The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites

We have recently found that in the presence, but not in the absence, of foetal calf serum, spermine inhibits the production of nitric oxide (NO) in cultured J774.2 macrophages stimulated with bacterial endotoxin (lipopolysaccharide; LPS) or with 7‐interferon (IFN), showing that polyamines may act as suppressants of NO‐mediated immune functions. Here, we have studied the mechanisms and the specificity of this inhibitory action. Other polyamines, as well as spermine, inhibit the formation of NO in cultured J774.2 macrophages, with the order of potency being spermine > spermidine > > putrescine = cadaverine. This inhibition of NO formation is not due to any cytotoxic effect of these agents for they neither reduced mitochondrial respiration nor increased the release of lactate dehydrogenase into the supernatant. Spermine is not a direct inhibitor of the activity of iNOS in induced J774.2 cells as measured by its lack of effect on the conversion of L‐arginine to L‐citrulline in homogenates. Neither spermine, nor its metabolites, interfere with the production of nitrite from NO or act as scavengers of NO. Thus, spermine is an inhibitor of the induction of iNOS. Spermine inhibits nitrite formation in the presence of foetal, newborn or adult bovine serum, but not rat or human serum. The effect of sper mine on nitrite production can be prevented by isoniazid, hydrazine or hydroxy‐lamine, inhibitors of spermine oxidase, as well as by phenylhydrazine, an aldehyde inhibitor. We have, therefore, tested the effects of spermine dialdehyde or malon dialdehyde on the induction of iNOS. Spermine dialdehyde (SDA, 10⁻⁵m) inhibits nitrite formation by IFN‐activated J774.2 cells in the absence of serum when given as a pretreatment but not when given 6 h after stimulation. In contrast, malon dialdehyde was ineffective. Thus, aldehyde metabolites of spermine, such as SDA, account for the inhibitory effect of polyamines on the induction of NOS in vitro. The inhibitory effect of polyamines on iNOS induction appears to be fairly specific to iNOS, for spermine does not inhibit LPS‐induced production of prostaglandin F_2α or tumour necrosis factor. 1994 British Pharmacological Society