Autoradiographic analysis of somatostatin SRIF1 and SRIF2 receptors in the human brain and pituitary

The distribution of somatostatin (SRIF) receptor sites was studied by in vitro receptor autoradiography in the human brain and pituitary using the SRIF₁ (sst₂) receptor selective [¹²⁵I]Tyr³-octreotide, the non-subtype selective [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) and the SRIF₂-receptor selective [¹²⁵I]CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing 120 mM Na⁺. SRIF receptor autoradiography was compared with mRNA expression of somatostatin receptors sst_1–5 as studied by in situ hybridisation in human brain. High levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide recognition sites were found in the deep layers of cerebral cortex and molecular layer of cerebellum of the human brain. The hypothalamus, choroid plexus, most areas of the brainstem and dentate nucleus were associated with low levels of binding. In contrast to [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide, no difference was observed for [¹²⁵I]CGP 23996 labelling in the various layers of cerebral cortex. The choroid plexus, substantia nigra and molecular layer of the cerebellum presented high densities of [¹²⁵I]CGP 23996 binding sites whereas no binding was observed in the hypothalamus and locus coeruleus using this radioligand. Both lobes of the human pituitary displayed low levels of [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide binding. By contrast, the anterior lobe of the pituitary displayed very high levels of [¹²⁵I]CGP 23996 labelled sites whereas intermediate levels were found in the posterior lobe. There was a partial overlap between sst₂ receptor mRNA and [¹²⁵I]Tyr³-octreotide binding, although the distribution of the binding sites was much wider than that of receptor mRNA. The same observation was made for sst₁ and/or sst₄ receptor mRNA and [¹²⁵I]CGP 23996 labelled sites. The present data show that SRIF₁ and SRIF₂ receptors are present in the human brain with different distributions, especially in the cerebral cortex and the pituitary. The very similar distribution of sites labelled with [¹²⁵I]LTT-SRIF-28 and [¹²⁵I]Tyr³-octreotide suggests (i) that sst₂ receptors are predominant within the SRIF₁ family in the human brain and (ii) that [¹²⁵I]LTT-SRIF-28 under the conditions used in the present study, does not significantly label SRIF₂ sites. Received: 8 August 1996 / Accepted: 8 November 1996     

Pharmacological characterisation of human cerebral cortex somatostatin SRIF1 and SRIF2 receptors

Radioligand binding studies were performed in membranes of human cerebral cortex using [¹²⁵I]Tyr³-octreotide in the presence of 5 mM MgCl₂, [¹²⁵I]SRIF-14 ([¹²⁵I]Tyr¹¹-SRIF-14) and [¹²⁵I]CGP 23996 ([¹²⁵I]c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) both in the presence of 120 mM NaCl, to characterise the nature of the somatostatin (SRIF) receptors. The pharmacological profile of human brain SRIF recognition sites was compared with that of recombinant human SRIF₁ (sst₂-sst₃-sst₅) or SRIF₂ receptors (sst₁-sst₄) and with that of native rat sst₁, sst₂ and sst₄ receptors. [¹²⁵I]Tyr³-octreotide labelled binding sites in human cerebral cortex: B _max = 238 ± 36 fmol/mg protein and pK_d = 9.73 ± 0.08. The pharmacological profile of [¹²⁵I]Tyr³-octreotide labelled sites correlated very significantly with that of recombinant human sst₂ receptors (r = 0.98) and much less with those of recombinant human sst₃ (r = 0.65) or sst₅ receptors (r = 0.72). The correlation between [¹²⁵I]Tyr³-octreotide binding to native sst₂ receptors in human and rat cerebral cortex was also highly significant (r = 0.97). [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding (performed in the presence of 120 mM NaCl) in the human cerebral cortex identified very similar populations of sites B _max = 44 ± 7 and 36 ± 5 fmol/mg protein and pK_d = 9.44 ± 0.08 and 9.48 ± 0.10, respectively. The pharmacological profiles of the sites labelled with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 correlated highly significantly with those of recombinant human sst₁ (r = 0.97–0.99) or sst₄ receptors (r = 0.91–0.94). Similarly, the correlations between [¹²⁵I]SRIF-14 or [¹²⁵I]CGP 23996 binding in human cortex and [¹²⁵I]SRIF-14 binding to native sst₁ sites in rat cerebral cortex were also highly significant (r = 0.97 and 0.94, respectively). Finally, the pharmacological profile of native rat lung sst₄ sites determined with [¹²⁵I]LTT-SRIF-28 ([Leu⁸,D-Trp²²,¹²⁵I-Tyr²⁵]SRIF-28) correlated with [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 binding in human cortex; r = 0.91 and 0.87, respectively. The present data show that in human cerebral cortex, [¹²⁵I]Tyr³-octreotide labels SRIF₁ receptor sites which are best characterised as of the sst₂ type, whereas [¹²⁵I]SRIF-14 and [¹²⁵I]CGP 23996 (both in the presence of 120 mM NaCl), label sites which fit almost equally well with sst₁ or sst₄ receptors and therefore are best described as of the SRIF₂ type. Under the conditions used, there was no evidence that either of these ligands would label sst₃ or sst₅ receptors in human cerebral cortex. Received: 8 August 1996 / Accepted: 8 November 1996     

