The phosphatase inhibitor 2,3-diphosphoglycerate interferes with phospholipase D activation in rabbit peritoneal neutrophils.
In the present study, we examined the ability of the phosphatase inhibitors p-nitrophenyl phosphate and 2,3-diphosphoglycerate (DPG) to inhibit phospholipase D (PLD) activation in the rabbit peritoneal neutrophil. Also assessed were choline, a product of PLD-catalyzed hydrolysis of phosphatidylcholine, and its metabolite phosphocholine. PLD activity was determined by measuring the accumulation, in the presence of ethanol, of [3H]phosphatidylethanol ([3H]PEt) in neutrophils prelabeled with 1-O-[3H]octadecyl-2-lyso-snglycero-3-phosphocholine. Of the compounds tested, only DPG interfered with PLD activation by N-formyl-Met-Leu-Phe (fMLP) in a dose- and time-dependent manner. In contrast, it augmented fMLP-stimulated levels of [3H]inositol phosphates in myo-[3H]inositol-labeled neutrophils. DPG also prevented PLD activation by the calcium ionophore ionomycin and by phorbol 12-myristate 13-acetate. The suppression of PLD activation by DPG appeared to arise from direct interaction with the enzyme, as evidenced by a DPG competitive pattern of inhibition (Ki = 9.0 +/- 1.5 mM) for PLD from Streptomyces chromofuscus. These results suggest that DPG may be a useful tool for investigating the role of PLD in physiological function in a wide variety of cell types. Interestingly, DPG inhibited fMLP-induced N-acetyl-beta-glucosaminidase release and O2- generation by the cytochalasin B-primed neutrophils in a dose-dependent manner, whereas it had minimal effect (at concentrations up to 5 mM) on O2- generation induced by fMLP in nonprimed cells. These results suggest that PLD plays an important role in fMLP stimulation of both N-acetyl-beta-glucosaminidase release and O2- generation in the primed neutrophils, but that a PLD-independent pathway plays the primary role in O2- generation by the nonprimed neutrophils.
Department of Biochemistry, Gifu University School of Medicine, Japan.
Y
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Kanaho
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Nakai
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Katoh
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Nozawa
D019794:2,3-Diphosphoglycerate; D000118:Acetylglucosaminidase / Q000097:blood; D000818:Animals; D004163:Diphosphoglyceric Acids / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D020404:Glycerophospholipids; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000097:blood; D010739:Phospholipase D / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D013302:Streptomyces / Q000201:enzymology; D013481:Superoxides / Q000097:blood*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004163:Diphosphoglyceric Acids; D020404:Glycerophospholipids; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D013481:Superoxides; D019794:2,3-Diphosphoglycerate; C008644:nitrophenylphosphate; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010739:Phospholipase D; D000118:Acetylglucosaminidase; D013755:Tetradecanoylphorbol Acetate