Xanthine oxidase activity in rat pulmonary artery endothelial cells and its alteration by activated neutrophils

S. H. Phan, David E Gannon, J. Varani, U. S. Ryan, P. A. Ward

Research output: Contribution to journalArticle

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Abstract

The possibility that endothelial cell-derived oxidants could contribute to neutrophil-mediated endothelial cell injury and cytotoxicity has been a subject of speculation. Rat pulmonary artery endothelial cells (RPAECs) were examined for the presence of xanthine oxidase (XO) activity, a well-known source of O2-. Using a sensitive assay based on measurements of radioactive xanthine conversion to uric acid by high performance liquid chromatography (HPLC), RPAEC extracts were found to contain both XO and xanthine conversion to uric acid by high performance liquid chromatography (HOLC), RPAEC extracts were found to contain both XO and xanthine dehydrogenase (XD) activities. Extracts from early passage cells have 55.3 ± 11.7 (mean ± SE) units/106 cells of total (XO + XD) activity, one unit of activity being defined as the conversion of 1% of substrate to product in 30 minutes of incubation. XO comprised 31.6 ± 3.1% of this total activity. Addition of human neutrophils stimulated with phorbol myristate acetate (PMA) caused a rapid and dose-dependent increase in RPAEC XO activity from 31.6 ± 3.1% to 71.7 ± 4.8% of total without altering total (XO + XD) activity. The neutrophil dose-response curve for increase in XO paralleled closely the curve for neutrophil-mediated RPAEC cytotoxicity. The basal XO and XD activities and the neutrophil-induced increase in XO activity were inhibited by treating RPAECs with allopurinol, oxypurinol, and lodoxamide, which also inhibited cytotoxicity, but not by catalase, superoxide dismutase, or deferoxamine. Addition of H2O2 failed to cause an increase in RPAEC XO activity or XD to XO conversion. The results suggest that during neutrophil-mediated injury, rapid conversion of RPAEC XD to XO occurs, resulting in increased XO-catalyzed endogenous oxidant production, which may contribute to the oxidant burden in the killing mechanism initiated by activated neutrophils. Although the mechanism for conversion of XD to XO is uncertain, it appears that neutrophil-derived H2O2 is not sufficient to cause this phenomenon. Furthermore, neither O2- nor chelatable iron is required for neutrophil-induced XD to XO conversion. Supernatant fluids from activated neutrophils failed to induce XD to XO conversion. Supernatant fluids from activated neutrophils failed to induce XD to XO conversion in RPAECs. This in vitro system provides an opportunity to define the cellular and molecular mechanisms underlying the in vivo phenomenn of XD to XO conversion associated with ischemic/reperfusion or inflammatory tissue injury.

Original languageEnglish (US)
Pages (from-to)1201-1211
Number of pages11
JournalAmerican Journal of Pathology
Volume134
Issue number6
StatePublished - Jan 1 1989

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Xanthine Oxidase
Pulmonary Artery
Xanthine Dehydrogenase
Neutrophils
Endothelial Cells
Oxidants
Xanthine
Uric Acid
Cell Extracts
Wounds and Injuries
Oxypurinol
High Pressure Liquid Chromatography
Allopurinol
Deferoxamine

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Xanthine oxidase activity in rat pulmonary artery endothelial cells and its alteration by activated neutrophils. / Phan, S. H.; Gannon, David E; Varani, J.; Ryan, U. S.; Ward, P. A.

In: American Journal of Pathology, Vol. 134, No. 6, 01.01.1989, p. 1201-1211.

Research output: Contribution to journalArticle

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