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Gastrin-releasing peptide: binding and functional studies in mouse pancreatic islets
M A Wahl1, E A Landsbeck, H P Ammon
1Department of Pharmacology, University of Tübingen, Germany.
Insights
Gastrin-releasing peptide (GRP) specifically binds to mouse pancreatic islets, influencing insulin secretion. This binding involves a high-affinity site and triggers intracellular signaling pathways, including phosphatidylinositol breakdown and calcium release, impacting glucose metabolism.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Gastrin-releasing peptide (GRP) is a neuropeptide with known physiological roles.
- Pancreatic islets are crucial for glucose homeostasis and insulin secretion.
- The specific interactions of GRP with pancreatic islets and its downstream effects require further elucidation.
Purpose of the Study:
- To investigate the specific binding characteristics of GRP to mouse pancreatic islets.
- To explore the functional consequences of GRP binding on insulin secretion and intracellular signaling.
- To identify the role of GRP in modulating glucose-stimulated insulin release.
Main Methods:
- Radioligand binding assays using 125I-GRP to determine binding affinity and specificity.
- Competitive inhibition studies with various GRP analogs and unrelated peptides.
- Measurement of phosphatidylinositol breakdown and intracellular calcium (Ca2+) efflux in response to GRP stimulation.
Main Results:
- Specific, saturable, high-affinity binding sites for GRP were identified on mouse pancreatic islets (Kd1 = 0.81 nM; Bmax1 = 12.8 fmol/50 islets).
- The C-terminus of GRP-1-27 is essential for high-affinity binding, while GRP-1-16 is ineffective.
- GRP-1-27 enhanced insulin secretion, increased phosphatidylinositol breakdown to Ins-1,4,5-P3, and promoted Ca2+ efflux from intracellular stores.
Conclusions:
- Mouse pancreatic islets possess specific GRP binding sites.
- GRP potentiates the insulinotropic effect of glucose.
- Ins-1,4,5-P3 likely acts as a second messenger in GRP-mediated biological effects, involving intracellular calcium mobilization.
Abstract:
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.