Relationship between receptor binding and biopotency of somatostatin-14 and somatostatin-28 in mouse pituitary tumor cells.
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. In this study we have investigated the relationship between receptor-binding affinities of S-14 and S-28 and their biopotency in these cells. 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-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 (10(-7) 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) whereas 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. These results demonstrate that 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Srikant
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Heisler
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D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin; D000242:Cyclic AMP / Q000096:biosynthesis; D004224:Diterpenes / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D053561:Somatostatin-28 / Q000031:analogs & derivatives
D004224:Diterpenes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D017481:Receptors, Somatostatin; D005576:Colforsin; D013004:Somatostatin; C032367:somatostatin, Tyr(11)-; D053561:Somatostatin-28; C045259:somatostatin 28, Leu(8)-Trp(22)-iodo-Tyr(25)-; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP