PMID- 8340389
OWN - NLM
STAT- MEDLINE
DCOM- 19930830
LR  - 20240109
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 268
IP  - 21
DP  - 1993 Jul 25
TI  - Extracellular production of singlet oxygen by stimulated macrophages quantified 
      using 9,10-diphenylanthracene and perylene in a polystyrene film.
PG  - 15649-54
AB  - The extracellular production of singlet oxygen (O2(1 delta g)) by stimulated 
      macrophages was measured using a modification of our quantitative method 
      initially developed to measure the intracellular production of O2(1 delta g) by 
      neutrophils (Steinbeck, M. J., Khan, A. U., and Karnovsky, M. J. (1992) J. Biol. 
      Chem. 267, 13425-13433). Glass coverslips were coated with the specific chemical 
      trap for O2(1 delta g), 9,10-diphenylanthracene (DPA) and perylene, which is an 
      internal standard, in a methylene chloride solution containing 0.3 mg/ml 
      polystyrene. On evaporation, the polystyrene formed an even coating of DPA and 
      perylene over the surface of a glass coverslip (PDP film). Unstimulated 
      macrophages or macrophages stimulated with 4 beta-phorbol 12-myristate 13-acetate 
      (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) were then added to the PDP 
      film in a darkened room and incubated at 37 degrees C for 30 min in a humidified 
      5% CO2 atmosphere. Both unstimulated and stimulated cells adhered to the PDP film 
      in approximately equivalent numbers. Only stimulated cells produced measurable 
      amounts of O2(1 delta g) in a dose-dependent response to either PMA or fMLP. The 
      production of O2(1 delta g) by macrophages stimulated with PMA was maximal in 
      response to 25 ng, 17.8 +/- 1.3 nmol of O2(1 delta g)/approximately 1.00 x 10(6) 
      cells. The maximal response for fMLP was at a concentration of 1 microM, 18.4 +/- 
      1.0 nmol of O2(1 delta g)/approximately 1.00 x 10(6) cells. The specific 
      detection of O2(1 delta g) by this method was confirmed by thermally releasing 
      O2(1 delta g) from the DPA-O2(1 delta g) reaction product, DPA-endoperoxide, 
      regenerating the original DPA compound. Production of O2(1 delta g) by the 
      stimulated cells was inhibited 80-89% by the addition of 60-120 micrograms of 
      superoxide dismutase, an enzyme that converts superoxide to hydrogen peroxide and 
      ground state molecular oxygen or 79-84% with the addition of 2 mM histidine, an 
      avid quencher of O2(1 delta g). Neither of these additions interfered with 
      adhesion of the cells to the PDP film. The ability of superoxide dismutase to 
      inhibit the production of O2(1 delta g) suggested that O2(1 delta g) was produced 
      via a superoxide-dependent route. The ability of an oxidase to produce O2(1 delta 
      g) secondary to superoxide production was substantiated further using a xanthine 
      oxidase-acetaldehyde system. Purified xanthine oxidase produced both superoxide 
      and O2(1 delta g), and their production was inhibited by the addition of 
      superoxide dismutase.(ABSTRACT TRUNCATED AT 400 WORDS)
FAU - Steinbeck, M J
AU  - Steinbeck MJ
AD  - Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
FAU - Khan, A U
AU  - Khan AU
FAU - Karnovsky, M J
AU  - Karnovsky MJ
LA  - eng
GR  - DE-08507/DE/NIDCR NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Anthracenes)
RN  - 0 (Free Radicals)
RN  - 0 (Polystyrenes)
RN  - 51BQ8IYQ9U (9,10-diphenylanthracene)
RN  - 59880-97-6 (N-Formylmethionine Leucyl-Phenylalanine)
RN  - 5QD5427UN7 (Perylene)
RN  - BBX060AN9V (Hydrogen Peroxide)
RN  - DY38VHM5OD (Sodium Hypochlorite)
RN  - EC 1.17.3.2 (Xanthine Oxidase)
RN  - NI40JAQ945 (Tetradecanoylphorbol Acetate)
RN  - S88TT14065 (Oxygen)
SB  - IM
MH  - Animals
MH  - Anthracenes/*pharmacology
MH  - Cell Adhesion
MH  - Free Radicals
MH  - Hydrogen Peroxide/chemistry
MH  - *Macrophage Activation
MH  - Macrophages/cytology/drug effects/*metabolism
MH  - Male
MH  - N-Formylmethionine Leucyl-Phenylalanine/pharmacology
MH  - Oxygen/*metabolism
MH  - Perylene/*pharmacology
MH  - Polystyrenes
MH  - Rats
MH  - Rats, Sprague-Dawley
MH  - Sodium Hypochlorite/chemistry
MH  - Tetradecanoylphorbol Acetate/pharmacology
MH  - Xanthine Oxidase/metabolism
EDAT- 1993/07/25 00:00
MHDA- 1993/07/25 00:01
CRDT- 1993/07/25 00:00
PHST- 1993/07/25 00:00 [pubmed]
PHST- 1993/07/25 00:01 [medline]
PHST- 1993/07/25 00:00 [entrez]
AID - S0021-9258(18)82305-5 [pii]
PST - ppublish
SO  - J Biol Chem. 1993 Jul 25;268(21):15649-54.