1. α-Melanocyte-Stimulating Hormone, MSH 11–13 KPV and Adrenocorticotropic Hormone Signalling in Human Keratinocyte Cells 
Richard J. Elliott, Marika Szabo, Mark J. Wagner, E. Helen Kemp, Sheila MacNeil, John W. Haycock 
Journal of Investigative Dermatology 
Volume 122, Issue 4, Pages (April 2004)
DOI: /j X x
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Acute intracellular calcium responses measured in Fura2-AM-loaded human HaCaT keratinocytes and normal human keratinocytes detected by optical fluorimetry: (a) HaCaT keratinocytes stimulated with ACTH, ACTH 1–17, α-MSH, KPV, or KP-D-V alone (10−9 M) did not lead to acute increases in intracellular calcium; (b and c) co-stimulation of HaCaT (b) and normal human keratinocytes (c) with PIA (10−5 M) and MSH/ACTH peptides lead to rapid and acute intracellular calcium release, returning to baseline values typically within 60 s. PIA was always added at the same time as the peptides as indicated by position of individual vertical arrows. α-MSH, α-melanocyte-stimulating hormone; KPV, L-Lys-L-Pro-L-Val; KP-D-V, L-Lys-L-Pro-D-Val; ACTH, adrenocorticotropin hormone; PIA, N6-(L-2-phenyl isopropyl) adenosine.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
An acute intracellular calcium response in Fura2-AM-loaded HaCaT keratinocytes detected by fluorescence microscopy time-lapse imaging. In this example, HaCaT cells were co-stimulated with PIA (10−5 M) and α-melanocyte-stimulating hormone (α-MSH) (10−12 M) at the point indicated by the vertical arrow: (a) illustrates the complete cycle of intracellular calcium release and recovery for an individual cell; (b) intracellular calcium signalling for a population of HaCaT cells in a single field-of-view. Images were captured every 2 s and artificially colored using graphical software.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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4. Figure 3
Acute intracellular calcium responses in Fura2-AM-loaded wild-type CHO-K1 cells and CHO-K1 cells stably transfected with the MC-1 receptor, detected by optical fluorimetry. No acute increases in intracellular calcium were observed for wild-type CHO-K1 cells. CHO-K1 cells transfected with the MC-1 receptor demonstrate acute increases in intracellular calcium responses when co-stimulated with α-MSH, KPV, KP-D-V, and PIA at 10−5 M. PIA was always added at the same time as the peptides as indicated by position of individual vertical arrows. In this illustration all peptides were used at 10−8 M. α-MSH, α-melanocyte-stimulating hormone; KPV, L-Lys-L-Pro-L-Val; KP-D-V, L-Lys-L-Pro-D-Val; ACTH, adrenocorticotropin hormone; PIA, N6-(L-2-phenyl isopropyl) adenosine.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
The effect of elevating intracellular calcium on the cytokine (tumor necrosis factor (TNF-α))-stimulated activation of nuclear factor-κB (NF-κB) in HaCaT keratinocytes. (a) NF-κB was measured according to p65 immunolabelling and assessment of intracellular localization. Light bars=no ionomycin, dark bars=ionomycin present at 10−6 M. Ionomycin had no effect on control-unstimulated levels of NFκB activation. Cells stimulated with 200 U per mL of TNF-α showed NF-κB activation, which was inhibited by ionomycin compared to TNF-α alone (p=0.026). α-MSH (at 10−7 M) did not inhibit TNF-α-stimulated NF-κB activity but co-stimulation with ionomycin (10−6 M) and α-melanocyte-stimulating hormone (α-MSH) (10−7) significantly inhibited NF-κB (p=0.005) compared to TNF-α and α-MSH alone (p=0.026) and TNF-α alone. (b) Intracellular calcium response of Fura2-AM-loaded HaCaT keratinocytes to addition of ionomycin (at 10−6 M, used for NF-κB measurement) detected by optical fluorimetry. NS, not significant; *, p<0.05; **>, p<0.01; ***>, p<0.001.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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6. Figure 5
cAMP elevation measured in HaCaT keratinocytes and normal human keratinocytes. (a) HaCaT keratinocytes and (b) normal human keratinocytes were incubated with α-MSH (▪) or KPV (▴) from 10−12 to 10−6 M for 20 min. Cell extracts were prepared and cAMP measured according toMichelangeli et al (1994). (c) cAMP elevation measured in HaCaT keratinocytes in response to α-MSH, KPV, KP-D-V, and ACTH at 10−8 and 10−6 M for 24 h. Forskolin was used as a positive control at 10−4 and 10−5 M. α-MSH, α-melanocyte-stimulating hormone; KPV, L-Lys-L-Pro-L-Val; KP-D-V, L-Lys-L-Pro-D-Val; ACTH, adrenocorticotropin hormone.
Journal of Investigative Dermatology  , DOI: ( /j X x)
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7. Figure 6
Cultured B16C1F10 pigmentary murine melanoma cells were incubated with: (a) α-MSH (▪), KPV (▴) or KP-D-V (♦) and (b) ACTH (▴) or ACTH 1—17 (•) for 24 h and extracts measured for dopa oxidase activity. α-MSH, α-melanocyte-stimulating hormone; KPV, L-Lys-L-Pro-L-Val; KP-D-V, L-Lys-L-Pro-D-Val; ACTH, adrenocorticotropin hormone.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions