Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes

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Abstract
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-tocoarse 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.
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