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JCS ePress online publication date 12 Feb 2003
doi: 10.1242/jcs.00301

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Research Article

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


Burkhard Wiesner, Birgit Roloff, Klaus Fechner, and Andrzej Slominski*
* Author for correspondence (e-mail: aslominski{at}utmem.edu)

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.
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