The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin.
1. We have measured the breakdown of [3H]phosphatidylinositol in rabbit neutrophils prelabelled with [3H]glycerol by a pulse-chase procedure. With a view to defining a possible causal relationship between phosphatidylinositol breakdown and enzyme secretion in these cells, we have compared the characteristics of both these processes induced by either the receptor-directed agonist formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) or the Ca2+-ionophore ionomycin. 2. The dependence on fMet-Leu-Phe concentration of phosphatidylinositol breakdown and secretion is identical (half-maximal at 0.3 nM). This is 30-fold less than that required for half-maximal occupation of receptors. 3. Both secretion and breakdown of phosphatidylinositol due to fMet-Leu-Phe are modulated by extracellular Ca2+. The sensitivity to Ca2+ of both processes is enhanced by pretreatment to deplete cell Ca2+. The concentration of Ca2+ required to cause half-maximal effects of both processes in Ca2+-depleted cells on stimulation with 1nM-fMet-Leu-Phe is 100 microM. Ionomycin-stimulated secretion and breakdown of phosphatidylinositol are completely dependent on extracellular Ca2+ over similar concentration ranges. 4. Both secretion and phosphatidylinositol breakdown due to fMet-Leu-Phe approach completion by 10s. With ionomycin these processes are slower, terminating by 2 min. 5. In the presence of [32P]Pi, labelling of [32P]phosphatidic acid reaches a maximum 15 min after stimulation with either fMet-Leu-Phe or ionomycin. This precedes the labelling of [32P]phosphatidylinositol and shows the expected precursor-product relationship. 6. We conclude from these results that in rabbit neutrophils a rise in cytosol [Ca2+] is both sufficient and necessary to cause secretion and phosphatidylinositol breakdown. In cells depleted of Ca2+, the occupation of receptors by fMet-Leu-Phe is without effect on these two processes.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002630:Chemotactic Factors / Q000494:pharmacology*; D004987:Ethers / Q000494:pharmacology; D066298:In Vitro Techniques; D015759:Ionomycin; D007476:Ionophores / Q000494:pharmacology*; D008715:Methionine / Q000031:analogs & derivatives*; D009239:N-Formylmethionine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011817:Rabbits; D013997:Time Factors
D002630:Chemotactic Factors; D004987:Ethers; D007476:Ionophores; D009842:Oligopeptides; D010716:Phosphatidylinositols; D009239:N-Formylmethionine; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008715:Methionine; D002118:Calcium