic acid moves from the bloodstream into tissues, we assessed transfer of the vitamin across the barrier generated by EA.hy926 endothelial cells when these were cultured on semipermeable filter supports. Ascorbate transfer from the luminal to the abluminal compartment was time dependent, inhibited by anion channel blockers and by activation of protein kinase A, but was increased by thrombin. Ascorbate transfer occurred by a paracellular route, since it did not correlate with intracellular ascorbate contents and was not rectified or saturable. Nonetheless, intracellular ascorbate inhibited the transfer of both ascorbate and radiolabeled inulin across the endothelial barrier. The increase in barrier function due to ascorbate was dependent on its intracellular concentration, significant by 15 min of incubation, prevented by the cytoskeletal inhibitor colchicine, associated with F-actin stress fiber formation, and not due to collagen deposition. These results show that ascorbate traverses the endothelial barrier by a paracellular route that is regulated by cell metabolism, ion channels, and ascorbate itself. Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo.

Endothelial cells respond to hypoxia by decreased degradation of hypoxia-inducible factor 1alpha (HIF-1alpha), accumulation of which leads to increased transcription of numerous proteins involved in cell growth and survival. Ascorbic acid prevents HIF-1alpha stabilization in many cell types, but the physiologic relevance of such effects is uncertain. Given their relevance for angiogenesis, endothelial cells in culture were used to evaluate the effects of ascorbate on HIF-1alpha expression induced by hypoxia and the hypoxia mimic cobalt. Although EA.hy926 cells in culture under oxygenated conditions did not contain ascorbate, HIF-1alpha expression was very low, showing that the vitamin is not necessary to suppress HIF-1alpha. On the other hand, hypoxia- or cobalt-induced HIF-1alpha expression/stabilization was almost completely suppressed by what are likely physiologic intracellular ascorbate concentrations. Increased HIF-1alpha expression was not associated with significant changes in expression of the SVCT2, the major transporter for ascorbate in these cells. Cobalt at concentrations sufficient to stabilize HIF-1alpha both oxidized intracellular ascorbate and induced an oxidant stress in the cells that was prevented by ascorbate. Whereas the interaction of ascorbate and cobalt is complex, the presence of physiologic low millimolar concentrations of ascorbate in endothelial cells effectively decreases HIF-1alpha expression and protects against cobalt-induced oxidant stress., ((c) 2009 International Union of Biochemistry and Molecular Biology, Inc.)

Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

Objectives: The reducing capacity of erythrocytes has been used clinically as to estimate resistance to oxidant stress. In this work we targeted the antioxidant capacity of pyridine nucleotide disulfide reductases of these cells by measuring their ability to reduce the disulfide alpha-lipoic acid., Methods: Erythrocyte reduction of alpha-lipoic acid and related disulfides was measured as reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) outside the cells., Results: Lipoic acid-dependent DTNB reduction by human erythrocytes required d-glucose and consumed NADPH, but not NADH. This activity was inhibited by carmustine and phenylarsine oxide, as expected if alpha-lipoic acid is reduced by the glutathione and thioredoxin reductase systems. Reduction of hydroxyethyl disulfide, which provides an estimate of total erythrocyte disulfide reduction capacity, was similar to that of alpha-lipoic acid. Erythrocytes incubated with alpha-lipoic acid also reduced extracellular ferricyanide, although rates of dehydroascorbate reduction were several-fold greater, probably because intracellular GSH can recycle ascorbate but not alpha-lipoic acid in erythrocytes., Conclusion: These results show that alpha-lipoic acid-dependent DTNB reduction provides a simple method to selectively assess the capacity of pyridine nucleotide disulfide reductases of human erythrocytes. When coupled with other non-destructive assays, such as reduction of hydroxyethyl disulfide and ferricyanide, this assay provides a comprehensive approach to assessing erythrocyte reducing capacity in a variety of clinical conditions associated with oxidant stress.

Erythrocytes have the same intracellular concentration of ascorbate as plasma, which is much lower than that of nucleated cells. To determine why erythrocytes are unable to concentrate ascorbate, we tested for the presence of ascorbate transporters in these cells. Human erythrocytes had very low rates of uptake of radiolabeled ascorbate, which was accounted for by the lack of ascorbate transporter SVCT2 in immunoblots. Using a cell culture model of Friend virus-infected mouse erythroblasts, immunoblots showed that the SVCT2 was present in the erythroblast stages, but was lost following extrusion of the nucleus in the formation of the reticulocyte stage. Rates of specific ascorbate transport correlated with the presence of the SVCT2. These results show that mature erythrocytes fail to concentrate ascorbate due to the loss of SVCT2 during maturation in the bone marrow.

Mitochondria are the major source of potentially damaging reactive oxygen species in most cells. Since ascorbic acid, or vitamin C, can protect against cellular oxidant stress, we studied the ability of mitochondria prepared from guinea pig skeletal muscle to recycle the vitamin from its oxidized forms. Although ascorbate concentrations in freshly prepared mitochondria were only about 0.2 mM, when provided with 6 mM succinate and 1 mM dehydroascorbate (the two-electron-oxidized form of the vitamin), mitochondria were able to generate and maintain concentrations as high as 4 mM, while releasing most of the ascorbate into the incubation medium. Mitochondrial reduction of dehydroascorbate was strongly inhibited by 1,3-bis(chloroethyl)-1-nitrosourea and by phenylarsine oxide. Despite existing evidence that mitochondrial ascorbate protects the organelle from oxidant damage, ascorbate failed to preserve mitochondrial alpha-tocopherol during prolonged incubation in oxygenated buffer. Nonetheless, the capacity for mitochondria to recycle ascorbate from its oxidized forms, measured as ascorbate-dependent ferricyanide reduction, was several-fold greater than total steady-state ascorbate concentrations. This, and the finding that more than half of the ascorbate recycled from dehydroascorbate escaped the mitochondrion, suggests that mitochondrial recycling of ascorbate might be an important mechanism for regenerating intracellular ascorbate.

To assess the disulfide reduction capacity of intact cells, EA.hy926 endothelial cells were incubated with alpha-lipoic acid in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Alpha-lipoic acid was reduced within cells to dihydrolipoic acid, which could be quantified upon efflux from the cells as reduction of DTNB. Uptake of both alpha-lipoic acid and alpha-lipoamide occurred at least in part via a medium chain fatty acid transporter, based on inhibition by octanoate. Alpha-lipoic acid was reduced within cells by pyridine nucleotide-disulfide oxidoreductases, since it is not reduced by GSH and since its reduction was inhibited by carmustine. Nonetheless, reduction was also dependent on the cellular redox environment, since it was inhibited by the redox cycling of menadione, by decreasing intracellular GSH, and by reduction of dehydroascorbate. Together, these results show that alpha-lipoic acid-dependent DTNB reduction provides a simple method to assess the disulfide-reducing capacity of intact cells, especially as determined by pyridine nucleotide-disulfide oxidoreductases.

Neurons maintain relatively high intracellular concentrations of vitamin C, or ascorbic acid. In this work we studied the mechanisms by which neuronal cells in culture transport and maintain ascorbate, as well as how this system responds to oxidant stress induced by glutamate. Cultured SH-SY5Y neuroblastoma cells took up ascorbate, achieving steady-state intracellular concentrations of 6 mM and higher at extracellular concentrations of 200 microM and greater. This gradient was generated by relatively high affinity sodium-dependent ascorbate transport (Km of 113 microM). Ascorbate was also recycled from dehydroascorbate, the reduction of which was dependent on GSH, but not on D-glucose. Glutamate in concentrations up to 2 mM caused an acute concentration-dependent efflux of ascorbate from the cells, which was prevented by the anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Intracellular ascorbate did not affect radiolabeled glutamate uptake, showing absence of heteroexchange.

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.

Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 microM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 microM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.

Endothelial cells preserve vascular integrity in part by synthesizing type IV collagen for the basement membrane of blood vessels. Vitamin C, which at physiologic pH is largely the ascorbate mono-anion, both protects these cells from oxidant stress and is required for collagen synthesis. Therefore, cultured endothelial cells were used to correlate intracellular concentrations of ascorbate with its uptake and ability to stimulate collagen release into the culture medium. The kinetics and inhibitor specificity of ascorbate transport into EA.hy926 endothelial cells were similar to those observed in other cell types, indicative of a specific high affinity transport process. Further, transport of the vitamin generated intracellular ascorbate concentrations that were 80-100-fold higher than concentrations in the medium following overnight culture, and transport inhibition with sulfinpyrazone and phloretin partially prevented such ascorbate accumulation. On the other hand, low millimolar intracellular concentrations of ascorbate impaired its transport measured after overnight culture. Synthesis and release of type IV collagen into the culture medium was markedly stimulated by ascorbate in a time-dependent manner, and was saturable with increasing medium concentrations of the vitamin. Optimal rates of collagen synthesis required intracellular concentrations of the vitamin up to 2 mM. Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium.

