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Internalization of sst₂, sst₃, and sst₅ Receptors: Effects of Somatostatin Agonists and Antagonists

¹ Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, Berne, Switzerland; ² Institute of Pharmacology and Toxicology, Otto-von-Guericke University, Magdeburg, Germany; ³ Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California; ⁴ Nuklearmedizinische Klinik und Poliklinik, Technical University of Munich, Munich, Germany; ⁵ Endocrine Research, Beaufour-Ipsen Group, Milford, Massachusetts; ⁶ Division of Radiological Chemistry, University Hospital, Basel, Switzerland; and ⁷ Department of Integrative Biology and Pharmacology, University of Texas Health Science Center–Houston, Houston, Texas

View larger version (62K): [in a new window]   FIGURE 1.  sst2 internalization is induced by various agonists and is abolished by antagonist Coy-14. HEK-sst2 cells were treated for 30 min either with vehicle (no peptide [A]) or with various agonists at concentrations inducing submaximal internalization effect (100 nmol/L [B and C] and 10 nmol/L [D–F]). (H–L) Cells treated with same agonists at same concentrations as in B–F but in presence of excess of specific sst2-antagonist Coy-14 (5 [H], 10 [I], and 1 [J–L] µmol/L). Effect of antagonist alone (10 µmol/L [G]) is also shown. After incubation with peptides, cells were processed for immunocytochemistry. Clear punctate perinuclear staining is detectable for all tested agonists. This punctate staining is efficiently abolished by excess of antagonist Coy-14. Antagonist alone has no effect on internalization.

View larger version (53K): [in a new window]   FIGURE 2.  Dose response of agonist-induced sst2 internalization. HEK-sst2 cells were treated with 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 µmol/L, or 10 µmol/L of various agonists. Also included are cells treated with 100 nmol/L TOC plus 5 µmol/L Coy-14 or with 1 µmol/L TOC plus 50 µmol/L Coy-14. After 30 min of incubation with peptides, cells were processed for immunocytochemistry, and internalized sst2 was quantified. (A) Representative experiment with dose-response curves for various agonists inducing sst2 internalization. TOC, Y-DOTA-TATE, and I-Gal-S-TATE are considerably more potent in inducing sst2 internalization than is DTPA-octreotide. TOC-induced internalization is efficiently abolished by excess of Coy-14. (B) Representative immunofluorescence images of agonist-induced internalized sst2 using DTPA-octreotide and TOC. TOC is almost 2 orders of magnitude more potent in inducing sst2 internalization than is DTPA-octreotide.

View larger version (73K): [in a new window]   FIGURE 3.  sst3 internalization is induced by various agonists and is efficiently abolished by sst3-specific antagonist sst3-ODN-8. HEK-sst3 cells were treated either with vehicle (no peptide [A]) or with various agonists at concentrations inducing submaximal internalization effect (100 nmol/L [B–D]). Cells treated with same agonists at same concentrations as in B–D but in presence of excess of specific sst3-antagonist sst3-ODN-8 (5 µmol/L [F–H]). Effect of antagonist alone (50 µmol/L [E]) is also illustrated. After incubation with peptides, cells were processed for immunocytochemistry. Clear perinuclear staining is detectable for all agonists tested. This staining is efficiently abolished by 50-fold excess of antagonist sst3-ODN-8. Antagonist alone has no effect on internalization.

View larger version (16K): [in a new window]   FIGURE 4.  Dose response of agonist-induced sst3 internalization. HEK-sst3 cells were treated with 1 nmol/L, 10 nmol/L, 100 nmol/L, 1 µmol/L, or 10 µmol/L of somatostatin-28 or KE108. Also included are cells treated with 100 nmol/L somatostatin-28 plus 5 µmol/L sst3-ODN-8 or with 1 µmol/L somatostatin-28 plus 50 µmol/L sst3-ODN-8. After incubation with peptides, cells were processed for immunocytochemistry, and internalized sst3 was quantified. This experiment shows that somatostatin-28 and KE108 are of similar potency in inducing sst3 internalization. Somatostatin-28–induced internalization effect is efficiently abolished by excess of sst3-ODN-8

View larger version (99K): [in a new window]   FIGURE 5.  sst5 internalization is induced by native somatostatin-28 but not by high-affinity synthetic sst5 agonists. HEK-sst5 cells were treated either with vehicle (no peptide); with 1 nmol/L, 10 nmol/L, 100 nmol/L, or 1 µmol/L somatostatin-28; or with 1 µmol/L KE108, 1 µmol/L BIM-23244, or 1 µmol/L L-817,818. After incubation with peptides, cells were processed for immunocytochemistry. Dose-response experiment with somatostatin-28 shows that it can elicit sst5 internalization. This is, however, not true for sst5 agonists KE108, BIM-23244, and L-817,818. Note also presence of intracellular sst5 even when cells were treated with vehicle alone (no peptide). All images are composed of 2 single pictures.