Pillai S, Menon G K, Bikle D D, Elias P M
Department of Dermatology, Veterans Administration Medical Center, San Francisco, California.
J Cell Physiol. 1993 Jan;154(1):101-12. doi: 10.1002/jcp.1041540113.
Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle-bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo-1 and ion-capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80-120 nM, whereas Cai of differentiated keratinocytes was 200-300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60-160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium-containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium-containing precipitates appeared as fine-to-coarse electron-dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle-bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures.
钙在调节培养的角质形成细胞的生长和分化过程中起着至关重要的作用。然而，这种调节的机制尚不清楚。先前的研究表明，细胞内游离钙（Cai）随着角质形成细胞的分化而增加。在本研究中，为了评估胞质游离钙和细胞器结合钙在角质形成细胞分化中的作用，我们分别利用荧光探针indo-1和离子捕获细胞化学技术，对角质形成细胞中的钙池进行了定量和定位。未分化角质形成细胞的Cai为80 - 120 nM，而分化角质形成细胞的Cai为200 - 300 nM，这取决于分化程度。未分化集落中单个细胞的Cai是异质的（60 - 160 nM），较大的细胞显示出较高的Cai。使用细胞化学技术在分层角质形成细胞培养物的不同层中，在细胞内含钙沉淀物中也观察到了异质性。钙沉淀物在较低的细胞层中丰富，向顶端逐渐减少，最上层没有沉淀物。含钙沉淀物在质膜上、胞质溶胶内、线粒体、细胞核和液泡细胞器中表现为从细到粗的电子致密颗粒。虽然在细胞外钙存在的情况下离子霉素增加了细胞内钙沉淀物的数量，但EGTA从细胞器中去除了钙沉淀物。与完整表皮不同，角质形成细胞没有细胞外钙储存库。通过三价镧（La）结合可视化的假定钙结合位点，在基底样细胞的细胞膜和桥粒上丰富，但在上层细胞层中减少。这些研究揭示了游离离子钙（通过荧光技术测定）和细胞器结合钙（通过细胞化学技术测定）分布的差异。在完整表皮和培养的表皮细胞之间，在钙定位方面也观察到了显著差异。培养的角质形成细胞中钙的定位模式可能反映了这些细胞的增殖状态，如在银屑病表皮中，和/或这些浸没培养物中缺乏正常的渗透屏障。
