Calcium regulation of phosphatidyl inositol turnover in macrophage activation by formyl peptides

Abstract

Stimulation by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP) of the guinea pig alveolar macrophage gives rise to transient production of superoxide anion (O₂⁻). Components of the phosphatidyl inositol (PI) cycle (phosphatidic acid) (PA), phosphatidyl inositol-4,5-bisphosphate (TPI) and phosphatidyl inositol-4-phosphate (DPI) were monitored using ³²P in order to examine the possible association of this cycle with the FNLLP-stimulated production of O₂⁻. Macrophage stimulation by FNLLP led to an increased flux of metabolites through the PI cycle. The level of ³²P label in both TPI and DPI rapidly decreased upon exposure to FNLLP, followed by a 5-min period during which the ³²P label in TPI and DPI approached prestimulated levels. During this period, there was a fivefold increase in ³²P-PA. It is suggested that diacylglycerol (DAG) is the O₂⁻ production. The importance of continued cycling of PI in the stimulated mechanism is demonstrated by the inhibition by LiCl of the extent, but not the initial rate, of both O₂⁻ production and the formation of ³²P-PA upon peptide stimulation after 1-h preincuexamined. It has previously been demonstrated that intracellular availability of calcium can influence the rate and extent of O₂⁻ production. In cells preloaded with quin-2, which acts as a high-affinity sink for calcium in the cytosol, the initial rate of FNLLP-stimulated O₂⁻ production is inhibited in low (10 μM) extracellular calcium medium. High extracellular calcium (1 mM) completely reverses this inhibition and also significantly extends the time course of O₂⁻ production in both quin-2 and control cells (Stickle et al., 1984). In parallel with these effects on O₂⁻ production, varying calcium conditions is demonstrated to influence the rate and extent of PA formation. These same calcium conditions were found to have little or no effect on the initial unstimulated levels of TPI, DPI, and PA. These results indicate that the influence of an intracellular pool of calcium on O₂⁻ production may be via its influence on stimulated PI turnover.