Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 1. Characterization, quantitative aspects and structural requirements of binding sites.
Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. 1. Characterization, quantitative aspects and structural requirements of binding sites.
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血管活性肠肽与大鼠分离的肠上皮细胞的相互作用。
DOI：
10.1111/j.1432-1033.1979.tb13033.x
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
G. Rosselin
中科院分区：
文献类型：
--
作者：
J. Prieto;M. Laburthe;G. Rosselin
猪血管活性肠肽的特异性结合位点的特点是在分离的肠上皮细胞从大鼠使用125碘标记的血管活性肠肽和各种物理化学条件。在15°C下，肽的结合在90和240分钟孵育之间达到平台，并且是高达约8.8 × 105个细胞/ml的细胞浓度的线性函数。120分钟后肽和结合位点的失活分别不超过对照的30%和15%。结合的最佳pH为7.5左右。在30°C下，不能获得血管活性肠肽结合的表观稳态，并且肽和结合位点的失活高于15°C下的失活。天然血管活性肠肽在0.1 nM-0.1 μM范围内竞争性抑制125 I标记肽的结合;在约3.1 nM血管活性肠肽时观察到半数最大抑制，低至0.15 nM的浓度显示出显著作用。动力学研究和Scatchard分析表明存在两个功能独立的类结合位点。约有1.4 × 105个高亲和力位点/细胞，解离常数（Kd）为1.6 nM，1.1 × 106个低亲和力位点/细胞，Kd为74 nM。
鸡血管活性肠肽表现出约7倍以上的亲和力比猪肽在竞争的125 I标记的肽的结合肠上皮细胞，这表明可能的作用，氨基硫脲在残基28在增强亲和力。促胰液素抑制125 I标记的肽的结合在一个连续和平行的方式，以未标记的肽，但与70倍低的亲和力。竞争实验使用几种合成类似物的促胰液素表明，结合亲和力部分相关的存在下，在NH 2-末端六肽序列的血管活性肠肽分子的疏水性氨基酸。胰高血糖素和抑胃肽分别在10和0.1 μM时对125 I标记的血管活性肠肽无效。肠上皮细胞中的血管活性肠肽受体的这些性质，连同关于该肽的生物学作用的早期报道，强烈支持该肽在生理条件下作为小肠功能的调节剂起作用的概念。
Specific binding sites for porcine vasoactive intestinal peptide have been characterized in isolated intestinal epithelial cells from rat using 125I-labeled vasoactive intestinal peptide and a variety of physicochemical conditions. At 15°C binding of the peptide reached a plateau between 90 and 240 min incubation and was a linear function of the cell concentration up to about 8.8 × 105 cells/ml. Inactivation of the peptide and of binding sites after 120 min did not exceed 30% and 15% of the controls, respectively. Optimal pH for binding was around 7.5. At 30°C an apparent steady-state of vasoactive intestinal peptide binding could not be obtained, and the inactivation of both peptide and binding sites were higher than those at 15°C. Native vasoactive intestinal peptide competitively inhibited the binding of the 125I-labeled peptide in the range 0.1 nM–0.1 μM; half-maximal inhibition was observed at about 3.1 nM vasoactive intestinal peptide, and a concentration as low as 0.15 nM showed a significant effect. Kinetics studies and Scatchard analysis indicated the existence of two functionally independent classes of binding sites. There are approximately 1.4 × 105 high affinity sites/cell with a dissociation constant (Kd) of 1.6 nM and 1.1 × 106 low affinity sites/cell with a Kd of 74 nM.
Chicken vasoactive intestinal peptide exhibited about seven times more affinity than the porcine peptide in competing with the binding of the 125I-labeled peptide to intestinal epithelial cells, suggesting the possible role of the aminothreonine at residue 28 in enhancing the affinity. Secretin inhibited the binding of the 125I-labeled peptide in a continuous and parallel fashion as to the unlabeled peptide but with a seventy times lower affinity. Competition experiments using several synthetic analogs of secretin indicated that binding affinity is partially related to the presence of hydrophobic amino acids at the NH2-terminal hexapeptide sequence of the vasoactive intestinal peptide molecule. Glucagon and gastric inhibitory peptide were ineffective in displacing 125I-labeled vasoactive intestinal peptide at 10 and 0.1 μM, respectively. These properties of the vasoactive intestinal peptide receptors in intestinal epithelial cells, together with earlier reports on biological actions of the peptide, strongly support the concept that this peptide may act under physiological conditions as a regulator of small intestinal function.