Spin trapping of nitric oxide by Nitronylnitroxides: Measurement of the activity of NO synthase from rat cerebellum

Nitric oxide (NO) has been shown to be an important mediator in vasodilation, neurotransmission and cellular cytotoxicity. We investigated a new series of nitronylnitroxyl radicals (NNR) as spin traps for NO. It was found these radicals react with NO with rate constants of about 10⁴ M⁻¹c⁻¹ forming stable iminonitroxides with dramatic changes in EPR spectra. To overcome fast reduction of the radicals (a few seconds in rat cerebella cytosol), NNR with charged trimethylammoniophenyl group (Ib) was incorporated into the inner volume of large unilamellar phosphatidylcholine liposomes. In this case the reduction of the radical Ib in rat cerebella cytosol is slow (ca. 1% per min). The rate of NO production by NO synthase from rat cerebellum measured by NNR, Ib, is in a reasonable agreement with that obtained by spectrophotometric method.