Neuropeptide (Calcitonin Gene-Related Peptide) Induction of Nitric Oxide in Human Keratinocytes in vitro  Yaping E, Samantha C. Golden, Alan R. Shalita, Wei-Li S. Lee, Daniel H. Maes, Mary S. Matsui  Journal of Investigative Dermatology  Volume 126, Issue 9, Pages 1994-2001 (September 2006) DOI: 10.1038/sj.jid.5700349 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Time course of NO production by keratinocytes stimulated with CGRP. hTERT cells are shown as the primary graph and NHEK are shown as an inset. Average fold change values from five (hTERT) or three (NHEK) separate and independent experiments were combined. Cells either served as vehicle control or were treated with either (⋄) UVB, (□) 0.1nm, (▵) 1nm, (X) 10nm, (*) 100nm CGRP, (○) 100nm CGRP8-37, or (+) 100nm CGRP8-37+100nm CGRP, and were harvested at 0.25, 0.5, 1hour, 1.5, 2, 3, 6, 12, 24, and 48hours. Intracellular NO levels were determined by FACS analysis, and values are shown as average -fold change from vehicle control. Error bars represent the SD. Cellular NO content of vehicle-treated cells was also determined by Greiss assay and ranged from 5.09 to 9.90μm. Student's t-test was used to determine significance, and *P<0.05 compared to the vehicle control. CGRP did not alter keratinocyte apoptosis or cytotoxicity. Journal of Investigative Dermatology 2006 126, 1994-2001DOI: (10.1038/sj.jid.5700349) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of CGRP on iNOS level in hTERT keratinocytes. Cells were treated with either (vehicle control) 0, (⋄) 0.1nm, (□) 1nm, or (▵) 10nm CGRP for 1hour, 3, 6, 12, 24, and 48hours. iNOS levels were measured by a colorometric ELISA and expressed as –fold change from vehicle control. Data are the means of triplicate determinations±SD; *P<0.05 compared with vehicle control. Journal of Investigative Dermatology 2006 126, 1994-2001DOI: (10.1038/sj.jid.5700349) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of CGRP or UVB on NOS activity. Cells were treated as indicated, harvested 30 or 60minutes later, and NOS activity in whole-cell lysate determined. Error bars show SD, *indicates a significant increase, and P<0.05, compared with the vehicle control. Each data point is the average of duplicate determinations from three independent experiments (six total determinations). Journal of Investigative Dermatology 2006 126, 1994-2001DOI: (10.1038/sj.jid.5700349) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of CGRP or UVB on S-nitrosothiol levels in hTERT keratinocytes. hTERT cells are shown as the primary graph and NHEK are shown as an inset. Average –fold change values from five (hTERT) or three (NHEK) separate and independent experiments were combined. Cells either served as vehicle control or were treated with either (⋄) UVB, (□) 0.1nm, (▵) 1nm, (X) 10nm, (*) 100nm CGRP, (○) 100nm CGRP8-37, or (+) 100nm CGRP8-37+100nm CGRP, and were harvested at 0.25, 0.5, 1hour, 1.5, 2, 3, 6, 12, 24, and 48hours. S-nitrosothiol levels were determined by a fluorimetric method. SD are shown, and *P<0.05 compared to vehicle control. Journal of Investigative Dermatology 2006 126, 1994-2001DOI: (10.1038/sj.jid.5700349) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions