Calcium release activated calcium entry in a human skin derived cell line (HaCaT)

Using isolated cells from a spontaneously immortalized human keratinocyte cell line (HaCaT) loaded with Fura-2 the regulation of intracellular Ca2+ concentration ([Ca2+]i) was studied. The continuous presence of ATP induced a biphasic response in high external Ca2+. The first component reflected the release of calcium from intracellular stores since it was present after the removal of external calcium with ethylene-glycol-bis-N,N,N',N'-tetraacetic acid (EGTA). The second phase of [Ca2+]i increase was not detectable in the absence of external calcium and raising the extracellular [Ca2+] increased the rate of rise in [Ca2+]i suggesting that it was influenced by the external environment. Furthermore, after adenosine triphosphate (ATP) had emptied the intracellular store in a calcium-free milieu, the elevation of external Ca2+ induced a secondary increase in [Ca2+]i that did not require the presence of ATP. Depleting the intracellular calcium store with a Ca-ATP-ase inhibitor (cyclopiasonic acid, 10 microM) also induced calcium entry. The depletion induced calcium entry was inhibited by econazole (100 microM). These findings indicate the presence of a calcium release activated calcium influx pathway in HaCaT keratinocytes.