Activation of adenylate cyclase by human recombinant sst5 receptors expressed in CHO-K1 cells and involvement of Gαs proteins

1 The coupling of the human somatostatin sst(5) receptor recombinantly expressed in Chinese hamster ovary (CHO-K1) cells to adenylate cyclase was investigated using receptor selective ligands. 2 Forskolin (10 mu M)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation was inhibited by somatostatin-14 and a number of receptor-selective agonists with a rank order of agonist potency typical of the sst(5) receptor. L-362,855 and BIM-23056 behaved as full agonists. At higher somatostatin-14 concentrations there was sub-maximal inhibition resulting in a bell-shaped concentration-effect relationship. Pertussis toxin (PTx; 100 ng ml(-1), 18 h) pre-treatment abolished agonist-mediated inhibition of cyclic AMP accumulation and markedly enhanced stimulation of cyclic AMP at higher agonist concentrations. 3 The concentration of prostaglandin E-2 (PGE(2)) in the incubation media was raised 14 fold by 1 mu M somatostatin-14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. 4 Pre-treatment with cholera toxin (ChTx; 20 mu g ml(-1), 18 h) markedly inhibited sst(5) receptor-mediated increases in cyclic AMP formation in intact cells. Somatostatin-14-stimulated cyclic AMP accumulation was also observed in sst(5) receptor containing CHO-K1 membranes and was inhibited by the synthetic peptide G(alpha s)acetyl-354-372-amide (100 mu M) by 65.9 +/- 3.5%, implicating a G(alpha s) protein involvement in this response. 5 Activation of G(alpha s) proteins by somatostatin-14 could be demonstrated with [S-35]-guanosine 5'-[gamma-thio]triphosphate ([S-35]-GTP gamma S) binding and subsequent immunoprecipitation of S-35 labelled G(alpha s) proteins with anti-G(alpha s) serum. 6 These data show that the sst(5) receptor is very efficiently coupled in a negative manner to adenylate cyclase. However, at higher agonist concentrations the receptor can also mediate activation of adenylate cyclase by a mechanism apparently involving G(alpha s) protein activation.