title: Nitric oxide in the healing wound: a time-course study.
abstract: BACKGROUND
  We studied the time course of nitric oxide expression in the healing wound and the cell populations responsible for its synthesis.
  
  
  METHODS
  Twenty four Lewis rats underwent subcutaneous implantation of polyvinyl alcohol sponges. Rats were sacrificed in groups of three on days 1, 3, 5, 7, 10, 14, and 35 after wounding. The conversion of 3H-labeled arginine to 3H-labeled citrulline, with or without N(G)-L-monomethyl-arginine (L-NMMA) in harvested sponges, was measured. Nitrate/nitrite (NOx) in plasma and wound fluid was quantified by Greiss reaction. Inducible nitric oxide synthase (iNOS) gene expression was determined by Northern analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). Inducible NOS was identified in specific wound cell populations by dual-label flow cytometry.
  
  
  RESULTS
  Nitric oxide synthase (NOS) activity peaked at 24 h after wounding (37.7 +/- 0.9 micromol citrulline per milligram sponge), with a steady decline thereafter. Percentage inhibition of NOS activity by l-NMMA was highest on days 1-7 (70-80%). This declined to 50% by day 10 and to 25% by days 14-35. The iNOS gene expression paralleled NOS biochemical activity. RT-PCR confirmed low-level expression up to 10 days after wounding. Plasma NOx levels remained within a narrow range of 22.6 +/- 1.3 to 29.3 +/- 1.5 microM throughout the postwounding period, while corresponding levels in wound fluid (microM) increased steadily from 27 +/- 3.8 on day 1 to 107.2 +/- 10.0 on day 14. Inducible NOS expression was detectable by fluorescence-activated cell sorting in wound macrophages on days 1 and 3 after wounding.
  
  
  CONCLUSIONS
  Our findings suggest maximal NOS activity early in cutaneous wound healing, with sustained production up to 10 days after wounding. NOS biochemical activity was paralleled by iNOS gene expression. Plasma NOx remained constant, while wound fluid NOx increased steadily to peak at day 14. Wound macrophages appear to be a source of nitric oxide production in the early phase of wound healing.
authors.affiliation: Division of Plastic & Reconstructive Surgery, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
authors.forename: R H
authors.initials: RH
authors.lastname: Lee
authors.forename: D
authors.initials: D
authors.lastname: Efron
authors.forename: U
authors.initials: U
authors.lastname: Tantry
authors.forename: A
authors.initials: A
authors.lastname: Barbul
mesh_terms: D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D012867:Skin / Q000293:injuries* / Q000503:physiopathology*; D013997:Time Factors; D014945:Wound Healing / Q000502:physiology*; D014947:Wounds and Injuries / Q000503:physiopathology*; D019323:omega-N-Methylarginine / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009566:Nitrates; D009573:Nitrites; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat