Phospholipid Metabolism, Calcium

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA) . HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner . The chemotactic peptide-stimulated release of [t "C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors . Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated ['"C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx . These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids . Leukocytes respond with a directed migratory movement, chemotaxis, to a number ofpeptides derived from complement (1), crude bacterial factors (2), and well-defined synthetic formylated peptides (3, 4). The chemotactic response to the formylated peptides involves interaction with specific receptors on the cell surface (5) . However, the biochemical events translating receptor activation into directed cell movement remain poorly understood . It has been shown in this and other laboratories that some early events in chemotactic peptide-stimulated cell movement are: activation ofprotein carboxy-o-methylase (6), degradation of phosphatidylcholine synthesized by transmethylation of phosphatidylethanolamine (7), release of arachidonic acid (7, 8) which can be metabolized to hydroxyeicosatetraenoic acids that are also chemoattractants (9, 10) and influx and redistribution of calcium ions (11, 12, 13). Inhibition of any one of these processes inhibits receptoractivated neutrophil chemotaxis (7, 14, 15, 16, 17, 18) . However, little is known about the interrelationships between peptide receptor, methyltransferase reactions, calcium ion fluxes, and phospholipase A2 activation leading to induced neutrophil 690 migration . Studies on other receptor-mediated stimulations of T lymphocytes, mast cells, and rat basophilic leukemia cells have suggested that phospholipid methylation is involved in calcium ion movement, activation of phospholipase A2 , and the specific cellular response (19, 20, 21). We report here that methyltransferase inhibitors partially block receptor-mediated calcium influx, phospholipase A2 activation, and chemotaxis in rabbit neutrophils . Furthermore the chemotactic peptide fMetLeu-Phe and the calcium ionophore A23187 specifically stimulate the release of arachidonic acid and no other fatty acid examined. MATERIALS AND METHODS