Localization and pharmacological characterization of somatostatin sst2 sites in the rat cerebellum

Radioligand binding studies were performed in membranes of rat cerebellum using [¹²⁵I]-[Tyr³]octreotide ([¹²⁵I]204-090) to characterize the nature of cerebellar somatostatin receptors. Saturation experiments suggest the presence of a single class of binding sites with high affinity, pK_d = 9.53 ± 0.11, but low receptor density, B _max = 12.7 ± 1.0 fmol/mg protein. The pharmacological profile of [¹²⁵I]204-090 sites in cerebellar membranes was established using a range of ligands known to interact with SSTR-2 (now called sst₂) and other somatostatin (SRIF) receptors. SRIF analogues such as octreotide (SMS 201-995), seglitide (MK 678) and somatuline (BIM 23014) displayed very high affinity for cerebellar [¹²⁵I]204-090 binding sites. The data were compared to results obtained using the same ligand in rat cerebral cortex membranes known to represent sst₂ binding. The pharmacological characteristics of the cerebellar sites were in close correlation with those of the cerebral cortex (r = 0.976, n = 19, p < 0.001) and CHO-cells expressing human recombinant sst₂ receptor (r = 0.977, n = 19, p < 0.001). By contrast, there was very little correlation between cerebellar binding and published affinities for rat sst₅ receptors (r = 0.465), for which octreotide has also high affinity. In vitro autoradiographic studies performed in cerebellar slices using [¹²⁵I]204-090 demonstrated the presence of binding sites in the molecular layer of the rat cerebellum. In situ hybridization studies using sst₂ receptor mRNA selective oligoprobes confirmed the presence of sst₂ receptor mRNA in the rat cerebellum. Together, the present data demonstrate the presence of a low density of SRIF receptors in the molecular layer of the adult rat cerebellum which are best characterized as sst₂. This is the first pharmacological characterization and localization of sst₂ receptors in the adult rat cerebellum.     

Agonist properties of putative small-molecule somatostatin sst2 receptor-selective antagonists

The availability of antagonist ligands for somatostatin receptors is very limited, with those that are available often displaying agonist properties or limited receptor subtype selectivity. Hay et al. [Bioorg. Med. Chem. Lett. 11 (2001) 2731] recently described the development of small-molecule somatostatin receptor subtype 2 (sst(2)) selective compounds. This study investigates the binding affinity and functional characteristics of two of those antagonists (2 and 3) and the agonist compound, from which they were derived (1). In radioligand binding studies using the agonist radioligands [125I][Tyr(11)]SRIF-14 (Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]LTT-SRIF-28 ([Leu(8),DTrp(22),125I-Tyr(25)]SRIF-28; Ser-Ala-Asn-Ser-Asn-Pro-Ala-Leu-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-c[Cys-Lys-Asn-Phe-Phe-DTrp-Lys-Thr-(125I-Tyr)-Thr-Ser-Cys]-OH), [125I]CGP 23996 (c[Lys-Asu-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Thr-Ser]), [125I][Tyr(3)]octreotide (DPhe-c[Cys-(125I-Tyr)-DTrp-Lys-Thr-Cys]-Thr-OH) and [125I][Tyr(10)]cortistatin-14 (Pro-c[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-(125I-Tyr)-Ser-Ser-Cys]-Lys) at human recombinant somatostatin receptors expressed in Chinese hamster lung fibroblast (CCL39) cells and native rat cortex, the compounds bound with high affinity (pK(d) 6.8-9.7) and selectivity to human sst(2) receptors. Some affinity was also observed for sst(5) labelled by [125I][Tyr(3)]octreotide and [125I]CGP 23996. In functional studies at human sst(2) receptors expressed in Chinese hamster ovary (CHO) cells, both the agonist 1 and the two putative antagonists 2 and 3 concentration dependently inhibited forskolin-stimulated adenylate cyclase and stimulated luciferase reporter gene expression, with similar efficacy to the natural ligand somatotropin release inhibiting factor (SRIF)-14. Compound 1 had similar potency to SRIF-14, which was in the nanomolar range, whereas 2 and 3 were 10-100-fold less potent. The intrinsic activity of 2 and 3 was too high to allow antagonist studies to be carried out. In conclusion, in contrast to previous findings, all three compounds are potent agonists at recombinant human sst(2) receptors.     

Identification and characterisation of heterogeneous somatostatin binding sites in rat distal colonic mucosa

We have previously shown that the somatostatin (SRIF) sst₂ receptor-selective peptide, BIM-23027, is a potent antisecretory agent in rat isolated distal colonic mucosa (RDCM) and in radioligand binding studies in RDCM membranes, it only maximally inhibited approximately 40% of [¹²⁵I]-Tyr¹¹-SRIF-14 binding (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402–411). The aim of this study was to characterise the BIM-23027-sensitive and -insensitive SRIF binding sites in more detail and to compare their properties with those of the recombinant sst₂ receptor stably expressed in mouse fibroblast (Ltk^–) cells.SRIF-14, SRIF-28, CGP-23996 and D Trp⁸-SRIF-14 abolished [¹²⁵I]-Tyr¹¹-SRIF-14 binding (pIC₅₀ values, 8.7–9.7) but the competition curves had Hill slopes which were less than unity. Octreotide and L-362,855 inhibited binding over a wide concentration range (0.1 nM-1 M) and inhibition of binding was incomplete at the highest concentration studied. BIM-23056 (PIC₅₀ <6.5) was a weak inhibitor of [¹²⁵]-Tyr¹¹-SRIF-14 binding. GTPS decreased [¹²⁵I]-Tyr¹¹-SRIF-14 binding by 40%. Further binding experiments with [¹²⁵I]-Tyr¹¹-SRIF-14 were carried out in RDCM in the continuous presence of BIM-23027 (1 M). Under these conditions, seglitide had no effect on [¹²⁵I]-Tyr¹¹-SRIF-14 binding at concentrations up to 10 M, whilst SRIF-14 and SRIF-28 abolished specific [¹²⁵I]-Tyr¹¹-SRIF-14 binding in a manner which was consistent with the ligand binding to two sites. SRIF-14 and SRIF-28 displayed high affinity (pIC₅₀ values of 9.8 and 9.3 respectively) for approximately 70% of these binding sites and low affinity (pIC₅₀ values of 7.8 and 7.3) for the remaining sites. Octreotide, L-362,855 and BIM-23056 were weak inhibitors of [¹²⁵I]-Tyr¹¹-SRIF-14 binding (PIC₅₀ <6.5). [¹²⁵I]-BIM-23027 labelled a single population of SRIF binding sites in RDCM membranes and mouse fibroblast (Ltk^–) cells stably expressing the human recombinant sst₂ receptor. There was a significant correlation between the affinitestimates of a range of SRIF analogues at inhibiting [¹²⁵I]-BIM-23027 binding in RDCM membranes and binding to the recombinant sst₂ receptor in Ltk^– cells, suggesting that the sites labelled by [¹²⁵I]-BIM-23027 in RDCM are similar to the sst₂ receptor. GTPS (100 M) decreased [¹²⁵I]-BIM-23027 binding in RDCM by 60%.The results from these studies demonstrate that [¹²⁵I]-Tyr¹¹-SRIF-14 labels a heterogeneous population of high affinity SRIF binding sites in RDCM membranes. The majority of these sites are insensitive to GTPS and display negligible affinity for the cyclic hexapeptides, BIM-23027 and seglitide. The remaining high affinity binding sites can be selectively labelled with [¹²⁵I]-BIM-23027, are sensitive to GTPS and show similar characteristics to the recombinant sst₂ receptor which appears to mediate the antisecretory effects of SRIF in the mucosa (McKeen ES, Feniuk W, Humphrey PPA (1995) Naunyn-Schmiedeberg's Arch Pharmacol 352:402–411).     

