Hill D J, McDonald T J
MRC Group in Fetal and Neonatal Health and Development, Lawson Research Institute, St. Joseph's Health Centre, London, Ontario, Canada.
Endocrinology. 1992 May;130(5):2811-9. doi: 10.1210/endo.130.5.1572294.
Gastrin-releasing polypeptide (GRP) has been implicated in the development of the human fetal lung. To determine whether GRP has a wider role in fetal development, its actions on DNA synthesis and cell replication by isolated epiphyseal growth plate chondrocytes obtained from ovine fetuses between 35 days gestation and near term (145 days) were examined. Chondrocytes were isolated using collagenase from the proximal tibia and cultured in monolayer. Synthesis of DNA was assessed from the incorporation of [3H]thymidine into previously growth-restricted cells after incubation in medium supplemented with GRP1-27 (40-1280 nM). Increase in cell number was assessed after incubation with test medium for 1 week. GRP caused a dose-dependent increase in both cell number and DNA synthetic rate compared to control incubations. Cell number was increased by 50% in the presence of a maximally effective 160 nM GRP and DNA synthesis by up to 800% utilizing chondrocytes obtained from animals of 75-80 days gestation. The mean (+/- SEM) half-maximal concentration of GRP for the stimulation of DNA synthesis was 97 +/- 12 nM (5 separate fetuses). Concentrations of GRP in excess of 160 nM caused a sharp reduction in both cell replication and DNA synthesis. To determine where within the cell cycle GRP exerted its mitogenic action, synchronized chondrocytes were transiently exposed to fetal bovine serum and cultured with GRP for increasing periods of time before pulse labeling with [3H]thymidine during S phase. GRP was as effective in stimulating DNA synthesis when present for the initial 4 h of G1 as when present for the entire G1 period. Since isolated fetal growth plate chondrocytes release insulin-like growth factor II (IGF II) and basic fibroblast growth factor (basic FGF) the possible mediation of GRP action by the release of these peptides or synergistic interactions were examined. Specific antibodies shown to negate the mitogenic actions of exogenous IGFs or basic FGF on chondrocytes did not alter GRP-stimulated DNA synthesis. The release of radioimmunoassayable IGF II by chondrocytes was not altered in the presence of GRP. Coincubation of GRP with submaximal concentrations of IGF I or basic FGF showed additive effects on DNA synthesis. When the actions of galanin were examined it was found to inhibit basal DNA synthesis by chondrocytes at a concentration of 167 nM. However, 66 nM or greater galanin was able to render 160 nM GRP inactive as a mitogen. These results suggest that GRP may potentially influence skeletal development in the ovine fetus and may interact with locally released peptide growth factors or other neuropeptides.
胃泌素释放肽（GRP）与人类胎儿肺的发育有关。为了确定GRP在胎儿发育中是否具有更广泛的作用，研究了其对从妊娠35天至接近足月（145天）的绵羊胎儿分离的骨骺生长板软骨细胞的DNA合成和细胞复制的作用。使用胶原酶从胫骨近端分离软骨细胞，并在单层中培养。在补充有GRP1 - 27（40 - 1280 nM）的培养基中孵育后，通过将[3H]胸苷掺入先前生长受限的细胞中来评估DNA合成。在与测试培养基孵育1周后评估细胞数量的增加。与对照孵育相比，GRP导致细胞数量和DNA合成速率呈剂量依赖性增加。在存在最大有效浓度160 nM GRP的情况下，细胞数量增加了50%，利用妊娠75 - 80天动物的软骨细胞，DNA合成增加了高达800%。刺激DNA合成的GRP的平均（±SEM）半数最大浓度为97±12 nM（5个不同的胎儿）。超过160 nM的GRP浓度导致细胞复制和DNA合成急剧减少。为了确定GRP在细胞周期的哪个阶段发挥其促有丝分裂作用，将同步化的软骨细胞短暂暴露于胎牛血清中，并在S期用[3H]胸苷脉冲标记之前与GRP培养不同的时间。当在G1期的最初4小时存在时，GRP刺激DNA合成的效果与在整个G1期存在时一样有效。由于分离的胎儿生长板软骨细胞释放胰岛素样生长因子II（IGF II）和碱性成纤维细胞生长因子（碱性FGF），因此研究了这些肽的释放或协同相互作用对GRP作用的可能介导。显示可消除外源性IGF或碱性FGF对软骨细胞有丝分裂作用的特异性抗体并没有改变GRP刺激的DNA合成。在存在GRP的情况下，软骨细胞释放的可通过放射免疫测定的IGF II没有改变。GRP与亚最大浓度的IGF I或碱性FGF共同孵育对DNA合成显示出相加作用。当检查甘丙肽的作用时，发现其在浓度为167 nM时抑制软骨细胞的基础DNA合成。然而，66 nM或更高浓度的甘丙肽能够使160 nM GRP作为有丝分裂原失去活性。这些结果表明，GRP可能潜在地影响绵羊胎儿的骨骼发育，并可能与局部释放的肽生长因子或其他神经肽相互作用。
