Abstract. Lactating mammary epithelial cells secrete high levels of caseins and other milk proteins. The extent to which protein secretion from these cells occurs in a regulated fashion was examined in experiments on secretory acini isolated from the mammary glands of lactating mice at 10 d postpartum . Protein synthesis and secretion were assayed by following the incorporation or release, respectively, of [35S]methionine-labeled TCAprecipitable protein. The isolated cells incorporated [35S]methionine into protein linearly for at least 5 h with no discernible lag period. In contrast, protein secretion was only detectable after a lag of -1 h, consistent with exocytotic secretion of proteins immediately after passage through the secretory pathway and package into secretory vesicles . The extent of protein secretion was unaffected by the phorbol ester PMA, 8-bromo-CAMP, or 8-bromo-cGMP but was doubled by the Call ionophore ionomycin . In a pulse-label protocol in which proteins were prelabeled for 1 h before P ROTE1N secretion by exocytosis occurs in many different cell types and is the mechanism involved in the secretion ofa wide range of molecules. Exocytotic secretion occurs either immediately after synthesis and packaging of proteins into secretory vesicles (constitutive exocytosis) or, following variable periods ofstorage in secretory vesicles or granules, in response to cell activation (regulated exocytosis) (Burgess and Kelly, 1987). Lactating mammary epithelial cells actively secrete large quantities of caseins and other milk proteins by an exocytotic mechanism (Franke et al., 1976;Linzell and Peaker, 1971;Saacke and Heald, 1974) in an apparently constitutive fashion . The possibility that mammary epithelial cells secrete caseins via a regulated secretory pathway has not been rigorously examined and much is still to be learned about the cell biology of secretion from this cell type.The signaling pathways involved in the differentiation of mammary epithelial cells to a secretory phenotype have been examined in detail, but less is known about the factors that may regulate exocytosis in lactating cells . Relatively little work has been carried out in recent years on the effect of sec-

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.