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

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Abstract
The coupling of the human somatostatin sst5 receptor recombinantly expressed in Chinese hamster ovary (CHO‐K1) cells to adenylate cyclase was investigated using receptor selective ligands. Forskolin (10 μM)‐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 sst5 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. The concentration of prostaglandin E2 (PGE2) in the incubation media was raised 14 fold by 1 μM somatostatin‐14 but was insufficient to stimulate adenylate cyclase activity via endogenous prostanoid receptors. Pre‐treatment with cholera toxin (ChTx; 20 μg ml−1, 18 h) markedly inhibited sst5 receptor‐mediated increases in cyclic AMP formation in intact cells. Somatostatin‐14‐stimulated cyclic AMP accumulation was also observed in sst5 receptor containing CHO‐K1 membranes and was inhibited by the synthetic peptide Gαsacetyl‐354‐372‐amide (100 μM) by 65.9±3.5%, implicating a Gαs protein involvement in this response. Activation of Gαs proteins by somatostatin‐14 could be demonstrated with [35S]‐guanosine 5′‐[γ‐thio]triphosphate ([35S]‐GTPγS) binding and subsequent immunoprecipitation of 35S labelled Gαs proteins with anti‐Gαs serum. These data show that the sst5 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 Gαs protein activation. British Journal of Pharmacology (1999) 126, 1221–1229; doi:10.1038/sj.bjp.0702401

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