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First published online 12 February 2003
doi: 10.1242/jcs.00301

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

¹ Institute of Molecular Pharmacology, D-13125 Berlin, Germany
² ASCA Angewandte Synthesechemie Adlershof GmbH, 12489 Berlin, Germany
³ Department of Pathology, University of Tennessee Health Science Center, Memphis, TN 38163, USA

View larger version (45K): [in a new window]   Fig. 1. Relative cytosolic fluorescence intensities of the Ca2+ indicators (mean and s.d.; control-fluorescence intensity before the application of the peptide=100%) at the final values of the time-dependent response (see Fig. 6) in HaCaT keratinocytes (A,B), neonatal normal keratinocytes (C,D), human melanoma cells (E,F) and neonatal normal melanocytes (G,H) after stimulation with different concentrations of CRF (A,C,E,G) and with different extracellular mediums and preincubation regimes (B,D,F,H). *Statically significant differences (P<0.05, unpaired t-test) from the control; +, statistically significant differences (P<0.05, unpaired t-test) from the effect after stimulation with 10-10 M CRF. Number of cells recorded (n) is shown in the respective columns.

View larger version (30K): [in a new window]   Fig. 2. Relative cytosolic fluorescence intensities of the Ca2+ indicators (mean and s.d.; control=fluorescence intensity before the application of the peptide=100%) at the maximal values of the time-dependent response (Fig. 6) in HaCaT keratinocytes (A,B) and human melanocytes (C,D) after stimulation with different concentrations of URC (A,C) and using different extracellular medium and preincubation conditions (B,D). *Statistically significant differences (P<0.05, unpaired t-test) from the control; +, statistically significant differences (P<0.05, unpaired t-test) from the effect after stimulation with 10-10 M URC. Number of cells recorded (n) is shown in the respective columns.

View larger version (19K): [in a new window]   Fig. 3. Relative cytosolic fluorescence intensities of the Ca2+ indicators (mean and s.d.; control=fluorescence intensity before the application of the peptide=100%) at maximal (URC) or final (CRF) values of the time-dependent response (see Fig. 6) in human melanocytes after preincubation with the specific antagonist -helical-CRF(9-41) (2) and stimulation with CRF (shaded, C) and URC (unshaded, U). *Statistically significant differences (P<0.05, unpaired t-test) from the control (control=100%); +, statistically significant differences (P<0.05, unpaired t-test) from the effects after stimulation with either peptide in the absence of antagonist (1). Number of cells recorded (n) is shown in the respective columns.

View larger version (25K): [in a new window]   Fig. 4. Ratio of the relative fluorescence intensities (nucleus/cytosol; mean and s.d.) of the Ca2+ indicators at maximal (URC) or final (CRF values of the time-dependent response (see Fig. 6) in HaCaT keratinocytes (4A) and human malignant melanocytes (4B) after stimulation with URC (U, filled bars) and CRF (C, unfilled bars) and in neonatal keratinocytes (K, unfilled) and normal melanocytes (NM, filled) after stimulation with CRF (4C). *Statistically significant differences (P<0.05, unpaired t-test) between the ratios after stimulation with URC and with CRF; +, statistically significant differences (P<0.05, unpaired t-test) between the ratios in keratinocytes and normal melanocytes. Number of cells recorded (n) is shown in the respective columns.

View larger version (103K): [in a new window]   Fig. 5. Time galleries of fluorescence images of the Ca2+ indicators in HaCaT after stimulation with CRF (10-7 M, left panel) and URC (10-7 M, right panel). The intensity range (8 bit) is indicated below the images galleries.

View larger version (31K): [in a new window]   Fig. 6. (A,B) Time course of the relative fluorescence intensities (unbroken line, cytosol; broken line, nucleus) of the Ca2+ indicators in two single HaCaT cells after stimulation with CRF (A, 10-7 M) and URC (B, 10-7 M). (C) Dose-dependent frequency of the oscillations in HaCaT-cells after stimulation with URC (mean and s.d.). Number of cells recorded (n) is shown in the respective columns. (D) Dose-dependent numbers of HaCaT cells with oscillations after stimulation with URC (percentage of total number of cells recorded). Number of cells recorded with oscillations (n) is shown in the respective columns.