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 (sst1-5), 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'-Omicron-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) 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 sst2/5 receptor in AtT-20 cell membranes. SRIF-14 and SRIF-28 produced a concentration-dependent increase in [35S]GTPgammaS binding (pEC50=6.72 and 7.45; Emax=79 and 74.9, respectively) which was completely abolished by pertussis toxin. sst2/5 receptor-selective ligands caused a concentration-dependent increase in [35S]GTPgammaS binding (pEC50=7.74-5.84; Emax=76.6-20.2) while sst1/3/4 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]GTPgammaS binding profiles (r=0.862 and 0.874, respectively). The effects of the sst2 receptor-preferring agonists Tyr3-octreotide and BIM 23027 on [35S]GTPgammaS binding, but not those of SRIF-14 and the sst5/1 receptor selective-agonist L-817,818, were competitively antagonised by the sst2 receptor antagonist d-Tyr8-CYN 154806 (pKB=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.

The Role of Somatostatin Analogs in Cushing's Disease

“…Second, both SOM230 and Oct, as well BIM-23268, an sst 5 -preferring SRIF analog (Table 2), potently inhibited CRH-induced ACTH release, while maximal inhibition by SOM230 and BIM-23268 compared with Oct was significantly higher. Both sst 2 and sst 5 are known to be involved in the regulation of ACTH and cAMP production by AtT-20 cells [54] but, apparently, sst 5 seems to have functional superiority over sst 2 , which has been suggested by others as well [55].…”

“…Chronic glucocorticoid treatment of GH 4 C 1 cells, a clonal strain of rat pituitary tumor cells, and AtT20/D16 cells, a clonal strain of mouse corticotroph tumor cells, caused a 20-40% decrease in [ 125 I-Tyr 1 ]-SRIF binding [51]. Furthermore, with respect to SRIF and SRIF-analog mediated inhibitory effects on ACTH release by corticotroph tumor cells, numerous reports have been published in which consensus is found that SRIF, as well as sst 2 -and sst 5 -preferring SRIF analogs, lower ACTH release in mouse corticotroph tumor cells [52][53][54][55][56][57]. The consequences of SRIF receptor down-regulation by glucocorticoids on the biological responsiveness of corticotroph adenoma cells was demonstrated in primary cultures of human corticotroph adenomas, in which Oct-induced inhibition of basal and CRH-induced ACTH release, was abolished if the cells were pretreated with hydrocortisone [21].…”

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

“…Among synthetic ligands, we used the peptidyl multiligand analog SOM230, which binds with high affinity to sst 1,2,3,5 [42][43][44], and the peptidyl multiligand analog KE108, which binds with high affinity to all five SRIF receptors [44,45]. The sst 1 -selective peptidyl agonist CH-275 [35,41,46,47], the sst 2 -preferring peptiydyl agonist SMS 201-995 (also known as sandostatin or octreotide) [38,41,46,48,49], and the sst 2 -selective nonpeptidyl agonist L-779,976 [46, 49 -51] were also used in the absence or presence of the sst 1 -selective nonpeptydil antagonist SRA-880 [46,52,53] and the sst 2 -selective peptydil antagonist CYN [36,38,48,49,54]. All compounds were applied at 1 M, which from previous studies, is a concentration giving maximal re- ceptor occupancy [2,41].…”

Somatostatin and somatostatin receptors in Cushing's disease

“…IC 50 values of inhibition suggested a sst 5 -like response of ACTHinhibition. Both sst 2 and sst 5 are involved in the inhibition of ACTH release and cAMP production in AtT20 cells [56] and a functional superiority of sst 5 over sst 2 has been suggested by others as well [57,58]. On the other hand, sst 2 receptors, albeit at low level, are co-expressed with sst 5 receptors in corticotroph adenomas as well.…”