1. Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes 
Masaki Yoshida, Yoshito Takahashi, Shintaro Inoue 
Journal of Investigative Dermatology 
Volume 114, Issue 2, Pages (February 2000)
DOI: /j x
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Histamine and other melanogens induced morphologic changes in human melanocytes. Melanocytes were cultured as described in Materials and Methods. They were treated by (a) control, (b) 10 nM endothelin-1, (c) 100 nM α-MSH, and (d) 1 μM histamine for 3 d. Scale bar: 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Histamine increased the melanin content and tyrosinase activity of human melanocytes. (a) Melanin content was determined by measuring the absorbance at 475 nm of a melanocyte cell lysate cultured for 4 d in the presence or absence of 5 μM histamine, as described in Materials and Methods. (b) Cells were treated with various concentrations of histamine for 60 h. The cells were incubated with 1.0 μCi [3H]tyrosine per ml for the last 36 h. Tyrosinase activity was determined as 3H2O release into culture media, as described in Materials and Methods. (c) Tyrosinase activity of each cell extract was determined as 3H2O release, as described in Materials and Methods. Each value of melanin content is the mean ± SEM of four determinations and tyrosinase activity is the mean ± SEM of three determinations. **p < 0.01 compared with the control by Dunnett’s test.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Comparison of the effects of antagonists on the morphologic changes and tyrosinase activity stimulated by histamine. (a) Human melanocytes were stimulated by 1 μM histamine for 72 h in the presence of (A) control, (B) mepyramine (H1 antagonist), (C) famotidine (H2 antagonist), and (D) thioperamide (H3 antagonist). Each antagonist was used at 1 μM. Scale bar: 50 μm. (b) Human melanocytes were stimulated by 1 μM histamine for 60 h in the presence of each antagonist described above. The cells were incubated with 1.0 μCi [3H]tyrosine per ml for the last 36 h. Tyrosinase activity was determined as 3H2O release into culture media as described in Materials and Methods. (c) Tyrosinase activity of human melanocytes in the absence (▪) or presence (□) of 1 μM histamine was determined as above with various concentrations of famotidine. (d) Tyrosinase activity of each cell extract was determined as 3H2O release, as described in Materials and Methods. Each value of tyrosinase activity is the mean ± SEM of three to nine determinations. **p < 0.01 as compared with the control by Dunnett’s test.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Effects of agonists on the morphology and tyrosinase activity of human melanocytes. (a) Human melanocytes were treated with histamine agonists for 72 h: (A) control; (B) betahistine (H1 agonist); (C) dimaprit (H2 agonist); and (D) imetit (H3 agonist). Each agonist was used at 1 μM. Scale bar: 50 μm. (b) Human melanocytes were treated with histamine agonists for 60 h. The cells were incubated with 1.0 μCi [3H]tyrosine per ml for the last 36 h. Tyrosinase activity was determined as described in Materials and Methods. (c) Human melanocytes were treated with various concentrations (0.1–10 μM) of dimaprit, and tyrosinase activity was measured. (d) Tyrosinase activity of each cell extract was determined as 3H2O release, as described in Materials and Methods. Each value of tyrosinase activity is the mean ± SEM of three to nine determinations. **p < 0.01, *p < 0.05 as compared with the control by Dunnett’s test.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Detection of mRNA encoding H2 receptors by RT-PCR. Gel electrophoresis of the RT-PCR reaction products of melanocytes. RT-PCR was performed using the primers and cycling conditions outlined in Materials and Methods. PCR products corresponding to human H2 receptor (330 bp) and G3PDH (450 bp) transcript were detected in lane H2 and lane G, respectively. Takara’s φX174 Hinc II digest was run in lane M to confirm the molecular size of the amplification product.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
cAMP accumulation of human melanocytes via H2 receptors. (a) Human melanocytes in 12 well plates were stimulated by various concentrations of histamine for 30 min in 500 μl MGM medium containing 0.1 mM IBMX. Ice-cold 100% ethanol (1 ml per well) was added to stop the reaction and the cultures were harvested. cAMP contents were determined as described in Materials and Methods. (b) Human melanocytes were incubated with various concentrations of famotidine for 10 min before stimulation by 1 μM histamine. After stimulation for 30 min, ice-cold 100% ethanol (1 ml per well) was added. cAMP contents in the absence (▪) or presence (□) of 1 μM histamine were determined as above. Each value of cAMP is the mean ± SEM of three determinations. **p < 0.01, *p < 0.05 as compared with the control by Dunnett’s test. Figure 7. Suppression of histamine action by H-89. (a) Morphologic changes of human melanocytes were observed after treatment with 1 μM histamine for 24 h together with (A) control, (B) 1 μM H-89. Scale bar: 50 μm. (b) and (c) Human melanocytes were cultured in the absence (▪) or presence (□) of 1 μM histamine for 60 h with various concentrations of H-89 or Go6976. The cells were incubated with 1.0 μCi [3H]tyrosine per ml for the last 36 h. Tyrosinase activity was determined as described in Materials and Methods. Each value of tyrosinase activity is the mean ± SEM of three determinations. **p < 0.01, *p < 0.05 as compared with the control by Dunnett’s test.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Suppression of histamine action by H-89. (a) Morphologic changes of human melanocytes were observed after treatment with 1 μM histamine for 24 h together with (A) control, (B) 1 μM H-89. Scale bar: 50 μm. (b) and (c) Human melanocytes were cultured in the absence (▪) or presence (□) of 1 μM histamine for 60 h with various concentrations of H-89 or Go6976. The cells were incubated with 1.0 μCi [3H]tyrosine per ml for the last 36 h. Tyrosinase activity was determined as described in Materials and Methods. Each value of tyrosinase activity is the mean ± SEM of three determinations. **p< 0.01, *p< 0.05 as compared with the control by Dunnett’s test.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

9. Figure 8
Induction of melanogenesis by dbcAMP. (a) Morphologic changes of human melanocytes were observed after treatment with (A) control and (B) 0.1 mM dbcAMP. Scale bar: 50 μm. (b) Human melanocytes were stimulated by various concentrations of dbcAMP for 60 h. The cells were incubated with 1.0 μCi [3H]tyrosine per ml for the last 36 h. Tyrosinase activity was determined as described in Materials and Methods. Each value of tyrosinase activity is the mean ± SEM of three determination. **p < 0.01, *p < 0.05 as compared with the control by Dunnett’s test.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions