Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin

Annong Huang, Joseph A. Vita, Richard C. Venema, John F. Keaney

Research output: Contribution to journalArticle

274 Citations (Scopus)

Abstract

Ascorbic acid enhances NO bioactivity in patients with vascular disease through unclear mechanism(s). We investigated the role of intracellular ascorbic acid in endothelium-derived NO bioactivity. Incubation of porcine aortic endothelial cells (PAECs) with ascorbic acid produced time- and dose- dependent intracellular ascorbic acid accumulation that enhanced NO bioactivity by 70% measured as A23187-induced cGMP accumulation. This effect was due to enhanced NO production because ascorbate stimulated both PAEC nitrogen oxide (NO2/- + NO3/-) production and L-arginine to L-citrulline conversion by 59 and 72%, respectively, without altering the cGMP response to authentic NO. Ascorbic acid also stimulated the catalytic activity of eNOS derived from either PAEC membrane fractions or baculovirus-infected Sf9 cells. Ascorbic acid enhanced bovine eNOS V(max) by O50% without altering the K(m) for L-arginine. The effect of ascorbate was tetrahydrobiopterin (BH4)- dependent, because ascorbate was ineffective with BH4 concentrations >10 μM or in PAECs treated with sepiapterin to increase intracellular BH4. The effect of ascorbic acid was also specific because A23187-stimulated cGMP accumulation in PAECs was insensitive to intracellular glutathione manipulation and only ascorbic acid, not glutathione, increased the intracellular concentration of BH4. These data suggest that ascorbic acid enhances NO bioactivity in a BH4-dependent manner by increasing intracellular BH4 content.

Original languageEnglish (US)
Pages (from-to)17399-17406
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number23
DOIs
StatePublished - Jun 9 2000

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Nitric Oxide Synthase Type III
Ascorbic Acid
Endothelial cells
Bioactivity
Swine
Endothelial Cells
Calcimycin
Glutathione
Arginine
Sf9 Cells
sapropterin
Nitrogen Dioxide
Citrulline
Baculoviridae
Cell membranes
Vascular Diseases
Endothelium
Catalyst activity
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. / Huang, Annong; Vita, Joseph A.; Venema, Richard C.; Keaney, John F.

In: Journal of Biological Chemistry, Vol. 275, No. 23, 09.06.2000, p. 17399-17406.

Research output: Contribution to journalArticle

Huang, Annong ; Vita, Joseph A. ; Venema, Richard C. ; Keaney, John F. / Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 23. pp. 17399-17406.
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