Samuels E R, Harrower T, Kahlon S, Oulton M R, MacDonald J, Siauw C, Scott J E
Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Canada.
Pediatr Pulmonol. 1999 Feb;27(2):95-103. doi: 10.1002/(sici)1099-0496(199902)27:2<95::aid-ppul5>3.0.co;2-5.
The fetal lung secretes significant quantities of surfactant during late gestation to prepare for initiation of respiration at birth. However, the mechanism by which this occurs has not been determined. Since Ca2+-phosphatidylserine (PS)-dependent protein kinase C has been implicated in surfactant secretion in adult lung, the present study was done to determine whether this enzyme is also involved in the initiation of surfactant release from fetal type II cells. Type II cells isolated from gestational day-24 fetal rabbits were used. Cells were prelabelled with [32P] and [3H]choline and exposed to 4beta phorbol ester (10(-5) M) for 2 h. Secretion product and subcellular fractions were isolated by removing the culture medium, mixing with homogenate from adult rabbit lung, and subfractionating by centrifugation on a sucrose gradient. Samples of secretion product were also prepared for electron microscopy. Ca2+-PS-dependent protein kinase C was also assayed in some samples, and an add-back technique was used to determine whether enzyme activity in the intracellularly stored surfactant fraction was due to contamination. The results showed that material released by fetal type II cells after exposure to phorbol ester coprecipitated with adult rabbit lung lamellar bodies and microsomes. Morphologically, a range of forms, including lamellar-body-like structures, was detected. The released material originated largely from the lamellar body compartment of the fetal type II cells and displayed immunoreactivity with antibody to surfactant protein A (SP-A) at 35 and 70 kDa apparent molecular mass. Assay of protein kinase C in fetal type II cells showed that exposure to conditioned medium, which induces differentiation, increased activity. Incubation with phorbol ester induced translocation of activity to the microsomal fraction. Add-back assays suggested that protein kinase C activation by treatment with phorbol ester induced translocation of enzyme activity to the lamellar body fraction; none was detected prior to treatment. These results support a role for Ca2+-PS-dependent protein kinase C in initiation of surfactant release by interaction with the developing lamellar body compartment in fetal type II cells.
胎儿肺在妊娠晚期分泌大量表面活性剂，为出生时开始呼吸做准备。然而，这一过程发生的机制尚未确定。由于钙 - 磷脂酰丝氨酸（PS）依赖性蛋白激酶C与成年肺表面活性剂分泌有关，本研究旨在确定该酶是否也参与胎儿II型细胞表面活性剂释放的起始过程。使用从妊娠第24天的胎儿兔分离的II型细胞。细胞先用[32P]和[3H]胆碱预标记，然后暴露于4β佛波酯（10^(-5) M）2小时。通过去除培养基、与成年兔肺匀浆混合并在蔗糖梯度上离心进行亚分级分离来分离分泌产物和亚细胞组分。还制备了分泌产物样品用于电子显微镜检查。在一些样品中也检测了钙 - PS依赖性蛋白激酶C，并使用回补技术来确定细胞内储存的表面活性剂组分中的酶活性是否是由于污染所致。结果表明，胎儿II型细胞在暴露于佛波酯后释放的物质与成年兔肺板层小体和微粒体共沉淀。在形态学上，检测到一系列形式，包括类似板层小体的结构。释放的物质主要源自胎儿II型细胞的板层小体区室，并在表观分子量为35和70 kDa时与表面活性剂蛋白A（SP - A）抗体显示免疫反应性。对胎儿II型细胞中蛋白激酶C的检测表明，暴露于诱导分化的条件培养基会增加活性。与佛波酯孵育会诱导活性向微粒体组分易位。回补试验表明，用佛波酯处理激活蛋白激酶C会诱导酶活性向板层小体组分易位；处理前未检测到。这些结果支持钙 - PS依赖性蛋白激酶C通过与胎儿II型细胞中发育中的板层小体区室相互作用在表面活性剂释放起始过程中发挥作用。
