Characterization of endothelium-derived relaxing factor/nitric oxide synthase from bovine cerebellum and mechanism of modulation by high and low oxygen tensions1

A. Rengasamy, Roger A Johns

Research output: Contribution to journalArticle

Abstract

Experiments were designed to investigate the role of oxygen tension on modulation of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthase activity. EDRF/NO synthase from bovine cerebellum was confirmed to have cofactor and kinetic characteristics similar to that reported in endothelium and other tissues. The effect of oxygen tension on EDRF/NO synthase activity as assessed by L-[3H]citrulline production was investigated. Hypoxia markedly inhibited EDRF/NO synthase activity whereas hyperoxia increased the initial rate of enzyme activity. The inhibition of EDRF/NO synthase activity by hypoxia was reversed by normoxia as well as by hyperoxia. The K(m) values for L-arginine in hyperoxia, normoxia and hypoxia were 7 ± 0.7, 4.8 ± 0.4 and 7 ± 1.3 μM whereas the V(max) values were 94 ± 8, 66 ± 7, and 32 ± 2 pmol/min/mg of protein, respectively. The effect of oxygen tension on EDRF/NO synthase activity as determined by L-[3H]citrulline production was correlated with EDRF/NO production using a bioassay in which an EDRF/NO synthase preparation was incubated in wells of cultured vascular smooth muscle and cyclic GMP production was measured. Hypoxia almost inhibited the production of cyclic GMP completely, which was comparable to its inhibition of L-[3H]citrulline production. Hyperoxia, however, showed partial inhibition of cyclic GMP accumulation with no significant effect on L-[3H]citrulline production. This cyclic GMP inhibition by hyperoxia was reversed partially by superoxide dismutase. We conclude that hypoxia inhibits EDRF/NO synthase activity primarily through depletion of oxygen, one of the substrates for the enzyme. In hyperoxia, the initial rate of EDRF/NO synthase activity (V(max)) is significantly enhanced with no significant change in enzyme activity at longer time intervals. EDRF/NO once formed is partially inactivated by superoxide radical.

Original languageEnglish (US)
Pages (from-to)310-316
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume259
Issue number1
StatePublished - 1991
Externally publishedYes

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Endothelium-Dependent Relaxing Factors
Nitric Oxide Synthase
Cerebellum
Oxygen
Hyperoxia
Citrulline
Cyclic GMP
Nitric Oxide
Enzymes
Vascular Smooth Muscle
Superoxides
Biological Assay
Superoxide Dismutase
Endothelium
Arginine
Hypoxia

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Characterization of endothelium-derived relaxing factor/nitric oxide synthase from bovine cerebellum and mechanism of modulation by high and low oxygen tensions1",
abstract = "Experiments were designed to investigate the role of oxygen tension on modulation of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthase activity. EDRF/NO synthase from bovine cerebellum was confirmed to have cofactor and kinetic characteristics similar to that reported in endothelium and other tissues. The effect of oxygen tension on EDRF/NO synthase activity as assessed by L-[3H]citrulline production was investigated. Hypoxia markedly inhibited EDRF/NO synthase activity whereas hyperoxia increased the initial rate of enzyme activity. The inhibition of EDRF/NO synthase activity by hypoxia was reversed by normoxia as well as by hyperoxia. The K(m) values for L-arginine in hyperoxia, normoxia and hypoxia were 7 ± 0.7, 4.8 ± 0.4 and 7 ± 1.3 μM whereas the V(max) values were 94 ± 8, 66 ± 7, and 32 ± 2 pmol/min/mg of protein, respectively. The effect of oxygen tension on EDRF/NO synthase activity as determined by L-[3H]citrulline production was correlated with EDRF/NO production using a bioassay in which an EDRF/NO synthase preparation was incubated in wells of cultured vascular smooth muscle and cyclic GMP production was measured. Hypoxia almost inhibited the production of cyclic GMP completely, which was comparable to its inhibition of L-[3H]citrulline production. Hyperoxia, however, showed partial inhibition of cyclic GMP accumulation with no significant effect on L-[3H]citrulline production. This cyclic GMP inhibition by hyperoxia was reversed partially by superoxide dismutase. We conclude that hypoxia inhibits EDRF/NO synthase activity primarily through depletion of oxygen, one of the substrates for the enzyme. In hyperoxia, the initial rate of EDRF/NO synthase activity (V(max)) is significantly enhanced with no significant change in enzyme activity at longer time intervals. EDRF/NO once formed is partially inactivated by superoxide radical.",
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N2 - Experiments were designed to investigate the role of oxygen tension on modulation of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) synthase activity. EDRF/NO synthase from bovine cerebellum was confirmed to have cofactor and kinetic characteristics similar to that reported in endothelium and other tissues. The effect of oxygen tension on EDRF/NO synthase activity as assessed by L-[3H]citrulline production was investigated. Hypoxia markedly inhibited EDRF/NO synthase activity whereas hyperoxia increased the initial rate of enzyme activity. The inhibition of EDRF/NO synthase activity by hypoxia was reversed by normoxia as well as by hyperoxia. The K(m) values for L-arginine in hyperoxia, normoxia and hypoxia were 7 ± 0.7, 4.8 ± 0.4 and 7 ± 1.3 μM whereas the V(max) values were 94 ± 8, 66 ± 7, and 32 ± 2 pmol/min/mg of protein, respectively. The effect of oxygen tension on EDRF/NO synthase activity as determined by L-[3H]citrulline production was correlated with EDRF/NO production using a bioassay in which an EDRF/NO synthase preparation was incubated in wells of cultured vascular smooth muscle and cyclic GMP production was measured. Hypoxia almost inhibited the production of cyclic GMP completely, which was comparable to its inhibition of L-[3H]citrulline production. Hyperoxia, however, showed partial inhibition of cyclic GMP accumulation with no significant effect on L-[3H]citrulline production. This cyclic GMP inhibition by hyperoxia was reversed partially by superoxide dismutase. We conclude that hypoxia inhibits EDRF/NO synthase activity primarily through depletion of oxygen, one of the substrates for the enzyme. In hyperoxia, the initial rate of EDRF/NO synthase activity (V(max)) is significantly enhanced with no significant change in enzyme activity at longer time intervals. EDRF/NO once formed is partially inactivated by superoxide radical.

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