Y. Kanaho et al., THE PHOSPHATASE INHIBITOR 2,3-DIPHOSPHOGLYCERATE INTERFERES WITH PHOSPHOLIPASE-D ACTIVATION IN RABBIT PERITONEAL NEUTROPHILS, The Journal of biological chemistry, 268(17), 1993, pp. 2492-2497
In the present study, we examined the ability of the phosphatase inhib
itors p-nitrophenyl phosphate and 2,3-diphosphoglycerate (DPG) to inhi
bit phospholipase D (PLD) activation in the rabbit peritoneal neutroph
il. Also assessed were choline, a product of PLD-catalyzed hydrolysis
of phosphatidylcholine, and its metabolite phosphocholine. PLD activit
y was determined by measuring the accumulation, in the presence of eth
anol, of [H-3]phosphatidylethanol ([H-3]PEt) in neutrophils prelabeled
with [H-3]octadecyl-2-lyso-sn-glycero-3-phosphocholine. Of the compou
nds tested, only DPG interfered with PLD activation by N-formyl-Met-Le
u-Phe (fMLP) in a dose- and time-dependent manner. In contrast, it aug
mented fMLP-stimulated levels of [H-3]inositol phosphates in myo-[H-3]
inositol-labeled neutrophils. DPG also prevented PLD activation by the
calcium ionophore ionomycin and by phorbol 12-myristate 13-acetate. T
he suppression of PLD activation by DPG appeared to arise from direct
interaction with the enzyme, as evidenced by a DPG competitive pattern
of inhibition (K(i) = 9.0 +/- 1.5 mM) for PLD from Streptomyces chrom
ofuscus. These results suggest that DPG may be a useful tool for inves
tigating the role of PLD in physiological function in a wide variety o
f cell types. Interestingly, DPG inhibited fMLP-induced N-acetyl-beta-
glucosaminidase release and O2- generation by the cytochalasin B-prime
d neutrophils in a dose-dependent manner, whereas it had minimal effec
t (at concentrations up to 5 mM) on O2- generation induced by fMLP in
nonprimed cells. These results suggest that PLD plays an important rol
e in fMLP stimulation of both N-acetyl-beta-glucosaminidase release an
d O2- generation in the primed neutrophils, but that a PLD-independent
pathway plays the primary role in O2- generation by the nonprimed neu
trophils.