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

Abstract

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.

Corticotropin releasing hormone and the skin

“…CRH also stimulated IP 3 production in all the tested skin cells (melanocytes, keratinocytes and fibroblasts) (22,23). Taken together, the data (6,7,12,(22)(23)(24)(25)39) indicate that increases in intracellular Ca 2+ concentrations are induced by two mechanisms: a) stimulation of IP 3 , which activates release of Ca 2+ from intracellular stores; and b) direct coupling to voltage-activated Ca 2+ ion channels, whose opening leads to rapid cytosolic Ca 2+ influx. We also found that CRH induced the production of cAMP in skin cells expressing CRH-R1alpha.…”

“…In the case of normal human epidermal keratinocytes, the lack of CRH/ urocortin-induced production of cAMP (22) together with lack of effect of PKA inhibitors on CRH induced keratinocyte differentiation program (23) indicates poor coupling of CRHR1alpha to G s in these cells. In SKMEL-188 melanoma cells, which only express the CRHR1d isoform, the lack of coupling to cAMP with overt stimulation of Ca 2+ flux suggests that the CRH-R1d signal transduction pathway is only coupled to either voltage-activated Ca 2+ ion channels or PLC (12,15,22,25,39). 5.2.3.…”

“…Exposure of skin cells to CRH or related peptides results in the rapid activation of the CRH-R1alpha receptor, coupling to G alpha s followed by cAMP synthesis or coupling to G alpha q with activation of IP 3 signaling (6,22,23), similarly to other systems (1,2). Ligand activation of CRH-R1alpha in skin cells also induces Ca 2+ influx (24); this requires opening of voltage-activated Ca 2+ ion channels separate from the cyclic nucleotide-gated ion channels (25). Thus, in the same skin cell CRH-R1alpha can be coupled to different signal transduction pathways (22), in agreement with results obtained with COS cells transfected with CRH-R1alpha.…”

Differential expression of HPA axis homolog in the skin

“…In the human skin, CRH-R1 is predominantly expressed in the epidermis with CRH-R1α being the major isoform present (Slominski et al, 2004a). The cutaneous CRH-R1 is activated by CRH or urocortin and its intracellular signal transduction pathways are coupled to cAMP, IP3 or Ca (Fazal et al, 1998;Slominski et al, 2006b;Slominski et al, 1999c;Slominski et al, 2000e;Wiesner et al, 2003;Zbytek and Slominski, 2005). UV radiation and factors raising intracellular cAMP increase CRH-R1α expression and change the pattern of isoform expression Slominski et al, 2001).…”

Expression of Urocortin 2 and its Inhibitory Effects on Intracellular Ca2+ Via L-Type Voltage-Gated Calcium Channels in Rat Pheochromocytoma (PC12) Cells

“…However, our results are inconsistent with some previous reports. It was found that urocortin, another Urocortin 2 inhibits [Camember of CRF family, stimulated Ca 2 + uptake in skin cells (Wiesner et al, 2003). In addition, urocortin-induced increase of ANP and BNP secretion was completely abolished by a-helical CRF, a specific CRFR2 antagonist, and partially inhibited by diltiazem, another L-type VGCC blocker in cultured neonatal cardiomyocytes (Ikeda et al, 1998).…”