Native somatostatin sst2 and sst5 receptors functionally coupled to Gi/o-protein, but not to the serum response element in AtT-20 mouse tumour corticotrophs

Abstract

Of the five cloned somatostatin (SRIF: somatotropin release inhibitory factor) receptors (sst_1–5), only sst₂ and sst₅ receptors appear to be endogenously expressed and functionally active in AtT-20 mouse anterior pituitary tumour cells. In this study, the presence and the functional coupling of SRIF receptors to G-protein in AtT-20 cells was evaluated by receptor autoradiography and guanosine-5'-Ο-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding, respectively. In addition, transcriptional effects via the serum response element (SRE) were assessed in AtT-20-SRE-luci cells, engineered to express constitutively SRE upstream of the luciferase reporter gene. [¹²⁵I]LTT-SRIF-28, [¹²⁵I]CGP 23996 and [¹²⁵I]Tyr³-octreotide binding illustrates the high level of sst_2/5 receptor in AtT-20 cell membranes. SRIF-14 and SRIF-28 produced a concentration-dependent increase in [³⁵S]GTPγS binding (pEC₅₀ = 6.72 and 7.45; E_max = 79 and 74.9, respectively) which was completely abolished by pertussis toxin. sst_2/5 receptor-selective ligands caused a concentration-dependent increase in [³⁵S]GTPγS binding (pEC₅₀ = 7.74–5.84; E_max = 76.6–20.2) while sst_1/3/4 receptor-selective ligands were devoid of activity. The binding profiles of [¹²⁵I]LTT-SRIF-28 and the inhibition of cAMP accumulation correlated highly significantly with their corresponding [³⁵S]GTPγS binding profiles (r=0.862 and 0.874, respectively). The effects of the sst₂ receptor-preferring agonists Tyr³-octreotide and BIM 23027 on [³⁵S]GTPγS binding, but not those of SRIF-14 and the sst_5/1 receptor selective-agonist L-817,818, were competitively antagonised by the sst₂ receptor antagonist d-Tyr⁸-CYN 154806 (pK_B = 7.36 and 7.72, respectively; slope factors not significantly different from unity). In AtT-20-SRE-luci cells, which carry a SRE-luciferase construct functioning in a very efficient manner, SRIF and its analogues did not affect luciferase activity. Taken together, these results demonstrate that in AtT-20 cells the expression of sst₂ and sst₅ receptors fit with their functional coupling to G_i/o-proteins. The pharmacological implications of the existence of different ligand/receptor complexes are discussed. However, the intracellular pathways coupled to the activation of sst₂ and sst₅ receptors appear not to modulate the SRE-mediated transcriptional activity, suggesting that SRIF effects on gene expression coupled to mechanisms that have promoters other than SRE.