We studied an involvement of various cellular
ceramide pools in signaling of immunoreceptor FcγII
(FcγRII). The cell surface ceramide level was
assessed by a technique based on binding of
ceramide probes to intact cells. Total cellular ceramide
was estimated by radioactive measurements. The
activity of sphingomyelinases was measured by NBDceramide
release while immunoprecipitation and
immunoblotting were applied to analyze protein
tyrosine phosphorylation. A complex pattern of protein
phosphorylation was found to accompany FcγRII
activation and the phosphorylation was either
diminished by imipramine or increased by B13,
modulators of acid sphingomyelinase and acid
ceramidase activity. The effects of the drugs on the
phosphorylation of FcγRII and NTAL were prominent and correlated with a reduction of the cell surface
ceramide production by imipramine and an
augmentation of the ceramide generation by B13.
The ceramide generation followed activation of acid
sphingomyelinase and preceded that of neutral
sphingomyelinase. The level of cell surface ceramide
was additionally elevated by exogenous bacterial
sphingomyelinase, but only at later stages of the
receptor activation. The total mass of ceramide was
diminished in the course of receptor activation
pointing to an engagement of enzymes metabolizing
ceramide. The data indicate that FcγRII activates
enzymes of the sphingomyelin cycle which affect
various sphingomyelin/ceramide pools in a cell.