The metabolism of glyceryl trinitrate to nitric oxide in the macrophage cell line J774 and its induction by Escherichia coli lipopolysaccharide

Daniela Salvemini, Alessandra Pistelli, Vincenzo Mollace, Erik Änggård, John Vane

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Abstract

The metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) was studied in the mouse macrophage cell line J774 and in the human monocytic cell line U937 in the absence or presence of Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in J774 cells. In addition, NO produced from GTN by cells or by cellular fractions was measured as nitrite (NO-2) one of its breakdown products. J774 cells (1.25 × 105 cells) treated with indomethacin (10 μM) enhanced the platelet inhibitory activity of GTN (22-352 μM) but not that of sodium nitroprusside (4 μM). This effect was abrogated by co-incubation with oxyhaemoglobin (oxyHb, 10 μM) indicating release of NO from GTN. U937 cells (up to 60 × 105) did not metabolize GTN to NO. LPS (0.5 μg/mL for 18 hr) enhanced at least 2-fold the capacity of J774 cells but not that of U937 cells to form NO from GTN and this enhancement was attenuated when cycloheximide (10 μg/mL) was incubated together with LPS. In the absence of LPS stimulation, cycloheximide had no effect. Furthermore, when incubated with GTN (200 μM), J774 cells treated with LPS released more NO from GTN as indicated by a 3-fold greater increase in their level of cGMP which was prevented by oxyHb (10 μM). Incubation of J774 cells with GTN (75-600 μM) for 30 min led to a concentration-dependent increase in NO-2 which was substantially reduced when the cells were boiled. The microsomal fraction was more potent than the cytosol in producing NO-2 from GTN (1.2-2.4 mM). Release of NO-2 from GTN by J774 cells was not affected by treating the cells with the NO synthase inhibitor, NG-monomethyl-l-arginine (MeArg, 300 μM). In J774 cells made tolerant to GTN, potentiation of the anti-platelet effects of GTN (11-352 μM) and release of NO-2 from GTN was reduced. Thus, J774 cells but not U937 cells convert GTN to NO. This enzymic pathway (present mainly in the microsomal fraction of the J774 cells) is induced by LPS and is not regulated by endogenous NO released from L-Arg by the enzyme NO synthase. Furthermore, when compared to normal cells, tolerant J774 cells metabolize GTN to NO less effectively as assessed by a reduced capacity to potentiate the anti-platelet effect of GTN and to release NO-2. The presence of this enzyme system and its induction by LPS in cells other than smooth muscle cells and endothelial cells has therapeutic implications.

Original languageEnglish
Pages (from-to)17-24
Number of pages8
JournalBiochemical Pharmacology
Volume44
Issue number1
DOIs
Publication statusPublished - Jul 7 1992

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Macrophages
Nitroglycerin
Metabolism
Escherichia coli
Lipopolysaccharides
Nitric Oxide
Cells
Cell Line
Platelets
U937 Cells
Blood Platelets
Cycloheximide
Nitric Oxide Synthase
Oxyhemoglobins
Bioassay
Guanylate Cyclase
Endothelial cells
Nitroprusside
Enzymes
Nitrites

ASJC Scopus subject areas

  • Pharmacology

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The metabolism of glyceryl trinitrate to nitric oxide in the macrophage cell line J774 and its induction by Escherichia coli lipopolysaccharide. / Salvemini, Daniela; Pistelli, Alessandra; Mollace, Vincenzo; Änggård, Erik; Vane, John.

In: Biochemical Pharmacology, Vol. 44, No. 1, 07.07.1992, p. 17-24.

Research output: Contribution to journalArticle

Salvemini, Daniela ; Pistelli, Alessandra ; Mollace, Vincenzo ; Änggård, Erik ; Vane, John. / The metabolism of glyceryl trinitrate to nitric oxide in the macrophage cell line J774 and its induction by Escherichia coli lipopolysaccharide. In: Biochemical Pharmacology. 1992 ; Vol. 44, No. 1. pp. 17-24.
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