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst(5) receptor

The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst(5) receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst(5) receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve).The msst(5) receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [(125)I]LTT-SRIF-28 ([Leu(8), D-Trp(22), (125)I-Tyr(25)]-SRIF-28), [(125)I]Tyr(10)-CST, [(125)I]CGP 23996, and [(125)I]Tyr(3)-octreotide labelled msst(5) receptors with high affinity (pK(d) values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [(125)I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [(125)I]Tyr(3)-octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [(125)I]LTT-SRIF-28, [(125)I]CGP 23996 and [(125)I]Tyr(10)-CST correlated highly significantly (r(2) =0.88-0.99), whereas [(125)I]Tyr(3)-octreotide binding was rather divergent (r(2) =0.77). Also, human and mouse sst(5) receptor profiles are very different, e. g. r(2) =0.385 for [(125)I]Tyr(10)-CST and r(2) =0.323 for [(125)I]LTT-SRIF-28-labelled sites.Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst(5) receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%.The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst(5) receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst(2) selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst(5) receptors than SRIF-14.     

Somatostatin sst2 receptor knock-out mice: localisation of sst1-5 receptor mRNA and binding in mouse brain by semi-quantitative RT-PCR, in situ hybridisation histochemistry and receptor autoradiography

The peptide hormone/neurotransmitter somatostatin (somatotropin release inhibiting factor; SRIF) and its receptors (sst(1)-sst(5)) appear to regulate many physiological functions in the CNS. Semi-quantitative analysis of the densities of mRNA expression for sst(1-5) receptors and SRIF receptor binding sites were established in sst(2) receptor knock-out (KO) mice. Patterns of sst(1-5) receptor mRNA expression were largely conserved for sst(1,3,4) and sst(5) selective oligonucleotide probes; whereas sst(2) signals were completely absent in KO mouse brain. Autoradiographic analysis demonstrated [(125)I]LTT SRIF(28), [(125)I]CGP 23996 (two radioligands known to label all five recombinant SRIF receptors) and [(125)I]Tyr(3)-octreotide (sst(2) and sst(5) receptor selective) binding in wild type (WT) mouse brain sections; yet no specific binding of [(125)I]Tyr(3)-octreotide in KO mice. In contrast, [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 binding was still present in a number of brain areas in KO mice, although to a lesser degree than in those regions where [(125)I]Tyr(3)-octreotide binding was found, in WT animals. The present data suggest first, that both sst(2) receptor protein and mRNA were completely absent in the brain of these KO animals. Second, there was little evidence of compensatory regulation, at the mRNA level, of the other SRIF receptors as a consequence of the sst(2) KO. Third, the absence of any [(125)I]Tyr(3)-octreotide binding, in KO mice, suggests that this particular ligand is selective for the sst(2) receptor subtype (under the conditions utilised); or that sst(5) receptors are only marginally expressed in brain. Fourth, there were regions where the binding of [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 were moderately affected by the sst(2) KO, suggesting that additional SRIF receptors may well contribute to the binding of the aforementioned radioligands. Finally, since the relative distribution of these two ligands were not entirely superimposable, it suggests that their respective selectivity profiles towards the different SRIF receptor subtypes in situ are not identical.     

Somatostatin receptors in the ventral pallidum/substantia innominata modulate rat locomotor activity

Rational Somatostatin and its receptors (sst₁ and sst₂) have been localized in brain nuclei implicated in motor control, such as the nucleus accumbens, ventral pallidum (VP) and substantia innominata (SI). Objectives The objective of the study is to investigate the effect of somatostatin and selective sst₁ and sst₂ analogs infused in the VP/SI on the locomotor activity of the rat. Methods Somatostatin (15, 30, 60, 120 and 240 ng/0.5 μl/side), CH275 (sst₁ analog; 60, 180, 240 and 480 ng/0.5 μl/side), MK678 (sst₂ analog; 120, 240 and 480 ng/0.5 μl/side), L-809,087 (sst₄ agonist, 240 ng/0.5 μl/side) or saline (vehicle) were infused bilaterally in the VP/SI of the rat and locomotor activity measured for 60 min. The effect of SRA-880 (sst₁ antagonist) and CYN-154806 (sst₂ antagonist) on somatostatin-, CH275- and MK678-mediated locomotor activity was also ascertained. Results Somatostatin decreased locomotor activity in the first 30 min after its infusion in the VP/SI and in a dose-dependent manner. The sst₁ and sst₂ antagonists, SRA-880 and CYN-154806, respectively, reversed the somatostatin effect. The sst₁ and sst₂ agonists CH275 and MK678, respectively, mimicked somatostatin’s actions, while the selective sst₄ agonist L-809,087 had no effect. Moreover, SRA-880 and CYN-154806 reversed the respective agonist action on locomotor activity. Conclusion The present study provides functional evidence for the presence of sst₁ and sst₂ receptors in the VP/SI and their implication in motor control. The mechanism via which somatostatin and agonists mediate the attenuation of locomotor activity is presently being investigated.     

