Singh G, Eng J, Raufman J P
Gastrointestinal Cell Biology Laboratory, State University of New York-Health Science Center at Brooklyn 11203-2098.
Regul Pept. 1994 Aug 31;53(1):47-59. doi: 10.1016/0167-0115(94)90158-9.
We synthesized and iodinated an exendin-4 analogue, [Y39]exendin-4 (700 Ci/mmol), for use as a radioligand to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig. Binding of this bioactive radioligand was rapid, temperature-dependent and specific (not inhibited by other pancreatic or gastric secretagogues). Measurement of the ability of exendin-4 to inhibit the binding of 125I-[Y39]exendin-4 indicated the presence of two classes of receptors. Pancreatic acini had 12.5.10(10) binding sites/mg acinar protein of which 6% were high affinity (Kd = 0.5 nM) and 94% were low affinity (Kd = 0.1 microM). Chief cells had 3370 binding sites/cell of which 9% were high affinity (Kd = 0.3 nM) and 91% were low affinity (Kd = 0.2 microM). Washing with 0.2 M acetic acid (pH 2.5), 0.2 M glycine (pH 10.5), or trypsin (100 micrograms/ml) after 30 min incubation at 37 degrees C, indicated that 63 and 49% of radioligand was internalized in acini and chief cells, respectively. Truncated glucagon-like peptide-1 (tGLP-1), a mammalian peptide sharing 53% homology with exendin-4, inhibited radioligand binding at the same concentrations that altered secretion from acini and chief cells. Glucagon, GLP-1 and GLP-2 inhibited 125I-[Y39]exendin-4 binding only at concentrations > or = 100 nM. Exendin(9-39)NH2, a specific exendin-receptor antagonist, potently inhibited 125I-[Y39] exendin-4 binding (IC50 = 6.1 and 3.5 nM in acini and chief cells, respectively). In pancreatic acini and gastric chief cells from guinea pig, exendin-3, exendin-4 and tGLP-1 increase cellular cAMP and modulate enzyme secretion by interacting with high-affinity exendin receptors. 125I-[Y39] exendin-4 is a useful radioligand for studying exendin receptors.
我们合成并碘化了一种艾塞那肽-4类似物，[Y39]艾塞那肽-4（700 Ci/mmol），用作放射性配体来鉴定豚鼠分散胰腺腺泡和胃主细胞上的艾塞那肽受体。这种生物活性放射性配体的结合迅速、依赖温度且具有特异性（不受其他胰腺或胃促分泌素抑制）。测定艾塞那肽-4抑制125I-[Y39]艾塞那肽-4结合的能力表明存在两类受体。胰腺腺泡每毫克腺泡蛋白有12.5×10¹⁰个结合位点，其中6%为高亲和力（Kd = 0.5 nM），94%为低亲和力（Kd = 0.1 μM）。主细胞每个细胞有3370个结合位点，其中9%为高亲和力（Kd = 0.3 nM），91%为低亲和力（Kd = 0.2 μM）。在37℃孵育30分钟后，用0.2 M乙酸（pH 2.5）、0.2 M甘氨酸（pH 10.5）或胰蛋白酶（100微克/毫升）洗涤，表明分别有63%和49%的放射性配体在腺泡和主细胞中被内化。截短的胰高血糖素样肽-1（tGLP-1），一种与艾塞那肽-4有53%同源性的哺乳动物肽，在改变腺泡和主细胞分泌的相同浓度下抑制放射性配体结合。胰高血糖素、GLP-1和GLP-2仅在浓度≥100 nM时抑制125I-[Y39]艾塞那肽-4结合。艾塞那肽(9 - 39)NH2，一种特异性艾塞那肽受体拮抗剂，有效抑制125I-[Y39]艾塞那肽-4结合（在腺泡和主细胞中的IC50分别为6.1和3.5 nM）。在豚鼠的胰腺腺泡和胃主细胞中，艾塞那肽-3、艾塞那肽-4和tGLP-1通过与高亲和力艾塞那肽受体相互作用增加细胞内cAMP并调节酶分泌。125I-[Y39]艾塞那肽-4是研究艾塞那肽受体的一种有用的放射性配体。
