SYNTHESIS OF PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE IN PERMEABILIZED NEUTROPHILS REGULATED BY RECEPTORS AND G-PROTEINS

Formylated Met-Leu-Phe (fMLP), platelet-activating factor (PAF), ATP, and various nonhydrolyzable guanine nucleotides stimulated accumulatio n of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4 5)P3) in int act human neutrophils. A protocol was devised to selectively inhibit t he capacity of the nucleotide-sensitive receptors to elicit accumulati on of PtdIns (3,4,5)P3. This enabled study of the regulation of phosph oinositide 3OH-kinase (PI3K) activities in permeabilized neutrophils f ree from interference due to activation of cell-surface nucleotide rec eptors. FMLP, PAF, and nonhydrolyzable GTP analogues stimulated an inc rease in the concentration, and rate of synthesis, of PtdIns(3,4,5)P3 in permeabilized neutrophils by increasing the rate of a PtdIns(4,5)P2 -directed PI3K-catalyzed reaction. A number of characteristics of thes e responses, including their relative sensitivities to inhibition by p ertussis toxin and guanosine 5'-beta(thio)diphosphate, suggested that fMLP and PAF increased this PI3K activity via the actions of heterotri meric G-proteins. Basal and guanosine 5'-gamma(thio)triphosphate/fMLP- stimulated increases in PI3K activity were resistant to changes in fre e calcium concentrations, staurosporine, acute treatment with phorbol esters, and evidently to permeabilization. This, in conjunction with o ther work, indicates that the PAF and fMLP-induced increase in PtdIns( 4,5)P2-directed PI3K activity is not being produced via activation of a currently defined G-protein regulated effector enzyme, or a protein tyrosine kinase coordinated mechanism of a type already known to regul ate PI3K activities.