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+ greater than 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 (Cai) are associated with these early signaling events, digital image analysis of fura-2-loaded keratinocytes was used to measure Cai in individual cells. Basal keratinocytes in 0.05 mM Ca2+ display a biphasic Cai increase when exposed to greater than 0.1 mM Ca2+ in serum-containing medium. These separate phases were controlled by different media components. Initial peak Cai occurred rapidly (within 60 s), was transient (lasting less than 5 min), and resulted from release of 10-20% of total intracellular Ca2+ stores. Peak Cai depended on serum concentration and was independent of extracellular Ca2+. This transient Cai response was lost as keratinocytes differentiated. Plateau Cai level was sustained (greater than 24 h) and depended on extracellular Ca2+, but not serum. The magnitude of plateau Cai increased incrementally following increases in extracellular Ca2+ as small as 0.02 mM. A similar biphasic Cai 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 Cai was not sensitive to small changes in extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)