and i"Depart1l1ent of Biomedical Sc iences. Univ ersiry of Bradfo rd. Un.ited Kingdom Human keratinocytes have the capacity to synthesize catecholamines from L-tyrosine, which in turn is produced from L-phenylalanine Ilia phenylalanine hydroxylase. This enzyme activity is controlled by the supply of the essential cofactor/electron donor (6R)5,6, 7 ,8 tetrahydrobiopterin (6-BH4)' Undifferentiated keratinocytes express high levels of the rlttelimiting enzymes for the de 110110 synthesis of 6-BH4'

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... i.e., GTP-cyclohydrolase-l, and for its recycling, Le., 4a-hydroxytetrahydrobiopterin dehydratase. As a consequence of 6-BH. synthesis, phenylalanine hydroxylase is activated, yielding L-tyrosine, which in the presence of excess 6-BH 4 turns on the biosynthesis of catecholamines Ilia the rate-limiting enzynJe tyrosine hydroxylase. Therefore, undifferentiated keratinocytes contain high levels of the catecholamine R ecently it h as bee n shown tha t the hum an epide rmis h as the cap acity for total ca techolamine biosynthesis (Fig 1) . Tyrosine h ydroxylase (T H) , th e key e n zym e, and phenylethanolamin e-N-meth y l tran sferase (PNMT), the e n zym e for con ve rsio n o f norepine phrine to epine phrine, h ave b een identified in keratinocytes in epide rm al suction blister roofs and in full-thickn ess skin [1]. Monoclonal antibo di es aga.inst T H and PNMT co nfimled tb e locatio n of b oth en zym es in keratinocytes (A.J. Thody. p e rson a l communicatio n) . T h e biosynth esis of th e catecholamin es d ep e nds o n the availa bility of L-tyrosin e synth es ized fr o m the essenti al amino acid Lphen ylalan ine. This reaction is controlled by pheny lalanine h ydl:"o ;'{y lase (PAH) and its essenti al cofacto r/e lectron donor (6R)5, 6, 7 ,8 tetrahydro biopterin (6-BH.,). 6-BH 4 is pro du ced de 11 0110 from GYP Il in the rate-limiting en zym e GTP-cyclohydrolase 1 (GTP-CH-l ) and recycled IIin the activities of 4a-h ydroxytetrahydro biop terU1 d e hydra tase (DH) together with dih ydroptel;dine re du ctase. DH is th e rate-1.imiting en zym e for the recyding of 6-BH 4 [2-4] (Fi~ 2) . On ly rece ntl y, it has b een reported that hum an undifFe re ntiated keratinocytes and ceIl extracts fro m epidermal su ction blisters express con stituti ve levels of PAH with sp ecifi c activities con.,p a-Manu script receivcd February 3, 1994; final revision received Decclbbc r 29, '1994; accepted for publi cation Janu ary 18, 1995. Reprint req uests to: Dr. K.U. SchaUreuter, Dcpa rtment of DerInatology, Universiry of Hamburg. Martinistra sse 52. 20246 Hamburg. Germany. Abbrev iations : 6-BH.I' (6R.)5,6,7,8 tetrahydrobiopteri n; DH, 4a-hYdroxytetrahydrobioptcrin dehydratase; GTP-CH-:I, GTP cyclohydrolase_ 1; PAH, phenylalanine hydroxylase; PNMT, phenylethanolamine-N-metbyl transfcrase; T H, tyrosin c hydro"-ylase. system yielding sufficient levels of norepinephrine and epinephrine, required for the induction of beta-2-adrenoceptors. Stimulation of beta-2-adrenoceptors by epinephrine causes a rise in intracellular calcium Ilia extracellular influx. This event corresponds with keratinocyte differentiation. In differentiated keratinocytes, all enzyme activities involved in 6-BH4' L-tyrosine, and epinephrine biosynthesis are decreased, resulting in significantly lower levels of epinephrine and a concomitant decrease in the expression of beta-2-adrenoceptors. These data strongly suggest a connection between catecholamine biosynthesis, beta-2-adrenoceptor expression, calcium flux, and the differentiation ofkeratinocytes in human epidermis. K ey IIIol·d: beta-2-advelloceptovs. ",est Del'11latol :1,04:953-957, 1995 [able to those of PAH found in liver and n e uro nal tissues, w h e reas difFe re ntiate d keratinocytes h ave barely d etectable levels [3, 4] . In addition, it h as b een shown that keratinocytes and epid e m 131 cell e xtracts h ave the cap acity for de 11 0110 synth esis and recycling of 6-BH4' th e rate-limiting cofactor/electron donor for PAH and TH [3, 4] (Fig 2) . T h e biosyn tll es is of epinephrine in th e e pidermis lead s to ill villo express ion of a h.i gh d en sity of b eta-2-adren oceptors in keratinocytes, as d etermine d by radio li gand binding and au toradiograph.ic techniques [5-7] . Upon adre n ergic stim ulati o n witl1 epinephrine, a signifi can t increase in both intracellular cal cium and cAMP occurs [7-9J. Cyclic A MP h as b een shown to induce the TH gen e, leading to an in crease in TH mRNA . It h as b een su ggested that the b eta-2adrenoceptor sys tem in ke ratinocytes primarily controls calcium hom eostasis during difFer entiation . The purpose of this study was to exa mine the relation between th e biosynth esis of e pinep hrin e togetller wi th the expression of b eta-2-adre n oceptors and the calcium flu x during th e difFerentiation of k e ratino cytes und er ill vitro conditi o n s [8 -1 2]. M.ATERIALS AND METH ODS 6-BH'I' 6,7 dimcthyl-BH •. and 6-biopterin were obtained from Schircks Oona, Switzerland). All othcr enzym es and reagents were purchased from Sigma (St. Louis, MO). I·C UL L-phenylaJanin c (51. 3 m Ci/ mm( 1) was fro m IC N isotopes (CA) . and 3H-methyl-S-adenosylmethion.inc (1.06 mC i/ mmol) was from Amersham Radiochemicals (England). (-)-["'H) CG P 12177 (55 mCi/mmol) was purchascd from Amersham Buchl er (Braunschweig, Germany). 45Ca.lcium was fro m New England Nuclear (Dupont, Boston, MA) (200 mCi / ml) . Dowex AG50 (WX8) was from Biorad. Monoclonal mouse anti-human cytokeratin antibody (CK 10) was obtained fro m DAKO A / S (Denmark). Monoclonal mouse anti-human cyto keratin 5/ 6 antibody was fro m Boehringer (Mannheim, Germany). 0022-202X/95/S09.50 • SSD I0022-202X (95)00169-L •