Human Keratinocytes Release ATP and Utilize Three Mechanisms for Nucleotide Interconversion at the Cell Surface

Abstract

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F1F0 ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F1F0 ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F1F0 ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing. Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F1F0 ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F1F0 ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F1F0 ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing. Extracellular nucleotides, such as ATP and ADP, are now recognized as important autocrine and paracrine factors involved in the regulation of many cellular processes in a wide range of tissues. They act via activation of the P2 family of receptors of which there are two subgroups: the P2X receptors (ligand-gated ion channels) and the P2Y receptors (G protein-coupled receptors) as determined by their molecular structure, transduction pathways, and pharmacological properties (1Abbracchio M.P. Boeynaems J.M. Barnard E.A. Boyer J.L. Kennedy C. Miras-Portugal M.T. King B.F. Gachet C. Jacobson K.A. Weisman G.A. Burnstock G. Trends Pharmacol. Sci. 2003; 24: 52-55Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). There are currently seven members of the P2X subgroup (P2X1-7) (2Khakh B.S. Burnstock G. Kennedy C. King B.F. North R.A. Seguela P. Voigt M. Humphrey P.P. Pharmacol. Rev. 2001; 53: 107-118PubMed Google Scholar) and eight members of the P2Y subgroup (P2Y1,2,4,6,11-14) identified in mammalian cell types (3Abbracchio M.P. Burnstock G. Pharmacol. Ther. 1994; 64: 445-475Crossref PubMed Scopus (993) Google Scholar). In the epidermis, P2 receptors are involved in the regulation of proliferation, differentiation, and apoptosis, and thus subtypes are expressed in different regions. Differential expression of multiple P2 receptor subtypes is species- and cell type-dependent (4Pillai S. Bikle D.D. J. Clin. Invest. 1992; 90: 42-51Crossref PubMed Scopus (107) Google Scholar, 5Reimer W.J. Dixon S.J. Am. J. Physiol. 1992; 263: C1040-C1048Crossref PubMed Google Scholar, 6Ralevic V. Burnstock G. Pharmacol. Rev. 1998; 50: 413-492PubMed Google Scholar, 7Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Br. J. Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (100) Google Scholar). Although P2Y2 receptor expression is almost entirely confined to the proliferative basal layer (7Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Br. J. Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (100) Google Scholar, 8Greig A.V. Linge C. Terenghi G. McGrouther D.A. Burnstock G. J. Invest. Dermatol. 2003; 120: 1007-1115Crossref PubMed Scopus (109) Google receptors are expressed in keratinocytes in the of differentiation, the layer, and receptors are confined to the differentiated of the layer A.V. Linge C. Terenghi G. McGrouther D.A. Burnstock G. J. Invest. Dermatol. 2003; 120: 1007-1115Crossref PubMed Scopus (109) Google Scholar, M. Burnstock G. 1999; PubMed Scopus Google Scholar). In normal human keratinocytes express for and and the P2Y2 receptor to Bowler W.B. Gallagher J.A. Sharpe G.R. J. Invest. Dermatol. 2003; 120: Full Text Full Text PDF PubMed Scopus Google Scholar). expression of P2Y2 and receptor is Bowler W.B. Gallagher J.A. Sharpe G.R. J. Invest. Dermatol. 2003; 120: Full Text Full Text PDF PubMed Scopus Google Scholar) in with In in the HaCaT cell cell P. J. A. J. PubMed Scopus Google multiple P2Y receptors are and with the receptor the Bowler W.B. Gallagher J.A. Sharpe G.R. J. Invest. Dermatol. 2003; 120: Full Text Full Text PDF PubMed Scopus Google Scholar) and the in culture the of the are nucleotides are important in normal epidermal homeostasis in the of and are to the epidermal nucleotides are by as of the reaction with Sci. S. A. PubMed Scopus Google Scholar) and by or at of these the of above that of normal thus healing. are not or by or are by keratinocytes (7Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Br. J. Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (100) Google Scholar). Although the for ATP release is in PubMed Scopus Google Scholar) and Sci. PubMed Scopus Google the by which nucleotides are by cell mechanisms G. Sci. S. A. 90: PubMed Scopus Google Scholar, D.A. J. 1994; Full Text PDF PubMed Google Scholar, G.R. W.B. Full Text PDF PubMed Scopus Google Scholar, D.A. Am. J. Physiol. PubMed Google Scholar, B.F. J. 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Although in nucleotide localized, in the extracellular of these P2 are to due to the nucleotide nucleotide conversion is this have for P2 receptor are by which P2Y receptors are of not previously in keratinocytes or the skin, have the mechanisms for nucleotide conversion at the cell surface in a cell is the of conversion by to a at which of extracellular nucleotides the that conversion In this study, we have for ATP via kinase, ATP synthase, and in the cell medium and in were nucleotides, diadenosine and were was and were to the of ATP synthase and were receptor was was and were HaCaT cell was a of and in of ATP was in the of a and was to the was used in and was by for with activity was by at for of ATP and ADP were and was to that activity ATP and ADP the were to were in with of and cell was in with and were at in a of and HaCaT were at and were with for with in for were not the with were in and at a of were and were in at a of and or ATP were of Extracellular was HaCaT in were in to addition of of were for at to that the medium, were to the and of medium were almost into nucleotide was this was were these were to the to addition of nucleotides and were at with the each of and was and the were into a were for a of was by a the range to were and were of nucleotides in medium alone the of and and of medium were and for a of each of these of was and the ATP was as ATP were of and of were into ATP of ADP, of each was into the of and the ATP was by addition of of of was each of of and of kinase of or of were and the was a was and the ATP for the was a the of for the of of and of kinase was a and the was was the ADP, and the were into ADP a were at a of and were to were with three for each to culture were with and a confocal were for with and in to in were in for and in for were in for for in in and in for a were for in the of to the of ATP synthase or to in and in in for each to and in the with for ATP synthase or for in in for at were in for each to were in to in for and in for were for with to epidermal receptor with to the of ATP in and in were in for each to and in the with in for at were in for each to was with in and a of with as NDPK this was to and in and for human was in for and were and in a with and were the for cell surface of keratinocytes were to the of ATP synthase and to and in used were for of and for of ATP was the was with of with the determined by a ATP in the (7Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Br. J. Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (100) Google of normal human keratinocytes have to release ATP in However, this not previously in the human cell release in keratinocytes to cell HaCaT were into at to and to for medium was with of medium, and were for a ATP was of medium the ATP of the medium the with cell this that HaCaT release ATP this release was in the HaCaT cell with at the medium HaCaT ATP that HaCaT and the cell were at and to and for medium was and with of medium were and ATP ADP, GTP, and UTP the ATP of have previously to express activity K.A. J.M. Dillon J.P. Gallagher J.A. J. 2003; PubMed Scopus Google this the that conversion of extracellular nucleotides in HaCaT HaCaT in and with P2 receptor culture medium was and the ATP was Although addition of UDP, and the of ATP in the culture medium by of nucleotides in the of ADP, GTP, and UTP elevated ATP in the medium by and and the in ADP in the of ATP in the culture medium K.A. J.M. Dillon J.P. Gallagher J.A. J. 2003; PubMed Scopus Google Scholar). in ATP addition of ADP to HaCaT was and was that in K.A. J.M. Dillon J.P. Gallagher J.A. J. 2003; PubMed Scopus Google Scholar). a of ATP is by ATP is to HaCaT the ATP by of ADP ATP to with and is at the and were as the in the of and to in the of and ATP in the culture medium in a of ADP were to of HaCaT of culture medium were and the ATP was ATP ADP in the of were and in the of were in and are of two are S. There are currently three by which ADP to ATP at the cell via adenylate kinase M. Am. J. PubMed Scopus Google Scholar, C.J. Br. J. Pharmacol. 