Intracellular calcium measurements of single human skin cells after stimulation with corticotropin-releasing factor and urocortin using confocal laser scanning microscopy

Using confocal laser scanning microscopy we investigated the Ca²⁺ distribution in single corticotropin releasing factor- and urocortin-stimulated human skin cells. The models tested included melanoma cells, neonatal melanocytes and keratinocytes, and immortalized HaCaT keratinocytes. The changes in intracellular Ca²⁺ signal intensities observed after stimulation of different cell types with corticotropin releasing factor and urocortin showed that: (1) the increase of intracellular Ca²⁺ concentration was caused by a Ca²⁺ influx(inhibition by EGTA); (2) this Ca²⁺ influx took place through voltage-activated Ca²⁺ ion channels (inhibition by d-cis-diltiazem,verapamil) and (3) cyclic nucleotide-gated ion channels were not involved in this process (no effect of Mg²⁺). The effects were also observed at very low peptide concentrations (10^-13 M) with no apparent linear correlation between peptide dosage and increase of fluorescence intensity,which implied co-expression of different corticotropin releasing factor receptor forms in the same cell. Immortalized (HaCaT) keratinocytes exhibited the strongest differential increases of a Ca²⁺ fluorescence after peptide-stimulation. Corticotropin releasing factor induced Ca²⁺flux into the cytoplasm, while urocortin Ca²⁺ flux into the nucleus with a remarkable oscillatory effect. The latter indicated the presence of an intracellular urocortin-induced signal transduction pathway that is unique to keratinocytes.