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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.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 fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist.

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.


fMLP induces a decrease in extracellular etheno-NAD cleaving activity of human neutrophils, but not of human monocytes. (A) Human neutrophils and (B) human monocytes were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml and incubated in the CO2 humidified chamber for 10 min. Then vehicle or fMLP of different concentration was treated. 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. **p<0.001; ***p<0.0001.
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Figure 3: fMLP induces a decrease in extracellular etheno-NAD cleaving activity of human neutrophils, but not of human monocytes. (A) Human neutrophils and (B) human monocytes were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml and incubated in the CO2 humidified chamber for 10 min. Then vehicle or fMLP of different concentration was treated. 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. **p<0.001; ***p<0.0001.

Mentions: Next, we investigated whether the scant extracellular etheno-NAD cleaving activity of human neutrophils is regulated following activation with fMLP, a chemoattractant for neutrophils. Since fMLP-like molecules could be abundant at bacterial infection sites, we investigated whether the extracellular etheno-NAD cleaving activity of intact human neutrophils are affected following fMLP stimulation. As shown in Fig. 3A, stimulation with fMLP induced a concentration-dependent decrease in the extracellular etheno-NAD cleaving activity of intact human neutrophils, which became significant at 100 nM, and was more marked at 1 µM concentration. In the next experiment, it was of interest that whether fMLP can affect extracellular etheno-NAD cleaving activity in other cells such as monocytes. Monocytes, however, was found to be unaffected by fMLP (Fig. 3B).


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 induces a decrease in extracellular etheno-NAD cleaving activity of human neutrophils, but not of human monocytes. (A) Human neutrophils and (B) human monocytes were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml and incubated in the CO2 humidified chamber for 10 min. Then vehicle or fMLP of different concentration was treated. 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. **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 3: fMLP induces a decrease in extracellular etheno-NAD cleaving activity of human neutrophils, but not of human monocytes. (A) Human neutrophils and (B) human monocytes were suspended in HBSS and seeded to 96 well-plate at a cell density of 1×107 cells/ml and incubated in the CO2 humidified chamber for 10 min. Then vehicle or fMLP of different concentration was treated. 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. **p<0.001; ***p<0.0001.
Mentions: Next, we investigated whether the scant extracellular etheno-NAD cleaving activity of human neutrophils is regulated following activation with fMLP, a chemoattractant for neutrophils. Since fMLP-like molecules could be abundant at bacterial infection sites, we investigated whether the extracellular etheno-NAD cleaving activity of intact human neutrophils are affected following fMLP stimulation. As shown in Fig. 3A, stimulation with fMLP induced a concentration-dependent decrease in the extracellular etheno-NAD cleaving activity of intact human neutrophils, which became significant at 100 nM, and was more marked at 1 µM concentration. In the next experiment, it was of interest that whether fMLP can affect extracellular etheno-NAD cleaving activity in other cells such as monocytes. Monocytes, however, was found to be unaffected by fMLP (Fig. 3B).

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.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 fMLP-mediated decrease in the extracellular etheno-NAD cleaving activity is reversed by WRW4, a potent FPRL1 antagonist.

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.