| Research Abstract |
(1). Chemoattractants such as N-formyl-Met-Leu-Phe (fMLP), leukotriene B_4, and platelet-activating factor, stimulated phospholipase D(PLD) activity as well as enzyme release of rabbit peritoneal neutrophils. In the presence of ethanol, enzyme release and phosphatidic acid production by PLD in response to these chemoattractants were inhibited. However, the extent of the inhibition of the former was less than that of the latter. These results indicate that PLD activation is, at least in part, involved in the enzyme release from neutrophils.
(2). PLD activation by fMLP of rabbit neutrophils was augmented by the pretintment with protein Kinase C(PKC) inhibitors, staurosporine and H-7. PKC activation by treatment with PMA inhibited PLD activation by fMLP, NaF and ionomycin. The inhibitory effect of PMA was abolished by staurosporine treatment prior to the PMA treatment. These results conclude that in rabbit neutrophils PKC rather negatively regulates PLD activity.
(3). PLD activation by fMLP and ionomycin of neutrophils absolutely required Ca^<2+>. The activation by these stimuli was inhibited by inhibitors of calmodulin (CaM) and of myosin light chain kinase (MLCK), W-7 and ML-7, respectively. From these results, it is suggested that fMLP stimulates influx of extracellular Ca^<2+> to activate CaM/MLCK pathway, thereby activating PLD.
(4). We have published that 0.1-0.2 % of Triton X- 1 00 activates PLD of rat and rabbit brain membranes and higher concentrations (0.5-1 %) of Triton X- 1 00 solubilizes more than 70% of PLD activity from membranes. These results indicate that Triton X- 100 may be useful to purify PLD from membranes. However, PLD was completely inactivated 2-3 days after solubilization by Triton X-100. Therefore, it is necessary to purify the enzyme under conditions that PLD is stable.
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