title: Regulation of phospholipase D in HL-60 granulocytes. Activation by phorbol esters, diglyceride, and calcium ionophore via protein kinase- independent mechanisms.
abstract: It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.
authors.affiliation: Department of Allergy and Inflammation, Schering-Plough Corporation, Bloomfield, New Jersey 07003.
authors.forename: M M
authors.initials: MM
authors.lastname: Billah
authors.forename: J K
authors.initials: JK
authors.lastname: Pai
authors.forename: T J
authors.initials: TJ
authors.lastname: Mullmann
authors.forename: R W
authors.initials: RW
authors.lastname: Egan
authors.forename: M I
authors.initials: MI
authors.lastname: Siegel
mesh_terms: D000001:Calcimycin / Q000494:pharmacology*; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D005989:Glycerides / Q000494:pharmacology*; D006098:Granulocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D004075:Diglycerides; D005989:Glycerides; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D010740:Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate