title: A reassessment of guanine nucleotide effects on catecholamine secretion from permeabilized adrenal chromaffin cells.
abstract: The role of guanine nucleotides in catecholamine secretion was investigated in alpha-toxin-permeabilized chromaffin cells. The stable GTP analogues, GTP-gamma-S (guanosine 5'-(gamma-thio)triphosphate) and GMP-PNP (guanosine 5'-(beta,gamma-imido)triphosphate), potentiated calcium-evoked catecholamine release in a dose-dependent manner. This effect was reversed by GDP-beta-S (guanosine 5'-(beta-thio)diphosphate) indicating that a GTP-binding protein plays a modulatory role in the calcium-dependent secretory process in chromaffin cells. Calcium and the phosphorylating nucleotide ATP were both necessary for secretion, even in the presence of GTP analogues, suggesting that the activation of a GTP-regulatory protein alone does not trigger exocytosis in these cells. TPA (12-O-tetradecanoylphorbol-13-acetate), a direct activator of protein kinase C, was found to mimic the effects of the GTP analogues, inducing a dose-dependent potentiation of the calcium-evoked release in alpha-toxin-permeabilized cells. Treatment of the permeabilized cells with sphingosine, a potent inhibitor of protein kinase C, completely abolished the stimulatory effects of both TPA and GTP-gamma-S. Moreover, long term incubation of chromaffin cells with TPA, a treatment which depletes cells of protein kinase C activity, suppressed the stimulatory effects of GTP-gamma-S. Protein kinase C is activated when it becomes membrane-bound in the presence of calcium and diacylglycerol; here, GTP-gamma-S was found to enhance the calcium-induced translocation of protein kinase C to membranes in alpha-toxin-permeabilized cells. These results suggest that guanine nucleotides modulate secretion by activating protein kinase C-linked events in chromaffin cells. Furthermore, the potentiation of calcium-induced secretion in alpha-toxin-permeabilized cells following activation of protein kinase C either directly with TPA or indirectly with GTP analogues provides additional support for the concept that protein kinase C may exert a positive control directly on the intracellular exocytotic machinery.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale Unité U44, Strasbourg, France.
authors.forename: M F
authors.initials: MF
authors.lastname: Bader
authors.forename: J M
authors.initials: JM
authors.lastname: Sontag
authors.forename: D
authors.initials: D
authors.lastname: Thiersé
authors.forename: D
authors.initials: D
authors.lastname: Aunis
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002463:Cell Membrane Permeability; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006460:Hemolysin Proteins; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology; D007700:Kinetics; D009498:Neurotoxins / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D013110:Sphingosine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D001427:Bacterial Toxins; D006150:Guanine Nucleotides; D006460:Hemolysin Proteins; D007295:Inositol Phosphates; D009498:Neurotoxins; D013873:Thionucleotides; C021091:staphylococcal alpha-toxin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D009638:Norepinephrine