In-situ generation of peroxynitrite from 3-morpholinosydnonimine decay in pulmonary cell culture medium

Abstract

Background: Peroxynitrite (ONOO-) is a strong oxidising and nitrating agent generally implicated in oxidative stress. Its cellular action is linked with pulmonary artery cell hyper-proliferation and vascular remodelling seen in pulmonary hypertensive diseases. It is thus vital to elucidate the biological actions of peroxynitrite; however, working with the anion is challenging. Whether supplied commercially or prepared extemporaneously, ONOO- is stabilised and stored under strongly alkaline conditions and the exposure of cells to this form of ONOO- will in tandem increase culture media pH. Accordingly, increasing number of studies are seeking alternative means of generating peroxynitrite and have utilised 3-morpholinosydnonimine (SIN-1) the active metabolite of the vasodilatory drug molisdomine to generate peroxynitrite in-situ. Even so, it is not clear how much authentic ONOO- is generated under these conditions and for how long. Aim: To establish the formation of peroxynitrite and to determine its decay kinetics following SIN-1 decomposition in a medium formulated for the culture of primary human and bovine pulmonary artery cells. Results: The half-life of authentic peroxynitrite was determined to be 1.38s in pulmonary artery cell culture medium. Formation of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition was continuously monitored from the loss in fluorescence associated with the ONOO- oxidation of nicotinamide adenine dinucleotide (NADH) to NAD+. SIN-1 decayed by 1st order kinetics and 20μM SIN-1 generated ONOO- at the rate of 0.11 μM min-1. SIN-1 decomposition in cell culture medium was associated with the formation of a stable intermediate product SIN-1C with absorbance (λ_max) at 279±2nm. The SIN-1 ➝ SIN-1C transformation was oxygen dependent and the result of -OH catalyzed hydrolytic decomposition. Stopped-flow spectroscopic evidence revealed that SIN-1 can be deprotonated in a pH dependent manner during the phase of the reaction leading up to SIN-1A formation. Conclusion: The formation of ONOO- was demonstrated by the qualitative and quantitative determination of its insitu generation from the decay of 3-morpholinosydnonimine (SIN-1). This will enable relevant correlations of the life of peroxynitrite in culture conditions to its actions in pulmonary cells.

Keywords: Peroxynitrite, from SIN-1, culture medium

Biokemistri 28(1): 1–15