The metabolism of glyceryl trinitrate to nitric oxide in the macrophage cell line J774 and its induction by Escherichia coli lipopolysaccharide.
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 (NO2-) one of its breakdown products. J774 cells (1.25 x 10(5) cells) treated with indomethacin (10 microM) enhanced the platelet inhibitory activity of GTN (22-352 microM) but not that of sodium nitroprusside (4 microM). This effect was abrogated by co-incubation with oxyhaemoglobin (oxyHb, 10 microM) indicating release of NO from GTN. U937 cells (up to 60 x 10(5)) did not metabolize GTN to NO. LPS (0.5 micrograms/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 micrograms/mL) was incubated together with LPS. In the absence of LPS stimulation, cycloheximide had no effect. Furthermore, when incubated with GTN (200 microM), 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 microM). Incubation of J774 cells with GTN (75-600 microM) for 30 min led to a concentration-dependent increase in NO2- which was substantially reduced when the cells were boiled. The microsomal fraction was more potent than the cytosol in producing NO2- from GTN (1.2-2.4 mM). Release of NO2- from GTN by J774 cells was not affected by treating the cells with the NO synthase inhibitor, NG-monomethyl-L-arginine (MeArg, 300 microM). In J774 cells made tolerant to GTN, potentiation of the anti-platelet effects of GTN (11-352 microM) and release of NO2- 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 NO2-.(ABSTRACT TRUNCATED AT 400 WORDS)
Salvemini
D
D
William Harvey Research Institute, St Bartholomew's Hospital Medical College, London, U.K.
Pistelli
A
A
Mollace
V
V
Anggård
E
E
Vane
J
J
D000594:Amino Acid Oxidoreductases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006152:Cyclic GMP / Q000032:analysis; D003513:Cycloheximide / Q000494:pharmacology; D004361:Drug Tolerance; D004926:Escherichia coli; D006358:Hot Temperature; D006801:Humans; D008070:Lipopolysaccharides; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000378:metabolism; D005996:Nitroglycerin / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology
D008070:Lipopolysaccharides; D009573:Nitrites; D010108:Oxyhemoglobins; D010975:Platelet Aggregation Inhibitors; D009569:Nitric Oxide; D003513:Cycloheximide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D005996:Nitroglycerin; D006152:Cyclic GMP