REGULATION OF INTRACELLULAR FREE CALCIUM IN NORMAL MURINE KERATINOCYTES

Cultured normal murine keratinocytes maintain a basal cell phenotype in medium with a Ca2+ concentration of 0.05 mM and differentiate when exposed for 28-48 h to medium supplemented with extracellular Ca2+ >0.10 mM. Previous studies have documented Ca2+ activation of signaling pathways in the plasma membrane and tightly regulated cellular responses to small incremental changes in extracellular Ca2+. To determine if changes in free cytosolic calcium (Ca(i)) are associated with these early signaling events, digital image analysis of fura-2-loaded keratinocytes was used to measure Ca(i) in individual cells. Basal keratinocytes in 0.05 mM Ca2+ display a biphasic Ca(i) increase when exposed to >0.1 mM Ca2+ in serum-containing medium. These separate phases were controlled by different media components. Initial peak Ca(i) occurred rapidly (within 60 s), was transient (lasting <5 min), and resulted from release of 10-20% of total intracellular Ca2+ stores. Peak Ca(i) depended on serum concentration and was independent of extracellular Ca2+. This transient Ca(i) response was lost as keratinocytes differentiated. Plateau Ca(i) level was sustained (>24 h) and depended on extracellular Ca2+, but not serum. The magnitude of plateau Ca(i) increased incrementally following increases in extracellular Ca2+ as small as 0.02 mM. A similar biphasic Ca(i) increase was noted in cultures of murine dermal fibroblasts stimulated by 1.2 mM Ca2+ and serum. However, fibroblasts did not lose the serum response in high-Ca2+ medium, and plateau Ca(i) was not sensitive to small changes in extracellular Ca2+. The sensitivity of keratinocytes to stimulation of a prolonged Ca(i) plateau by small increases of extracellular Ca2+ may contribute to the induction of differentiation-specific genes and to terminal differentiation in this cell type.