Regulatory role of the GTP-binding protein, G(o), in the mechanism of exocytosis in adrenal chromaffin cells.
To elucidate the possible involvement of GTP-binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and antibodies against the G proteins (Gi and G(o)) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine-evoked catecholamine release. High K(+)-, histamine-, or gamma-aminobutyric acid-evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10(-4) M acetylcholine was decreased significantly in PTX-treated cells. In digitonin-permeabilized cells, PTX pretreatment resulted in a decrease of the half-maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP-gamma-S (a nonhydrolyzable GTP analogue) inhibited Ca(2+)-dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the alpha subunit of the PTX-sensitive G protein, G(o). Treatment with similar antibodies against the alpha subunit of Gi had no effect. These findings suggest that G(o) directly controls the Ca(2+)-triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.
Ohara-Imaizumi
M
M
Life Science Institute, Sophia University, Tokyo, Japan.
Kameyama
K
K
Kawae
N
N
Takeda
K
K
Muramatsu
S
S
Kumakura
K
K
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D015151:Immunoblotting; D037342:Pertussis Toxin; D011188:Potassium / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002395:Catecholamines; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005680:gamma-Aminobutyric Acid; D006632:Histamine; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D004072:Digitonin; D011188:Potassium; D002118:Calcium