Enhancement by cimetidine of chemotactic peptide-stimulated ATP release and chemiluminescence in human neutrophils
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As determined by luciferase-luciferin, we recently found that the H2-blocker CIM considerably increased the ATP release from fMLP-stimulated PMN. This observation correlates well with our previous report  regarding the enhancement of superoxide output (chemiluminescence) in human neutrophils by CIM plus fMLP.
In order to compare the ATP release from PMN of different donors, a standard procedure has been developed consisting of the determination of the ATP present initially in the cell suspension (without stimulation), ATP release after stimulation with fMLP, and ATP release in the presence of CIM plus fMLP. The whole ATP content per neutrophil was determined after ultrasonication of the cells as well. The mean value of the initially present ATP was 0.45×10−17 mol/cell in the suspension.
Stimulation with fMLP plus CIM yielded within 5–10 minutes considerably higher ATP amounts than fMLP alone. The corresponding and statistically significantly different mean values were 2.46×10−17 mol/PMN (s.d.=1.047) and 1.38×10−17 mol/PMN (s.d.=0.55), respectively.
The whole ATP per neutrophil was found to be 1.22×10−15 mol (mean; s.d.=0.60) and thus, the stimulation with CIM plus fMLP released about 2.0 per cent, with fMLP alone about 1.0 per cent of the whole ATP.
CIM without fMLP did not enhanced the ATP release during the reaction time applied. On the other hand, fMLP-stimulated, lucigenin-amplified chemiluminescence determinations were carried out in the presence of CIM as well; contrarily to our previous method, CIM was dissolved in PBS without DMSO, because DMSO inhibited the chemiluminescence slightly. Our previous results showing an enhancement of the chemiluminescence by CIM could be justified.
KeywordsReaction Time DMSO Superoxide Cell Suspension Standard Procedure
Dulbeccos phosphate balanced salt solution
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