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• W2042578571 abstract "Hyperpigmentation frequently accompanies chronic or acute inflammation. A number of inflammatory mediators have been shown to stimulate melanin synthesis in human melanocytes. Although histamine is ubiquitous as an inflammatory factor, its involvement in pigmentation remains obscure. In this work, we examined the effects of histamine on cultured human melanocytes. Treatment of human melanocytes with 0.1–10 μM histamine evoked morphologic changes and increases in tyrosinase activity. The concomitant increases in melanin content of the histamine-treated melanocytes indicated an elevation of melanin synthesis by tyrosinase activation. These stimulatory effects of histamine were completely inhibited by an H2 antagonist, famotidine, whereas H1 and H3 antagonists had no inhibitory effect whatsoever. In addition, an H2 agonist, dimaprit, induced the same degree of melanogenesis as histamine at concentrations of 0.1–10 μM. We observed an increase in the intracellular cAMP contents of human melanocytes induced by histamine via the H2 receptors. We know that this cAMP accumulation and subsequent protein kinase A activation plays a critical role in histamine-induced melanogenesis, because a specific protein kinase A inhibitor, H-89, completely suppressed these stimulatory effects of histamine, and because dibutylic cAMP, a specific protein kinase A activator, stimulated human melanocytes as potently as histamine. Taken together, we show here that histamine induces melanogenesis of human cultured melanocytes by protein kinase A activation via H2 receptors. Hyperpigmentation frequently accompanies chronic or acute inflammation. A number of inflammatory mediators have been shown to stimulate melanin synthesis in human melanocytes. Although histamine is ubiquitous as an inflammatory factor, its involvement in pigmentation remains obscure. In this work, we examined the effects of histamine on cultured human melanocytes. Treatment of human melanocytes with 0.1–10 μM histamine evoked morphologic changes and increases in tyrosinase activity. The concomitant increases in melanin content of the histamine-treated melanocytes indicated an elevation of melanin synthesis by tyrosinase activation. These stimulatory effects of histamine were completely inhibited by an H2 antagonist, famotidine, whereas H1 and H3 antagonists had no inhibitory effect whatsoever. In addition, an H2 agonist, dimaprit, induced the same degree of melanogenesis as histamine at concentrations of 0.1–10 μM. We observed an increase in the intracellular cAMP contents of human melanocytes induced by histamine via the H2 receptors. We know that this cAMP accumulation and subsequent protein kinase A activation plays a critical role in histamine-induced melanogenesis, because a specific protein kinase A inhibitor, H-89, completely suppressed these stimulatory effects of histamine, and because dibutylic cAMP, a specific protein kinase A activator, stimulated human melanocytes as potently as histamine. Taken together, we show here that histamine induces melanogenesis of human cultured melanocytes by protein kinase A activation via H2 receptors. α-melanocyte-stimulating hormone conventional protein kinase C dibutylic 3′,5′-cyclic adenosine monophosphate protein kinase A reverse transcription polymerase chain reaction Ultraviolet (UV) exposure or inflammation frequently evokes skin pigmentation (Prunieras, 1986Prunieras M. Melanocytes, melanogenesis & inflammation.Int J Dermatology. 1986; 25: 624-628Crossref PubMed Scopus (15) Google Scholar) via activation of melanin synthesis and distribution of melanin to keratinocytes. Melanin synthesis is triggered by melanogens, a large group of melanocyte-stimulating factors, such as endothelin-1 (Imokawa et al., 1995Imokawa G. Miyagishi M. Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.J Invest Dermatology. 1995; 105: 32-37Crossref PubMed Scopus (229) Google Scholar) and α-melanocyte-stimulating hormone (α-MSH) (Hunt et al., 1994Hunt G. Todd C. Kyne S. Thody A.J. ACTH stimulates melanogenesis in cultured human melanocytes.J Endocrinol. 1994; 140: R1-R3Crossref PubMed Scopus (71) Google Scholar), and several inflammatory factors (Tomita et al., 1992Tomita Y. Maeda K. Tagami H. Melanocyte-stimulating properties of arachidonic acid metabolites: possible role in postinflammatory pigmentation.Pigment Cell Research. 1992; 5: 357-361Crossref PubMed Scopus (184) Google Scholar;Morelli and Norris, 1993Morelli J.G. Norris D.A. Influence of inflammatory mediators and cytokines on human melanocyte function.J Invest Dermatology. 1993; 100: 191S-195SCrossref PubMed Scopus (107) Google Scholar). Although histamine is ubiquitous as an inflammatory mediator (Leurs et al., 1995Leurs R. Smit J. Timmerman H. Molecular pharmacological aspects of histamine receptors.Pharmacol Ther. 1995; 66: 413-463Crossref PubMed Scopus (343) Google Scholar), its involvement in pigmentation remains obscure. Several researchers reported increases in the melanin synthesis of cultured melanoma cells and melanocytes on the addition of histamine (Bernd et al., 1986Bernd A. Altmeyer P. Schafer G. Marsch W.C. Holzmann H. Bleomycin enhances the tyrosinase activity of human malignant melanoma cells in culture.Pharmacol Res Communications. 1986; 18: 1075-1091Crossref PubMed Scopus (6) Google Scholar;Tomita et al., 1988Tomita Y. Maeda K. Tagami H. stimulatory effect of histamine on normal human melanocytes in vitro.Tohoku J Expermental Med. 1988; 155: 209-210Crossref PubMed Scopus (18) Google Scholar,Tomita et al., 1993Tomita Y. Maeda K. Tagami H. Histamine stimulates normal human melanocytes in vitro: one of the possible inducers of hyperpigmentation in urticaria pigmentosa.J Dermatol Science. 1993; 6: 146-154Abstract Full Text PDF PubMed Scopus (26) Google Scholar;Whitehead et al., 1988Whitehead R.J. Taylor D.J. Evanson J.M. Hart I.R. Woolley D.E. Demonstration of histamine H2 receptors on human melanoma cells.Biochem Biophys Res Comms. 1988; 151: 518-523Crossref PubMed Scopus (34) Google Scholar). As histamine antagonists did not prevent the melanogenic activity of histamine in these studies, however, it appears that the histamine receptors did not mediate the action of histamine in human melanocytes. Tomita et al. speculated that histamine metabolites stimulated human melanocytes, rather than histamine itself, through imidazoline/guanidinium receptor sites, a group of nonspecific receptor sites for imidazoles that induce intracellular cAMP accumulation (Montefiori and Kline, 1981Montefiori D.C. Kline E.L. Regulation of cell division and of tyrosinase in B16 melanoma cells by imidazole: possible role for the concept of metabolite gene regulation in mammalian cells.J Cellular Physiology. 1981; 106: 283-291Crossref PubMed Scopus (23) Google Scholar;Lanier et al., 1993Lanier S.M. Ivkovic B. Singh I. Neumeyer J.L. Bakthavachalam V. Visualization of multiple imidazolie/guanidium-receptive sites.J Biol Chemistry. 1993; 268: 16047-16051PubMed Google Scholar). For these reasons, histamine has not been regarded as a melanogen. On the other hand, McEvans et al. reported inhibitory effects of histamine agonists on the melanogenesis of human melanoma cells (McEwan and Parsons, 1991McEwan M.T. Parsons P.G. Regulation of tyrosinase expression and activity in human melanoma cells via histamine receptors.J Invest Dermatology. 1991; 97: 868-873Abstract Full Text PDF PubMed Scopus (19) Google Scholar;Fechner et al., 1994Fechner G.A. Jacobs J.J. Parsons P.G. Dimaprit analogues inhibit tyrosinase via a disulphide breakdown product independently of the histamine H2 receptor.Biochem Biophys Res Comms. 1994; 201: 687-693Crossref PubMed Scopus (3) Google Scholar) and an H2-agonist-induced reduction in the melanin synthesis of human melanocytes and melanoma cells. In a more recent study, however, they found that the inhibitory action of H2 agonists on melanogenesis was mediated by imidazoline/guanidinium receptor sites (Gros et al., 1994Gros G.L. Zhang X.-M. Parsons P.G. Alternation of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands.Melanoma Reserch. 1994; 4: 359-364Crossref PubMed Scopus (7) Google Scholar). Thus, at this juncture, there are no reports detailing the action of histamine via histamine receptors in human melanocytes. In consideration of a report by Reynolds et al. that suggested the existence of H1 and H2 receptors on the surface of human melanoma cells and melanocytes (Reynolds et al., 1996Reynolds J.L. Akhter J. Morris D.L. In vitro effect of histamine and histamine H1 and H2 receptor antagonists on cellular proliferation of human malignant melanoma cell lines.Melanoma Res. 1996; 6: 95-99Crossref PubMed Scopus (62) Google Scholar), we decided that these previous investigations were not sufficient to evaluate the action of histamine on human melanocytes. In this study, we attempted to evaluate the effect of histamine on the melanogenesis of human cultured melanocytes via histamine receptors. To our surprise, we found that the melanogenic activity of histamine is specifically mediated by histamine H2 receptors. Moreover, our findings were confirmed by an investigation of intracellular cAMP accumulation and subsequent protein kinase A (PKA) activation in histamine-treated melanocytes. Human melanocytes and bovine pituitary extracts (BPE) were purchased from Kurabo (Osaka, Japan). MCDB153 medium, bovine insulin, and trichloroacetic acid were obtained from Wako Pure Chemical Industries (Osaka, Japan). Hydrocortisone was purchased from Merck (Whitehouse Station, NJ). Basic fibroblast growth factor (bFGF) was purchased from Intergen (New York). Phorbol 12-myristate 13-acetate (PMA), melanin, epidermal growth factor, mepyramine, famotidine, thioperamide, 3-isobutyl-1-methylxantine (IBMX), ethidium bromide, and betahistine were purchased from Sigma-Aldrich (St. Louis, MO). The cAMP assay kit and [3H]tyrosine were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Charcoal was obtained from Kanto Chemical (Tokyo, Japan). Dimaprit dihydrochloride, α-MSH, and imetit dihydrobromide were purchased from Funakoshi (Tokyo, Japan). H-89 and Go6976 were purchased from Calbiochem-Novabiochem (CA). Dibutylic 3′,5′-cyclic adenosine monophosphate (dbcAMP) and agarose were purchased from Nacarai Tesque (Kyoto, Japan). The reverse transcription polymerase chain reaction (RT-PCR) kit, Oligotex-dT30<Super> kit, and φX174 Hinc II digest were purchased from Takara (Shiga, Japan), and Dowex-50 resin was purchased from Dow Chemical. Human melanocytes were maintained in MCDB 153 supplemented with 0.4% (vol/vol) BPE, 1 ng recombinant human bFGF per ml, 5 μg insulin per ml, 500 ng hydrocortisone per ml, and 10 ng PMA per ml (Yada et al., 1991Yada Y. Higuchi K. Imokawa G. Effects of endothelins on signal transduction and proliferation in human melanocytes.J Biol Chemistry. 1991; 266: 18352-18357PubMed Google Scholar). The cells were incubated in MCDB 153 supplemented with 1 ng recombinant human bFGF per ml, 5 μg purified mouse epidermal growth factor per ml, and 5 μg insulin per ml (MGM) for at least 3 d before the experiment. Melanocytes were seeded in 100 mm dishes at a density of 1.2 × 106 cells per dish and cultured. After incubation in MGM for 3 d, the cells were treated with various concentrations of histamine. Next, the cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), suspended in 500 μl PBS, and counted. The cells were then solubilized by adding 500 μl of 10N NaOH and incubating for 30 min at room temperature. Absorbance at 475 nm was measured and compared with a standard curve for synthetic melanin. To determine the tyrosinase activity of the cells, the activity of tyrosine hydroxylation was measured using the following method. Melanocytes were seeded in 12 well culture trays at a density of 1.2 × 105 cells per well and cultured. After incubation in MGM for 3 d, the cells were treated with histamine or another reagent for 60 h. Over the last 36 h of treatment, the cells were incubated with 1.0 μCi [3H]tyrosine per ml. Next, 700 μl of 10% trichloroacetic acid containing 20% charcoal (charcoal solution) was added to the medium (700 μl), and the mixture was mixed in a vortex for 30 s and then centrifuged at 10,000 rpm with an MRX-150 centrifuge (Tomy Seiko, Tokyo, Japan) for 10 min. Seven hundred microliters of the supernatant was transferred into a new tube and treated twice with the charcoal solution. The radioactivity of the final supernatant was determined in a liquid scintillation counter (LSC1000, Aloka). Determination of tyrosinase activity of the cell extract was performed as described by Pawlek et al. (Pawlek, 1978Pawlek J. Melanoma in culture.Meth Enzymol. 1978; 58: 564-570Crossref Scopus (30) Google Scholar;Chakraborty et al., 1998Chakraborty A.K. Funasaka Y. Komoto M. Ichihashi M. Effect of arbutin on melanogenic proteins in human melanocytes.Pigment Cell Research. 1998; 11: 206-212Crossref PubMed Scopus (117) Google Scholar), with a slight modification. After incubation in MGM for 3 d, melanocytes were treated with histamine or another reagent for 60 h. Cells were collected and washed twice with PBS, and then lyzed in 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X-100. After determining the protein content in the cell extract, 10 μg of each extract was incubated in 100 μl of 10 mM sodium phosphate buffer (pH 6.8) containing 1.0 μCi [3H]tyrosine per ml, 5 μg L-dihydroxyphenylalanine, and 1% Triton X-100 for 15 min at 37°C. After adding 900 μl of charcoal solution, the samples stood for 20 min at 4°C and then were centrifuged at 10,000 rpm in an MRX-150 centrifuge for 10 min. The supernatants were applied to a 0.2 ml Dowex-50 column equilibrated in 10% trichloroacetic acid and washed with 0.5 ml of 10% trichloroacetic acid, after which the radioactivity of the effluents was determined in the liquid scintillation counter. Melanocytes were seeded in a 225 cm2 flask at a density of 1.2 × 106 cells per flask and cultured. After incubation in MGM for 4 d, the cells were harvested by trypsinization, washed with PBS, suspended in 500 μl of PBS, and counted. Cells were collected by centrifugation at 800 rpm with an MRX-150 centrifuge (Tomy Seiko) for 5 min, and then solubilized by adding 2 ml of TRIzol reagent. The cell lysates were passed through a pipette several times, and then incubated at room temperature for 5 min. After adding 0.4 ml of chloroform, the samples were vigorously mixed by inverting the tubes. Then, the samples were centrifuged at 12,000 × g for 10 min at 4°C. Following centrifugation, the aqueous phase was transferred into new tubes. The RNA was precipitated from the aqueous phase by the addition of isopropanol, and the pellet was dissolved in water. Poly(A)+ RNA was purified from the total RNA by oligo (dT) cellulose chromatography with an Oligotex-dT30<Super> kit. The concentration of poly(A)+ RNA was determined by measuring OD260. Synthesis of the first strand cDNA and subsequent PCR reaction was carried out using the RT-PCR kit reagents in a PCR DNA thermal cycler (Perkin-Elmer, Sweden). One hundred nanograms of each RNA sample was reverse transcribed to cDNA in a 20 μl reaction volume in the presence of 1 × reverse transcriptase buffer (50 mM KCl, 10 mM Tris(hydroxymethyl)aminomethane-HCl, pH 8.3), 5 mM MgCl2, 1 mM deoxyribonucleoside triphosphate, 50 pmol oligo (dT) primers, and 50 units AMV reverse transcriptase. The samples were incubated at 42°C for 50 min, followed by 70°C for 15 min, and then immediately cooled on ice. The sense and antisense primer oligonucleotides were designed as described byJansen-Olesen et al., 1997Jansen-Olesen I. Ottosson A. Cantera L. et al.Roles of endothelium and nitric oxide in histamine-induced resposes in human cranial arteries and detection of mRNA encoding H1- and H2-receptors by RT-PCR.Br J Pharmacology. 1997; 121: 41-48Crossref PubMed Scopus (45) Google Scholar The sequence of the oligonucleotide primers to amplify the 330 bp fragment corresponding to the region of human histamine H2 receptors was as follows: sense primer 5′-TCGTGTCCTTGGCTATCAC-3′ corresponding to nucleotides 170–188 and antisense primer 5′- CCTTGCTGGTCTCGTTCCT-3′ complementary to nucleotides 481–499. Purchased sense and antisense primers for amplifications of the 450 bp fragment corresponding to the G3PDH region were used as a control. The PCR amplification reaction was composed of 6 μl of first strand cDNA reaction mixture and 94 μl of master mix containing 1 × PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.3)], 1 mM MgCl2, and 20 pmol of each specific sense and antisense specific primer for human histamine H2 receptors, or G3PDH. The reaction mixture was subjected to 30 cycles of PCR amplification. After an initial denaturation step at 94°C for 1 min, the cycle profile included denaturation for 1 min at 94°C, annealing for 1 min at 58°C, and extension at 72°C for 1 min. After the final cycle, the temperature was maintained at 72°C for 7 min to allow for completion of synthesis of the amplified products. Twenty microliters from each PCR amplified product was electrophoresed in a 1% agarose gel. Each DNA band was visualized by staining with ethidium bromide. The cells were washed twice with PBS, and then an aliquot (400 μl) of MGM containing 0.1 mM IBMX and various concentrations of famotidine was placed into the wells. After incubation for 10 min at 37°C, 100 μl of MGM containing 0.1 mM IBMX and various concentrations of histamine were added, and then the cells were incubated for 30 min at 37°C. Ice-cold 100% ethanol (1 ml per well) was added to stop the incubation, and the mixture, including the scraped cells, was transferred to a new glass tube, placed on ice for 30 min, and then centrifuged for 10 min at 1900 ×g. The supernatant was dried in a Speed Vac Concentrator (Tomy Seiko) and resuspended in 300 μl of 50 mM Tris-HCl and 2.0 mM ethylenediamine tetraacetic acid. The extracted cAMP was measured with a cAMP assay kit. A probability level of p < 0.05 was considered significant when results were subjected to Dunnett’s multiple comparison procedure. Melanogens, such as endothelin-1 (10 nM) and α-MSH (100 nM), changed the configuration of the melanocytes to an activated form with an increased number of dendrites and enlarged cell area compared with the control (compare Figure 1a with Figure 1b, c). These morphologic changes of the melanocytes treated with typical melanogens were completely consistent with a previous report (Hara et al., 1995Hara M. Yaar M. Gilchrest A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatology. 1995; 105: 744-748Crossref PubMed Scopus (122) Google Scholar). Histamine (1 μM) also changed the morphology of human melanocytes into an activated form as potently as the other melanogens (compare Figure 1d with Figure 1b, c). These morphologic changes were observed after approximately 10 h of treatment with histamine (data not shown). With the morphologic changes of the histamine-treated melanocytes, the activity of tyrosinase, a rate-limiting enzyme for melanin synthesis, increased 1.5–2.5-fold (Figure 2a). These results showed the elevation of tyrosinase activity of each cell, because the number of melanocytes was not significantly affected by histamine throughout the assay (data not shown). The measurement of tyrosinase activity by another method using the melanocyte extract also showed the upregulation of tyrosinase activity by histamine (Figure 2b). Measured tyrosinase activities by these two methods showed similar results in all of our experiments, as noted below (Figure 3, Figure 4). These results show that histamine induced the same degree of tyrosinase activity in human melanocytes as that by other melanogens discussed in previous reports (Hunt et al., 1994Hunt G. Todd C. Kyne S. Thody A.J. ACTH stimulates melanogenesis in cultured human melanocytes.J Endocrinol. 1994; 140: R1-R3Crossref PubMed Scopus (71) Google Scholar;Imokawa et al., 1995Imokawa G. Miyagishi M. Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis.J Invest Dermatology. 1995; 105: 32-37Crossref PubMed Scopus (229) Google Scholar). Moreover, the increase of melanin synthesis by tyrosinase activation was confirmed by measuring the elevated melanin contents of histamine-treated melanocytes (Figure 2c). These findings, demonstrating histamine-induced stimulation of human melanocytes and melanin synthesis, concur with a previous report regarding the melanogenesis of histamine-treated melanocytes (Tomita et al., 1993Tomita Y. Maeda K. Tagami H. Histamine stimulates normal human melanocytes in vitro: one of the possible inducers of hyperpigmentation in urticaria pigmentosa.J Dermatol Science. 1993; 6: 146-154Abstract Full Text PDF PubMed Scopus (26) Google Scholar).Figure 3Comparison 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.View Large Image Figure ViewerDownload (PPT)Figure 4Effects 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.View Large Image Figure ViewerDownload (PPT) The actions of histamine on cells are mediated by three distinct receptors, H1, H2, and/or H3 (Arrang, 1994Arrang J.