Phorbol ester induces rapid actin assembly in neutrophil leucocytes independently of changes in [Ca2+]i and pHi

Summary The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), at nanomolar concentrations, induces rapid (t_1/2 approximately 30 s) protrusion of multiple petal-shaped lamellae by neutrophil leucocytes. Lamellae are richly endowed with actin filaments as determined by the localization of rhodamine-phalloidin, suggesting extensive assembly at the cell cortex. Direct measurement of the proportion of total cell actin which is polymerized, by using a deoxyribonuclease I inhibition assay, indicates that the proportion of polymerized actin approximately doubles, and that assembly initiated by 30nm TPA occurs with no obvious lag phase and with a t_1/2 of about 30 s. A half-maximal response was induced at 2nm TPA. Since both actin assembly and protrusion of lamellae are completely inhibited by 10^–6m cytochalasin D, protrusion of lamellae is presumably dependent on actin filament assembly.To examine whether TPA induces actin assembly via changes in [Ca²⁺]_i or pH_i, these parameters were monitored in cells loaded with the fluorescent indicators quin2 and quene1 respectively. Addition of TPA caused no change in [Ca²⁺]_i but a biphasic change in pH_i. To examine further the potential role of ionic changes in regulation of actin assembly, the morphological responses of cells to TPA were monitored in severely Ca²⁺-depleted cells, or cells in which pH_i had been experimentally raised or lowered by simultaneous additions of a weak base (NH₄Cl) or weak acid (CH₃COONa) respectively. The protrusion of lamellae induced by TPA was completely unaffected by these experimental manipulations indicating that TPA can directly regulate actin assembly, and hence morphology, by mechanisms essentially independent of [Ca²⁺]_i and pH_i. Since TPA is believed to mimic diacylglycerol, which is putatively generated by cleavage of phosphatidylinositol-4, 5-bisphosphate in a number of cellular responses involving actin filament assembly, these results implicate diacylglycerol, either acting directly, or via protein kinase C-mediated phosphorylation, as a common step in the receptor-mediated regulation of cortical actin filament assembly in cells.