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MOLECULAR PHARMACOLOGY 51:882-888 (1997).

3-Morpholino-Sydnonimine-Induced Suppression of Human Neutrophil Degranulation is Not Mediated by Cyclic GMP, Nitric Oxide or Peroxynitrite: Inhibition of the Increase in Intracellular Free Calcium Concentration by N-Morpholino- iminoacetonitrile, a Metabolite of 3-Morpholino-Sydnonimine

Hannu Kankaanranta, Richard G. Knowles, Pauli Vuorinen, Outi Kosonen, Päivi Holm, and Eeva Moilanen

Medical School, University of Tampere, FIN-33101 Tampere, Finland (H.K., O.K., P.H., E.M.), Glaxo-Wellcome Research and Development, Stevenage, SG1 ZNY Hertfordshire, UK (R.G.K.), and Departments of Clinical Microbiology (P.V.) and Clinical Chemistry (E.M.), Tampere University Hospital, FIN-33521 Tampere, Finland

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 µM) inhibited degranulation (-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 µM) did not inhibit, but at high concentrations (1000-2350 µM), it potentiated FMLP-induced -glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 µM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 µM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.