Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands.
Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands.
复制标题
离体胃腺体中 H -K -ATP 酶活性的刺激相关重新分布。
DOI：
10.1152/ajpgi.1987.252.4.g458
复制
发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Forte,JG
中科院分区：
文献类型：
--
作者：
Urushidani,T;Forte,JG
本研究的目的是建立一种在兔离体胃腺体外模型系统中研究刺激依赖性膜重分布和H+-K+- atp酶特性的方法。刺激腺(10（-4)M组胺+ 10(-5)M福斯克林）和静息腺（10(-4)M甲胺）均质，分为PO （40 g， 5分钟）、P1 （400 g， 10分钟）、P2 （14,500 g， 10分钟）、P3 （48,200 g， 90分钟）和上清S3。我们的H+-K+- atp酶（K+-对硝基苯磷酸酶）活性标记物的分布发生了显著变化：P3活性降低，P1代偿性增加。P3显示缬霉素（Val）依赖性的囊泡H+摄取，而P1的H+摄取与Val无关。三磷酸腺苷酶的直接测定表明，P3的H+-K+-三磷酸腺苷酶活性是Val依赖性的，并因刺激而降低；P1的H+-K+-ATPase活性与Val无关，刺激后升高。进一步对P1进行密度梯度纯化，发现比17% Ficoll轻的膜具有更高的特异性H+-K+-ATPase活性，并且观察到H+-K+-ATPase与刺激相关的增加更为明显。此外，受刺激P1的较轻组分具有较高的潜在H+-K+- atp酶活性，这被正辛基葡萄糖苷所掩盖。离体腺膜组分的性质与体内实验结果一致：静息腺中P3的高H+-K+- atp酶活性对应于缺乏KCl转运途径的细胞质管泡；受刺激腺体分泌的高活性P1对应于顶质膜囊泡，除了H+-K+- atp酶外，还含有KCl运输，并且完全具备生成HCl的能力。
The objective of this work is to establish a procedure to study the stimulation-dependent membrane redistribution and properties of H+-K+-ATPase in an in vitro model system, rabbit isolated gastric glands. Stimulated (10(-4) M histamine plus 10(-5) M forskolin) and resting (10(-4) M metiamide) glands were homogenized and fractionated into PO (40 g, 5 min), P1 (400 g, 10 min), P2 (14,500 g, 10 min), P3 (48,200 g, 90 min), and supernatant, S3. Significant changes occurred in the distribution of our marker for H+-K+-ATPase (K+-p-nitrophenyl phosphatase) activity: a reduction in activity of P3 and a compensatory increment in P1. P3 showed valinomycin (Val)-dependent vesicular H+ uptake, while H+ uptake in P1 was Val independent. Direct measurements of ATPase revealed that H+-K+-ATPase activity of P3 was Val dependent and decreased by stimulation; H+-K+-ATPase activity of P1 was Val independent and increased by stimulation. Further density gradient purification of P1 showed that membranes lighter than 17% Ficoll contained higher specific H+-K+-ATPase activity, and the observed increase in H+-K+-ATPase associated with stimulation was more pronounced. Also, the lighter fractions from stimulated P1 had much latent H+-K+-ATPase activity that was unmasked by n-octylglucoside. The properties of membrane fractions from isolated glands were consistent with results obtained in vivo: high H+-K+-ATPase activity of P3 from resting glands corresponds to cytoplasmic tubulovesicles lacking KCl transport pathways; high activity of P1 from stimulated glands corresponds to apical plasma membrane vesicles containing KCl transport in addition to the H+-K+-ATPase, and full competency for the generation of HCl.