Mallampalli R K, Mathur S N, Warnock L J, Salome R G, Hunninghake G W, Field F J
Pulmonary Division, Department of Veterans Affairs Medical Center, Iowa City, IA, USA.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):333-41. doi: 10.1042/bj3180333.
Glucocorticoids appear to play an integral role in stimulating surfactant synthesis by activating the rate-regulatory enzyme for phosphatidylcholine synthesis, CTP:cholinephosphate cytidylyltransferase (CT). The activity of liver CT, in vitro, has been shown to be inhibited by the sphingomyelin hydrolysis product, sphingosine. In order to investigate the mechanisms by which glucocorticoids alter CT activity, in vivo, we administered betamethasone (1 mg/kg intraperitoneally) sequentially to adult male rats for 5 days. Betamethasone increased CT activity 2-fold relative to control in whole lung. The hormone also increased membrane-bound activity, but did not affect cytosolic enzyme activity. Betamethasone modestly increased CT mRNA as determined by the reverse-transcription PCR and Southern analysis of PCR products, but did not alter the levels of immunoreactive enzyme in lung membranes as demonstrated by Western blotting. The hormone did, however, produce a nearly 3-fold increase in membrane-associated sphingomyelin, and co-ordinately a substantial decrease in the levels of sphingosine in lung membranes. Sphingosine, but not sphinganine, was a competitive, reversible inhibitor of lung CT with respect to the enzyme activator, phosphatidylglycerol. Betamethasone decreased the activities of the sphingomyelin hydrolases: acid sphingomyelinase by 33% and of alkaline ceramidase by 21%. The hormone also inhibited the generation of sphingosine from lysosphingomyelin in lung membranes. There was no significant effect of the hormone on serine palmitoyltransferase activity, the first committed enzyme for sphingolipid biosynthesis. Further, administration of L-cycloserine, an inhibitor of sphingosine formation, was shown to stimulate CT activity by 74% and increase disaturated phosphatidylcholine in alveolar lavage by 52% relative to control. These observations suggest that glucocorticoids up-regulate surfactant synthesis at the level of a key regulatory enzyme by significantly altering the availability of inhibitory metabolites resulting from sphingomyelin hydrolysis.
糖皮质激素似乎在刺激表面活性剂合成中发挥着不可或缺的作用，它通过激活磷脂酰胆碱合成的速率调节酶——CTP：胆碱磷酸胞苷转移酶（CT）来实现。体外实验表明，肝脏CT的活性会受到鞘磷脂水解产物鞘氨醇的抑制。为了研究糖皮质激素在体内改变CT活性的机制，我们对成年雄性大鼠连续5天腹腔注射倍他米松（1mg/kg）。与对照组相比，倍他米松使全肺CT活性增加了2倍。该激素还增加了膜结合活性，但不影响胞质酶活性。通过逆转录PCR和PCR产物的Southern分析确定，倍他米松适度增加了CT mRNA，但如Western印迹所示，它并未改变肺膜中免疫反应性酶的水平。然而，该激素使肺膜中与膜相关的鞘磷脂增加了近3倍，同时肺膜中鞘氨醇水平大幅下降。相对于酶激活剂磷脂酰甘油，鞘氨醇而非二氢鞘氨醇是肺CT的竞争性、可逆抑制剂。倍他米松降低了鞘磷脂水解酶的活性：酸性鞘磷脂酶降低了33%，碱性神经酰胺酶降低了21%。该激素还抑制了肺膜中溶血鞘磷脂生成鞘氨醇。该激素对丝氨酸棕榈酰转移酶活性（鞘脂生物合成的首个关键酶）没有显著影响。此外，相对于对照组，给予鞘氨醇形成抑制剂L-环丝氨酸可使CT活性提高74%，并使肺泡灌洗中的二饱和磷脂酰胆碱增加52%。这些观察结果表明，糖皮质激素通过显著改变鞘磷脂水解产生的抑制性代谢物的可用性，在关键调节酶水平上上调表面活性剂合成。
