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Characterization of binding sites for oxyntomodulin on a somatostatin-secreting cell line (RIN T3)
L Gros1, E Demirpence, C Jarrousse
1Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Abstract:
Oxyntomodulin (OXM), a glucagon-containing peptide extended at its C-terminal end by an octapeptide, is a potent inhibitor of gastric acid secretion in rat and man. OXM appears to act on gastric mucosa at least partially through a stimulation of gastric somatostatin release. We have investigated the effects of OXM on a somatostatin-secreting cell line (RIN T3) derived from a radiation-induced rat insulinoma and characterized specific binding sites for this peptide. OXM increased somatostatin release with an ED50 of 2.3 nM. OXM also stimulated the cAMP accumulation in intact RIN T3 cells and adenylate cyclase activity in RIN T3 cell membranes with ED50 values of 0.5 and 11 nM, respectively. On these parameters, glucagon was 10-30 times less potent than OXM. Forskolin, isobutylmethylxanthine, and 8-bromo-cAMP mimicked the effect of OXM on somatostatin release. Specific binding for mono-[125I]OXM was dependent upon time and membrane concentration. Binding of mono-[125I]OXM was inhibited by OXM and glucagon in a concentration-dependent manner, with dissociation constants (Kd) of 4.5 and 43 nM, respectively. The nonhydrolyzable analogs of GTP (guanosine 5',3-O-(thio)triphosphate and guanosine 5' (beta,gamma-imino)triphosphate decreased the binding of mono-[125I]OXM to its binding sites. Covalent cross-linking of mono-[125I]OXM or mono-[125I]glucagon to RIN T3 cell membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single radiolabeled band at 63,000 mol wt, which differed from that observed after cross-linking with liver plasma membranes (55,000 mol wt). These results demonstrate the presence of specific high affinity binding sites for OXM in a somatostatin-secreting cell line (RIN T3) and their coupling to adenylate cyclase via guanine nucleotide-binding proteins.
Insights
Oxyntomodulin (OXM) stimulates somatostatin release and cAMP accumulation in rat insulinoma cells. Specific OXM binding sites are identified and linked to adenylate cyclase via G proteins.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Oxyntomodulin (OXM), a peptide hormone, inhibits gastric acid secretion.
- OXM's mechanism involves stimulating somatostatin release.
- The RIN T3 cell line, derived from rat insulinoma, secretes somatostatin.
Purpose of the Study:
- To investigate the effects of OXM on the somatostatin-secreting RIN T3 cell line.
- To characterize specific binding sites for OXM in these cells.
- To elucidate the signaling pathway involved in OXM's action.
Main Methods:
- Stimulation of somatostatin release and cAMP accumulation assays.
- Radioligand binding studies using mono-[125I]OXM.
- Guanine nucleotide analog experiments.
- Covalent cross-linking and SDS-PAGE analysis.
Main Results:
- OXM significantly increased somatostatin release (ED50 = 2.3 nM) and cAMP accumulation (ED50 = 0.5 nM) in RIN T3 cells.
- Specific high-affinity binding sites for OXM (Kd = 4.5 nM) were identified, distinct from glucagon binding sites.
- OXM-induced effects were mediated through adenylate cyclase coupled to guanine nucleotide-binding proteins.
- Cross-linking revealed a 63,000 mol wt OXM binding protein in RIN T3 cells.
Conclusions:
- RIN T3 cells possess specific, high-affinity binding sites for OXM.
- These sites are coupled to adenylate cyclase via G proteins, mediating OXM's effects on somatostatin release.
- OXM's action provides a molecular mechanism for its role in regulating gastric function.