Evidence for free radical involvement in the hydroxylation of proline: Inhibition by nitro blue tetrazolium

Bhatnagar, Rajendra S.; Liu, Tsan Z.

doi:10.1016/0014-5793(72)80535-0

Cited by 37 publications

(9 citation statements)

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“…It should be noted that ascorbate is the cofactor used commonly in the in vitro assay of prolyl hydroxylase. Although the precise role of ascorbate in this reaction is not clearly understood, the generation of superoxide has been implicated (22,40). The data presented in Table 2 demonstrate that the effect of DHF was mediated through the generation of superoxide since scavengers of superoxide abolished the effect.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, superoxide anion has been shown to stimulate collagen synthesis in cultured cells which are normally programmed to synthesize connective tissue components. Chemically generated superoxide in the medium induced prolyl hydroxylase and caused enhanced synthesis of collagen polypeptide in WI-38 cells and in lung organ cultures (22)(23)(24). The purpose of the present study is to evaluate the effect of exogenous superoxide on collagen synthesis in normal cultured hepatocytes.…”

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confidence: 97%

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Increased prolyl hydroxylase activity and collagen synthesis in hepatocyte cultures exposed to superoxide

Hussain¹,

Watson²,

Bhatnagar³

1987

Hepatology

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Primary monolayer cultures of rat hepatocytes at confluence were exposed to an exogenously added source of superoxide, and its influence on collagen synthesis was examined. Superoxide was generated by the addition of dihydroxyfumarate to the culture medium. Exposure of hepatocytes to dihydroxyfumarate greatly stimulated the activity of prolyl hydroxylase and the synthesis of collagen. A significant increase in prolyl hydroxylase activity was observed with 5 micrograms per ml dihydroxyfumarate in 24 hr relative to that in the untreated cultures. Maximum stimulation of greater than 3-fold compared to the control value was elicited by 25 micrograms per ml dihydroxyfumarate. When scavengers of superoxide such as superoxide dismutase and Cu(Lys)2 were added in the medium, the increase in prolyl hydroxylase activity induced by dihydroxyfumarate was nearly abolished. Experiments with actinomycin D indicated that synthesis of new RNA was involved in the stimulation of prolyl hydroxylase activity. Analysis of collagen synthesis in cultures exposed to dihydroxyfumarate also showed a marked increase compared to that of the untreated cultures. The presence of superoxide dismutase in the medium significantly reduced the increase in collagen synthesis. Our results indicate that superoxide mediates the stimulation of collagen synthesis in hepatocytes. These findings may provide a possible explanation for excess collagen formation during induced liver fibrosis.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 97%

Increased prolyl hydroxylase activity and collagen synthesis in hepatocyte cultures exposed to superoxide

Hussain¹,

Watson²,

Bhatnagar³

1987

Hepatology

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“…The inhibition caused by Nitro Blue Tetrazolium was more pronounced in the presence of ascorbate than in its absence, whereas the opposite would have been expected if it inhibited the enzymic reaction by oxidizing°2 -". Bhatnagar & Liu (1972) found that prolyl hydroxylase, which is also stimulated by Fe2+ and ascorbate, may be inhibited by Nitro Blue Tetrazolium. These authors suggest that a free radical, possibly superoxide radical, may be generated at a 'reducing site' and serve to reduce Fe3+, which was presumed to be formed from Fe2+ during incorporation of oxygen atoms into the substrates.…”

Section: Discussionmentioning

confidence: 99%

Does superoxide anion participate in 2-oxoglutarate-dependent hydroxylation?

