Exocytosis from permeabilized lactating mouse mammary epithelial cells. Stimulation by Ca2+ and phorbol ester, but inhibition of regulated exocytosis by guanosine 5′-[γ-thio]triphosphate

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Abstract
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.