-M. Pharmacological properties of histamine receptor subtypes.Cell Mol Biol. 1994; 40: 273-279Google Scholar). To determine the involvement of each, we investigated the effect of selective antagonists on the action of histamine. The pA2 values of these antagonists indicate their blocking ability against histamine. The reported pA2 values for mepyramine, famotidine, and thioperamide (9.0, 7.8, and 8.3, respectively;Leurs et al., 1995Leurs R. Smit J. Timmerman H. Molecular pharmacological aspects of histamine receptors.Pharmacol Ther. 1995; 66: 413-463Crossref PubMed Scopus (343) Google Scholar), demonstrate that each antagonist, at a 1 μM concentration, is sufficient to block the action of 1 μM histamine. One micromolar famotidine, an H2 antagonist, significantly prevented the morphologic changes by 1 μM histamine, but the H1 and H3 antagonists demonstrated no preventative effects (Figure 3a). Moreover, histamine-induced tyrosinase activation was specifically suppressed by famotidine, though famotidine had no effect on the activity of the histamine-untreated melanocytes (Figure 3b, c). These effects by the antagonists on histamine-induced tyrosinase activation were also observed by another method for measuring tyrosinase activity with cell extracts (Figure 3d). Another H2 receptor antagonist, ranitidine, showed effects similar to famotidine in suppressing the histamine action on melanocytes (data not shown). These findings suggest the involvement of H2 receptors in histamine action on human melanocytes. To confirm the involvement of H2 receptors, we investigated the effects of histamine agonists on human melanocytes. As expected, only dimaprit, an H2 agonist, induced morphologic changes (Figure 4a) and increased the tyrosinase activity of human melanocyte cultures and extracts (Figure 4b–d). These stimulatory activities of dimaprit were observed at 0.1–10 μM. In contrast, a much higher concentration (100 μM or more) of dimaprit reduced tyrosinase activity (data not shown). We speculate that the decrease of tyrosinase activity was due to the side-effect of dimaprit via the imidazoline/guanidinium receptor sites, as reported byGros et al., 1994Gros G.L. Zhang X.-M. Parsons P.G. Alternation of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands.Melanoma Reserch. 1994; 4: 359-364Crossref PubMed Scopus (7) Google Scholar. To determine the existence of H2 receptors, we performed RT-PCR with poly(A)+ RNA from human melanocytes (Figure 5). The specific 330 bp band for H2 receptors was detected, although the band was not detected without reverse-transcriptase treatment (data not shown). The size of the band completely agreed with a previous report (Jansen-Olesen et al., 1997Jansen-Olesen I. Ottosson A. Cantera L. et al.Roles of endothelium and nitric oxide in histamine-induced resposes in human cranial arteries and detection of mRNA encoding H1- and H2-receptors by RT-PCR.Br J Pharmacology. 1997; 121: 41-48Crossref PubMed Scopus (45) Google Scholar) and digestion by several restriction enzymes generated the expected length of DNA fragments (data not shown). Thus, we concluded that mRNA for H2 receptors was expressed in human melanocytes. These findings suggested the existence of H2 receptors in human melanocytes. cAMP accumulation followed by PKA activation is the major signaling pathway mediated by H2 receptors. To clarify the existence of functional H2 receptors, we examined histamine-induced cAMP accumulation. When human melanocytes were treated with 0.1–10 μM histamine in the presence of 0.1 mM IBMX for 30 min, the intracellular cAMP contents were increased dose-dependently, up to 9-fold with 10 μM, compared with the controls. Whereas famotidine completely prevented this histamine-induced cAMP accumulation (Figure 6a), it had no affect on the cAMP contents of untreated melanocytes (Figure 6b). These results show that functional H2 receptors exist on the cell surfaces of human melanocytes and that histamine induces cAMP accumulation only via H2 receptors. Considering the above results, we determined that histamine induces morphologic changes and melanin