SYNTHESIS AND CHARACTERIZATION OF A NEW LABELED GASTRIN LIGAND, I-125BH-[LEU(15)]-GASTRIN-(5-17), ON BINDING TO CANINE FUNDIC MUCOSAL CELLS AND JURKAT CELLS

In the course of our study concerning gastrin and cholecystokinin (CCK ) receptors, we have synthesized and characterized a new labeled gastr in ligand, I-125-BH-[Leu(15)]-gastrin-(5-17) hydroxyphenyl)propionyl-[ Leu(15)]-gastrin-(5-17)]. Binding of I-125-BH-[Leu(15)]-gastrin-(5-17) to isolated canine fundic mucosal cells was specific, saturable and o f high affinity. I-125-BH-[Leu(15)]-gastrin-(5-17) and I-125-BH-CCK-8 [(3-[I-125]iodo-4-hydroxyphenyl)-propionyl-CCK-8] interact with isolat ed canine fundic mucosal cells with small differences in maximal bindi ng capacities and affinities, 3800 +/- 900 binding sites/cell(K-d = 0. 52 +/- 0.23 nM) and 6200 +/- 1100 binding sites/cell (Kd = 0.31 +/- 0. 18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing 125I-BH-[Leu(15)]-gastrin-(5-17) binding cor related well with those obtained using I-125-BH-CCK-8. Selective CCK/g astrin antagonists L-364,718 (MK-329) and L-365,260 also inhibited I-1 25-BH-[Leu(15)]-gastrin-(5-17) binding. These results indicate that I- 125-BH-[Leu(15)]-gastrin-(5-17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized I-125-BH- [Le u's]-gastrin-(5-l7) binding to the human Jurkat lymphoblastic cell lin e (Jurkat cells) known to express the CCK-B/gastrin receptor. Saturati on experiments have shown that both I-125-BH-[Leu(15)]-gastrin-(5-17) and I-125-BH-CCK-8 interact with a single class of high-affinity bindi ng sites in the Jurkat cell line. Binding characteristics of I-125-BH- [Le(15)S]-gastrin-(5-17) and I-125-BH-CCK-8 were estimated to be about 2500 +/- 400 binding sites/cell (Kd = 0.19 +/- 0.19 nM) and 2400 +/- 350 binding sites/cell (Kd = 0.06 +/- 0.02 nM), respectively. Furtherm ore, the apparent affinities of CCK analogs and selective antagonists MK-329 and L-365,260 for the sites labeled by both probes were identic al. The biological activity of cold I-127-BH-[Leu(15)]-gastrin-(5-17) and [Leu's]-gastrin-(5-l7) was demonstrated by their ability to increa se intracellular calcium concentration in Jurkat cells. All these expe riments showed that I-125-BH-[Leu(15)]-gastrin-(5-17) provides a conve nient ligand for gastrin receptor binding studies. (C) Munksgaard 1993 .