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, F 1 F 0 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, F 1 F 0 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, F 1 F 0 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 proteincoupled receptors) as determined by their molecular structure, transduction pathways, and pharmacological properties (1). There are currently seven members of the P2X subgroup (P2X 1-7 ) (2) and eight members of the P2Y subgroup (P2Y 1,2,4,6,11-14 ) identified in mammalian cell types (3).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 (4 -7). Although P2Y 2 receptor expression is almost entirely confined to the proliferative basal layer (7, 8), P2X 5 receptors are expressed in keratinocytes in the early stages of differentiation, i.e. the spinous layer, and P2X 7 receptors are confined to the terminally differentiated cells of the cornified layer (8, 9). In vitro, normal human keratinocytes express mRNA for P2Y 1 , P2Y 2...

The AKU mouse was established as a model of both the plasma biochemistry of AKU and its associated arthropathy. Early-stage treatment of AKU patients with nitisinone could prevent the development of associated joint arthropathies. The cellular pathology of ochronosis in AKU mice is identical to that observed in early human ochronosis and thus is a model in which the early stages of joint pathology can be studied and novel interventions evaluated.

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We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.