Koster H P, Hartog A, van Os C H, Bindels R J
Department of Cell Physiology, University of Nijmegen, The Netherlands.
Am J Physiol. 1996 Jan;270(1 Pt 2):F53-60. doi: 10.1152/ajprenal.1996.270.1.F53.
Rabbit connecting tubule and cortical collecting duct cells were isolated by immunodissection and cultured to confluence on permeable filters and on glass coverslips. Extracellular ATP dose-dependently reduced transcellular Na+ and Ca2+ transport (half-maximal inhibitory concentration, IC50, of 0.5 +/- 0.2 and 3.2 +/- 0.5 microM), with a maximal inhibition of 57 +/- 5 and 43 +/- 4%, respectively. Purinergic receptor agonists inhibited transport with the following rank order of potency: UTP = ATP > ADP; this suggests involvement of P2u purinoceptors. ATP also caused a dose-dependent (50% effective dose, EC50, of 1.5 +/- 0.2 microM) transient increase in intracellular Ca2+ concentration ([Ca2+]i), which decreased to a sustained elevated level. In the absence of extracellular Ca2+, a similar Ca2+ transient occurred, but the sustained response was abolished. Preloading the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) completely prevented the ATP-induced Ca2+ transients, but not the ATP-induced inhibition of Na+ and Ca2+ absorption. Activation of protein kinase C (PKC) by the cell-permeable diacylglycerol analogue, 1,2-dioctanoyl-en-glycerol, mimicked ATP-induced inhibition of Na+ and Ca2+ absorption. The inhibitory effects of ATP were no longer observed after culturing cells in the presence of phorbol ester (12-O-tetradecanoylphorbol-13-acetate) for 5 days, which resulted in downregulation of cellular PKC activity.
通过免疫解剖分离出兔连接小管和皮质集合管细胞，并将其培养至在可渗透滤膜和玻璃盖玻片上汇合。细胞外ATP剂量依赖性地降低跨细胞Na⁺和Ca²⁺转运（半数最大抑制浓度，IC50，分别为0.5±0.2和3.2±0.5微摩尔），最大抑制率分别为57±5%和43±4%。嘌呤能受体激动剂抑制转运的效力顺序如下：UTP = ATP > ADP；这表明P2u嘌呤受体参与其中。ATP还引起细胞内Ca²⁺浓度（[Ca²⁺]i）剂量依赖性（半数有效剂量，EC50，为1.5±0.2微摩尔）的短暂升高，随后降至持续升高水平。在无细胞外Ca²⁺的情况下，出现类似的Ca²⁺瞬变，但持续反应消失。用Ca²⁺螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）预加载细胞可完全阻止ATP诱导的Ca²⁺瞬变，但不能阻止ATP诱导的Na⁺和Ca²⁺吸收抑制。细胞可渗透的二酰基甘油类似物1,2-二辛酰-sn-甘油激活蛋白激酶C（PKC）可模拟ATP诱导的Na⁺和Ca²⁺吸收抑制。在佛波酯（12-O-十四酰佛波醇-13-乙酸酯）存在下培养细胞5天后，不再观察到ATP的抑制作用，这导致细胞PKC活性下调。
