Gastrin-releasing peptide: binding and functional studies in mouse pancreatic islets

The binding of GRP (gastrin-releasing peptide) to mouse pancreatic islets was studied. Binding of 100 pM 125I-GRP to collagenase-prepared isolated islets at 22 degrees C was one-half maximal after 15 min and maximal at 60 min. At 60 min, total binding was 1.62% of total radioactivity per 50 islets; nonspecific binding (presence of 1 microM unlabeled GRP-1-27) was 0.05-0.61% of total radioactivity. GRP binds specifically to a high-affinity site (Kd1 = 0.81 nM; Bmax1 = 12.8 fmol/50 islets). The specific binding is saturable. Hormones with the intact C-terminus of GRP-1-27, such as N-acetyl-GRP-20-27 and neuromedin C (GRP-18-27), possess the same inhibition curve as GRP-1-27. GRP-1-16, with a cleaved C-terminus, does not inhibit binding of 125I-GRP. However, hormones that virtually are not structurally related to GRP, such as eledoisin, galanin, and VIP (vasoactive intestinal peptide) do not compete for GRP binding. The rank order of GRP analogs such as GRP-1-27, N-acetyl-GRP-20-27, and GRP-1-16 is similar though not identical with respect to inhibition of 125I-GRP binding and insulin secretory potency. We found that 1 and 10 nM GRP-1-27, at a stimulatory glucose concentration, increases the breakdown of phosphatidylinositol to Ins-1,4,5-P3, the biological relevant isomer of Ins-P3; 10 nM GRP-1-27 is effective even at a nonstimulatory glucose concentration in this respect. In a virtually Ca(2+)-free medium, 5 nM GRP-1-27 increases the 45Ca2+ efflux from 45Ca(2+)-prelabeled islets. These data indicate that (a) specific binding sites for GRP are present in mouse pancreatic islets; (b) GRP superimposes the maximal insulinotropic effect of glucose; and (c) Ins-1,4,5-P3 is probably involved as a second messenger in the biological effects of GRP-1-27, which is underlined by the efflux of Ca2+ from intracellular stores but is not a sufficient signal by itself.