Pharmacological characterisation of native somatostatin receptors in AtT-20 mouse tumour corticotrophs

1. The mouse corticotroph tumour cell line AtT-20 is a useful model to investigate the physiological role of native somatostatin (SRIF, Somatotropin release inhibitory factor) receptor subtypes (sst(1) - sst(5)). The objective of this study was to characterise the pharmacological features and the functional effects of SRIF receptors expressed by AtT-20 cells using radioligand binding and cAMP accumulation. 2. [(125)I]LTT-SRIF-28, [(125)I]CGP 23996, [(125)I]Tyr(10)-cortistatin-14 and [(125)I]Tyr(3)-octreotide labelled SRIF receptor binding sites with high affinity and in a saturable manner (B(max)=315, 274, 239 and 206 fmol mg(-1), respectively). [(125)I]LTT-SRIF-28 labels significantly more sites than [(125)I]Tyr(10) -cortistatin-14 and [(125)I]Tyr(3) -octreotide as seen previously in cells expressing pure populations of sst(2) or sst(5) receptors. 3. SRIF analogues displaced the binding of the four radioligands. sst(2/5) receptor-selective ligands showed much higher affinity than sst(1/3/4) receptor-selective ligands. The binding profile of [(125)I]Tyr(3)-octreotide was different from that of [(125)I]LTT-SRIF-28, [(125)I]CGP 23996 and [(125)I]Tyr(10)-cortistatin-14. The sst(5/1) receptor-selective ligand L-817,818 identified two binding sites, one with subnanomolar affinity (sst(5) receptors) and one with micromolar affinity (sst(2) receptors); however, the proportions were different: 70 - 80% high affinity with [(125)I]LTT-SRIF-28, [(125)I]CGP 23996, [(125)I]Tyr(10)-cortistatin-14, but only 20% with [(125)I]Tyr(3)-octreotide. 4. SRIF analogues inhibited the forskolin-stimulated cAMP levels depending on concentration. sst(2/5) receptor-selective ligands were highly potent, whereas sst(1/3/4) receptor-selective ligands had no significant effects. The sst(2) receptor antagonist D-Tyr(8)-CYN 154806 competitively antagonised the effects of SRIF-14 and sst(2) receptor-preferring agonists, but not those of L-817,818. 5. The complex binding properties of SRIF receptor analogues indicate that sst(2) and sst(5) receptors are the predominant SRIF receptors expressed on AtT-20 cell membranes with no or only negligible presence of sst(1), sst(3) and sst(4) receptors. In the functional studies using cAMP accumulation, only sst(2) and sst(5) receptors appear to play a role. However, the "predominant" receptor appears to be the sst(2) receptor, although sst(5) receptors can also mediate the effect, when the ligand is not able to activate sst(2) receptors. This clearly adds flexibility to SRIF-mediated functional effects and suggests that the physiological role of SRIF and its analogues may be mediated preferentially via one subtype over another.     

