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

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Abstract
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 t1/2 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 10−6 m 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 pHi, 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 pHi. 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 pHi 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 pHi. 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.