Koh D S, Moody M W, Nguyen T D, Hille B
Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195-7290, USA.
J Gen Physiol. 2000 Oct;116(4):507-20. doi: 10.1085/jgp.116.4.507.
We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.
我们探讨了上皮细胞中胞吐作用及其调节机制是否与可兴奋细胞中的机制具有共同特征。将培养的犬胰腺导管上皮细胞装载可氧化的神经递质多巴胺或血清素，然后通过碳纤维安培法检测胞吐过程中这些外源性分子的释放。装载后的细胞表现出自发性胞吐作用，这可能代表组成型膜转运。单个囊泡融合诱导的量子安培事件具有快速的起始和衰减，类似于肾上腺嗜铬细胞和分泌血清素的水蛭神经元中的事件。量子事件之前常常有一个“足”，推测是递质通过瞬时融合孔泄漏，这表明这些细胞类型共享一种共同的融合机制。与神经元和内分泌细胞一样，使用离子霉素升高细胞质Ca(2+)可诱发上皮细胞中的胞吐作用。与神经元不同的是，高渗溶液会降低上皮细胞中的胞吐作用，并且偶尔会观察到由许多并发量子事件组成的巨大安培事件。已知能增加细胞内cAMP的试剂，如福斯可林、肾上腺素、血管活性肠肽或8-溴-cAMP，会增加胞吐作用速率。福斯可林的作用被蛋白激酶A的特异性拮抗剂cAMPS的Rp-异构体抑制，而PKA的特异性激动剂Sp-异构体则诱发胞吐作用。因此，PKA是cAMP的下游效应器。最后，佛波醇-12-肉豆蔻酸酯-13-乙酸酯激活蛋白激酶C也会增加胞吐作用。无活性类似物4α-佛波醇-12,13-十二烷酸酯不能模拟PMA的作用，且其作用被PKC拮抗剂双吲哚马来酰亚胺I阻断。福斯可林或PMA的作用不需要细胞内Ca(2+)升高。总之，上皮细胞中的胞吐作用可直接由Ca(2+)、PKA或PKC刺激，并且由与神经元和内分泌细胞中类似的物理机制介导。