2003; PubMed Scopus Google Scholar, M. J. 2003; Full Text Full Text PDF PubMed Scopus Google NDPK M. Am. J. PubMed Scopus Google Scholar, R.A. Am. J. Physiol. 2001; PubMed Google Scholar, S. J. 2001; PubMed Scopus (109) Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google and F1F0 ATP synthase P. S. Sci. S. A. 1999; PubMed Scopus Google Scholar, J.A. Sci. S. A. 2001; PubMed Scopus Google Scholar, A. 2003; Google Scholar, S. J.P. C. V. 2003; PubMed Scopus Google Scholar). the have to in a of different cell their role in the of extracellular ATP was in the HaCaT cell ADP to ATP in the kinase the ATP and is inhibited by V. PubMed Scopus Google Scholar). the of kinase ADP conversion to of to were to of HaCaT and the was for to addition of ADP ATP of of culture medium were the in addition of ADP, to due to adenylate kinase that conversion by However, elevated the ATP of culture medium in the of ADP not of were of ATP and ADP were not in with Br. J. 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Sci. 2003; PubMed Scopus Google and we have that F1F0 ATP synthase expression is suggesting that the ATP by is important this provide a of nucleotides are at the cell surface to the of the of GTP alone could not to NDPK as we the role of three was of oligomycin, and almost entirely the conversion of ADP or GTP to ATP suggesting that there are However, the activity of NDPK, F1F0 ATP synthase, or adenylate kinase, determined the nucleotides could not fully account for the ATP of the culture medium the nucleotides were conversion due to of the we postulate that this discrepancy is to due to the reverse reaction of adenylate kinase utilizing of the adenylate kinase adenylate S. PubMed Scopus Google Scholar) and M. A. J. 1999; PubMed Scopus Google Scholar) GTP as a phosphate in the reverse reaction in the of this for the in ATP GTP was in the of the adenylate kinase that the HaCaT cell three mechanisms for nucleotide conversion not in the of nucleotides cellular processes are the of P2 receptor these are human keratinocytes express P2Y2 receptors and the HaCaT cell receptors Bowler W.B. Gallagher J.A. Sharpe G.R. J. Invest. Dermatol. 2003; 120: Full Text Full Text PDF PubMed Scopus Google which by UTP and conversion in HaCaT ADP ATP and thus the to the P2Y2 and receptor activation of P2 receptors is involved in proliferation, differentiation, and in the A.V. Linge C. Terenghi G. McGrouther D.A. Burnstock G. J. Invest. Dermatol. 2003; 120: 1007-1115Crossref PubMed Scopus (109) Google Scholar, Bowler W.B. Gallagher J.A. Sharpe G.R. J. Invest. Dermatol. 2003; 120: Full Text Full Text PDF PubMed Scopus Google release of nucleotides, and provide a these processes are the of and ATP was in normal human In with HaCaT of ATP synthase and NDPK the layer the In the basal layer, expression of was in the differentiated and the of was in the as for mitochondrial cell surface In the layer, the of was at the cell surface and was almost entirely the cell ATP synthase in the ATP is by the keratinocytes in the basal layer (7Dixon C.J. Bowler W.B. Littlewood-Evans A. Dillon J.P. Bilbe G. Sharpe G.R. Gallagher J.A. Br. J. Pharmacol. 1999; 127: 1680-1686Crossref PubMed Scopus (100) Google Scholar). ATP P2Y2 which are to in normal keratinocytes and HaCaT Bowler W.B. Gallagher J.A. Sharpe G.R. J. Invest. Dermatol. 2003; 120: Full Text Full Text PDF PubMed Scopus Google this is not for normal epidermal for wound healing. is that there is of cell surface in the basal layer there is of extracellular ATP are with ATP release as for and ATP release in G.R. J. 2003; Full Text Full Text PDF PubMed Scopus Google ATP to ADP, and Scopus Google Scholar) in the basal which are for could to the layer cell surface are There is the potential for generation of extracellular ATP in the important for that in the differentiated layer receptors are A.V. Linge C. Terenghi G. McGrouther D.A. Burnstock G. J. Invest. Dermatol. 2003; 120: 1007-1115Crossref PubMed Scopus (109) Google elevated of ATP are to activate this receptor that nucleotide is involved in the in the and that the generation of extracellular ATP We are to and for with the and and for the of