Simon F R, Sutherland E, Accatino L
J Clin Invest. 1977 May;59(5):849-61. doi: 10.1172/JCI108707.
Since phenobarbital administration produces a profound increase in bile flow without changing bile acid secretion, we examined whether this drug increases the activity of hepatic sodium-potassium-activated ATPase [Na+-K+)-ATPase], the postulated regulating enzyme in the secretion of bile salt independent bile flow. After freeze-thawing to increase substrate accessibility, (Na+-K+) ATPase activity was determined by ouabain inhibition of total ATPase activity. Its activity was highest in isolated liver surface membrane fractions enriched in bile canalicult. Phenobarbital administration significatly increased (Na+-K+)-ATPase activity in both liver surface membrane fractions as well as liver homogenates. This enhanced activity is apparently selective for other membrane phosphatases and the enzyme activity in other tissues is either unaltered or decreased. Kinetic analysis of (Ka+-K+)-ATPase indicates that phenobarbital treatment increased maximum velocity and half-maximum activation constant was unchanged, consistent with activation of latent molecules or an increased number of enzyme molecules. The latter process seems more likely because cycloheximide prevented phenobarbital induction and activators were not demonstrated in vitro. Examination of the full time course of phenobarbital induction to determine whether phenobarbital increased synthesis or decreased degradation was consistent with increased synthesis since the apparent degradation rates were similar with or without phenobarbital treatment. The apparent half-life for (Na+-K+)-ATPase was estimated to be approximately 2.5 days, consistent with liver surface membrane protein turnover. The correlation of changes in bile flow with (Na+-K+)-ATPase was examined under several experimental situations. Phenobarbital caused a parallel increase in each during the 1st 2 days of greatment: thereafter other factors become rate limiting for flow, since enzyme activity doesn't reach a new steady state until 4-days. Consistent with increased sodium-potassium exchange, bile sodium was unchanged while potasium concentrations were significantly reduced. Changes in both bile flow and (Na+-K+)-ATPase induced by phenobarbital are independent of thyroid hormone. These studies support the postulate that (Na+-K+)-ATPase is an important factor in regulation of bile flow. In addition, phenobarbital enhancement of both bile flow and (Na+-K+)-ATPase is dependent upon de novo protein synthesis.
由于给予苯巴比妥可使胆汁流量显著增加，而胆汁酸分泌却无变化，因此我们研究了该药物是否会增加肝钠钾激活的ATP酶（[Na⁺-K⁺]-ATP酶）的活性，该酶被认为是胆盐非依赖性胆汁流量分泌的调节酶。在进行冻融处理以增加底物可及性后，通过哇巴因抑制总ATP酶活性来测定（Na⁺-K⁺）ATP酶活性。其活性在富含胆小管的分离肝表面膜组分中最高。给予苯巴比妥显著增加了肝表面膜组分以及肝匀浆中的（Na⁺-K⁺）-ATP酶活性。这种增强的活性显然对其他膜磷酸酶具有选择性，并且其他组织中的酶活性要么未改变，要么降低。对（Na⁺-K⁺）-ATP酶的动力学分析表明，苯巴比妥处理增加了最大速度，而半最大激活常数未变，这与潜在分子的激活或酶分子数量的增加一致。后一种过程似乎更有可能，因为环己酰亚胺可阻止苯巴比妥的诱导作用，且在体外未证实有激活剂存在。对苯巴比妥诱导的整个时间进程进行检查以确定苯巴比妥是增加了合成还是减少了降解，结果表明是合成增加，因为无论有无苯巴比妥处理，表观降解速率相似。（Na⁺-K⁺）-ATP酶的表观半衰期估计约为2.5天，这与肝表面膜蛋白周转情况一致。在几种实验情况下，研究了胆汁流量变化与（Na⁺-K⁺）-ATP酶之间的相关性。在给予苯巴比妥的头2天内，胆汁流量和（Na⁺-K⁺）-ATP酶均呈平行增加：此后其他因素成为流量的限速因素，因为酶活性直到4天才达到新的稳态。与钠钾交换增加一致，胆汁中的钠含量未变，而钾浓度显著降低。苯巴比妥诱导的胆汁流量和（Na⁺-K⁺）-ATP酶的变化均与甲状腺激素无关。这些研究支持了（Na⁺-K⁺）-ATP酶是调节胆汁流量的重要因素这一假设。此外，苯巴比妥对胆汁流量和（Na⁺-K⁺）-ATP酶的增强作用依赖于从头合成蛋白质。
