Serotoninergic System in Hamster Skin

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Serotoninergic System in Hamster Skin Andrzej Slominski, MD, PhD, Alexander Pisarchik Journal of Investigative Dermatology Volume 119, Issue 4, Pages 934-942 (October 2002) DOI: 10.1046/j.1523-1747.2002.00156.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cloning and structural analysis of the open reading frame of hamster TPH gene. (A) Sequencing of hamster tryptophan hydroxylase. Open box represents a reading frame of gene. Solid lines: 5′ and 3′ untranslated regions; dashed lines: amplified regions. Primers used for sequencing and amplification are shown above by arrows. (B) Phylogenic analysis of TPH mRNA sequences from different organisms by DNAMLK program (DNA Maximum Likelihood program with molecular clock, version 3.5c, PHYLIP package). (C) Predicted amino acid sequence of hamster TPH (accession no. AY034600), and comparison with mouse (accession no. NM_009414), rat (Accession No. X53501) and human (accession no. XM_006391) sequences. Conserved amino acids are shadowed. The numbers in the right-hand column refer to the amino acid number. The single line marks regulatory domain of the protein over the amino acid sequence. The double line represents the catalytic domain and the dashed line represents the leucine zipper. Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Reverse transcription–PCR amplification of hamster TPH and AANAT mRNA. (A) Organization of hamster TPH and AANAT genes. Numbers correspond to exons. Arrows represent PCR primers. Dashed lines show the PCR fragments. (B) Detection of TPH mRNA in hamster tissues: 2, eye; 3, pituitary; 4, heart; 5, spleen; 6, skin; 7, melanoma Ma; 8, melanoma MI; 9, melanoma AbC1. Lane 1: DNA ladder. (C) Detection of AANAT in hamster tissues: 2, pituitary; 3, eye; 4, skin; 5, spleen; 6, 8, and 10 untreated AbC1 melanoma cell line; 9, melanized AbC1; 11, AbC1 irradiated by 5 mJ per cm2 of UVB; 12, AbC1 irradiated by 20 mJ per cm2 of UVB; 13, AbC1 irradiated by 50 mJ per cm2 of UVB. Lanes 1 and 7: DNA markers. Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Representative HPLC chromatograms obtained from reaction mixture in which hamster skin from ears was the enzyme source. Experimental incubation with acetyl-CoA and serotonin (A) or tryptamine (C), and (B,D) corresponding controls represented by incubation without acetyl-CoA. The numbers in the figure indicate the elution position of standards. (1) NAS (A,B) or N-acetyltryptamine (C,D); (2) serotonin (A,B) or tryptamine (C,D). Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Representative HPLC chromatograms obtained from reaction mixture in which Ab amelanotic melanoma transplantable in hamsters was the enzyme source. Experimental incubation with acetyl-CoA and serotonin (A) or tryptamine (C), and (B,D) corresponding control extracts without amine substrate. The numbers in the figure indicate the elution position of standards. (1) NAS (A,B) or N-acetyltryptamine (C,D); (2) serotonin (A,B) or tryptamine (C,D). Right insert: expanded scale showing detectable peaks with retention time corresponding to NAS and serotonin. Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 LC-MS analysis of the products of serotonin acetylation by skin extracts. SIM (selected ion monitoring) of adduct ion [M + H]+ with m/z 219 was used to detect the NAS. (A) Experimental incubation with acetyl-CoA and serotonin. (B) Inhibition of enzymatic activity by specific AANAT bisubstrate inhibitor CoA-S-N-acetyltryptamine (5 µM). (C) Sample incubated without serotonin (enzymatic reaction control). (D) Acetyl CoA and serotonin incubated without enzyme source (chemical acetylation control). Arrow identifies the compound with retention time (retention time) 11.4 min and with m/z 219 (calculated mass was 218) corresponding to NAS standard. Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of the specific AANAT bisubstrate inhibitor CoA-S-N-acetyltryptamine (BSI) on the enzyme activity in hamster skin. Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 LC-MS identification of NAS in hamster AbC-1 melanoma cells. Shown in the center is an adduction (M + H)+ at m/z 219.5, and the corresponding retention time (14.5 min) is in the right insert. Left insert shows (M + H)+ ion at m/z of 219 of NAS standard. Journal of Investigative Dermatology 2002 119, 934-942DOI: (10.1046/j.1523-1747.2002.00156.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions