Serotoninergic System in Hamster Skin
2002; Elsevier BV; Volume: 119; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2002.00156.x
ISSN1523-1747
AutoresAndrzej Słomiński, Alexander Pisarchik, Igor Semak, Trevor W. Sweatman, Andre Szczesniewski, Jacobo Wortsman,
Tópico(s)Olfactory and Sensory Function Studies
ResumoWe have cloned the tryptophan hydroxylase cDNA from hamster pituitary and demonstrated its expression in the skin, melanotic and amelanotic melanomas, spleen, heart, and the eye. We further demonstrated that skin, melanomas, spleen, pituitary, and eye but not heart expressed arylalkylamine N-acetyltransferase mRNA. The cutaneous expression of the arylalkylamine N-acetyltransferase gene was accompanied by enzymatic activity for the conversion of serotonin and tryptamine to N-acetylserotonin and N-acetyltryptamine, respectively. There was marked regional variation in the serotonin N-acetyltransferase activity, which was higher in ear skin than in corpus skin, and was lower in melanomas than in normal skin. Serotonin N-acetyltransferase activity was significantly inhibited by Cole bisubstrate at low concentration (≤ 1 µM); this evidence in conjunction with arylalkylamine N-acetyltransferase mRNA expression implies an involvement of arylalkylamine N-acetyltransferase in serotonin metabolism in the skin. We also documented both the in vitro transformation of serotonin to N-acetylserotonin using liquid chromatography/mass spectrometry and the generation/storage of N-acetylserotonin in cultured melanoma cells. Thus, we have uncovered a cutaneous pathway displaying capabilities for serotonin biosynthesis and/or its metabolism to N-acetylserotonin in rodent skin. As serotonin has powerful vasodilator, immunomodulator, and growth factor actions, this pathway could be involved in skin physiology and/or pathology. We have cloned the tryptophan hydroxylase cDNA from hamster pituitary and demonstrated its expression in the skin, melanotic and amelanotic melanomas, spleen, heart, and the eye. We further demonstrated that skin, melanomas, spleen, pituitary, and eye but not heart expressed arylalkylamine N-acetyltransferase mRNA. The cutaneous expression of the arylalkylamine N-acetyltransferase gene was accompanied by enzymatic activity for the conversion of serotonin and tryptamine to N-acetylserotonin and N-acetyltryptamine, respectively. There was marked regional variation in the serotonin N-acetyltransferase activity, which was higher in ear skin than in corpus skin, and was lower in melanomas than in normal skin. Serotonin N-acetyltransferase activity was significantly inhibited by Cole bisubstrate at low concentration (≤ 1 µM); this evidence in conjunction with arylalkylamine N-acetyltransferase mRNA expression implies an involvement of arylalkylamine N-acetyltransferase in serotonin metabolism in the skin. We also documented both the in vitro transformation of serotonin to N-acetylserotonin using liquid chromatography/mass spectrometry and the generation/storage of N-acetylserotonin in cultured melanoma cells. Thus, we have uncovered a cutaneous pathway displaying capabilities for serotonin biosynthesis and/or its metabolism to N-acetylserotonin in rodent skin. As serotonin has powerful vasodilator, immunomodulator, and growth factor actions, this pathway could be involved in skin physiology and/or pathology. arylalkylamine N-acetyltransferase tryptophan hydroxylase liquid chromatography mass spectrometry N-acetylserotonin Accumulated evidence indicates the expression of neuroendocrine activities in the skin (Slominski and Wortsman, 2000Slominski A. Wortsman J. Neuroendocrinology of the skin.Endocrine Rev. 2000; 21: 457-487Crossref PubMed Scopus (601) Google Scholar). Thus, the skin has been found to produce locally some of the mediators involved in the systemic response to stress, including the proopiomelanocortin-derived peptides melanocyte-stimulating hormone, adrenocorticotropic hormone, and β-endorphin, corticotropin-releasing hormone, and urocortin (Slominski et al., 2000Slominski A. Wortsman J. Luger T. Paus R. Salomon S. Corticotropin releasing hormone and proopiomelanocortin involvement in the cutaneous response to stress.Physiol Rev. 2000; 80: 979-1020Crossref PubMed Scopus (599) Google Scholar, Slominski et al., 2001Slominski A. Wortsman J. Pisarchik A. Zbytek A. Linton E.A. Mazurkiewicz J. Wei E.T. Cutaneous expression of corticotropin releasing hormone (CRH), urocortin and CRH receptors.FASEB J. 2001; 15: 1678-1693Crossref PubMed Scopus (229) Google Scholar). In addition to that, the skin also participates in the activation of steroid hormones, e.g., the conversion of testosterone to 5α-dihydrotestosterone or to estradiol (reviewed inSlominski and Wortsman, 2000Slominski A. Wortsman J. Neuroendocrinology of the skin.Endocrine Rev. 2000; 21: 457-487Crossref PubMed Scopus (601) Google Scholar); and, epidermal keratinocytes specifically produce parathyroid hormone-related protein and vitamin D (Holick, 1994Holick M.F. McCollum Award Lecture, Vitamin D—new horizons for the 21st century.Am J Clin Nutr. 1994; 60: 619-630Crossref PubMed Scopus (521) Google Scholar;Philibrick et al., 1996Philibrick W.M. Wysolmerski J.J. Glabraith S. et al.Defining the roles of parathyroid hormone-related protein in normal physiology.Physiol Rev. 1996; 76: 127-173PubMed Google Scholar). Moreover, epidermal neurotransmitters have also been detected as documented by Schallreuter et al., 1995Schallreuter K.U. Lemke K.R. Pittelkow M.R. Wood J.M. Korner C. Malik R. Catecholamines in human keratinocyte differentiation.J Invest Dermatol. 1995; 104: 953-957Crossref PubMed Scopus (102) Google Scholar for the catecholaminergic system, and by Grando and Horton, 1997Grando S.A. Horton R.M. The keratinocyte cholinergic system with acetylcholine as an epidermal cytotransmitter.Cur Opin Dermatol. 1997; 4: 268-462PubMed Google Scholar for the cholinergic system. Serotonin is another neurotransmitter involved in control neuroendocrine pathways, and has been already detected in normal human melanocytes (Johansson et al., 1998Johansson O. Lui P.-Y. Bondesson L. et al.A serotonin-like immunoreactivity is present in human cutaneous melanocytes.J Invest Dermatol. 1998; 111: 1010-1014Crossref PubMed Scopus (29) Google Scholar). Serotonin is the product of a multistep metabolic pathway that starts with the hydroxylation of the aromatic amino acid L-tryptophan by tryptophan hydroxylase (TPH: E.C. 1.14.16.4; Mockus and Vrana, 1998Mockus S.M. Vrana K.E. Advances in the molecular characterization of tryptophan hydroxylase.J Mol Neurosci. 1998; 10: 163-179Crossref PubMed Scopus (76) Google Scholar. The resulting metabolite hydroxytryptophan is decarboxylated by an enzyme ubiquitously present in peripheral tissues, aromatic L-amino acid decarboxylase (AAADC: E.C. 4.1.1.28) to generate the active neurohormone serotonin (Yu and Reiter, 1993Yu H.S. Reiter R.J. Melatonin Biosynthesis, Physiological Effects, and Clinical Implications. CRC Press, Boca Raton1993Google Scholar). Besides its specific actions, serotonin also serves as a precursor to melatonin (Yu and Reiter, 1993Yu H.S. Reiter R.J. Melatonin Biosynthesis, Physiological Effects, and Clinical Implications. CRC Press, Boca Raton1993Google Scholar). Acetylation of serotonin by arylalkylamine N-acetyltransferase (AANAT: E.C. 2.3.1.87), generates N-acetylserotonin (NAS), and after methylation, by hydroxyindole-O-methyltransferase (E.C. 2.1.1.4) melatonin (Sugden et al., 1987Sugden D. Cena V. Klein D.C. Hydroxyindole O-methyltransferase.Methods Enzymol. 1987; 142: 590-596Crossref PubMed Scopus (47) Google Scholar;Yu and Reiter, 1993Yu H.S. Reiter R.J. Melatonin Biosynthesis, Physiological Effects, and Clinical Implications. CRC Press, Boca Raton1993Google Scholar). We recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human skin (Slominski et al., 2002Slominski A. Pisarchik A. Semak I. et al.Serotoninergic and melatoninergic systems are fully expressed in human skin.FASEB J. 2002; 16 (April, 23): 896-898https://doi.org/10.1096/fj.0.1–0952fjeCrossref PubMed Google Scholar). We found that human skin cells show expression of the genes coding for the biosynthetic enzymes TPH, AANAT, and hydroxyindole-O-methyltransferase, as well as expressing the corresponding enzymatic activities (Slominski et al., 2002Slominski A. Pisarchik A. Semak I. et al.Serotoninergic and melatoninergic systems are fully expressed in human skin.FASEB J. 2002; 16 (April, 23): 896-898https://doi.org/10.1096/fj.0.1–0952fjeCrossref PubMed Google Scholar). As we previously showed metabolism of serotonin to NAS in the hamster skin (Gaudet et al., 1993Gaudet S.J. Slominski A. Etminan M. Pruski D. Paus R. Namboordiri M.A.A. Identification and characterization of two isozymic forms of arylamine N-acetyltransferase in Syrian hamster skin.J Invest Dermatol. 1993; 101: 660-665Abstract Full Text PDF PubMed Google Scholar;Slominski et al., 1996Slominski A. Baker J. Rosano T. Guisti L.W. Ermak G. Grande M. Gaudet S.J. Metabolism of serotonin to N-acetylserotonin, melatonin, and 5-methoxytryptamine in hamster skin culture.J Biol Chem. 1996; 271: 12281-12286Crossref PubMed Scopus (97) Google Scholar), we investigated whether a serotoninergic system was also expressed in full in this experimental model. As necessary step for this research we had to sequence the reading frame of hamster TPH (the enzyme catalyzing the rate of the limiting step in serotonin synthesis) obtained from pituitary tissue; we then used molecular and biochemical methods, including liquid chromatography mass spectrometry (LC-MS) for the analysis. Trizol reagent, Superscript preamplification system was purchased from Gibco-BRL (Gaithersburg, MD), pGEM-Teasy cloning vector and Taq polymerase were from Promega (Madison, WI), DNA marker and nucleotides from Fermentas (Hanover, MD), GFX polymerase chain reaction (PCR) DNA and gel band purification kit from Amersham-Pharmacia-Biotech (Piscataway, NJ), plasmid purification kit from Qiagen (Valencia, CA). SMART RACE cDNA amplification kit was from Clontech (Palo Alto, CA). Primers were synthesized by Integrated DNA Technology Inc. (Coralville, IA). Cell culture media included Ham's F10 medium, Dulbecco's modified essential medium, fetal bovine serum, anti-mycotic/antibiotic mixture and phosphate-buffered saline (all from Gibco-BRL). All reagents used for enzymatic assays and for high-performance liquid chromatography (HPLC) separations were of the highest purity. L-[5-3H]tryptophan was from Amersham Life Sciences Inc (Arlington Heights, IL). All chemicals for biochemical assays not listed above, including dye reagents for protein assay and chemicals for cell culture or molecular assays were purchased from Sigma (St Louis, MO). Cole bisubstrate inhibitor was obtained from Dr David Klein (National Institute of Health, Bethesda, MD). Syrian hamsters (males 3 months old) were purchased from Charles River Laboratory (Wilmington, MA) and housed in community cages at the animal facilities of the Albany Medical College (AMC), Albany NY or were derived from an in-house colony at the Belarus State University, Minsk, Belarus. The animals were killed under pentobarbital anesthesia and selected organs as well as back skin were collected following protocols routinely used in our laboratory (Slominski et al., 1996Slominski A. Baker J. Rosano T. Guisti L.W. Ermak G. Grande M. Gaudet S.J. Metabolism of serotonin to N-acetylserotonin, melatonin, and 5-methoxytryptamine in hamster skin culture.J Biol Chem. 1996; 271: 12281-12286Crossref PubMed Scopus (97) Google Scholar). Tissue specimens were frozen rapidly in liquid nitrogen. Hamster tissue samples consisted of pituitary, eye, heart, skin, and spleen. Hamster Bomirski Ma melanotic, MI hypomelanotic, and Ab amelanotic melanomas were propagated in male Syrian hamsters by subcutaneous inoculation of tissue suspension as described previously (Bomirski et al., 1988Bomirski A. Slominski A. Bigda J. The natural history of a family of transplantable melanomas in hamsters.Cancer Metastasis Rev. 1988; 7: 95-119Crossref PubMed Scopus (84) Google Scholar). After killing the animals tumor tissue was freed from connective and necrotic tissues and frozen rapidly in liquid nitrogen. Tissue specimens were stored at -80°C until analysis. The original research protocol was approved by the Institutional Animal Care and Use Committee at AMC, and a similar protocol was approved at the University of Tennessee Health Science Center (Memphis, TN). Bomirski hamster AbC-1 melanoma cells were grown in Ham's F10 medium as described previously; the media was supplemented with 10% fetal bovine serum and antibiotics (Slominski et al., 1988Slominski A. Moellman G. Kuklinska E. Bomirski A. Pawelek J. Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway: L-tyrosine and L-dopa.J Cell Sci. 1988; 89: 287-296PubMed Google Scholar). To induce melanin pigmentation the cells were cultured for 3 d in Dulbecco's modified essential medium (Slominski et al., 1988Slominski A. Moellman G. Kuklinska E. Bomirski A. Pawelek J. Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway: L-tyrosine and L-dopa.J Cell Sci. 1988; 89: 287-296PubMed Google Scholar). After washing with PBS, melanoma cells were detached using Ca and Mg free Tyrode's solution containing 1 mM ethylenediamine tetraacetic acid. The cells were centrifuged and pellets were used for analyses. Total RNA was extracted using Trizol isolation kit. The synthesis of first-strand cDNA was performed using the Superscript pre-amplification system. An aliquot (5 µg) of total RNA per reaction were reverse transcribed according to the manufacturer's protocol using oligo(dT) as the primer. For this analysis, all samples were standardized by amplification of the housekeeping gene GAPDH as described previously by Robbins and McKinney, 1992Robbins M. McKinney M. Transcriptional regulation of neuromodulin (GAP-43) in mouse neuroblastoma clone N1E-115 as evaluated by the RT/PCR method.Mol Brain Res. 1992; 13: 83-92Crossref PubMed Scopus (53) Google Scholar. PCR amplifications were performed under standard conditions with modifications where indicated. Thus, the reaction mixture (25 µl) contained 2.5 mM MgCl2, 2.5 mM of each deoxyribonucleoside triphosphate, 0.4 µM of each primer, 75 mM Tris–HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, and 1.25 units of Taq polymerase. The mixture was heated to 94°C for 2.5 min and then amplified for 35 or 30 cycles as specified: 94°C for 30 s (denaturation), 55°C for 45 s (annealing) for hamster tryptophan hydroxylase or 60°C for serotonin N-acetyltransferase and 72°C for 1 min (extension). A full-length sequence of hamster tryptophan hydroxylase mRNA was obtained from alignment of two separate fragments. The first fragment was amplified by RACE method (random amplification of cDNA ends). Amplification of pituitary cDNA was performed according to the manufacturer's protocol (SMART RACE cDNA amplification kit, Clontech). For the 3′RACE we used primer P129 (5′-TCCGTCCTGTGGCTGGTTACCTCTC-3′). Amplified fragment was sequenced. A second anti-sense primer P151 (5′-GAACAGTGTCCTCTGACGCTCCAAG-3′) was designed. This primer did not produce a single band in 5′RACE. Therefore, using mouse gene sequence we designed primer P176 (5′-GATTGAAGACAACAAGGAGAACAAAG-3′) with a sequence homologous to the region starting from the initiation codon of the mouse tryptophan hydroxylase (Stoll et al., 1990Stoll J. Kozak D.A. Goldman D. Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line.Genomics. 1990; 7: 88-96Crossref PubMed Scopus (59) Google Scholar). Amplification of hamster pituitary cDNA by primers P151 and P176 produced a single band. This band corresponded to the 5′ fragment of the hamster gene. Amplification products were separated by agarose electrophoresis and visualized by ethidium bromide staining, according to a standard protocol used in our laboratory (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.FASEB J. 2001; 15 (full text October 2715), 2710.1096/fj.2701–0487fje: 2754-2756PubMed Google Scholar), and the corresponding fragments sequenced. The identified PCR products were excised from the agarose gel and purified by GFX PCR DNA and gel band purification kit (Amersham-Pharmacia-Biotech). PCR fragments were cloned in pGEM-T easy vector system and purified by a plasmid purification kit. Sequencing was performed in the Molecular Resource Center at the University of Tennessee Health Science Center (Memphis, TN) using Applied Biosystems 3100 Genetic Analyzer and BigDye™ Terminator Kit (Foster City, CA). Amplification of hamster tryptophan hydroxylase was conducted by a single PCR using primers P134 (5′-CAGACACCTGCCATGAACTC-3′) and P135 (5′CAAAGACTCTCAGCTGTCCATC-3′). These primers were designed specifically for the hamster gene. PCR conditions were the same as described above, except that the annealing temperature was 55°C. Hamster serotonin N-acetyltransferase was amplified