Burrell Helen E, Wlodarski Brenda, Foster Brian J, Buckley Katherine A, Sharpe Graham R, Quayle John M, Simpson Alec W M, Gallagher James A
Department of Human Anatomy & Cell Biology, School of Biomedical Sciences, University of Liverpool, The Sherrington Buildings, UK.
J Biol Chem. 2005 Aug 19;280(33):29667-76. doi: 10.1074/jbc.M505381200. Epub 2005 Jun 15.
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.
P2受体的核苷酸激活在许多组织的自分泌和旁分泌调节中起着重要作用。在表皮中，核苷酸参与细胞增殖、分化和凋亡。在本研究中，我们结合使用荧光素-荧光素酶发光法、药理学抑制剂和共聚焦显微镜，证明HaCaT角质形成细胞将ATP释放到培养基中，并且存在三种核苷酸相互转化机制，导致在细胞表面产生ATP。向培养基中添加ADP、GTP或UTP可提高ATP浓度。二磷酸腺苷五磷酸、寡霉素和UDP分别抑制ADP向ATP的转化，提示细胞表面腺苷酸激酶、F(1)F(0)ATP合酶和核苷二磷酸激酶（NDPK）参与其中，免疫组织化学也支持这一点。同时添加ADP和GTP可使ATP浓度高于单独添加每种核苷酸时的浓度，表明GTP作为磷酸供体。然而，NDPK、F(1)F(0)ATP合酶的活性或腺苷酸激酶的正向反应不能完全解释培养基中ATP的含量。我们推测这种差异是由于腺苷酸激酶利用AMP的逆向反应所致。在正常人体皮肤中，F(1)F(0)ATP合酶和NDPK的定位不同，线粒体在基底层表达，细胞表面在分化层表达。我们和其他人之前已经证明角质形成细胞表达多种P2受体。在本研究中，我们现在确定了激活这些受体所需的细胞外ATP的潜在来源，并更好地理解了核苷酸在正常表皮稳态和伤口愈合中的作用。