Holme

Lindstedt

et al. 1982

Biochemical Journal

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The possible role of superoxide anion in 2-oxoglutarate-coupled dioxygenase reactions has been investigated. γ-Butyrobetaine hydroxylase (EC 1.14.11.1) was inhibited by human erythrocyte superoxide dismutase (EC 1.15.1.1), probably due to release of Cu2+ or Zn2+, as the inhibition was more pronounced after heat-inactivation of the dismutase and as Cu2+ was a potent inhibitor. Bovine superoxide dismutase and the Mn2+-containing superoxide dismutase from Escherichia coli were not inhibitory. Superoxide anion generated from xanthine/xanthine oxidase was not stimulatory and could not replace ascorbate. Thymine 7-hydroxylase (EC 1.14.11.6) and thymidine 2′-hydroxylase (EC 1.14.11.3) were not inhibited by erythrocyte superoxide dismutase or stimulated by superoxide anion. γ-Butyrobetaine hydroxylase was inhibited by a number of low-molecular-weight compounds, such as tetranitromethane, Nitro Blue Tetrazolium, adrenaline and Tiron, which may act as scavengers of superoxide anion. Involvement of this radical in other oxygenase reactions has been inferred from the findings that they were inhibitory for the respective enzymes. Several of these compounds also inhibited γ-butyrobetaine hydroxylase. It could be concluded from these experiments, however, that mechanisms other than disposal of superoxide anion might equally well be operative, such as hydrophobic interaction with the enzyme protein and interaction with compounds required for full enzymic activity, e.g. iron and ascorbate. The results appear to rule out a requirement for superoxide anion generated in free solution, and have not yielded evidence for participation of enzyme-bound superoxide anion in 2-oxoglutarate-dependent hydroxylations.

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“…How curious that the first three are also players in the ROS game! It was known in 1972 that nitroblue tetrazolium, a free radical scavenger, would inhibit proline hydroxylation [164]. Both lysyl and prolyl hydroxylases were reported to be inhibited by several low molecular weight copper chelates with SOD-like activity [165].…”

Section: Collagenmentioning

confidence: 99%

Role of Nitric Oxide and Reactive Oxygen Species in Arthritis

Cuzzocrea

2006

CPD

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A vast amount of circumstantial evidence implicates oxygen-derived free radicals, especially reactive oxygen species and nitric oxide as mediators of inflammation and/or tissue destruction in inflammatory and arthritic disorders. The aim of the current article is to overview the recent developments in this field, as it relates to the roles of nitric oxide (NO) and reactive oxygen species in the pathogenesis of this condition. The first part of the review focuses on the biochemical impact of NO and reactive oxygen species. The second part of the review deals with the novel findings related to the recently identified regulatory roles of the inducible isoform of nitric oxide synthase (iNOS) in the expression of pro-inflammatory mediators in inflammation. Reactive oxygen species can initiate a wide range of toxic oxidative reactions. These include initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, inactivation of glyceraldehyde-3phosphate dehydrogenase, inhibition of membrane sodium/potassium ATP-ase activity, inactivation of membrane sodium channels, and other oxidative modifications of proteins. All these toxicities are likely to play a role in the pathophysiology of inflammation. Reactive oxygen species are all potential reactants capable of initiating DNA single strand breakage, with subsequent activation of the nuclear enzyme poly (ADP ribose) synthetase (PARS), leading to eventual severe energy depletion of the cells, and necrotic-type cell death. Recently it has been demonstrated that iNOS inhibitor prevents the activation of poly (ADP ribose) synthetase, and prevents the organ injury associated with inflammation. Although the severity and duration of inflammation may dictate the timing and extent of NOS expression, it is now evident that the up-regulation of NOS can take place during sustained inflammation. Thus, induced nitric oxide, in addition to being a "final common mediator" of inflammation, is essential for the up-regulation of the inflammatory response. Furthermore, a picture of a pathway is evolving that contributes to tissue damage both directly via the formation of reactive oxygen species, with them associated toxicities, and indirectly through the amplification of the inflammatory response.

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Evidence for free radical involvement in the hydroxylation of proline: Inhibition by nitro blue tetrazolium

Cited by 37 publications

References 9 publications

Increased prolyl hydroxylase activity and collagen synthesis in hepatocyte cultures exposed to superoxide

Increased prolyl hydroxylase activity and collagen synthesis in hepatocyte cultures exposed to superoxide

Does superoxide anion participate in 2-oxoglutarate-dependent hydroxylation?

Role of Nitric Oxide and Reactive Oxygen Species in Arthritis

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