by nested PCR. Primers were designed according to the already published sequence (GenBank accession no. AF092100). The first round of amplification was performed with primers P242(5′-CCAGCGAGTTCCGTTGCCTTAC-3′) and P243(5′-GCCTGTGCAGTGTCAGTGACTC-3′). An aliquot of the mixture from the first PCR round was transferred into a new tube and amplified again with primers P244(5′-CGTGTTTGAGATTGAGCGTGAAG-3′) and P245(5′-CTTGTCCCAAAGTGAGCCGATG-3′). The resulting 163 bp long PCR band represented exons 2 and 3 of the hamster gene. Ultraviolet (UV) irradiation of hamster melanoma cells was performed as described previously (Pisarchik and Slominski, 2001Pisarchik A. Slominski A. Alternative splicing of CRH-R1 receptors in human and mouse skin: identification of new variants and their differential expression.FASEB J. 2001; 15 (full text October 2715), 2710.1096/fj.2701–0487fje: 2754-2756PubMed Google Scholar). After irradiation PBS was substituted by standard culture medium, cells were incubated for 24 h, and then detached, collected, and processed for RNA isolation. The radiometric assay for TPH (kindly performed by Dr Wilfred Pinto) followed the methodology described by Beevers et al., 1983Beevers S.J. Knowles R.G. Pogson C.I. A sensitive radiometric assay for tryptophan hydroxylase applicable to crude extracts.J Neurochem. 1983; 40: 894-897Crossref PubMed Scopus (23) Google Scholar with minor modifications. Cells pelleted by low speed centrifugation (≈ 1000 × g) were homogenized in 3–5 volumes of ice-cold 50 mM Tris–HCl buffer (pH 7.6 at room temperature) with a Tekmar tissuemizer (Tekmar Co., Cincinnati, OH) at high setting (13,500 r.p.m., 10 s). The homogenizing buffer as well as the assay buffer contained 2 mM dithiothreitol in order to maintain the enzyme in the active conformation. The crude cell homogenate was utilized as the source material for the tryptophan hydroxylase assays. The assay mixture contained, in a final volume of 200 µl, 50 mM Tris buffer containing 2 mM dithiothreitol, 100 µM L-tryptophan, ≈10 nM L-[5–3H]tryptophan as radioactive tracer (≈ 100 000 cpm, 31 Ci per mmol, Amersham Life Sciences Inc.), 0.5 mM 6-methyl-5,6,7,8-tetrahydropterine, and crude cell homogenate containing 30–50 µg protein. The pterin cofactor dissolved in 0.1 M HCl to a concentration of 5 mM was stored frozen in multiple aliquots at -80°C until the assay. Adding the crude cell homogenate to the prepared assay mixture, and mixing the contents by vortexing initiated the reaction. Background "blank" tubes were prepared substituting the cell extract for an equivalent volume of assay buffer. Following incubation for 120 min at 37°C, 40 µl of 60% perchloric acid (HClO4) were added to the assay mixture to the final HClO4 concentration of 12%; the contents were then mixed and incubated for an additional 30 min at 37°C. The assay contents were allowed to reach room temperature before the addition of 250 µl of a Norit-Dextran T70 slurry (50 mg each per ml H2O). After vortexing the samples remained at room temperature for 5 min and then centrifuged for 5 min at 10,000 × g. Subsequently, 200 µl aliquots of the supernatant were added to minivials containing scintillation cocktail (Scintiverse, Fisher Scientific Co., Fair Lawn, NJ) and counted in a Beckman LS7000 liquid scintillation spectrometer (Beckman Coulter Co., Fullerton, CA). Serotonin N-acetyltransferase activity was measured in samples of hamster skin and melanoma cell lines by a modification of the method described by Thomas et al., 1990Thomas K.B. Zawilska J. Iuvone P.M. Arylalkylamine (serotonin) N-acetyltransferase assay using high-performance liquid chromatography with fluorescence or electrochemical detection of N-acetyltryptamine.Anal Biochem. 1990; 184: 228-234Crossref PubMed Scopus (49) Google Scholar and further detailed by Slominski et al., 2002Slominski A. Pisarchik A. Semak I. et al.Serotoninergic and melatoninergic systems are fully expressed in human skin.FASEB J. 2002; 16 (April, 23): 896-898https://doi.org/10.1096/fj.0.1–0952fjeCrossref PubMed Google Scholar. All preparatory procedures were performed at 0–4°C. The samples of melanoma tumors and melanoma cell line were homogenized with a glass homogenizer in an ice-cold 0.25 M potassium phosphate buffer (pH 6.8) containing 1 mM dithiothreitol, 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid, protease inhibitor cocktail (2 µl per ml homogenization mixture) and 0.625 mM acetyl-coenzyme A (acetyl-CoA). The homogenization medium for the samples of hamster skin contained the same compounds with the exception of absence of acetyl-CoA. Homogenates were centrifuged at 15,000 r.p.m. (5,000 g) in microcentrifuge (Hermle Z 231M, Labnet, Woodbridge, NJ) for 10 min at 4°C. When melanoma tumors and melanoma cells were the enzyme source, the final concentrations of acetyl-CoA and amine substrate were 0.5 mM and 1 mM, respectively. When assaying AANAT activity in hamster skin, the final concentration of serotonin, tryptamine, and acetyl coenzyme were 0.55 mM, 1 mM, and 0.1 mM, respectively. The enzymatic reactions were run at 37°C and after various time stopped by the addition of HClO4 (Slominski et al., 2002Slominski A. Pisarchik A. Semak I. et al.Serotoninergic and melatoninergic systems are fully expressed in human skin.FASEB J. 2002; 16 (April, 23): 896-898https://doi.org/10.1096/fj.0.1–0952fjeCrossref PubMed Google Scholar).After centrifugation the supernatant was subjected to HPLC in a system equipped with a Novapak C18 reverse-phase column (100 × 5 mm, i.d.) and fluorimetric detector (Waters, Milford, MA). The detector was calibrated with excitation and emission wavelengths set at 285 and 360 nm, respectively. The elution was carried out isocratically at ambient temperature with a flow rate 1.5 ml per min for the different mobile phases depending upon the amine substrate used. The mobile phase contained 4 mM sodium 1-octane sulfonate as an ion-pairing agent, 50 mM ammonium formate (pH 4.0) vs methanol (80 : 20) for serotonin and (75 : 25) for tryptamine. Elution peaks of NAS and N-acetyltryptamine were identified by retention time. Their identity was verified by their coelution with authentic standards. Comparing the peak areas with known concentrations of standards produced the quantitative determination of NAS and N-acetyltryptamine. For the background controls, the reaction mixture was incubated without substrates or without an enzyme source. To identify unknown peaks, standards of assorted indole compounds were added to the samples. To test the effect of Cole bisubstrate inhibitor CoA-S-N-acetyltryptamine (BSI) the samples of hamster skin were homogenized with a glass homogenizer in an ice-cold 0.25 M potassium phosphate buffer (pH 6.8) containing 1 mM dithiothreitol, 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid and 0.125 mM acetyl-CoA. Homogenates were centrifuged at 13,000 r.p.m. (11,500 g) in centrifuge (Biofuga Fresco, Heraeus, Kendro Laboratory Products, Germany) for 10 min at 4°C. Aliquots of supernatant were mixed with serotonin in 0.25 M potassium phosphate buffer (pH 6.8) and incubated for 1 h at 37°C. The final concentrations of acetyl-CoA and serotonin were 0.1 mM and 10 mM, respectively. Inhibition of AANAT activity was determined using the specific AANAT bisubstrate inhibitor CoA-S-N-acetyltryptamine (BSI) at five concentrations ranging from 0.1 to 10 µM. The enzymatic reaction was stopped by the addition of 20 µl of 6 M HClO4. After centrifugation at 13,000 r.p.m. (11,500 g) in a centrifuge (Biofuga Fresco, Heraeus, Kendro Laboratory Products) for 5 min at 4°C, 20 µl of the supernatants were separated on a LC-MS-QP8000α (Shimadzu, Japan) through Restec Allure C18 reverse-phase column (150 × 4.6 mm; 5 µm particle size; 60 Å pore size). The elution was carried out isocratically at a flow rate of 0.3 ml per min at 30°C by mobile phases consisted of 20% acetonitrile and 0.1% trifluoroacetic acid. The effluent from the HPLC system was routed to the MS electrospray interface used in positive mode. Nitrogen was used as nebulizing gas. The MS parameters were as follows: the nebulizer gas flow rate was 4.5 l per min; the electrospray voltage was set to 4.5 kV; the CDL (curved desolvation line) heater temperature was 250°C. SIM (selected ion monitoring) mode was applied and ions with m/z = 219 was detected. The LCMS workstation Class-8000 software was used for system control and data acquisition (Shimadzu, Japan). For the controls, a reaction mixture