title: Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O.
abstract: The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5'-[beta gamma-imido]-triphosphate greater than guanosine 5'-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.
authors.affiliation: Department of Experimental Pathology, School of Medicine, University College London.
authors.forename: J
authors.initials: J
authors.lastname: Stutchfield
authors.forename: S
authors.initials: S
authors.lastname: Cockcroft
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects*; D005966:Glucuronidase / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013301:Streptolysins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D013301:Streptolysins; D013873:Thionucleotides; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D005966:Glucuronidase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium