Limits...
Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1.

Hasan MA, Sultan MT, Ahn WG, Kim YJ, Jang JH, Hong CW, Song DK - Korean J. Physiol. Pharmacol. (2014)

Bottom Line: Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences.The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist.These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon 200-702, Korea.

ABSTRACT
Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N(6)-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

No MeSH data available.


Related in: MedlinePlus

fMLP-induced decrease in extracellular etheno-NAD cleaving activity of intact human neutrophils is reversed by WRW4, an FPRL1 antagonist. Human neutrophils were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Then, the cells were treated with vehicle or WRW4 (1 µM (A) or 10 µM (B)) for 5 min before the treatment of vehicle or fMLP (1 µM). Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed at 37℃ for 15 min. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. ns, no significant difference; **p<0.001; ***p<0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4296039&req=5

Figure 4: fMLP-induced decrease in extracellular etheno-NAD cleaving activity of intact human neutrophils is reversed by WRW4, an FPRL1 antagonist. Human neutrophils were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Then, the cells were treated with vehicle or WRW4 (1 µM (A) or 10 µM (B)) for 5 min before the treatment of vehicle or fMLP (1 µM). Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed at 37℃ for 15 min. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. ns, no significant difference; **p<0.001; ***p<0.0001.

Mentions: Since fMLP, at micromolar concentration showed the maximal inhibition in the extracellular etheno-NAD cleaving activity of intact human neutrophils (Fig. 3A), we hypothesized that fMLP-induced decrease in the extracellular etheno-NAD cleaving activity might occur through FPRL1. To test this hypothesis, the effect of WRW4, a potent antagonist for FPRL1, on the fMLP-induced effect was examined. WRW4 at 10 µM but not at 1 µM reversed the fMLP-induced decrease in the extracellular etheno-NAD cleaving activity of intact human neutorphils (Fig. 4), suggesting that fMLP-induced decrease in the extracellular etheno-NAD cleaving activity of intact human neutrophils occur via FPRL1.


Scant Extracellular NAD Cleaving Activity of Human Neutrophils is Down-Regulated by fMLP via FPRL1.

Hasan MA, Sultan MT, Ahn WG, Kim YJ, Jang JH, Hong CW, Song DK - Korean J. Physiol. Pharmacol. (2014)

fMLP-induced decrease in extracellular etheno-NAD cleaving activity of intact human neutrophils is reversed by WRW4, an FPRL1 antagonist. Human neutrophils were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Then, the cells were treated with vehicle or WRW4 (1 µM (A) or 10 µM (B)) for 5 min before the treatment of vehicle or fMLP (1 µM). Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed at 37℃ for 15 min. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. ns, no significant difference; **p<0.001; ***p<0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4296039&req=5

Figure 4: fMLP-induced decrease in extracellular etheno-NAD cleaving activity of intact human neutrophils is reversed by WRW4, an FPRL1 antagonist. Human neutrophils were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml. Then, the cells were treated with vehicle or WRW4 (1 µM (A) or 10 µM (B)) for 5 min before the treatment of vehicle or fMLP (1 µM). Substrate etheno-NAD (final concentration 20 µM) was added following 5 min pre-read. Cleavage of etheno-NAD was continuously followed at 37℃ for 15 min. Activity was defined as the fluorescence change (▴F)/min/2×106 cells. ns, no significant difference; **p<0.001; ***p<0.0001.
Mentions: Since fMLP, at micromolar concentration showed the maximal inhibition in the extracellular etheno-NAD cleaving activity of intact human neutrophils (Fig. 3A), we hypothesized that fMLP-induced decrease in the extracellular etheno-NAD cleaving activity might occur through FPRL1. To test this hypothesis, the effect of WRW4, a potent antagonist for FPRL1, on the fMLP-induced effect was examined. WRW4 at 10 µM but not at 1 µM reversed the fMLP-induced decrease in the extracellular etheno-NAD cleaving activity of intact human neutorphils (Fig. 4), suggesting that fMLP-induced decrease in the extracellular etheno-NAD cleaving activity of intact human neutrophils occur via FPRL1.

Bottom Line: Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences.The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist.These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon 200-702, Korea.

ABSTRACT
Extracellular nicotinamide adenine dinucleotide (NAD) cleaving activity of a particular cell type determines the rate of the degradation of extracellular NAD with formation of metabolites in the vicinity of the plasma membrane, which has important physiological consequences. It is yet to be elucidated whether intact human neutrophils have any extracellular NAD cleaving activity. In this study, with a simple fluorometric assay utilizing 1,N(6)-ethenoadenine dinucleotide (etheno-NAD) as the substrate, we have shown that intact peripheral human neutrophils have scant extracellular etheno-NAD cleaving activity, which is much less than that of mouse bone marrow neutrophils, mouse peripheral neutrophils, human monocytes and lymphocytes. With high performance liquid chromatography (HPLC), we have identified that ADP-ribose (ADPR) is the major extracellular metabolite of NAD degradation by intact human neutrophils. The scant extracellular etheno-NAD cleaving activity is decreased further by N-formyl-methionine-leucine-phenylalanine (fMLP), a chemoattractant for neutrophils. The fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist. These findings show that a much less extracellular etheno-NAD cleaving activity of intact human neutrophils compared to other immune cell types is down-regulated by fMLP via a low affinity fMLP receptor FPRL1.

No MeSH data available.


Related in: MedlinePlus