was incubated without substrate or without an enzyme source. Quantitative determination of NAS was made by comparing its peak areas with those given by known concentrations of NAS standard. Protein concentration was determined by a dye-binding method with BSA as the standard (Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205570) Google Scholar). The experimented samples were analyzed by LC-MS using a model M-8000 LC/3DQ-MS quadropole ion trap mass spectrometer (Hitachi Instruments Inc., San Jose, CA) in the tandem mode. Briefly, AbC-1 hamster melanoma cell pellets were suspended in 0.03 M perchloric acid containing 40 µM pargyline at a density of 17–20 × 107 cells per ml. The extract was sonicated, centrifuged for 10 min at 15 000 × g and supernatants were filtered through a Millex-LH filter (0.45 µm pore size). Fifty microliter aliquots were separated by reverse phase-HPLC on a Hitachi 7000 System through a C18 Wydac column (250 × 2 mm; 5 µm particle size; 300 Å pore size; cat. no. 218TP52) (Hesperia, CA) with a mobile phase A (water with 0.2% acetic acid), and separating phase B (methanol with 0.2% acetic acid). Separation was performed with methanol at the following gradients: 5% (0–5 min), 90% (5–40 min), and 90% (40–45 min), while maintaining the flow rate at 0.3 ml per min. The effluent from the HPLC system was routed to the MS through atmospheric pressure chemical ionization. The atmospheric pressure chemical ionization conditions were as follow: nebulizer temperature 220°C; desolvator temperature 250°C; aperture 1 temperature 170°C; aperture 2 temperature 120°C; needle voltage 4 kV; drift voltage 30 V; focus voltage 30 V. The NAS standard was analyzed by LC-MS under the same conditions. The cloning strategy for the hamster pituitary is shown in Figure 1(a) A full-length sequence of hamster TPH mRNA was obtained by alignment of two separate fragments. First, we amplified a fragment of 933 bp by spanning one-half of the coding sequence and the 3′-untranslated sequence by the 3′-RACE (Figure 1a). The amplified fragment was then sequenced and used to design an anti-sense primer (P151). As this initial primer did not produce a clear band in 5′RACE, we then designed another primer, P176, homologous to mouse TPH. Amplification of hamster pituitary cDNA with primers P151 and P176 did produce a single band of 906 bp. The complete coding sequence of composite hamster TPH cDNA was retrieved by alignment of the two fragments. The open reading frame contained 1338 bp and encoded a protein composed of 446 amino acids with a predicted molecular weight of 48.5 kDa (accession no. AY034600). We then compared the sequence of the hamster gene with that of TPH genes available from the GenBank database using the DNAMLK program from the PHYLIP package (Figure 1b). Mouse and rat genes appeared to be the closest to hamster. The hamster cDNA sequence had 91%, 91%, and 86% identity with mouse, rat, and human sources, respectively. Comparison of deduced TPH amino acid sequences between hamster and mouse, rat, and human proteins showed an even higher degree of identity (92%, 92%, and 87%, respectively) (Figure 1c). TPH is organized into N-terminal regulatory and C-terminal catalytic domains; the latter contains a leucine zipper involved in the formation of the tetrameric holoenzyme (Mockus and Vrana, 1998Mockus S.M. Vrana K.E. Advances in the molecular characterization of tryptophan hydroxylase.J Mol Neurosci. 1998; 10: 163-179Crossref PubMed Scopus (76) Google Scholar). Additional inspection of the amino acid sequences showed that the structure most conserved was the catalytic domain (94–97% homology) with intermediate amino acid homology for the N-terminal regulatory domain (84–86%), and lowest identity for the C-terminal fragment containing the leucine zipper (67%) (Figure 1c). That the catalytic domain would be the most conserved portion is not surprising as that is critical for enzyme activity. We used hamster-specific primers located at exons 7 and 8 for TPH and exons 2 and 3 for AANAT for the reverse transcription–PCR assays performed on RNA from hamster skin, melanomas, eye, pituitary, heart, and spleen (Figure 2). Representative amplifica
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