Guanine nucleotides reduce the free calcium requirement for secretion of granule constituents from permeabilized human neutrophils

Abstract

Human neutrophils can be permeabilized with the cholesterol complexing agent digitonin and then induced to secrete lysosomal constituents by increases in free Ca2+ alone. In order of increasing requirements for Ca2+, vitamin B-12 binding protein, lysozyme and [beta]-glucuronidase were released. A variety of guanine nucleotides were examined with respect to their abilities to modulate this response. GTP, along with its analogues 5'-guanylylimidodiphosphate (Gpp[NH]p) and guanosine-5'-O-[3-thio]-triphosphate (GTP[[gamma]S]) decreased the Ca2+ requirements for secretion of all three granule constituents by one third to one order of magnitude. This synergy was dependent upon the concentration of guanine nucleotides employed. The effects of Gpp[NH]p could be blocked with the inactive derivative GDP[[beta]-S]. The active guanine nucleotides, particularly GTP, served as stimuli in their own right. At high concentrations of Ca2+ and GTP, degranulation was strikingly inhibited; inhibition was also achieved with high concentrations of guanylyl[[beta],[gamma]-methylene]diphosphate (Gpp[CH2]p). Both GDP and GMP were without any effect. When neutrophils were pretreated with pertussis toxin, granule discharge induced by fMet-Leu-Phe was almost completely blocked, as reported by others. If the neutrophils pretreated with pertussis toxin were then permeabilized with digitonin, the synergy between Ca2+ and the stimulatory guanine nucleotides was maintained. These data suggest the involvement of G-proteins in secretion induced by Ca2+; however, this response either uses a different G-protein or a different pool of G-proteins from those responses triggered by fMet-Leu-Phe.