Brönnegård M, Okret S
Department of Medical Nutrition, Huddinge University Hospital, Sweden.
J Steroid Biochem. 1988 Nov;31(5):809-17. doi: 10.1016/0022-4731(88)90290-7.
Glucocorticoid receptor (GR) from fetal rat lung cytosol was characterized during development. A gradual increase in receptor concentration without an apparent change in ligand affinity was observed during ontogenesis (16-20 days of gestation). GR was present at least 2 days prior to gestational day 18, from which day maternal betamethasone administration stimulated choline chloride incorporation into phosphatidylcholine, the major phospholipid in surfactant. Gel permeation analysis of lung cytosolic GR from fetuses of different gestational ages showed a gradual disappearance of a 3.6 nm GR seen in day 16 cytosol and to the appearance of a 5.8 nm GR in cytosol from day 19. The differences in Stokes' radii of GR were not due to transcriptional or posttranscriptional modifications of the GR transcript, since both day 16 and day 19 fetal lung contained a 7 kb GR mRNA similar to that in adult rat lung. Mixing experiments showed that the 3.6 nm GR was generated by an increased proteolytic activity in day 16 lung tissue. Preservation of a normal size 5.8 nm in day 16 fetal lung upon extraction could only be achieved by preincubating and homogenizing the lung tissue in the presence of protease inhibitors. No protease activity was found in day 16 cytosol suggesting the presence of a rapidly inactivated protease(s). The protease activity responsible for GR degradation was probably of a serine protease type, since proteolytic activity could be inactivated by diisopropylfluorophosphate alone, a potent inhibitor of serine proteases. From these results we conclude that: (i) the observed differences in Stokes' radii between GR from fetal lung of different developmental stages is attributable to proteolysis following extraction, most likely by a rapidly inactivated serine protease. This activity diminished during fetal lung development. However, in intact lung cells, GR is physicochemically identical throughout development; (ii) the lack of glucocorticoid stimulation or surfactant synthesis on day 16 and 17 in fetal rat lung despite the presence of low concentration of GR is therefore not explained by any differences in GR structure.
对胎鼠肺胞质溶胶中的糖皮质激素受体（GR）在发育过程中的特性进行了研究。在个体发育过程中（妊娠16 - 20天），观察到受体浓度逐渐增加，而配体亲和力无明显变化。在妊娠第18天前至少2天就已存在GR，从这一天起，母体给予倍他米松可刺激氯化胆碱掺入磷脂酰胆碱，磷脂酰胆碱是表面活性物质中的主要磷脂。对不同胎龄胎儿的肺胞质溶胶GR进行凝胶渗透分析表明，在第16天胞质溶胶中可见的3.6纳米GR逐渐消失，而在第19天的胞质溶胶中出现了5.8纳米的GR。GR斯托克斯半径的差异并非由于GR转录本的转录或转录后修饰，因为第16天和第19天的胎肺均含有与成年大鼠肺中相似的7千碱基GR mRNA。混合实验表明，3.6纳米的GR是由第16天肺组织中蛋白水解活性增加产生的。只有在蛋白酶抑制剂存在下对肺组织进行预孵育和匀浆，才能在提取时使第16天胎肺中的5.8纳米正常大小得以保留。在第16天的胞质溶胶中未发现蛋白酶活性，提示存在一种快速失活的蛋白酶。负责GR降解的蛋白酶活性可能属于丝氨酸蛋白酶类型，因为蛋白水解活性可仅被二异丙基氟磷酸酯（一种丝氨酸蛋白酶的有效抑制剂）灭活。根据这些结果我们得出结论：（i）不同发育阶段胎肺GR之间观察到的斯托克斯半径差异可归因于提取后的蛋白水解作用，最有可能是由一种快速失活的丝氨酸蛋白酶所致。这种活性在胎肺发育过程中降低。然而，在完整的肺细胞中，GR在整个发育过程中物理化学性质相同；（ii）尽管存在低浓度的GR，但胎鼠肺在第16天和第17天缺乏糖皮质激素刺激或表面活性物质合成，因此不能用GR结构的任何差异来解释。