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB(+)) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB(+). Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB(+) <5 microM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB(+)-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB(+)-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB(+), which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB(+), that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB(+) reduction may correspond to MB(+)-dependent NAD(P)H reductase activity in erythrocyte ghosts.

Nitrite is a breakdown product of nitric oxide that in turn is oxidized to nitrate in cells. In this work, we investigated whether reactive oxidant species mightbe generated during nitrite metabolism in cultured EA.hy926 endothelial cells. Nitrite was taken up by the cells in a time- and concentration-dependent manner and oxidized to nitrate, which accumulated in cells to concentrations almost 10-fold those of nitrite. Conversion of low millimolar concentrations of nitrite to nitrate was associated with increased oxidant stress in the cells. This manifested as increased oxidation of dihydrofluorescein in tandem with depletion of both GSH and ascorbate. Further, loading cells with ascorbate or treatment with desferrioxamine prevented nitrite-induced dihydrofluorescein oxidation. Nitrite within cells also increased the fluorescence of 4-amino-5-methylamino-2',7'-difluorofluorescein and inhibited the activity of cellular glyceraldehyde 3-phosphate dehydrogenase, which are markers of intracellular nitrosation reactions. Intracellular ascorbate partially prevented both of these effects of nitrite. Although ascorbate can reduce nitrite to nitric oxide at low pH, in endothelial cells loaded with ascorbate, its predominant effect at high nitrite concentrations is to prevent potentially damaging nitrosation reactions.

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.

The thiazine dye methylene blue has long been used to stimulate cellular redox metabolism. To determine the extent to which it also generates oxidant stress in cells, its effects in cultured human-derived endothelial cells were studied. As expected, low concentrations of the dye (2-20 microM) activated the pentose phosphate pathway and oxidized both NADPH and NADH. Methylene blue enhanced extracellular ferricyanide reduction, indicating that the reduced form of the dye was present outside the cells. This reduction was greater when ferricyanide was added just before rather than 15 min after methylene blue, confirming that the dye is at least initially reduced at the cell surface. In the absence of glucose, methylene blue at concentrations above 5 microM increased intracellular oxidant stress, as manifest by oxidation of dihydrofluorescein and cellular GSH. Inclusion of glucose protected against these effects. In cells that had been loaded with ascorbate, the dye caused progressive oxidation of ascorbate, even in the presence of D-glucose. Loading cells with ascorbate also partially prevented oxidation of dihydrofluorescein by methylene blue. These results suggest that concentrations of the dye above 5 microM generated intracellular reactive oxygen species that were scavenged by ascorbate and GSH. Further, although D-glucose enhanced reduction of methylene blue, it ameliorated the oxidant stress generated by the dye.

Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 microM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3-1.6 mM generated intracellular DHA concentrations of 20-30 microM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms.

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.

A viral disease with dwarfing symptoms is associated with severe damage of different cereal crops including rice, maize, wheat and sorghum grown in China. It is believed that the pathogenic agent of the disease on rice and sorghum is rice black streaked dwarf virus (RBSDV), however, the cause of maize dwarf disease in China is still inconclusive. In this report, dsRNA was isolated from virus particles obtained from the diseased plants of rice, maize, wheat and sorghum from two Chinese provinces. Full-length cDNAs of genome segments 9 (S9) and 10 (S 10) were obtained through a RT-PCR approach. Sequence analysis showed that the S9 sequences of Chinese isolates and Japanese RBSDV isolate were very similar to each other (89.1-89.6% identity at the nucleotide level, 92.3-92.9% and 95.8-98.6% identity at the amino acid level for ORF1 and ORF2, respectively). In addition, the S10 sequences of Chinese isolates and Japanese RBSDV were very similar to each other (93.0-95.4% identical nucleotides and 96.2-97.0% identical amino acids, respectively). However, there were lower similarities for S9 and S10 sequences between Chinese isolates and an Italian Maize Rough Dwarf Virus (MRDV) isolate. Phylogenetic analysis indicates that Chinese viral isolates found to infect rice, maize, wheat and sorghum and leading to similar cereal dwarfing manifestations could be grouped to the same virus species, RBSDV.

