AHMET YILMAZ ÇOBAN, BORA EKİNCİ, ASUMAN BİRİNCİ, Belma DURUPINAR, Murat ERTÜRK

The effects of low pH on nitric oxide (NO) synthesis from human monocyte derived macrophages

Çalışmada, asidik karakterde çevresel pH değişikliklerinin makrofajlann nitrik oksit (NO) sentezine etkilerinin araştırılması amaçlanmıştır. İnsan monosit derive makrofajlar sağlıklı gönüllülerin periferal kanlarından Ficoll-Hypaque density graident metod kullanılarak elde edilmiştir. İzole edilen monositler doku kültür flasklarına dağıtıldıktan sonra %10 fetal-calf serumlu (FCS) RPMI (Roswell Park Memorial Institute)-l640 besiyerinde 7 gün 370C ve %5 CO2 içeren ortamda inkübe edil-miştir. Oluşan makrofajlar 96 kuyucuklu plaklara l O5 hücre/kuyucuk olacak şekilde dağıtılmıştır. Tüm kuyucuklara pH 7.4, 7.0 ve 6.8 olan %10 FCS1Iu RPMI 1640 ilave edil-miştir. 3, 6, 24 ve 48 saat sonra kuyucuklardan alınan örneklerdeki nitrit kon-santrasyonları tespit edilmiştir. Farklı pH'daki besiyerleri ile adherent makrofajların besiyerleri değiştirildiğinde NO üretiminin bir göstergesi olan nitrit birikiminde değişiklik olmaktadır. pH daha asidik olunca kültür ortamında daha çok nitrit tespit edilmektedir. Sonuçlar göstermektedir ki, makrofajların maruz kaldığı pH'ın durumu NO sentezinde belirgin bir artışa neden ol-maktadır.

Anahtar Kelimeler:

Nitrik oksit, Hidrojen-iyon konsantrasyonu, Makrofajlar

İnsan monosit-derive makrofajların nitrik oksit (NO) sentezine düşük pH'ın etkileri

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 370C 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.

Keywords:

Nitric Oxide, Hydrogen-Ion Concentration, Macrophages,

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