Endothelin receptors in the human coronary artery,ventricle and atrium

Summary In the present experiments we investigated endothelin (ET) receptors in the human coronary artery, and in ventricular and atrial muscle using quantitative receptor autoradiography. Displacement of [¹²⁵I]Sf6b (Sarafotoxin S6b) (30 pM)- and [¹²⁵I]ET-1 (30 pM)-labeled binding sites was studied using ET-1, the ET_A receptor selective ligand BQ-123 (cyclo[D-Asp-L-Pro-D Val-L-Leu-D-Trp-]), and the ETB receptor selective ligand [Ala^1,3,11,15]ET-1.Specific binding was more dense in atrium and coronary artery (relative optical density (r.o.d.): 0.14±0.01 and 0.16±0.01, respectively) than in ventricular muscle (r.o.d.: 0.10±0.01). In the coronary artery, binding was especially dense in the media. ET-1 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b monophasically in atrium, ventricle and coronary artery. [Ala^1,3,11,15]ET 1 and BQ-123 displaced [¹²⁵I]ET-1 and [¹²⁵I]Sf6b-labeled sites biphasically in the ventricle and in the atrium. In the human coronary artery, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I]ET-1-labeled sites monophasically (pIC₅₀): ET-1 (9.72±0.12) > BQ-123 (6.84±0.08) > [Ala^1,3,11,15]ET-1 (6.40±0.12). In contrast, [Ala^1,3,11,15]ET-1 and BQ-123 displaced [¹²⁵I] Sf6b-labeled coronary artery sites biphasically (high affinity pIC₅₀: BQ-123, 9.03±0.25;[Ala^1,3,11,15]ET-1, 8.40±0.14; low affinity pIC₅₀: BQ-123, 7.24±0.14; [Ala^1,3,11,15]ET-1, 6.99±0.09).These data indicate that both [¹²⁵I]ET-1 and [¹²⁵I] Sf6b-labeled ET_A and ET_B binding sites in human ventricular and atrial muscle. In the human coronary artery, both radioligands labeled ET_A binding sites, but [¹²⁵I] Sf6b also labeled a non-ET_A, non-ET_B binding site with relatively high affinity for both BQ-123 and [Ala^1,3,11,15] ET-1. Correspondence to W. A. Bax, at the above address     

Differential effects of somatostatin and angiopeptin on cell proliferation

1. Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst₂/sst₅ receptor-selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO-K1 cells stably transfected with the human or rat recombinant sst₂ or sst₅ receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors.
2. In CHO-K1 cells, expressing either human or rat recombinant sst₂ or sst₅ receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1–1000 nM) had no effect on basal re-growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml⁻¹) stimulated re-growth of these cells.
3. SRIF (0.1–1000 nM) caused a concentration-dependent inhibition of the bFGF-stimulated re-growth in CHO-K1 cells expressing human sst₂ (h sst₂) or sst₅ (h sst₅) receptors (pIC₅₀=8.05±0.03 and 8.56±0.12, respectively). In contrast, angiopeptin (0.1–1000 nM) acted as a partial agonist at the h sst₂ receptor (44.6±2.7% inhibition of the bFGF-stimulated re-growth at 100 nM; pIC₅₀=8.69±0.25) but was devoid of any agonist activity at the h sst₅ receptor.
4. In CHO-K1 cells stably expressing rat recombinant sst₂ (r sst₂) or sst₅ (r sst₅) receptors, SRIF (0.1–1000 nM) was able to inhibit the bFGF-stimulated re-growth (pIC₅₀=7.98±24 and 8.50±0.12, respectively). Angiopeptin (0.1–1000 nM) caused a concentration-dependent inhibition of bFGF-stimulated re-growth at the r sst₂ receptor (pIC₅₀=8.08±0.24) but acted as a partial agonist at the r sst₅ receptor (maximum response=57.7±3.6% inhibition of bFGF-stimulated re-growth at 100 nM; pIC₅₀=8.60±0.16).
5. Although angiopeptin was inactive as an agonist at the h sst₅ receptor, 100 nM angiopeptin potently antagonized the SRIF-induced inhibition of proliferation in CHO h  sst₅ (estimated pK_B=10.4±0.3). 5-Hydroxytryptamine (0.1 nM–10 μM) also inhibited bFGF-stimulated re-growth (pIC₅₀=8.36±0.11) and angiopeptin had no effect on this response (pK_B<7).
6. SRIF (0.1–1000 nM) caused a concentration-dependent (pIC₅₀=8.04±0.08) inhibition of bFGF-stimulated re-growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF-stimulated re-growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration-effect curve to SRIF with an estimated pK_B value of 7.70±0.12.
7. These findings suggest that the low intrinsic activity of angiopeptin at the h sst₂ receptor, combined with its lack of agonist activity at the h sst₅ receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

     

