title: Calcium rather than protein kinase C is the major factor to activate phospholipase D in FMLP-stimulated rabbit peritoneal neutrophils. Possible involvement of calmodulin/myosin L chain kinase pathway.
abstract: In the present study, we first investigated which of the factors, protein kinase C (PKC) or Ca2+, plays an important role in activation of phospholipase D (PLD) of rabbit peritoneal neutrophils stimulated by the chemoattractant FMLP. PLD activity was assessed by measuring [3H]phosphatidylethanol ([3H]PEt), the unambiguous marker of PLD, generated by [3H]lyso platelet-activating factor-prelabeled neutrophils in the presence of ethanol. PKC inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl-2-methylpiperazine dihydrochloride, augmented the plateau level of [3H]PEt produced in FMLP-stimulated cells, although they had no effect on the initial rate of the formation. Furthermore, it was found that the FMLP-stimulated [3H]PEt formation was inhibited by pretreatment of cells with PMA, a PKC activator, and exposure of cells to staurosporine before PMA pretreatment moderately blocked the PMA inhibition. Ca2+ ionophore ionomycin, as well as FMLP, stimulated [3H]PEt formation, accompanied by a decrease in [3H]phosphatidylcholine, in a time- and concentration-dependent manner. Both FMLP and ionomycin absolutely required extracellular Ca2+ to increase [3H]PEt formation. These results imply that elevated intercellular Ca2+ by FMLP stimulation is the major factor for PLD activation and that PKC rather negatively regulates the enzyme activity. Interestingly, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide, and a myosin L chain kinase inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-1H-h exahydro-1,4-diazepine hydrochloride, both inhibited the ionomycin- and FMLP-stimulated [3H]PEt formation in a concentration-dependent manner. Results obtained in this study suggest that, in FMLP-stimulated rabbit peritoneal neutrophils, increased intracellular Ca2+ activates PLD through calmodulin/myosin L chain kinase pathway and, thereafter, the enzyme activation is turned off by simultaneously activated PKC.
authors.affiliation: Department of Biochemistry, Gifu University School of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kanaho
authors.forename: A
authors.initials: A
authors.lastname: Nishida
authors.forename: Y
authors.initials: Y
authors.lastname: Nozawa
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D004789:Enzyme Activation; D066298:In Vitro Techniques; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000201:enzymology*; D010529:Peritoneal Cavity / Q000166:cytology; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010739:Phospholipase D / Q000032:analysis*; D011493:Protein Kinase C / Q000502:physiology*; D011817:Rabbits; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D010714:Phosphatidylethanolamines; D013449:Sulfonamides; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C017967:W 7; D011493:Protein Kinase C; D009219:Myosin-Light-Chain Kinase; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium