somatostatin--tyr(11)- and Adenoma--Islet-Cell

Previous studies with heterogeneous populations of pancreatic cells have provided evidence for the presence of somatostatin (SRIF) receptors in cytosol and secretion vesicles, as well as the plasma membrane. To examine the distribution of SRIF receptors between soluble and membrane fractions in a homogeneous pancreatic islet cell population, we have used the clonal RINm5F insulinoma cell line. These cells contain specific, high affinity binding sites for [125I-Try11]SRIF on the cell surface, and occupancy of these sites by SRIF and SRIF analogs correlates with inhibition of insulin secretion. Stable, steady state binding was achieved using both intact cells and membranes by performing binding incubations with [25I-Tyr11]SRIF at 22 C. Half-maximal inhibition of [125I-Tyr11]SRIF binding occurred with 0.21 +/- 0.11 nM SRIF in membranes and 0.35 +/- 0.30 nM SRIF in cells. In contrast, the binding of [125I-Tyr11]SRIF to cytosolic macromolecules was not reduced by concentrations of SRIF as high as 100 nM, demonstrating that this binding was of much lower affinity. RINm5F membranes were further purified using a Percoll gradient to prepare a microsomal fraction, which was enriched in adenylate cyclase activity, and a secretory granule fraction, which was enriched in insulin. [125I-Tyr11]SRIF binding to the microsomal fraction (3.8 +/- 0.3 fmol/mg) was 3 times higher than to secretion granules (1.2 +/- 0.2 fmol/mg). Thus, high affinity SRIF binding sites were most abundant in microsomal membranes and were low or undetectable in secretory granules and cytosol. To determine whether translocation of SRIF receptors to the plasma membrane accompanied insulin secretion, we examined the effects of various insulin secretagogues on [125I-Tyr11]SRIF binding to intact cells. Leucine (20 mM), glyceraldehyde (15 mM), forskolin (1 microM), and glucagon (1 microM) stimulated insulin release 1.5- to 4.0-fold in different experiments. However, these secretagogues did not increase [125I-Tyr11]SRIF binding. In summary, our results indicate that high affinity SRIF receptors in RINm5F cells are located primarily on the plasma membrane and that the concentration of SRIF receptors at the cell surface is independent of the secretory activity of the cells.

Topics: Adenoma, Islet Cell; Animals; Cell Fractionation; Cell Membrane; Colforsin; Cytoplasmic Granules; Cytosol; Glucagon; Glyceraldehyde; Insulin; Insulin Secretion; Insulinoma; Iodine Radioisotopes; Kinetics; Leucine; Microsomes; Pancreatic Neoplasms; Rats; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin; Tumor Cells, Cultured

Somatostatin (SRIF) is a neuropeptide which inhibits secretion from a variety of target cells including pancreatic beta-cells. In this study we have used the RINm5F rat insulinoma cell line to characterize high affinity receptors for SRIF. The binding of 0.03 nM [125I-Tyr11]SRIF to RINm5F cells reached a plateau level within 4 h at 37 C at which time 80% of the total binding could be displaced by 100 nM unlabeled SRIF. In contrast, 100 nM concentrations of eight structurally unrelated peptides did not inhibit [125I-Tyr11]SRIF binding. Scatchard analysis indicated that RINm5F cells contained a single class of noninteracting binding sites (910 +/- 190 sites per cell) with high affinity for [125I-Tyr11]SRIF [equilibrium dissociation constant (Kd) = 0.04 +/- 0.01 nM]. Competition experiments with SRIF analogs showed that the binding affinity for [I-Tyr11]SRIF (Kd = 0.03 +/- 0.02 nM) was higher than that for either SRIF (0.24 +/- 0.04 nM) or [Tyr11]SRIF (0.27 +/- 0.04 nM) and that reduced SRIF analogs bound poorly (Kd greater than 50 nM). These results demonstrate that RINm5F cells possess specific, high affinity binding sites for SRIF. Insulin release stimulated by 20 mM leucine or 15 mM glyceraldehyde was inhibited as much as 80% by maximal concentrations (100 nM) of SRIF. The IC50 for SRIF inhibition of leucine-stimulated insulin secretion was 0.43 +/- 0.15 nM, in good agreement with the apparent Kd for binding. In fact, this close correlation between binding affinity and potency to inhibit insulin release was observed for six SRIF analogs, indicating that the characterized binding sites are the receptors which mediate the biological actions of SRIF in RINm5F cells.

Topics: Adenoma, Islet Cell; Animals; Binding, Competitive; Cell Line; Glyceraldehyde; Insulin; Insulin Secretion; Insulinoma; Kinetics; Leucine; Pancreatic Neoplasms; Rats; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin

The peptide somatostatin (SRIF) is secreted by delta cells of the endocrine pancreas and inhibits the secretion of insulin from pancreatic beta cells. We have previously shown that [125I-Tyr11]SRIF binds to specific, high affinity receptors on RINm5F insulinoma cells and that these receptors mediate the action of SRIF to inhibit insulin release. In the present study we investigated the processing of receptor-bound [125I-Tyr11]SRIF in this clonal cell line. Surface-bound and internalized peptides were distinguished by the ability of an acid/salt solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to dissociate only exposed ligand-receptor complexes. Surprisingly, greater than 80% of saturably bound [125I-Tyr11]SRIF was removed by this acid wash independent of the time or temperature of the binding incubation. In contrast, the processing of receptor-bound [125I]EGF (epidermal growth factor) in RINm5F cells was markedly temperature-dependent. Although over 90% of saturably bound [125I]EGF was dissociated by acid after a 4 degrees C binding incubation, less than 10% was removed by acid treatment after 37 degrees C binding. The radioactivity released upon dissociation of receptor-bound [125I-Tyr11]SRIF was analyzed by high performance liquid chromatography and shown to consist of a mixture of intact peptide (40%) and [125I]tyrosine (60%). However, neither the rate of [125I-Tyr11]SRIF dissociation nor its degradation were affected by NH4Cl, methylamine, or leupeptin at concentrations which inhibited the lysosomal degradation of [125I] EGF. Of 11 other protease inhibitors tested, only the metalloendoprotease inhibitor, phosphoramidon, substantially reduced the degradation of receptor-bound [125I-Tyr11]SRIF. These data indicate that, unlike [125I] EGF, receptor-bound [125I-Tyr11]SRIF is not rapidly internalized by RINm5F cells and is degraded by a nonlysosomal process which may involve a metalloendoprotease.

Topics: Adenoma, Islet Cell; Ammonium Chloride; Animals; Cells, Cultured; Deoxyglucose; Epidermal Growth Factor; Glycopeptides; Insulinoma; Iodine Radioisotopes; Lysosomes; Methylamines; Pancreatic Neoplasms; Protease Inhibitors; Rats; Receptors, Cell Surface; Receptors, Somatostatin; Somatostatin; Temperature