Yang C M, Tsai Y J, Pan S L, Tsai C T, Wu W B, Chiu C T, Luo S F, Ou J T
Department of Pharmacology, Chang Gung College of Medicine and Technology, Tao-Yuan, Taiwan.
Naunyn Schmiedebergs Arch Pharmacol. 1997 Jul;356(1):1-7. doi: 10.1007/pl00005015.
Extracellular nucleotides, acting through P2-purinoceptors, have been implicated in the regulation of ion transport in epithelia, including Madin-Darby canine kidney (MDCK) cells. In this study, experiments were conducted to characterize the P2-purinoceptor subtype on MDCK cells responsible for stimulating inositol phosphate (IP) accumulation using a range of nucleotide analogues. In Ca2+- and Mg2+-free Krebs-Henseleit solution (KHS), ATP, UTP, and ATPgammaS caused an increase in IP accumulation as a function of concentration with comparable kinetics. The order of potency for the nucleotide analogues was UTP = ATPgammaS > ATP = 2-chloro ATP (Cl-ATP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = 2-methylthio ATP (2MeSATP). Selective agonists for P1-, P2X- and P2Y-purinoceptors, such as N6-cyclopentyl adenosine, AMP, alpha,beta-MeATP, and 2MeSATP, had little effect. Stimulation of MDCK cells with maximally effective concentrations of ATP and UTP showed no additive effect and furthermore, ATP, UTP, and ATPgammaS induced cross-desensitization of the IP response, suggesting that ATP and UTP act upon a common nucleotide receptor, i.e. a P2U-purinoceptor. In Ca2+- and Mg2+-containing KHS, the concentration-response curves of ATP, UTP, and ATPgammaS were shifted to the right of those obtained in Ca2+- and Mg2+-free buffer, and asymptotic maxima were not reached, indicating that ATP4- and not MgATP2- or CaATP2- was the active agonist. Pretreatment of MDCK cells with pertussis toxin (PTX) inhibited ATP- and UTP-induced IP accumulation in a concentration-dependent fashion but did not completely abolish the IP accumulation, indicating that a PTX-sensitive G protein was partially involved in the IP response. In conclusion, ATP- and UTP-stimulated IP accumulation in MDCK cells appears to be mediated through the activation of P2U-purinoceptors coupled to a G protein that is partially sensitive to PTX. A form of nucleotide uncomplexed with divalent ions such as ATP4- seems to be the preferential agonist form for the purinoceptors on MDCK cells.
细胞外核苷酸通过P2嘌呤受体发挥作用，参与上皮细胞（包括Madin-Darby犬肾细胞，即MDCK细胞）中离子转运的调节。在本研究中，使用一系列核苷酸类似物进行实验，以表征MDCK细胞上负责刺激肌醇磷酸（IP）积累的P2嘌呤受体亚型。在不含Ca2+和Mg2+的Krebs-Henseleit溶液（KHS）中，ATP、UTP和ATPγS导致IP积累随浓度增加，动力学相似。核苷酸类似物的效力顺序为UTP = ATPγS > ATP = 2-氯ATP（Cl-ATP） >> α,β-亚甲基ATP（α,β-MeATP） = 2-甲硫基ATP（2MeSATP）。P1、P2X和P2Y嘌呤受体的选择性激动剂，如N6-环戊基腺苷、AMP、α,β-MeATP和2MeSATP，作用甚微。用最大有效浓度的ATP和UTP刺激MDCK细胞未显示出相加效应，此外，ATP、UTP和ATPγS诱导IP反应的交叉脱敏，表明ATP和UTP作用于共同的核苷酸受体，即P2U嘌呤受体。在含Ca2+和Mg2+的KHS中，ATP、UTP和ATPγS的浓度-反应曲线向右移动，且未达到渐近最大值，表明ATP4-而非MgATP2-或CaATP2-是活性激动剂。用百日咳毒素（PTX）预处理MDCK细胞以浓度依赖方式抑制ATP和UTP诱导的IP积累，但未完全消除IP积累，表明PTX敏感的G蛋白部分参与IP反应。总之，ATP和UTP刺激MDCK细胞中IP积累似乎是通过激活与部分对PTX敏感的G蛋白偶联的P2U嘌呤受体介导的。一种未与二价离子络合的核苷酸形式，如ATP4-，似乎是MDCK细胞上嘌呤受体的优先激动剂形式。