A salt-induced fructose-1,6-diphosphate (FDP) aldolase cDNA (DsALDP) in Dunaliella salina was cloned by suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis of DsALDP revealed that the 1520 bp cDNA had an open reading frame (ORF) of 327 amino acid residues. BLAST Search showed that DsALDP shared an amino acid identity (73-66%) with AldP in other plants. Alignment with homologues in other plants indicated that all the conserved substrate-specific binding sites could also be found in DsALDP. Phylogenetic analysis further confirmed the deduced amino acid sequence of the D. salina DsALDP gene belonged to the same subfamily to AldP of other green algae. Southern blot analysis suggested possible presence of the D. salina DsALDP gene as a few copies and Northern blot analysis confirmed salt-induced expression pattern at the transcriptional level. A 62 kDa fusion protein generated by adding a Trx-His.tag at the N-terminal of DsALDP was induced by IPTG in Escherichia coli BL21. An improvement of salt tolerance in E. coli expressing DsALDP fusion protein was observed.

Alpha-lipoic acid, which becomes a powerful antioxidant in its reduced form, has been suggested as a dietary supplement to treat diseases associated with excessive oxidant stress. Because the vascular endothelium is dysfunctional in many of these conditions, we studied the uptake, reduction, and antioxidant effects of alpha-lipoic acid in cultured human endothelial cells (EA.hy926). Using a new assay for dihydrolipoic acid, we found that EA.hy926 cells rapidly take up and reduce alpha-lipoic acid to dihydrolipoic acid, most of which is released into the incubation medium. Nonetheless, the cells maintain dihydrolipoic acid following overnight culture, probably by recycling it from alpha-lipoic acid. Acute reduction of alpha-lipoic acid activates the pentose phosphate cycle and consumes nicotinamide adenine dinucleotide phosphate (NADPH). Lysates of EA.hy926 cells reduce alpha-lipoic acid using both NADPH and nicotinamide adenine dinucleotide (NADH) as electron donors, although NADPH-dependent reduction is about twice that due to NADH. NADPH-dependent alpha-lipoic acid reduction is mostly due to thioredoxin reductase. Pre-incubation of cells with alpha-lipoic acid increases their capacity to reduce extracellular ferricyanide, to recycle intracellular dehydroascorbic acid to ascorbate, to decrease reactive oxygen species generated by redox cycling of menadione, and to generate nitric oxide. These results show that alpha-lipoic acid enhances both the antioxidant defenses and the function of endothelial cells.

Several peptides that could bind specifically to the outer coat protein encoded by the S10 gene of Rice black streaked virus (RBSDV) were isolated from a phage-display random 12-mer peptide library. The sequence analysis showed that the amino acid motif (K)K**(*)P, the asterisk denoting any amino acid, might be the core sequence by which the peptides bind to the target protein. The peptide 1 that had a high affinity to RBSDV outer coat protein was synthesized by a chemical method and its fusion protein with glutathione-S-transferase (GST) was produced in an Escherichia coli expression system. The dot and Western blot analyses indicated that RBSDV could be detected with a high sensitivity in crude extracts of diseased plant leaves using a purified GST fusion protein. The circular dichroism (CD) spectroscopy revealed that the synthesized binding peptide but not a nonbinding peptide could bring about a marked change in the conformation of outer coat RBSDV protein. Since the protein functions only when it has correct conformation, the peptides binding specifically to it could possibly disturb the function of the virus outer coat protein and might be used to block the transmission pathway of the virus. Summing up, as these peptides showed a high specificity and sensitivity and diagnostic potential for RBSDV, they may represent the basis of a novel strategy for development of resistance to RBSDV.

Phages with high affinity to the P20 protein of rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of phage display screening. Nine different peptides from the enriched library were selected by enzyme-linked immunosorbent assay (ELISA). The P20 protein from raw extracts of rice leaves infected with RSV could be detected by those 9 peptides displayed on the phage, which suggested that a peptide could be an effective tool for diagnosis of RSV in rice and planthopper. Circular dichroism (CD) spectra of P20 fusion proteins with the binding phages and non-binding phages showed that the conformation of P20 protein was changed after binding to each of the 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of the P20 protein. Thereafter, those peptides might be used to develop plant resistance and disrupt virus transmission. Three of the 12-mer peptide genes were fused with the glutathione-S-transferase (GST) gene in the vector pGEX 3X. The fusion proteins were obtained from an Escherichia coli expression system and purified. The fusion proteins might have a potential to develop a plant peptide-based resistance to its pathogens and virus diagnosis. It also provided a tool (i) to confirm the inhibition of the function of P20 protein by the fusion peptides in vivo, and (ii) to detect the function of P20 protein and the interaction between the virus and its vector.

Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA.

This report describes isolation of virus particles from plants of rice, maize, wheat and sorghum with symptoms of dwarfing collected from two provinces of China, purification of double-stranded RNA (dsRNA) from the virus particles, and synthesis of full-length cDNAs of genome segments 9 (S9) and 10 (S10) by reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis showed that the S9 sequences of the Chinese isolates and a Japanese rice black-streaked disease virus (RBSDV) isolate were very similar (89.1-89.6% homology at nucleotide level and 92.3-92.9% and 95.8-98.6% homology at amino acid level for ORF1 and ORF2, respectively). Analogical similarity was found also for the S10 sequences of the isolates under comparison: 93.0-95.4% homology at nucleotide level and 96.2-97.0% homology at amino acid level. However, there was a relatively lower similarity for S9 and S10 segments ofthe Chinese isolates and an Italian maize rough dwarf virus (MRDV) isolate. The phylogenetic analysis indicated that the Chinese isolates that infect rice, maize, wheat and sorghum and cause similar symptoms could represent the same virus species, RBSDV.

Nitric oxide, when released into the bloodstream, is quickly scavenged by Hb in erythrocytes or oxidized to nitrite. Nitrite can also enter erythrocytes and oxidize Hb. The goals of this work were to determine the mechanism of erythrocyte nitrite uptake and whether this uptake causes oxidant stress in these cells. Erythrocytes took up 0.8 mM nitrite with a half-time of 11 min. Nitrite uptake was sensitive to temperature and to the pH and ionic composition of the medium but was not inhibited by the specific anion-exchange inhibitor DIDS. About 25% of nitrite uptake occurred on the sodium-dependent phosphate transporter and the rest as diffusion of nitrous acid or other species across the plasma membrane. Methemoglobin formation increased in proportion to the intracellular nitrite concentration. Nitrite reacted with erythrocyte ascorbate, but ascorbate loading of cells decreased nitrite-induced methemoglobin formation only at high nitrite concentrations. In conclusion, nitrite rapidly enters erythrocytes and reacts with oxyhemoglobin but does not exert a strong oxidant stress on these cells.

The antidiabetic drug troglitazone contains the active chromanol ring of alpha-tocopherol, which should give it antioxidant properties within cells. In these studies, the antioxidant effects of troglitazone were tested in human erythrocytes and in their ghosts. Troglitazone bound to erythrocyte ghosts in a linear manner and was retained even after centrifugation washes. In response to an oxidant stress generated by a water-soluble free radical initiator, troglitazone that was bound to erythrocyte ghosts was oxidized, but induced a lag-phase in the disappearance of endogenous alpha-tocopherol and in the appearance of lipid hydroperoxides. Troglitazone also delayed loss of endogenous alpha-tocopherol and hemolysis in washed intact erythrocytes in response to free radical-induced extracellular oxidant stress. To mimic exposure of erythrocytes to lipid hydroperoxides in vivo, erythrocytes were incubated with phospholipid liposomes that contained small amounts of preformed lipid hydroperoxides. This induced an oxidant stress in both the liposomes and cells. Troglitazone in concentrations above 4 microM almost completely prevented further appearance of lipid hydroperoxides in the liposomes, and also completely preserved alpha-tocopherol in the erythrocytes. The present results suggest that troglitazone will help to prevent peroxidative damage to erythrocytes in areas of excessive oxidant stress in the vascular bed.

We investigated the possibility that human erythrocytes can reduce extracellular ascorbate free radical (AFR). When the AFR was generated from ascorbate by ascorbate oxidase, intact cells slowed the loss of extracellular ascorbate, an effect that could not be explained by changes in enzyme activity or by release of ascorbate from the cells. If cells preserve extracellular ascorbate by regenerating it from the AFR, then they should decrease the steady-state concentration of the AFR. This was confirmed directly by electron paramagnetic resonance spectroscopy, in which the steady-state extracellular AFR signal varied inversely with the cell concentration and was a saturable function of the absolute AFR concentration. Treatment of cells N-ethylmaleimide (2 mM) impaired their ability both to preserve extracellular ascorbate, and to decrease the extracellular AFR concentration. These results suggest that erythrocytes spare extracellular ascorbate by enhancing recycling of the AFR, which could help to maintain extracellular concentrations of the vitamin., (Copyright 2000 Academic Press.)