Table 1

Ligand binding assays. Receptor-ligand binding assays were done with membranes isolated from CHO-K1 cells expressing each of the cloned human sst receptors. Results shown are expressed asK i values (in nanomoles). The ligand 3-[125I]iodotyrosyl25–somatostatin-28(Leu8, D-Trp22, Tyr25) was obtained from Amersham and was used at a final concentration of 0.1 nM. Assays were done in 96-well polypropylene plates in a final volume of 200 μl. The assay buffer consisted of 50 mM tris-HCl (pH 7.8), 1 mM EGTA, 5 mM MgCl2, leupeptin (10 μg/ml), pepstatin (10 μg/ml), bacitracin (200 μg/ml), and aprotinin (0.5 μg/ml). Test compounds were examined over a range of concentrations from 0.01 to 10,000 nM. CHO-K1 cell membranes, ligand, and test compound were incubated at room temperature for 45 min and then collected on Packard 96-well glass fiber filter plates treated with 0.1% polyethyleneimine. Plates were washed with cold 50 mM tris-HCl (pH 7.8), then dried before counting. K i values were calculated with the Cheng-Prussof equation (17).K d values for 3-[125I]iodotyrosyl25–somatostatin-28(Leu8,D-Trp22, Tyr25) with each of the five receptors were determined from Scatchard plots of saturation binding curves. The apparent dissociation constants were 1.5, 0.1, 0.4, 4.2, and 0.7 nM for receptors sst1 through sst5, respectively.

Compoundhsstr1hsstr2hsstr3hsstr4hsstr5
ss-140.40.040.71.72.3
L-363,37756640.53072>10,0002009
L-797,5911.4187522401703600
L-779,97627600.057293104260
L-796,7781255>10,0002486501200
L-803,087199472012800.73880
L-817,8183.35264820.4