       Document 0175
 DOCN  M95B0175
 TI    Human mononuclear phagocyte inducible nitric oxide synthase (iNOS):
       analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide
       production by blood monocytes and peritoneal macrophages.
 DT    9511
 AU    Weinberg JB; Misukonis MA; Shami PJ; Mason SN; Sauls DL; Dittman WA;
       Wood ER; Smith GK; McDonald B; Bachus KE; et al; Department of Medicine,
       Veterans Affairs, Medical Center, Durham,; NC.
 SO    Blood. 1995 Aug 1;86(3):1184-95. Unique Identifier : AIDSLINE
       MED/95345471
 AB    Nitric oxide (NO) is produced by numerous different cell types, and it
       is an important regulator and mediator of many processes including
       smooth muscle relaxation, neurotransmission, and murine
       macrophage-mediated cytotoxicity for microbes and tumor cells. Although
       murine macrophages produce NO readily after activation, human monocytes
       and tissue macrophages have been reported to produce only low levels of
       NO in vitro. The purpose of this study was to determine if stimulated
       human mononuclear phagocytes produce inducible nitric oxide synthase
       (iNOS) mRNA, protein, and enzymatic activity. By reverse
       transcriptase-polymerase chain reaction (RT-PCR) analysis, we show that
       human monocytes can be induced to express iNOS mRNA after treatment with
       lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma). By
       immunofluorescence and immunoblot analyses, we show monocytes and
       peritoneal macrophages contain detectable levels of iNOS antigen after
       stimulations with cytokines in vitro. Control monocytes or those
       cultured with LPS and/or various cytokines have low levels of NOS
       functional activity as measured by the ability of cell extracts to
       convert L-arginine to L-citrulline, and they produce low levels of the
       NO catabolites nitrite and nitrate. Peritoneal macrophages have
       significantly enhanced nitrite/nitrate production and NOS activity after
       treatment with LPS and/or IFN-gamma, whereas monocyte nitrite/nitrate
       production and NOS activity are not altered by the treatments. Monocytes
       cultured with various live or heat-killed bacteria, fungi, or human
       immunodeficiency virus (HIV)-1 do not produce high levels of
       nitrite/nitrate. Antibodies against transforming growth factor-beta
       (TGF-beta), a factor known to inhibit iNOS expression and NO production
       in mouse macrophages, do not enhance NO production in human monocytes or
       macrophages. Biopterin, an obligate cofactor of iNOS enzymatic activity,
       is undetectable in freshly isolated or cultured human monocytes and
       peritoneal macrophages. However, replenishment of intracellular levels
       of tetrahydrobiopterin by culture with the cell-permeable, nontoxic
       precursor sepiapterin does not enhance the abilities of the human
       mononuclear phagocytes to produce NO in vitro. Mixing experiments show
       no evidence of a functional NOS inhibitor in human mononuclear
       phagocytes. Thus, we demonstrate that human mononuclear phagocytes can
       produce iNOS mRNA and protein, and (despite this) their abilities to
       generate NO are very low.
 DE    Amino Acid Oxidoreductases/*BIOSYNTHESIS  Animal  Base Sequence
       Biopterin/ANALOGS & DERIVATIVES/METABOLISM  Cell Line  DNA
       Primers/CHEMISTRY  Enzyme Induction  Gene Expression  Human  Interferon
       Type II/PHARMACOLOGY  Lipopolysaccharides/PHARMACOLOGY  Macrophages,
       Peritoneal/*ENZYMOLOGY  Mice  Molecular Sequence Data
       Monocytes/*ENZYMOLOGY  Nitrates/METABOLISM  Nitrites/METABOLISM
       Pteridines/PHARMACOLOGY  RNA, Messenger/GENETICS  Support, Non-U.S.
       Gov't  Support, U.S. Gov't, Non-P.H.S.  Support, U.S. Gov't, P.H.S.
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

