| Abstract: | 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. |
