Exocytosis from permeabilized lactating mouse mammary epithelial cells. Stimulation by Ca2+ and phorbol ester, but inhibition of regulated exocytosis by guanosine 5'-[gamma-thio]triphosphate.
Lactating mouse mammary epithelial cells secrete large amounts of milk protein via constitutive or regulated exocytotic pathways. Secretion through both pathways was quantified by assaying the release of [35S]methionine-labelled trichloroacetic acid-precipitable proteins from digitonin-permeabilized secretory acini isolated from mammary glands of 10-day-post-partum lactating mice. Protein secretion from the isolated permeabilized cells was either Ca(2+)-dependent (regulated) or Ca(2+)-independent (constitutive). In both cases there was a requirement for ATP. Addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) caused a marked increase in the percentage protein secretion from the cells in a Ca(2+)-independent manner. However, the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused a partial inhibition of Ca(2+)-dependent exocytosis, while having no significant effect on Ca(2+)-independent exocytosis. Thus the GTP[S] is exerting its effect on the regulated pathway at a site subsequent to protein sorting and packaging into secretory vesicles at the trans-Golgi network.
Turner
M D
MD
Physiological Laboratory, University of Liverpool, U.K.
Wilde
C J
CJ
Burgoyne
R D
RD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects*; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D007700:Kinetics; D007774:Lactation / Q000502:physiology*; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D011506:Proteins / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D011506:Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium