Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

Somatostatin inhibits growth of rainbow trout

Implantation of rainbow trout Oncorhynchus mykiss with somatostatin-14 (SS-14) for 20 days resulted in reduced food conversion as well as significant growth retardation compared to controls. Relative growth as mass was reduced by 20%, whereas relative growth by length was reduced by 45%. A single intraperitoneal injection of SS-14 reduced plasma levels of growth hormone (GH), insulin-like growth factor-1 (IGF-I) and insulin. SS-14 injection also reduced [³⁵S]-sulphate incorporation into gill cartilage compared to saline-injected fish. In addition, in vitro incubation of gill cartilage with SS-14 reduced [³⁵S]-sulphate incorporation in a doserelated manner. These results indicate that SS-14 inhibits growth of rainbow trout and suggests that SS-14, in addition to influencing GH, may play an extra-pituitary role in the modulation of the GH-IGF-I axis.     

Zinc partitions insulin-like growth factors (IGFs) from soluble IGF binding protein (IGFBP)-5 to the cell surface receptors of BC3H-1 muscle cells

Zinc (Zn(2+)) is a multifunctional micronutrient. The list of functions for this micronutrient expanded with the recent discovery that Zn(2+) retains insulin-like growth factors binding proteins (IGFBPs) on the surface of cultured cells, lowers the affinity of cell-associated IGFBPs, and increases the affinity of the cell surface insulin-like growth factor (IGF)-type 1 receptor (IGF-1R). However, currently there is no information concerning the effect of Zn(2+) on soluble IGFBPs. In the current study, the soluble IGFBP-5 secreted by BC(3)H-1 cells is shown to bind approximately 50% more [(125)I]-IGF-II than [(125)I]-IGF-I at pH 7.4. Zn(2+) is shown to depress the binding of both IGF-I and IGF-II to soluble secreted IGFBP-5; [(125)I]-IGF-I binding is affected more so than [(125)I]-IGF-II binding. Zn(2+) acts by lowering the affinity (K(a)) of IGFBP-5 for the IGFs. Scatchard plots are non-linear indicating the presence of high and low affinity binding sites; Zn(2+) affects only binding to the high affinity site. In contrast, Zn(2+) increases the affinity by which either [(125)I]-IGF-I or [(125)I]-R(3)-IGF-I binds to the IGF-1R, but depresses [(125)I]-IGF-II binding to the IGF-type 2 receptor (IGF-2R) on BC(3)H-1 cells. By depressing the association of the IGFs with soluble IGFBPs, Zn(2+) is shown to repartition either [(125)I]-IGF-I or [(125)I]-IGF-II from soluble IGFBP-5 onto cell surface IGF receptors. Zn(2+) was active at physiological doses depressing IGF binding to IGFBP-5 and the IGF-2R at 15-20 microM. Hence, a novel mechanism is further characterized by which the trace micronutrient Zn(2+) could regulate IGF activity.     

Somatostatin inhibits hepatic growth hormone receptor and insulin-like growth factor I mRNA expression by activating the ERK and PI3K signaling pathways

Previously, we reported that somatostatins (SS) inhibit organismal growth by reducing hepatic growth hormone (GH) sensitivity and by inhibiting insulin-like growth factor I (IGF-I) production. In this study, we used hepatocytes isolated from rainbow trout to elucidate the mechanism(s) associated with the extrapituitary growth-inhibiting actions of SS. SS-14, a predominant SS isoform, stimulated tyrosine phosphorylation of several endogenous proteins, including extracellular signal-regulated kinase (ERK), a member the mitogen-activated protein kinase (MAPK) family, and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K). SS-14 specifically stimulated the phosphorylation of both ERK 1/2 and Akt in a concentration-dependent fashion. This activation occurred within 5-15 min, then subsided after 1 h. The ERK inhibitor U0126 retarded SS-14-stimulated phosphorylation of ERK 1/2, whereas the PI3K inhibitor LY294002 blocked SS-14-stimulated phosphorylation of Akt. SS-14-inhibited expression of GH receptor (GHR) mRNA was blocked by U0126 but not by LY294002. By contrast, U1026 had no effect on SS-14 inhibition of GH-stimulated IGF-I mRNA expression, whereas LY294002 partially blocked the inhibition of GH-stimulated IGF-I mRNA expression by SS-14. These results indicate that SS-14-inhibited GHR expression is mediated by the ERK signaling pathway and that the PI3K/Akt pathway mediates, at least in part, SS-14 inhibition of GH-stimulated IGF-I expression.     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

Insulin-like growth factors and binding proteins in the fetal rat: Alterations during maternal starvation and effects in fetal brain cell culture

Maternal malnutrition adversely affects fetal body and brain growth during late gestation. We utilized a fetal brain cell culture model to examine whether alternations in circulating factors may contribute to reduce brain growth during maternal starvation; we then used specific immunoassay and western blotting techniques, and purified peptides to investigate the potential role that altered levels of insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) may play in impaired growth during maternal nutritional restriction.Fetal, body, liver, and brain weight were reduced after 72 hr maternal starvation, and plasma from starved fetuses were less potent than fed fetal plasma in stimulating brain cell growth. Circulating levels of IGF-I were reduced in starved compared to fed fetuses, while levels of IGF-II were similar in both groups. In contrast, [¹²⁵I]-IGF-I binding assay demonstrated an increase in the availability of plasma IGFBPs following starvation. Western ligand blotting and densitometry indicated that levels of 32 Kd IGFBPs were 2-fold higher in starved compared to fed fetal plasma. Immunoblotting and immunoprecipitation with antiserum against rat IGFBP-1 confirmed that heightened levels of immunoreactive IGFBP-1 accounted for the increase in 32 Kd IGFBPs in starved plasma. Levels of 34 Kd BPs, representing IGFBP-2, were unaffected by starvation. Reconstitution experiments in cell culture showed that IGF-I promoted fetal brain cell growth, and that when they were supplemented with IGF-I, the growth promoting activity of starved fetal plasma was restored to fed levels. These changes were measured using MTT to assess mitochondrial reductase activity. Conversely, addition of physiological amounts of rat IGFBP-1 inhibited the effects of fed fetal plasma on brain cell growth, and bioactivity was reduced even further with higher concentrations of IGFBP-1. Based on these results, we conclude that reciprocal changes in circulating levels of IGFBP-1 (increased) and IGF-I (decreased) may combine to reduce the availability of IGF-I to this tissue and limit fetal brain cell growth when maternal nutrition is impaired.     

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the inhibition of DNA synthesis in proliferating mink lung epithelial cells by insulin-like growth factor binding protein-3

Insulin-like growth factor binding protein-3 (IGFBP-3) can inhibit cell growth by directly interacting with cells, as well as by forming complexes with IGF-I and IGF-II that prevent their growth-promoting activity. The present study examines the mechanism of inhibition of DNA synthesis by IGFBP-3 in CCL64 mink lung epithelial cells. DNA synthesis was measured by the incorporation of 5-bromo-2'-deoxyuridine, using an immunocolorimetric assay. Recombinant human IGFBP-3 (rh[N109D,N172D]IGFBP-3) inhibited DNA synthesis in proliferating and quiescent CCL64 cells. Inhibition was abolished by co-incubation of IGFBP-3 with a 20% molar excess of Leu(60)-IGF-I, a biologically inactive IGF-I analogue that binds to IGFBP-3 but not to IGF-I receptors. DNA synthesis was not inhibited by incubation with a preformed 1:1 molar complex of Leu(60)-IGF-I and IGFBP-3, indicating that only free IGFBP-3 inhibits CCL64 DNA synthesis. Inhibition by IGFBP-3 is not due to the formation of biologically inactive complexes with free IGF, since endogenous IGFs could not be detected in CCL64 conditioned media; any IGFs that might have been present could only have existed in inactive complexes, since endogenous IGFBPs were present in excess; and biologically active IGFs were not displaced from endogenous IGFBP complexes by Leu(60)-IGF-I. After incubation with CCL64 cells, (125)I-IGFBP-3 was covalently cross-linked to a major thick similar400-kDa complex. This complex co-migrated with a complex formed after incubation with (125)I-labeled transforming growth factor-beta (TGF-beta) that has been designated the type V TGF-beta receptor. (125)I-IGFBP-3 binding to the thick similar400-kDa receptor was inhibited by co-incubation with unlabeled IGF-I or Leu(60)-IGF-I. The ability of Leu(60)-IGF-I to decrease both the inhibition of DNA synthesis by IGFBP-3 and IGFBP-3 binding to the thick similar400-kDa receptor is consistent with the hypothesis that the thick similar400-kDa IGFBP-3 receptor mediates the inhibition of CCL64 DNA synthesis by IGFBP-3.     

Epidermal growth factor enhancement of insulin-like growth factor-I-induced down-regulation of insulin-like growth factor I receptor in cultured placental trophoblastic cells.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) synergistically stimulate placental lactogen (hPL) secretion by placental cells. To understand the mechanism of actions we have investigated a possible heterologous regulatory effect of EGF and IGF-I on each other's receptors. Pretreatment of the cells with IGF-I had no effect on [125I]-EGF binding or the down-regulation of EGF receptor. Pretreatment of the cells with EGF, concomitantly with IGF-I, had no effect on [125I]-IGF-I binding but it augmented the IGF-I down-regulation of IGF-I receptor. The time required to initiate the IGF-I-induced down-regulation of IGF-I receptor was reduced by 4 h in the presence of EGF. IGF-I-down-regulated decreased (P less than 0.05) receptor numbers were further decreased (p less than 0.05) in the presence of EGF. These results suggested that the synergistic effect of EGF and IGF-I seen in hPL secretion by placental cells is not due to direct heterologous hormone-receptor interactive effects. However, the effects seen may be due to a differentiating effect of EGF sensitizing the cells for responsiveness to IGF-I.     

Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Receptors for insulin-like growth factor-I (IGF-I) in myocardial capillary endothelium of the intact perfused heart

Isolated intact, beating hearts were perfused with HPLC-pure [125]-IGF-I (1 ng/ml) alone or [125]-IGF-I (1 ng/ml) plus varying concentrations of unlabeled IGF-I (10-3,000 ng/ml) or unlabeled insulin (1,000-100,000 ng/ml). After 1 min of perfusion with peptides, the hearts were rapidly fixed, sectioned and analyzed for radioautographic [125I] grain counts. Greater than 90% of [125I] grains were shown to represent intact [125I]-IGF. Maximal grain counts over capillaries occurred after perfusion with [125I]-IGF-I alone and decreased in a dose-dependent manner when unlabeled IGF-I was coperfused. Coperfusion of [125I]-IGF-I with unlabeled insulin also decreased 125I grains over capillaries but less potently than unlabeled IGF-I. EM radioautography demonstrated that [125I]-IGF-I grains were localized over capillary endothelial cells. Thus, specific IGF-I receptors are present in the capillary endothelium of the intact heart and have properties similar to IGF-I receptors in cultured capillary endothelial cells.     

Quantitative and functional expression of somatostatin receptor subtypes in human thymocytes

We recently demonstrated the expression of somatostatin (SS) and SS receptor (SSR) subtype 1 (sst1), sst2A, and sst3 in normal human thymic tissue and of sst1 and sst2A on isolated thymic epithelial cells (TEC). We also found an inhibitory effect of SS and octreotide on TEC proliferation. In the present study, we further investigated the presence and function of SSR in freshly purified human thymocytes at various stages of development. Thymocytes represent a heterogeneous population of lymphoid cells displaying different levels of maturation and characterized by specific cell surface markers. In this study, we first demonstrated specific high-affinity 125I-Tyr(11)-labeled SS-14 binding on thymocyte membrane homogenates. Subsequently, by RT-PCR, sst2A and sst3 mRNA expression was detected in the whole thymocyte population. After separation of thymocytes into subpopulations, we found by quantitative RT-PCR that sst2A and sst3 are differentially expressed in intermediate/mature and immature thymocytes. The expression of sst3 mRNA was higher in the intermediate/mature CD3+ fraction compared with the immature CD2+CD3- one, whereas sst2A mRNA was less abundant in the intermediate/mature CD3+ thymocytes. In 7-day-cultured thymocytes, SSR subtype mRNA expression was lost. SS-14 significantly inhibited [3H]thymidine incorporation in all thymocyte cultures, indicating the presence of functional receptors. Conversely, octreotide significantly inhibited [3H]thymidine incorporation only in the cultures of immature CD2+CD3- thymocytes. Subtype sst3 is expressed mainly on the intermediate/mature thymocyte fraction, and most of these cells generally die by apoptosis. Because SS-14, but not octreotide, induced a significant increase in the percentage of apoptotic thymocytes, it might be that sst3 is involved in this process. Moreover, sst3 has recently been demonstrated on peripheral human T lymphocytes, which derive directly from mature thymocytes, and SS analogs may induce apoptosis in these cells. Interestingly, CD14+ thymic cells, which are cells belonging to the monocyte-macrophage lineage, selectively expressed sst2A mRNA. Finally, SSR expression in human thymocytes seems to follow a developmental pathway. The heterogeneous expression of SSR within the human thymus on specific cell subsets and the endogenous production of SS as well as SS-like peptides emphasize their role in the bidirectional interactions between the main cell components of the thymus involved in intrathymic T cell maturation.     

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion

The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells.     

Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells

In a series of human corticotroph adenomas, we recently found predominant mRNA expression of somatostatin (SS) receptor subtype 5 (sst5). After 72 h, the multiligand SS analog SOM230, which has a very high sst5 binding affinity, but not Octreotide (OCT), significantly inhibited basal ACTH release. To further explore the role of sst5 in the regulation of ACTH release, we conducted additional studies with mouse AtT-20 cells. SOM230 showed a 7-fold higher ligand binding affinity and a 19-fold higher potency in stimulating guanosine 5'-O-(3-thiotriphosphate) binding in AtT-20 cell membranes compared with OCT. SOM230 potently suppressed CRH-induced ACTH release, which was not affected by 48-h dexamethasone (DEX) pretreatment. However, DEX attenuated the inhibitory effects of OCT on ACTH release, whereas it increased the inhibitory potency of BIM-23268, an sst5-specific analog, on ACTH release. Quantitative PCR analysis showed that DEX lowered sst(2A+2B) mRNA expression significantly after 24 and 48 h, whereas sst5 mRNA levels were not significantly affected by DEX treatment. Moreover, Scatchard analyses showed that DEX suppressed maximum binding capacity (B(max)) by 72% when 125I-Tyr3-labeled OCT was used as radioligand, whereas B(max) declined only by 17% when AtT-20 cells were treated with [125I-Tyr11]SS-14. These data suggest that the sst5 protein, compared with sst2, is more resistant to glucocorticoids. Finally, after SS analog preincubation, compared with OCT both SOM230 and BIM-23268 showed a significantly higher inhibitory effect on CRH-induced ACTH release. In conclusion, our data support the concept that the sst5 receptor might be a target for new therapeutic agents to treat Cushing's disease.