Rutten M J, Dempsey P J, Luttropp C A, Hawkey M A, Sheppard B C, Crass R A, Deveney C W, Coffey R J
Department of Surgery, Oregon Health Sciences University, Portland, USA.
Am J Physiol. 1996 Apr;270(4 Pt 1):G604-12. doi: 10.1152/ajpgi.1996.270.4.G604.
Binding and localization of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) were assessed using in vitro primary cultures of guinea pig gastric mucous epithelial cells (GMEC). GMEC were isolated and cultured in six-well plates with Dulbecco's modified Eagle's medium + 10% serum and then changed to serum-free medium for 24 h for binding studies. The binding time course of 125I-labeled EGF and 125I-TGF-alpha in GMEC cultures at 4 degrees C was saturable, reaching a plateau within 4-6 h. Competition-binding curves revealed that the amount of unlabeled EGF and TGF-alpha to reduce 125I-EGF binding by 50% was 0.35 and 0.23 nM, respectively. The amount of unlabeled EGF and TGF-alpha to decrease 125I-TGF-alpha binding by 50% was 0.30 and 0.21 nM, respectively. A Scatchard analysis of the data disclosed that a single class of high-affinity binding sites (dissociation constant = 0.24 nM) was present. The maximal binding capacity was approximately 20 fmol/10(6) cells or approximately 12,000 receptors per cell. The binding of 125I-EGF and 125I-TFG-alpha to GMEC cultures was maximal between pH 7.0 and 8.5. No specific binding of EGF or TGF-alpha could be detected below pH 5.0. The half-maximal pH dissociation value for EGF and TGF-alpha was pH 5.89 and pH 6.83, respectively. We found no difference in the final amounts of membrane-bound or internalized 125I-EGF and 125I-TGF-alpha. However, there was a significant difference (P < 0.05) at 5-30 min in the rate of dissociated and internalized 125I-EGF- and 125I-TGF-alpha. Immunofluorescence microscopy of GMEC cultures for EGF/TGF-alpha receptors showed increased fluorescence at the leading edges and around the perimeter of cells. Detection of an EGF/TGF-alpha receptor was also confirmed by Western blotting. Our findings demonstrate that guinea pig GMEC possess a specific EGF/TGF-alpha receptor, which further supports a physiological role for EFG and TFG-alpha as mitogens in these cells.
利用豚鼠胃黏液上皮细胞（GMEC）的体外原代培养物评估转化生长因子-α（TGF-α）和表皮生长因子（EGF）的结合及定位。将GMEC分离并接种于含 Dulbecco 改良 Eagle 培养基 + 10%血清的六孔板中培养，随后更换为无血清培养基培养24小时以进行结合研究。4℃下GMEC培养物中125I标记的EGF和125I-TGF-α的结合时间进程呈饱和状态，4 - 6小时内达到平台期。竞争结合曲线显示，未标记的EGF和TGF-α使125I-EGF结合减少50%的量分别为0.35和0.23 nM。未标记的EGF和TGF-α使125I-TGF-α结合减少50%的量分别为0.30和0.21 nM。对数据进行Scatchard分析表明存在一类单一的高亲和力结合位点（解离常数 = 0.24 nM）。最大结合容量约为20 fmol/10(6)个细胞或每个细胞约12,000个受体。125I-EGF和125I-TFG-α与GMEC培养物的结合在pH 7.0至8.5之间最大。在pH 5.0以下未检测到EGF或TGF-α的特异性结合。EGF和TGF-α的半数最大pH解离值分别为pH 5.89和pH 6.83。我们发现膜结合或内化的125I-EGF和125I-TGF-α的最终量没有差异，但在5 - 30分钟时，125I-EGF和125I-TGF-α的解离和内化速率存在显著差异（P < 0.05）。GMEC培养物中EGF/TGF-α受体的免疫荧光显微镜检查显示细胞前缘和周边的荧光增强。Western印迹也证实了EGF/TGF-α受体的检测。我们的研究结果表明豚鼠GMEC具有特异性的EGF/TGF-α受体，这进一步支持了EFG和TFG-α作为这些细胞中有丝分裂原的生理作用。
