小鼠胃和结肠H，K-ATP酶的药理学特性
Pharmacological profiles of the murine gastric and colonic H,K-ATPases.
作者信息
Shao Jiahong, Gumz Michelle L, Cain Brian D, Xia Shen-Ling, Shull Gary E, van Driel Ian R, Wingo Charles S
机构信息
Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
出版信息
Biochim Biophys Acta. 2010 Sep;1800(9):906-11. doi: 10.1016/j.bbagen.2010.05.002. Epub 2010 May 25.
BACKGROUND
The H,K-ATPase, consisting of α and ß subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα₁ and HKα₂ encoded by different genes. The ouabain-resistant gastric HKα₁-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα₂-H,K-ATPase from different species shows poor primary structure conservation of the HKα₂ subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα₁, HKα₂, or HKbeta genes.
METHODS
Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα₁, HKα₂, or HKß knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined.
RESULTS
The colonic H,K-ATPase demonstrated slightly greater affinity for K(+) than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα₂ KO but was observed in HKß KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα₁-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα₂-H,K-ATPase was not inhibited by these compounds.
CONCLUSIONS
These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride.
GENERAL SIGNIFICANCE
Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.
背景
H，K - ATP酶由α和β亚基组成，属于P型ATP酶家族。α亚基有两种同工型，即由不同基因编码的HKα₁和HKα₂。耐哇巴因的胃HKα₁ - H，K - ATP酶对Sch28080敏感。然而，来自不同物种的结肠HKα₂ - H，K - ATP酶在物种间HKα₂亚基的一级结构保守性较差，对哇巴因和Sch28080的药理敏感性也各不相同。本研究旨在利用HKα₁、HKα₂或HKβ基因靶向敲除的小鼠模型，确定每个基因对功能活性及其药理特性的贡献。
方法
提取野生型（WT）、HKα₁、HKα₂或HKβ基因敲除（KO）小鼠胃黏膜和结肠远端的膜囊泡。检测K - ATP酶活性和药理特性。
结果
结肠H，K - ATP酶对K⁺的亲和力略高于胃H，K - ATP酶。HKα₂基因敲除小鼠的结肠中未检测到这种K - ATP酶活性，但在HKβ基因敲除小鼠中观察到该活性，其特性与野生型无差异。哇巴因和Sch28080对野生型结肠K - ATP酶活性均无显著影响，但原钒酸盐可消除该活性。氨氯吡咪及其类似物苄甲氯铵和5 - N - 乙基 - N - 异丙基氨氯吡咪可抑制含HKα₁的H，K - ATP酶的K - ATP酶活性；这三种抑制剂的抑制剂量依赖性相似。相比之下，结肠HKα₂ - H，K - ATP酶不受这些化合物的抑制。
结论
这些数据表明，小鼠结肠H，K - ATP酶表现出对哇巴因和Sch28080不敏感、对原钒酸盐敏感的K - ATP酶活性。有趣的是，药理研究表明小鼠胃H，K - ATP酶对氨氯吡咪敏感。
一般意义
H，K - ATP酶药理特性的表征对于理解相关基因敲除动物以及考虑抑制剂的特异性很重要。
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