Basal poly-ADP ribosyl synthetase activity is involved in the regulation of the expression of the indicible isoform of ntric oxide synthase in cultured macrophages and conscious rats

Using 5-iodo-6-amino-1,2-benzopyrone (INHBP), a novel, potent inhibitor of polyADP ribosyl syntethase (PARS), we have investigated the regulation of the expression of the inducible isoform of NO synthase (iNOS) by basal PARS activity in vitro and in vivo. In J774 macrophages, pretreatment with INHBP (10-300 μM) inhibited nitrite production (measured at 24h) in response to bacterial lipopolysaccharide (LPS). This inhibition was markedly reduced when INHBP was added 6h after LPS (when the induction process has already started). Reduced iNOS expression after INHBP was confirmed by measurement of conversion of L-arginine to L-citrulline as well as by Western blotting in cell homogenates. In J774 cells, there was a basal PARS activity (as measured by ADP-ribosytation of nuclear proteins), which was due to basal Superoxide production in these cells, since Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable Superoxide dismutase mimetic dose-dependently inhibited it. MnTBAP also suppressed the induction of iNOS. NO or peroxynitrite production is not involved in the maintenance of basal PARS activity, since inhibition of iNOS did not diminish it. In rats, intraperitoneal injection of LPS (15 mg/kg) for 6h induced iNOS in many organs and increased plasma nitrate levels. Pretreatment with INHBP (10 mg/kg) inhibited the expression of iNOS in this model, whereas the inhibition significantly diminished when INHBP was given 2h after LPS treatment. Thus, basal PARS activity, maintained by Superoxide, is involved in the regulation of the induction of iNOS by endotoxin.