Relationship between Receptor Binding and Biopotency of Somatostatin-14 and Somatostatin-28 in Mouse Pituitary Tumor Cells*

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Abstract
Somatostatin-14 (S-14) acts via specific receptors to inhibit basal as well as hormone- and forskolin-stimulated ACTH secretion in tumor cells (AtT-20/D16-16) of mouse anterior pituitary. In addition S-14 inhibits the stimulated but not basal cAMP accumulation. The potency of somatostatin-28 (S-28) for regulating these processes in these tumor cells has not been reported. The relationship between receptor-binding affinities of S-14 and S-28 and their biopotency in these cells was investigated. Membrane receptors for S-14 characterized using [125I-Tyr11]S-14 as the radioligand [maximum binding capacity (Bmax) = 1.28 .+-. 0.1 pmol/mg; dissociation constant (Kd) = 1.1 .+-. 0.04 nM] bound S-28 with 3-fold greater affinity than S-14. Binding sites quantitated using an S-28 analog [Leu8, D-Trp22,125I-I-Tyr25]S-28 as radioligand (Bmax = 1.18 .+-. 0.15 pmol/mg; Kd = 0.08 .+-. 0.06 nM) also exhibited greater affinity for S-28 than S-14. Forskolin-stimulated cAMP accumulation and ACTH secretion in these cells were inhibited to a greater extent (4- and 9-fold, respectively) by S-28 than S-14. Preincubation of the cells with S-14 and S-28 (107 M) resulted in a marked decrease (36% and 71%, respectively) of S-14 receptor concentration. Coincubation of the cells with both S-14 and S-28 led to 56% decrease in S-14 receptor binding. The responsiveness of the cells to forskolin stimulation of ACTH secretion and cAMP accumulation was significantly enhanced by preincubation with S-14 (10-7 M); the responsiveness to forskolin was completely abolished by preincubation with S-28. Simultaneous exposure of the cells to both S-14 and S-28 resulted in a partial reversal of the inhibiting effect of S-28 on forskolin-stimulated cAMP accumulation in these cells but did not result in a partial reversal of the inhibitory effect of S-28 on forskolin-stimulated ACTH secretion in these cells. Apparently, S-28 is more potent than S-14 in AtT-20/D16-16 cells, its greater potency arising from its greater affinity for binding to S-14 receptors. The differential effects of these peptides after preincubation on the responsiveness of ACTH secretion and cAMP accumulation in these cells to forskolin stimulation suggests the possibility of existence of distinct S-14 and S-28 receptors, but these could not be identified by direct binding experiments using the S-14 and S-28 analogs employed in the study as radioligands. Alternatively the actions or these peptides may be mediated via more than 1 postreceptor mechanisms that are differentially sensitive to S-14 and S-28.