The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin

Abstract

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.

Cell surface expression of fMet-Leu-Phe receptors on human neutrophils. Correlation to changes in the cytosolic free Ca2+ level and action of phorbol myristate acetate.

“…The number of receptors were 2.8 X 104/cell (A) and 1.3 X 104/cell (B), respectively. The affinity and the number of fMet-Leu-Phe receptors in this study are in the same range as those previously reported (17,27). This analysis revealed that the depressed receptor expression after reduction in the [Ca2+]i is most readily explained by a decreased number of fMet-Leu-Phe receptors on the cell surface.…”

Role of Ca2+ in secretagogue-stimulated breakdown of phosphatidylinositol in rat pancreatic islets.

“…Recent investigations on stimulated lymphocytes (32), neutrophils (33), hepatocytes (34), and pancreas (35,36) have also demonstrated that removal of Ca2+ from the extracellular environment decreases the stimulation of PI metabolism and suggest (34) a distinction between changes in the availability of Ca2+ within the cell and the actual entry of Ca2+ through specific channels in the plasma membrane. Thus, the conclusions of several other studies support a model in which PI turnover is itself mediated by Ca2 .…”

Adenosine triphosphate-dependent calcium pump in the plasma membrane of guinea pig and human neutrophils.

“…There is increasing evidence that the intracellular concentration of ionized Ca may play a crucial role in modulating the activity of PMN, for example, the stimulation of the cell by Ca2" ionophores (4)(5)(6)(7) and the inhibition of cell function by Ca2" antagonists (8-1 1). Intracellular Ca-binding proteins such as gelsolin and calmodulin seem to be implicated in the regulation of PMN movement and metabolism.…”