GTP-induced fusion of isolated pancreatic microsomal vesicles is increased by acidification of the vesicle lumen.
Using the 'fusogen' polyethyleneglycol (PEG), Dawson et al. have concluded that both guanosine triphosphate (GTP)-induced calcium efflux and the enhancement of IP3-promoted calcium release from rat liver microsomal vesicles could be attributed to a GTP-dependent vesicle fusion. We have studied GTP-induced fusion of microsomal vesicles from rat exocrine pancreas using light scatter and fluorescence dequenching methods. In the presence of PEG (3%), GTP (10 microM) induced a decrease in light scatter and an increase in fluorescence in the fluorescence dequenching assay (GTP-effect) indicating fusion of the vesicles. Guanosine 5'-O-(3-thiotriphosphate) (10 microM) had no effect on its own and inhibited the GTP-induced signals. Preincubation of the vesicles with adenosine triphosphate (ATP) (4 mM) increased the GTP-effect by 80%, whereas bafilomycin B1, a specific inhibitor of vacuolar type H(+)-ATPases, and the protonophore CCCP (10 microM) inhibited only the ATP-dependent part of the GTP-effect. Inhibitors of the vacuolar type H(+)-ATPase, which are also SH-alkylating reagents such as N-ethylmaleimid (100 microM) and the tyrosine-, cysteine- and lysine-reactive reagent 7-chloro-4-nitrobenz-2-exa-1,3-diazole (10 microM), abolished the GTP-effect in the absence or presence of ATP. We conclude that GTP induces fusion of pancreatic microsomes which is increased by an H+ gradient established by a vacuolar type H(+)-ATPase.
Hampe
W
W
Max-Planck-Institut für Biophysik, Frankfurt (Main), FRG.
Zimmermann
P
P
Schulz
I
I
D000165:Acridine Orange; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002459:Cell Fusion / Q000187:drug effects*; D005453:Fluorescence; D006160:Guanosine Triphosphate / Q000494:pharmacology*; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008954:Models, Biological; D010179:Pancreas / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D051381:Rats
D006859:Hydrogen; D006160:Guanosine Triphosphate; D000251:Adenosine Triphosphatases; D000165:Acridine Orange