title: Exocytosis in electropermeabilized neutrophils. Responsiveness to calcium and guanosine 5'-[gamma-thio]triphosphate.
abstract: Electropermeabilized neutrophils were used to study the exocytotic response in rabbit neutrophils. Enzyme release from electropermeabilized neutrophils could be induced by elevating the Ca2+ concentration. Ca(2+)-induced secretion was significantly enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a concentration-dependent manner. The effect of GTP[S] could be blocked by guanosine 5'-[beta-thio]diphosphate (GDP[S]) and was not affected by pertussis toxin. GTP[S] did not induce enzyme release in the absence of Ca2+. Induction of an exocytotic response did not require addition of ATP. However, neutrophils permeabilized in the absence of ATP became refractory to stimulation due to a reduction in their affinity for Ca2+. Responsiveness to the effectors Ca2+ or Ca2+ + GTP[S] could be prolonged or restored by ATP. ATP was not the only agent that prolonged responsiveness; other nucleotides and inorganic phosphates were also effective. The protein kinase C inhibitors staurosporine and 1-O-hexadecyl-2-methyl-sn-glycerol did not inhibit exocytosis and had only a small effect on the prolongation and restoration of responsiveness by ATP. A hypothesis is presented suggesting that the loss of responsiveness is caused by dephosphorylation and that the restoration or prolongation of responsiveness is not mediated by protein kinase C. It is possible that an as yet unidentified Ca(2+)-binding protein is dephosphorylated, resulting in a decrease in Ca2+ affinity.
authors.affiliation: Department of Medical Biochemistry, University of Leiden, The Netherlands.
authors.forename: G J
authors.initials: GJ
authors.lastname: Boonen
authors.forename: J
authors.initials: J
authors.lastname: van Steveninck
authors.forename: T M
authors.initials: TM
authors.lastname: Dubbelman
authors.forename: P J
authors.initials: PJ
authors.lastname: van den Broek
authors.forename: J G
authors.initials: JG
authors.lastname: Elferink
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004558:Electric Stimulation; D005089:Exocytosis; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009711:Nucleotides / Q000502:physiology; D010710:Phosphates / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011817:Rabbits; D013301:Streptolysins / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D009711:Nucleotides; D010710:Phosphates; D013301:Streptolysins; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium