Distribution of somatostatin receptors in RINm5F insulinoma cells.
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.
D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology; D005985:Glyceraldehyde / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D007930:Leucine / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007328:Insulin; D007457:Iodine Radioisotopes; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D005576:Colforsin; D005985:Glyceraldehyde; D013004:Somatostatin; C032367:somatostatin, Tyr(11)-; D005934:Glucagon; D007930:Leucine