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 Ca(2+) 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 Ca(2+) signal intensities observed after stimulation of different cell types with corticotropin releasing factor and urocortin showed that: (1) the increase of intracellular Ca(2+) concentration was caused by a Ca(2+) influx (inhibition by EGTA); (2) this Ca(2+) influx took place through voltage-activated Ca(2+) ion channels (inhibition by d-cis-diltiazem, verapamil) and (3) cyclic nucleotide-gated ion channels were not involved in this process (no effect of Mg(2+)). 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 Ca(2+) fluorescence after peptide-stimulation. Corticotropin releasing factor induced Ca(2+) flux into the cytoplasm, while urocortin Ca(2+) 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.
Wiesner
Burkhard
B
Institute of Molecular Pharmacology, D-13125 Berlin, Germany.
Roloff
Birgit
B
Fechner
Klaus
K
Slominski
Andrzej
A
D001683:Biological Clocks / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008544:Melanocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism; D011930:Reaction Time / Q000187:drug effects / Q000502:physiology; D018019:Receptors, Corticotropin-Releasing Hormone / Q000187:drug effects / Q000378:metabolism; D013312:Stress, Physiological / Q000378:metabolism; D054832:Urocortins
D002121:Calcium Channel Blockers; D002614:Chelating Agents; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D003346:Corticotropin-Releasing Hormone; D002118:Calcium