Girolomoni G, Santantonio M L, Pastore S, Bergstresser P R, Giannetti A, Cruz P D
Department of Dermatology, University of Modena, Modena, Italy.
J Invest Dermatol. 1993 Mar;100(3):282-7. doi: 10.1111/1523-1747.ep12469769.
Extracellular adenosine 5'-triphosphate (ATPo) can induce pore formation in cell membranes, leading to cell permeabilization and eventual cell death. In this study, we examined the sensitivity of human epidermal Langerhans cells to ATP-induced permeabilization and tested the possibility that the Mg(++)- or Ca(++)-dependent plasma membrane ectonucleotidase (mATPase) on Langerhans cells provides protection against the cytotoxic effects of ATPo. Membrane permeability was assessed by using the fluorescent tracer propidium iodide, which confers red nuclear fluorescence to permeabilized cells. Langerhans cells were identified within human epidermal cell suspensions with fluorescein isothiocyanate-conjugated MoAb against CD1a or human leukocyte antigen-DR (HLA-DR) antigens. Cultured human keratinocytes and J774 macrophages were both highly sensitive to permeabilization induced by incubation with ATP (0.5 to 20 mM at 37 degrees C), whereas Langerhans cells were relatively resistant. The non-hydrolyzable ATP analog, adenosine 5'-(beta,gamma-imido) triphosphate, but not other nucleotides such as ADP, AMP, GTP, or UTP, was also able to induce permeabilization comparable to that of ATP, thereby suggesting that ATP hydrolysis is not required for this effect. ATP4- is the moiety most likely responsible for permeabilization, because propidium iodide uptake occurred only when the pH of the medium was > or = 7.4. Permeabilization induced by ATP was augmented by chelation of divalent cations with ethylene-diamine-tetraacetic acid and by the addition of lanthanum or cerium (0.01 to 1 mM). Finally, incubation with the adenosine analog, 5'-p-fluorosulfonylbenzoyl-adenosine (1 mM), inhibited mATPase staining of Langerhans cells in human epidermal sheets, but markedly augmented ATP-induced permeabilization of Langerhans cells. The results indicate that epidermal LC are resistant to the lytic effects of ATPo and that mATPase is involved in such resistance.
细胞外三磷酸腺苷（ATPo）可诱导细胞膜形成孔洞，导致细胞通透性增加并最终死亡。在本研究中，我们检测了人表皮朗格汉斯细胞对ATP诱导的通透性增加的敏感性，并测试了朗格汉斯细胞上依赖镁离子或钙离子的质膜外切核苷酸酶（mATPase）是否能提供保护，使其免受ATPo的细胞毒性作用。使用荧光示踪剂碘化丙啶评估膜通透性，碘化丙啶可使通透性增加的细胞呈现红色核荧光。在人表皮细胞悬液中，用异硫氰酸荧光素偶联的抗CD1a单克隆抗体或人白细胞抗原-DR（HLA-DR）抗原鉴定朗格汉斯细胞。培养的人角质形成细胞和J774巨噬细胞对在37℃下与ATP（0.5至20 mM）孵育诱导的通透性增加高度敏感，而朗格汉斯细胞相对耐受。不可水解的ATP类似物5'-（β，γ-亚氨基）三磷酸腺苷，但不是其他核苷酸如ADP、AMP、GTP或UTP，也能够诱导与ATP相当的通透性增加，从而表明这种效应不需要ATP水解。ATP4-最有可能是导致通透性增加的部分，因为只有当培养基的pH值≥7.4时才会发生碘化丙啶摄取。用乙二胺四乙酸螯合二价阳离子以及添加镧或铈（0.01至1 mM）可增强ATP诱导的通透性增加。最后，用腺苷类似物5'-对氟磺酰苯甲酰腺苷（1 mM）孵育可抑制人表皮片中朗格汉斯细胞的mATPase染色，但显著增强ATP诱导的朗格汉斯细胞通透性增加。结果表明表皮朗格汉斯细胞对ATPo的裂解作用具有抗性，且mATPase参与了这种抗性。
