Turner M D, Rennison M E, Handel S E, Wilde C J, Burgoyne R D
Department of Physiology, University of Liverpool, United Kingdom.
J Cell Biol. 1992 Apr;117(2):269-78. doi: 10.1083/jcb.117.2.269.
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 TCA-precipitable 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 approximately 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 Ca2+ ionophore ionomycin. In a pulse-label protocol in which proteins were prelabeled for 1 h before a chase period, constitutive secretion was unaffected by depletion of cytosolic Ca2+ but ionomycin was found to give a twofold stimulation of the secretion of presynthesized protein in a Ca(2+)-dependent manner. Ionomycin was still able to stimulate protein secretion after constitutive secretion had terminated. These results suggest that lactating mammary cells possess both a Ca(2+)-independent constitutive pathway and a Ca(2+)-activated regulatory pathway for protein secretion. The same proteins were secreted by both pathways. No ultrastructural evidence for apocrine secretion was seen in response to ionomycin and so it appears that regulated casein release involves exocytosis. Ionomycin was unlikely to be acting by disassembling the cortical actin network since cytochalasin D did not mimic its effects on secretion. The regulated pathway may be controlled by Ca2+ acting at a late step such as exocytotic membrane fusion.
泌乳期乳腺上皮细胞分泌高水平的酪蛋白和其他乳蛋白。在产后10天从泌乳期小鼠乳腺分离出的分泌腺泡的实验中，研究了这些细胞的蛋白质分泌在何种程度上以一种受调控的方式发生。通过分别追踪[35S]甲硫氨酸标记的三氯乙酸可沉淀蛋白的掺入或释放来测定蛋白质合成和分泌。分离出的细胞将[35S]甲硫氨酸线性掺入蛋白质中至少5小时，没有明显的延迟期。相比之下，蛋白质分泌在大约1小时的延迟后才检测到，这与蛋白质在通过分泌途径并包装成分泌小泡后立即进行胞吐分泌一致。蛋白质分泌的程度不受佛波酯PMA、8-溴-cAMP或8-溴-cGMP的影响，但被Ca2+离子载体离子霉素加倍。在一个脉冲标记方案中，蛋白质在追踪期之前预标记1小时，组成型分泌不受胞质Ca2+耗竭的影响，但发现离子霉素以Ca(2+)依赖的方式对预合成蛋白质的分泌有两倍的刺激作用。在组成型分泌终止后，离子霉素仍然能够刺激蛋白质分泌。这些结果表明，泌乳期乳腺细胞具有蛋白质分泌的Ca(2+)非依赖组成型途径和Ca(2+)激活调控途径。两条途径分泌相同的蛋白质。没有观察到离子霉素刺激下顶浆分泌的超微结构证据，因此似乎受调控的酪蛋白释放涉及胞吐作用。离子霉素不太可能通过分解皮质肌动蛋白网络起作用，因为细胞松弛素D不能模拟其对分泌的影响。受调控途径可能由Ca2+在诸如胞吐膜融合等后期步骤起作用来控制。
