The Effects of Low pH on Nitric Oxide (NO) Synthesis from Human Monocyte Derived Macrophages

Abstract

The aim of this study was to asses the effects of environmental pH changes to acidic range on nitric oxide (NO) synthesis from macrophages. Human monocyte derived macrophages were obtained from peripheral blood of healthy human volunteers by using Ficoll-Hypaque density gradient method. Isolated monocytes were seeded into tissue culture flasks and incubated at 37°C in a 5 %CO2 humidified incubator for 7 days in RPMI (Roswell Park Memorial Institute)-1640 medium with 10% Fetal-calf serum (FCS). Macrophages were then harvested and seeded in 96-well plates at 105 cells/well. RPMI 1640 containing 10% FCS at three different pH ranges of 7.4. 7.0 and 6.8 was added to all wells. Samples from each well were taken for determining nitrite concentration at 3, 6, 24 and 48 hours time. Substitution of the medium of adherent macrophages with media at different pH modified NO production as reflected by changes in nitrite accumulation; as the pH became more acidic, more nitrite was detected in the culture media. The results indicate that the state of pH to which macrophages exposed to causes significant improvement in NO synthesis.