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,N6-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.
Adenosine Diphosphate Ribose ; Animals ; Bone Marrow ; Cell Membrane ; Chromatography, Liquid ; Humans ; Lymphocytes ; Mice ; Monocytes ; NAD* ; Neutrophils* ; Receptors, Formyl Peptide

We fortuitously observed a human neutrophil intracellular free-calcium concentration ([Ca2+]i) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced Ca2+ infl ux in human neutrophils without any cytotoxic eff ect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) (30 microM) and SKF-96365 (20 microM) signifi cantly inhibited K49-PLA2-induced [Ca2+]i increase. These results suggest that K49-PLA2 modulates [Ca2+]i in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.
Calcium Channels ; Chromatography, High Pressure Liquid ; Crotalus* ; Electrophoresis, Polyacrylamide Gel ; Humans* ; Membranes ; Neutrophils ; Phosphodiesterase I ; Phospholipases A2 ; S Phase ; Venoms*

We investigated the utility of the duck-feet collagen extraction patching procedure in the traumatic tympanic membrane (TM) perforation via a comparison with spontaneous healing or paper patch. Fifty-six ears of adult male Sprague-Dawley rats, each weighing in the range of 250 to 300 g, were used for the animal studies. Sixteen rats had one-side ear in the control group and the opposite-side ear in the treated groups. The remaining twelve rats had a one-side ear with the duck-feet collagen patch and the opposite-side ear with a paper patch. The proliferating cell nuclear antigen (PCNA) expression cells were calculated among the 200 basal cells, and the expression percentage was identified as the labeling index. The healing of the perforation in the duck-feet collagen patch group was confirmed to be more rapid compared to the healing of the other groups. PCNA staining was observed in the migrating portion of PCNA enhanced cell to collagen scaffold in Post operative day (POD) 7 of collagen patch group. Thus, the adhesive effect of the duck-feet collagen patch to perforated margin was better than that of the paper patch. After completing the healing process, the collagen patch shrank and detached from the tympanic membrane (POD 14). In this study, we confirmed that the use of a duck-feet collagen patch had the advantage of early healing, inducing natural TM contour, and disappearing ability after the patch effect is complete.
Adhesives ; Adult ; Animals ; Collagen* ; Ducks* ; Ear ; Ear, Middle ; Fibroins ; Foot* ; Humans ; Male ; Proliferating Cell Nuclear Antigen ; Rats ; Rats, Sprague-Dawley ; Tympanic Membrane Perforation* ; Tympanic Membrane*