Pharmacological characterisation of the goldfish somatostatin sst5 receptor

Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst1-5). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst5 receptors (gfsst5) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [125I]-LTT-SRIF-28 ([Leu8, DTrp22, 125I-Tyr25]SRIF-28) and [125I][Tyr10]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (pKd: 9.99-9.71; Bmax: 300-350 fmol mg-1). 5'-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5-57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [125I]LTT-SRIF-28 and [125I][Tyr10]cortistatin-14 correlated significantly (r2=0.97, n=20). Pharmacological profiles of human and mouse sst5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst5 profile (r2=0.52-0.78, n > or = b16). Functional expression of the gfsst5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [35S]GTPgammaS ([35S]guanosine 5'-O-(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies (r2=0.81-0.93, n=20), although relative potency (pEC50) was reduced compared to pKd values. Relative efficacy profiles of luciferase expression and [35S]GTPgammaS binding, were rather divergent (r2=0.48, n=20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 (D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2) acted as an antagonist on the effects of SRIF-14 (pKB=6.74 +/- 0.23) on stimulation of [35S]GTPgammaS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [35S]GTPgammaS binding suggesting coupling of the receptor to G(i)/G(o) proteins. In summary, the present studies demonstrate that the gfsst5 receptor has a similar pharmacological profile and transductional properties to mammalian sst5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

Rat somatostatin sst2(a) and sst2(b) receptor isoforms mediate opposite effects on cell proliferation

We have investigated the actions of somatostatin (SRIF) and angiopeptin on cell proliferation of CHO-K1 cells expressing the recently cloned rat sst_2(b) receptor (CHOsst_2(b)) and compared these to their effects in cells expressing the sst_2(a) receptor (CHOsst_2(a)). In contrast to the sst_2(a) receptor, the sst_2(b) receptor did not mediate inhibition of bFGF (10 ng ml⁻¹)-stimulated re-growth and cell proliferation. Rather, SRIF (0.1–1000 nM) and angiopeptin (0.1–1000 nM) stimulated basal re-growth and proliferation of CHOsst_2(b) cells in a concentration-dependent manner (estimated pEC₅₀ values of 7.8 and 7.9, respectively). The opposite effects of SRIF on cell proliferation mediated through the two sst₂ receptor isoforms were both abolished by 18 h pre-treatment with pertussis toxin. The proliferative effect via the sst_2(b) receptor was also abolished by the tyrosine kinase inhibitor, genistein. In conclusion, the present study shows that the rat sst_2(a) and sst_2(b) receptor splice variants mediate opposite effects on cell proliferation.     

Characterisation of the fish sst3 receptor, a member of the SRIF1 receptor family: atypical pharmacological features

The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor = SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst3), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst3 receptors; [125I]LTT-SRIF28 ([Leu8, D-Trp22, 125I-Tyr25]-SRIF28), [125I]Tyr10-cortistatin, [125I]CGP 23996, and [125I]Tyr3-octreotide labelled the fsst3 receptor with high affinity (pKd values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different Bmax values; 4500, 4000, 3400 and 1500 fmol/mg, respectively. The affinities of SRIF peptides and analogues determined for fsst3 receptors displayed the following rank order of potency: seglitide = SRIF25 > SRIF14 = SRIF28 > cortistatin 14 > BIM 23014 > RC160 = L361,301 = octreotide > or = BIM 23052 > or = L362,855 > CGP23996 > BIM 23056 > BIM 23030 = cycloantagonist > SRIF22. The pharmacological profiles determined with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I]Tyr10-cortistatin correlated highly significantly (r = 0.96-0.99), whereas [125I]Tyr3-octreotide binding was rather divergent (r = 0.78-0.81). Further, [125I]Tyr3-octreotide- and [125I]CGP 23996-labelled sites showed higher affinity for the various peptides than [125I]LTT-SRIF28 and [125I]Tyr10-cortistatin-labelled sites, although there were exceptions. [125I]LTT-SRIF28-binding to fsst3 receptors and human sst1-5 receptors was compared; the fsst3 binding profile correlated better with the hsst5- than with the hsst3 receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst3 transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fsst3 receptor to Gialpha and/or Goalpha. [125I]LTT-SRIF28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [125I]LTT-SRIF28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst3 receptor, in agreement with expression of the fsst3 receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst3 receptor has a pharmacological profile compatible with that of a SRIF1 receptor, although the rank order of affinity of fsst3 is closer to that of hsst5 than hsst3 receptors, as may be found when comparing very distantly related species. The fsst3 receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian sst3 receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst3 receptor in brain as well as other yet to be defined SRIF receptor subtypes.     

