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1 Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y 2 receptors by human keratinocytes, the cells which comprise the epidermis. 2 Reverse transcriptase-polymerase chain reaction (RT ± PCR) revealed expression of mRNA for the G-protein-coupled, P2Y 2 receptor in primary cultured human keratinocytes. 3 In situ hybridization studies of skin sections revealed that P2Y 2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y 2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4 Increases in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5 In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgS were found to stimulate the proliferation of keratinocytes. 6 Using a real-time ®re¯y luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7 These ®ndings indicate that P2Y 2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

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...

Extracellular nucleotides are agonists at the family of receptors known as the P2 receptors, and in keratinocytes the P2Y2 subtype is known to elevate the intracellular free calcium concentration (Cai) and stimulate proliferation. In this study, we have investigated the presence of other functional members of the P2Y subgroup in both normal human keratinocytes and the HaCaT cell line. Using reverse transcription polymerase chain reaction, the expression of mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors was demonstrated in HaCaT cells and differentiated and undifferentiated normal human keratinocytes. Cai was monitored in response to a panel of P2Y receptor agonists. To couple mobilized Cai to a downstream cellular response, cell proliferation was also addressed. In both cell types, adenosine 5'-triphosphate and uridine 5'-triphosphate induced Cai transients of approximately equal duration, magnitude, and shape, confirming the presence of functional P2Y2 receptors. In HaCaT cells, additional characteristic responses were observed in a subpopulation of cells; adenosine 5'-triphosphate failed to elevate Cai in some cells responding to uridine 5'-triphosphate, indicating the presence of P2Y4 receptors, whereas the P2Y1-specific agonist 2-methylthio-5'-adenosine diphosphate was, again, only effective in a small subpopulation. Uridine 5'-diphosphate was ineffective, indicating the absence of functional P2Y6 receptors. Adenosine 5'-triphosphate and uridine 5'-triphosphate equally promoted cell growth in normal human keratinocytes in comparison with the control. In HaCaT cells, adenosine 5'-triphosphate, uridine 5'-triphosphate, and adenosine 5'-diphosphate significantly increased proliferation in comparison to the controls, with a 30% higher response to uridine 5'-triphosphate than with adenosine 5'-triphosphate. These data demonstrate that multiple P2Y receptors (P2Y1, P2Y2, and P2Y4 subtypes) are differentially involved in the regulation of proliferation in human keratinocytes and therefore may be important in wound healing.

The safety profile of intermittent or continuous long-term application of 0.1% tacrolimus ointment for up to 4 years was consistent with that which has been established from shorter studies and gave no reason for concern. In addition, 0.1% tacrolimus ointment demonstrated sustained efficacy as reflected by the expression of high satisfaction with treatment by both patients and investigators.