Use of 125I-[Y39]exendin-4 to characterize exendin receptors on dispersed pancreatic acini and gastric chief cells from guinea pig.
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.
Singh
G
G
Gastrointestinal Cell Biology Laboratory, State University of New York-Health Science Center at Brooklyn 11203-2098.
Eng
J
J
Raufman
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D000077270:Exenatide; D005753:Gastric Mucosa / Q000378:metabolism*; D005934:Glucagon / Q000494:pharmacology; D052216:Glucagon-Like Peptide 1; D053767:Glucagon-Like Peptide 2; D006168:Guinea Pigs; D007457:Iodine Radioisotopes; D008297:Male; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine; D014688:Venoms
D053767:Glucagon-Like Peptide 2; D007457:Iodine Radioisotopes; D010455:Peptides; D011964:Receptors, Gastrointestinal Hormone; D014688:Venoms; C074030:exendin receptor; D014443:Tyrosine; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000077270:Exenatide; D000242:Cyclic AMP