title: Effect of pertussis toxin and neomycin on G-protein-regulated polyphosphoinositide phosphodiesterase. A comparison between HL60 membranes and permeabilized HL60 cells.
abstract: The promyelocytic HL60 cell can be differentiated with dimethyl sulphoxide or dibutyryl cyclic AMP leading to the appearance of fMetLeuPhe receptors on the cell surface. G-protein-stimulated polyphosphoinositide phosphodiesterase (PPI-pde) activity was assessed in membranes prepared from both differentiated and non-differentiated HL60 cells. Both the extent of the response and the rank order of potency of the GTP analogues to stimulate PPI-pde activation (guanosine 5'-[gamma-thio]triphosphate (GTP[S]) greater than guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG) remains unchanged after differentiation with dimethyl sulphoxide. In comparison, differentiation by dibutyryl cyclic AMP leads to diminution of PPI-pde activity when stimulated by GTP[S] or fluoride, but not by millimolar concentrations of Ca2+. GTP[S]-stimulated PPI-pde in membranes is sensitive to the presence of Ca2+ (pCa 8-5). Pertussis-toxin pretreatment of intact HL60 cells leads to inhibition of both the secretory response and the formation of inositol phosphates when stimulated by fMetLeuPhe. In contrast, pertussis-toxin pretreatment has no effect on either GTP[S]- or fluoride-stimulated PPI-pde. Neomycin in a concentration-dependent manner inhibits both GTP[S] plus Ca2+ (pCa 5)-stimulated secretion and PPI-pde activation in streptolysin-O-permeabilized cells. The extent of PPI-pde activation in membranes compared with streptolysin-O-permeabilized cells reveals that the membrane preparation does not possess all the components that make up the inositide signalling system.
authors.affiliation: Department of Experimental Pathology, School of Medicine, University College London, U.K.
authors.forename: S
authors.initials: S
authors.lastname: Cockcroft
authors.forename: J
authors.initials: J
authors.lastname: Stutchfield
mesh_terms: D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007942:Leukemia, Experimental / Q000201:enzymology; D009355:Neomycin / Q000494:pharmacology*; D037342:Pertussis Toxin; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
chemical_list: D010566:Virulence Factors, Bordetella; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D019204:GTP-Binding Proteins; D009355:Neomycin; D002118:Calcium