nitrophenols and phosphatidylethanol

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.

Topics: 2,3-Diphosphoglycerate; Acetylglucosaminidase; Animals; Diphosphoglyceric Acids; Dose-Response Relationship, Drug; Enzyme Activation; Glycerophospholipids; In Vitro Techniques; Ionomycin; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitrophenols; Organophosphorus Compounds; Phosphatidic Acids; Phospholipase D; Rabbits; Streptomyces; Superoxides; Tetradecanoylphorbol Acetate