title: Ascorbate in aqueous humor augments nitric oxide production by macrophages.
abstract: Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting proinflammatory NO production by macrophages (MΦs). Consistent with previous observations, we observed that although MΦs stimulated in the presence of AqH expressed NO synthase 2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure of NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of L-arginine (L-Arg) into L-citrulline, was augmented in lysates of MΦs stimulated in the presence of AqH. These data suggested that intracellular L-Arg may have been limited by AqH. However, we observed increased mRNA expression of the L-Arg transporter, cationic amino acid transporter 2B, and increased L-Arg uptake in MΦs stimulated in the presence of AqH. Arginases were expressed by stimulated Ms, but competition for L-Arg with NOS2 was excluded. Expression of GTP cyclohydrolase, which produces tetrahydrobiopterin (H(4)B), an essential cofactor for NOS2 homodimerization, increased after M stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together, these data provided no evidence for inhibited NOS2 enzymatic activity by AqH, suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH, and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in MΦs via ascorbate, which limited degradation of H(4)B. Therefore, AqH may augment NO production in macrophages by stabilizing H(4)B and increasing intracellular L-Arg.
authors.affiliation: Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, USA. mckennakc@upmc.edu
authors.forename: Kyle C
authors.initials: KC
authors.lastname: McKenna
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Beatty
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Scherder
authors.forename: Fuwang
authors.initials: F
authors.lastname: Li
authors.forename: Huanbo
authors.initials: H
authors.lastname: Liu
authors.forename: Alex F
authors.initials: AF
authors.lastname: Chen
authors.forename: Arnab
authors.initials: A
authors.lastname: Ghosh
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000378:metabolism; D000818:Animals; D001082:Aqueous Humor / Q000737:chemistry / Q000378:metabolism*; D001119:Arginase / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D005136:Eye Proteins / Q000494:pharmacology; D005260:Female; D006136:GTP Cyclohydrolase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009414:Nerve Growth Factors / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D011817:Rabbits; D015843:Serpins / Q000494:pharmacology; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology; D000521:alpha-MSH / Q000494:pharmacology; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D026923:Amino Acid Transport Systems, Basic; D001119:Arginase; D001120:Arginine; D001205:Ascorbic Acid; D001708:Biopterins; D015740:Calcitonin Gene-Related Peptide; D005136:Eye Proteins; D006136:GTP Cyclohydrolase; D009414:Nerve Growth Factors; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D015843:Serpins; D053781:Transforming Growth Factor beta2; D000521:alpha-MSH; D000099174:Pigment Epithelium-Derived Factor; C087435:Slc7a2 protein, mouse; C003402:sapropterin