synthesis of human melanocytes via an H2-receptor-mediated pathway, and concluded that histamine is a melanogen. We also examined the involvement of the cAMP-PKA cascade in histamine-induced melanogenesis. A PKA inhibitor, H-89, completely suppressed the stimulatory effects of histamine on the morphology and tyrosinase activity of human melanocytes at concentrations without toxicity, whereas a specific conventional protein kinase C (cPKC) inhibitor, Go6976, had no effects on histamine-induced tyrosinase activation (Figure 7a–c). As we observed AP-1 activation by PMA and specific inhibition of this by Go6976 (data not shown), we concluded that PMA-responsive cPKC was restored in the melanocytes under those conditions and that cPKC inhibition by Go6976 did not affect the stimulation by histamine. Thus, we concluded that H-89 suppressed melanogenesis by inhibition of PKA, but not cPKC, because the reagent has a lesser activity on PKC activity at these concentrations (data not shown). In contrast to the above, dbcAMP, a specific PKA activator, induced morphologic changes and tyrosinase activation to the same extent as histamine (Figure 8a,b). These findings show that activation of the cAMP-PKA cascade is necessary and sufficient to evoke melanogenesis by histamine. Taken together, these findings indicate that histamine induces morphologic changes and melanin synthesis of human melanocytes by PKA activation via H2-receptor-mediated cAMP accumulation. Based on observations that changes in skin coloration are frequently accompanied by inflammatory reactions, a number of inflammatory factors have been reported as melanogens. The mechanism of hyperpigmentation accompanied by inflammation, however, has remained obscure. We demonstrated here that histamine, a ubiquitous inflammatory factor, has a melanogenic effect on human cultured melanocytes via H2 receptors. In human skin, histamine is mostly produced and released by dermal mast cells, and this histamine release from mast cells leads to the development of inflammatory reactions. Thus, we speculated that histamine may often participate in the hyperpigmentation of human skin. For example, hyperpigmentation after UV irradiation is a physiologic reaction combining acute inflammation and pigmentation. An elevation of histamine in UV-irradiated skin was reported byGilchrest et al., 1981Gilchrest B.A. Soter N.A. Stoff J.S. Mihm M.C. The human sunburn reaction: histologic and biochemical studies.J Am Acad Dermatol. 1981; 5: 411-422Abstract Full Text PDF PubMed Scopus (250) Google Scholar. In addition, hyperpigmentation is a frequent outcome of several diseases, with proliferation and infiltration of the mast cells in the skin, e.g., urticaria pigmentosa and atopic dermatitis. Thus, it is easy to infer that histamine levels are high at the infiltrating site of mast cells. Our findings may bring to light a new pharmacologic property of H2 antagonists that might be a remedy for these hyperpigmentations. Our results regarding the involvement of H2 receptors did not agree with a previous report byTomita et al., 1993Tomita Y. Maeda K. Tagami H. Histamine stimulates normal human melanocytes in vitro: one of the possible inducers of hyperpigmentation in urticaria pigmentosa.J Dermatol Science. 1993; 6: 146-154Abstract Full Text PDF PubMed Scopus (26) Google Scholar. They reported that cimetidine, an H2 antagonist, could not prevent the induction of tyrosinase activation by histamine. We speculated that the discrepancy is due to the melanogenic activity of cimetidine reported byGros et al., 1994Gros G.L. Zhang X.-M. Parsons P.G. Alternation of tyrosinase activity in human melanocytes and melanoma cells by histamine H2 and H3 ligands.Melanoma Reserch. 