Pettitt J M, Humphris D C, Barrett S P, Toh B H, van Driel I R, Gleeson P A
Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia.
J Cell Sci. 1995 Mar;108 ( Pt 3):1127-41. doi: 10.1242/jcs.108.3.1127.
The parietal cell of the gastric mucosa undergoes rapid morphological transformation when it is stimulated to produce hydrochloric acid. In chemically fixed cells, this process is seen as a reduction in number of cytoplasmic 'tubulovesicles' as the apical surface of the cell progressively invaginates to increase the secretory surface area. It is widely believed that the tubulovesicles represent stored secretory membrane in the cytoplasm of the unstimulated cell, which is incorporated into the apical membrane upon stimulation, because they share H+,K+-ATPase activity with the apical membrane. However, fusion of tubulovesicles with the apical membrane concomitant with parietal cell activation has never been convincingly demonstrated. We have used fast freeze-fixation and freeze-substitution to study stages of morphological transformation in these cells. Tubulovesicles were not seen in the cytoplasm of any of our cryoprepared cells. Instead, the cytoplasm of the unstimulated cell contained numerous and densely packed helical coils of tubule, each having an axial core of cytoplasm. The helical coils were linked together by connecting tubules, lengths of relatively straight tubule. Lengths of straight connecting tubule also extended from coils lying adjacent to the apical and canalicular surfaces and ended at the apical and canaliculus membranes. Immunogold labelling with alpha- and beta-subunit-specific antibodies showed that the gastric H+,K+-ATPase was localized to the membranes of this tubular system, which therefore represented the configuration of the secretory membrane in the cytoplasm of the unstimulated parietal cell. Stimulation of the cells with histamine and isobutylmethylxanthine lead to modification of the tubular membrane system, correlated with progressive invagination of the apical membrane. The volume of the tubule lumen increased and, as this occurred, the tight spiral twist of the helical coils was lost, indicating that tubule distension was accounted for by partial unwinding. This exposed the cores of cytoplasm in the axes of the coils as rod-shaped elements of a three-dimensional reticulum, resembling a series of microvilli in random thin sections. Conversely, treatment with the H2 antagonist cimetidine caused severe contraction of the tubular membrane system and intracellular canaliculi. Our results indicate that tubulovesicles are an artifact of chemical fixation; consequently, they cannot have a role in parietal cell transformation. From our findings we propose an alternative model for morphological transformation in the parietal cell. This model predicts cytoskeleton-mediated control over expansion and contraction of the tubular membrane network revealed by cryopreparation. The model is compatible with the localization of cytoskeletal components in these cells.
胃黏膜壁细胞在被刺激产生盐酸时会经历快速的形态转变。在化学固定的细胞中，随着细胞顶端表面逐渐内陷以增加分泌表面积，这个过程表现为细胞质中“微管泡”数量的减少。人们普遍认为，微管泡代表未受刺激细胞胞质中储存的分泌膜，在受到刺激时会整合到顶端膜中，因为它们与顶端膜共享H⁺，K⁺ - ATP酶活性。然而，微管泡与顶端膜的融合以及壁细胞的激活从未得到令人信服的证实。我们使用快速冷冻固定和冷冻置换来研究这些细胞形态转变的阶段。在我们所有的冷冻制备细胞的胞质中都未见到微管泡。相反，未受刺激细胞的胞质中含有大量紧密排列的螺旋状微管，每个微管都有一个细胞质轴芯。螺旋状微管通过连接微管连接在一起，连接微管是相对较直的微管段。直的连接微管段也从靠近顶端和小管表面的微管延伸出来，并终止于顶端和小管膜处。用α和β亚基特异性抗体进行免疫金标记显示，胃H⁺，K⁺ - ATP酶定位于这个管状系统的膜上，因此该管状系统代表未受刺激壁细胞胞质中分泌膜的结构。用组胺和异丁基甲基黄嘌呤刺激细胞会导致管状膜系统发生改变，这与顶端膜的逐渐内陷相关。微管腔的体积增加，随着这种情况发生，螺旋状微管的紧密螺旋扭曲消失，这表明微管扩张是由部分展开引起的。这使得微管轴芯中的细胞质以三维网状的杆状结构暴露出来，在随机薄切片中类似于一系列微绒毛。相反，用H₂拮抗剂西咪替丁处理会导致管状膜系统和细胞内小管严重收缩。我们的结果表明，微管泡是化学固定的假象；因此，它们在壁细胞转变中不可能起作用。根据我们的发现，我们提出了壁细胞形态转变的另一种模型。该模型预测由冷冻制备揭示的管状膜网络的扩张和收缩受细胞骨架介导的控制。该模型与这些细胞中细胞骨架成分的定位是一致的。
