Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.
Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent.
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T84 细胞中 cAMP 刺激分泌的顶端铵抑制是碳酸氢盐依赖性的。
DOI：
10.1152/ajpgi.00451.2004
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
Matthews,JeffreyB
中科院分区：
文献类型：
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作者：
Worrell,RogerT;Best,Alison;Crawford,OscarR;Xu,Jie;Soleimani,Manoocher;Matthews,JeffreyB
正常人结肠腔 (NH4+) 浓度 ([NH4+]) 范围为 ∼10 至 100 mM。然而，人们对结肠上皮中 NH4+ 对转运的影响以及 NH4+ 转运本身的影响性质知之甚少。我们在此阐明顶端 NH4+ 对结肠 T84 细胞中 cAMP 刺激的 Cl- 分泌的影响。在 HEPES 缓冲溶液中，10 mM 顶端 NH4+ 对 cAMP 刺激电流没有显着影响。相比之下，在 25 mM HCO3− 存在的情况下，10 mM 顶端 NH4+ 在 5 分钟内将电流降低至 61 ± 4%。电流抑制不仅仅是由于细胞外 K+ 样阳离子的增加，因为相对于 5 mM 顶端 K+，10 mM 顶端 K+ 的电流强度为 95 ± 5%，10 mM 顶端 NH4+ 的电流强度为 46 ± 3%。我们之前证明，在无 HCO3− 的条件下，基底外侧 NH4+ 会抑制 Cl− 分泌，并表现出异常的摩尔分数行为。相比之下，HCO3−缓冲液中电流的顶端 NH4+ 抑制没有表现出异常摩尔分数行为，并且遵循 K+-NH4+ 混合物中的绝对 [NH4+]，其中 K+ 浓度 + [NH4+] = 10 mM。 100 μM 醋甲唑胺并未阻止顶端 NH4+ 抑制作用，表明顶端碳酸酐酶没有作用。然而，用 500 μM DIDS、100 μM 4,4'-二硝基芪-2,2'-二磺酸 (DNDS) 或 25 μM 尼氟酸对根尖表面进行 10 分钟预处理，可以防止根尖 NH4+ 对电流的抑制，这表明 NH4+ 通过根尖阴离子交换剂发挥作用。通过 RT-PCR 以及 Northern 和 Western blot 检测 T84 细胞中顶端阴离子交换剂 SLC26A3 [在腺瘤 (DRA) 中下调] 和 SLC26A6 [假定的阴离子转运蛋白 (PAT1)] 的 mRNA 和蛋白质。 DRA 和 PAT1 似乎与顶膜中的 CFTR 相关。我们得出结论，顶端 NH4+ 分泌抑制的 HCO3−依赖性是由于 NH4+ 对顶端阴离子交换剂的作用。
Normal human colonic luminal (NH4+) concentration ([NH4+]) ranges from ∼10 to 100 mM. However, the nature of the effects of NH4+on transport, as well as NH4+transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH4+on cAMP-stimulated Cl−secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH4+had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH4+reduced current within 5 min to 61 ± 4% in the presence of 25 mM HCO3−. Current inhibition was not simply due to an increase in extracellular K+-like cations, in that the current magnitude was 95 ± 5% with 10 mM apical K+and 46 ± 3% with 10 mM apical NH4+relative to that with 5 mM apical K+. We previously demonstrated that inhibition of Cl−secretion by basolateral NH4+occurs in HCO3−-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH4+inhibition of current in HCO3−buffer did not show anomalous mole fraction behavior and followed the absolute [NH4+] in K+-NH4+mixtures, where K+concentration + [NH4+] = 10 mM. The apical NH4+inhibitory effect was not prevented by 100 μM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH4+inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 μM DIDS, 100 μM 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), or 25 μM niflumic acid, suggesting a role for NH4+action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO3−dependence of apical NH4+inhibition of secretion is due to the action of NH4+on an apical anion exchanger.