1994; 4: 359-364Crossref PubMed Scopus (7) Google Scholar. According to their report, some H2 antagonists have a melanogenic action on human melanoma cells and melanocytes at mM concentrations. In fact, we also observed a limited elevation of tyrosinase activity by cimetidine at a concentration much higher than 300 μM (data not shown). Gros et al. however, also surmised that these H2 antagonist effects were nonspecific effects mediated not by H2 receptors but by imidazoline/guanidinium receptor sites. Thus, we considered that the side-effects of these H2 antagonists might prevent them from finding the correct signaling pathway of histamine-induced melanogenesis. To paraphrase, we speculate that cimetidine in the previous reports might have induced melanogenesis via the imidazoline/guanidinium receptor sites, though it blocked the stimulation of histamine via H2 receptors. Thus, these results suggest the need for more thorough examinations of the choice and dosages of H2 antagonists used to remedy histamine-related pigmentation, even if the stimulation of histamine is mediated only by H2 receptors. We have demonstrated here that histamine has a melanogenic effect on cultured human melanocytes. As the abundant release of histamine in vivo does not always lead to pigmentation in the skin, however, there must be another mechanism to trigger the melanin synthesis in vivo. We later intend to discuss a possible mechanism to regulate the initiation of pigmentation. In this study, we carried out all of our experiments in the presence of bFGF, a mitogenic, but not melanogenic, cytokine for melanocytes, because it is generally understood that bFGF is one of the essential factors that maintains cultured melanocytes. As reported with other cAMP-accumulating agents, we could not observe histamine-induced melanogenesis in the absence of bFGF (Swope et al., 1995Swope V.B. Medrano E.E. Smalara D. Abdel-Malek Z.A. Long-term proliferation of human melanocytes is supported by the physiologic mitogens α-melanotropin, endothelin-1, and basic fibroblast growth factor.Exp Cell Research. 1995; 217: 453-459Crossref PubMed Scopus (120) Google Scholar) (data not shown). We speculate that the coupling of PKA activation with bFGF action is necessary for melanocyte stimulation, and that costimulators like bFGF might regulate the induction of in vivo hyperpigmentation by cAMP-elevating substances, such as histamine and α-MSH. This hypothesis regarding the regulatory mechanism by a costimulator can easily solve the conflict between the frequency of pigmentation and histamine release in the skin. Iwata et al. reported that histamine or α-MSH had no melanogenic effect on a human foreskin culture (Iwata et al., 1990Iwata M. Iwata S. Everett M.A. Fuller B.B. Hormonal stimulation of tyrosinase activity in human foreskin organ cultures.In Vitro Cellular Dev Biology. 1990; 26: 554-560Crossref PubMed Scopus (15) Google Scholar). We suppose that these results were due to the absence of bFGF in the culture media and cultured skin, however. It remains unclear whether bFGF, which has no signal peptide for secretion, is truly released in the skin under physiologic conditions; therefore the existence of bFGF in pigmented skin will have to be clarified to pursue this hypothesis. Moreover, there have been some reports noting that lethal damage to cells was required to evoke bFGF secretion (D’Amore, 1990D’Amore P.A. Modes of FGF release in vivo and in vitro.Cancer Metastasis Reviews. 1990; 9: 227-238Crossref PubMed Scopus (139) Google Scholar;Ku and D’Amore, 1995Ku P.-T. D’Amore P.A. Regulation of basic fibroblast growth factor (bFGF) gene and protein expression following its release from sublethally injured endothelial cells.J Cellular Biochemistry. 1995; 58: 328-343Crossref PubMed Scopus (75) Google Scholar). As a result, more investigations will be needed to determine if bFGF is a true costimulator in vivo. We cannot deny the presence of another costimulator at this time. To establish the melanogenic role of histamine or α-MSH in vivo, it will first be necessary to evaluate the presence of costimulators such as bFGF in pigmented skin. Studies are now in progress to determine the involvement of costimulators in pigmentation. The authors would like to thank Ms. Sayuri Yamaguchi for her technical assistance. We express our gratitude to Mr. Shingo Sakai and Mr. Minoru Sasaki for their kind and helpful advice." @default.
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• W2042578571 title "Histamine Induces Melanogenesis and Morphologic Changes by Protein Kinase A Activation via H2 Receptors in Human Normal Melanocytes" @default.
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