Long-term administration of m-chlorophenylpiperazine (mCPP) to rats induces changes in serotonin receptor binding,dopamine levels and locomotor activity without altering prolactin and corticosterone secretion

Meta-chlorophenylpiperazine (mCPP) is a serotonin (5-HT) agonist with antidepressant actions. In order to investigate the effects of chronic mCPP treatment the drug was administered to rats for 15 days (5 mg/kg twice daily). Controls were administered saline. Long-term mCPP treatment led to a 36% increase in [³H]8-hydroxy-2-(di-n-propylamino)tetralin ([³H]8-OH-DPAT) binding to 5-HT_1a receptors in hippocampus and a 74% decrease in [³H]ketanserin binding to 5-HT₂ receptors in cortex, while (-)[¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) binding to 5-HT_1b receptors in hypothalamus and striatum was unchanged. In hypothalamus, chronic mCPP treatment decreased the levels of dopamine (DA) but not 5-HT. The usual suppression of locomotor activity induced by acute mCPP administration was less after long-term mCPP treatment. Brain and plasma levels of mCPP following an acute dose were not different between controls and rats previously administered mCPP, suggesting that altered rate of metabolism of the drug did not explain the tolerance to the mCPP-induced decrease in locomotor activity. mCPP-induced prolactin (PRL) and corticosterone release were not changed by previous long-term mCPP administration. Thus, chronic mCPP administration to rats induced alterations in density of 5-HT receptor subtypes, hypothalamic levels of DA and locomotor behavior.     

[125I]-HEAT: Fifty percent of the ligand can bind to the alpha1-adrenoceptors with extremely high affinity

Summary [¹²⁵I]-HEAT,¹²⁵iodo-2-[-(4-hydroxyphenyl)-ethyl-aminomethyl]tetralone, is a novel alpha₁-adrenoceptor ligand which labels alpha₁-adrenoceptors in peripheral tissues as well as in the central nervous system. Using the technique of ligand saturation by receptors, we find that only 50% of the¹²⁵I-labeled HEAT molecules bind with high affinity to receptors from a variety of tissues.This was observed with partially purified rat brain membranes and highly purified rat liver plasma membranes in the absence or presence of sodium ion (as NaCl, 150 mM) which stimulated¹²⁵I-HEAT binding, by increasing the affinity. If the bindability of [¹²⁵I]-HEAT is taken into account,K _D values as low as 7–8 pM (at 30°C) are found in equilibrium binding experiments and optimally stimulating concentrations of sodium ion. The limited high affinity binding of [¹²⁵I]-HEAT could not be explained by radiochemical impurities. Instead, we suggest that only one enantiomer of the racemic ligand is preferentially bound to the receptors with aK _D in the picomolar range.Since the enantiomers are in dynamic equilibrium in solution (via keto-enol tautomerism) [¹²⁵I]-HEAT is a unique radioligand which makes it unlikely that the respective isomers can be separated by successive depletion with receptors.     

Binding and functional properties of the novel somatostatin analogue KE 108 at native mouse somatostatin receptors

Clinically used somatostatin (SRIF) analogs, octreotide and lanreotide, act primarily by binding to SRIF receptor subtype 2 (sst2). In contrast, the recently described multiligand SOM230 binds with high affinity to sst(1-3) and sst5 and KE 108 is characterised as a high affinity ligand for all five SRIF receptors. In tumoural mouse corticotrophs (AtT-20 cells) and in mouse hippocampus, binding and functional features of KE 108 were examined and compared to SRIF-14, octreotide and SOM230. In AtT-20 cells, KE 108 bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF-14, octreotide and SOM230. At the functional level, all four ligands increased guanosine-5'-O-(3-[35S]thio)-triphosphate binding and decreased cAMP accumulation or intracellular Ca2+ concentration through G(i/o) proteins. In hippocampal slices, KE 108, octreotide and SOM230 also bound with high affinity at [125I]LTT-SRIF-28-labelled sites similarly to SRIF-14, but KE 108, octreotide or SOM230 did not influence spontaneous epileptiform activity which was, in contrast, inhibited by SRIF-14. In conclusion, this study demonstrates that KE 108 has high affinity for native mouse SRIF receptors. Functionally, KE 108 mediates SRIF action at sst(2/5) in corticotrophs whereas it does not mimic the SRIF-induced inhibition of hippocampal excitation suggesting that the high potency and efficacy of a synthetic ligand to all known SRIF receptors may not reproduce entirely the effects of the natural SRIF.