Somatostatin receptor subtype 5 regulates insulin secretion and glucose homeostasis

Somatostatin (SRIF) regulates pancreatic insulin and glucagon secretion. In the present study we describe the generation of SRIF receptor subtype 5 knockout (sst(5) KO) mice to examine the role of SRIF receptor subtypes (sst) in regulating insulin secretion and glucose homeostasis. Mice deficient in sst(5) were viable, fertile, appeared healthy, and displayed no obvious phenotypic abnormalities. Pancreatic islets isolated from sst(5) KO mice displayed increased total insulin content as compared with islets obtained from wild-type (WT) mice. Somatostatin-28 (SRIF-28) and the sst(5)/sst(1)-selective agonist compound 5/1 potently inhibited glucose-stimulated insulin secretion from WT islets. SRIF-28 inhibited insulin secretion from sst(5) KO islets with 16-fold less potency while the maximal effect of compound 5/1 was markedly diminished when compared with its effects in WT islets. sst(5) KO mice exhibited decreased blood glucose and plasma insulin levels and increased leptin and glucagon concentrations compared with WT mice. Furthermore, sst(5) KO mice displayed decreased susceptibility to high fat diet-induced insulin resistance. The results of these studies suggest sst(5) mediates SRIF inhibition of pancreatic insulin secretion and contributes to the regulation of glucose homeostasis and insulin sensitivity. Our findings suggest a potential beneficial role of sst(5) antagonists for alleviating metabolic abnormalities associated with obesity and insulin resistance.     

Tricyclic pharmacophore-based molecules as novel integrin alphavbeta3 antagonists. Part IV: preliminary control of alphavbeta3 selectivity by meta-oriented substitution

To establish the in vivo efficacy of alphavbeta3/alphaIIbbeta3 dual antagonists possessing a tricyclic pharmacophore, a corresponding alphavbeta3-selective antagonist was required as a control. We initially took two synthetic approaches to obtain alphavbeta3-selective antagonists based on the RGD recognition pattern or on modification of the dihedral angle between the central benzene ring and the adjacent heterocycle, but both proved unsuccessful. However, synthesis of novel antagonists with meta-substitution of the central benzene ring generated weak selectivity for alphavbeta3 over alphaIIbbeta3 for the first time in the family of compounds with the tricyclic pharmacophore. Optimization of meta-oriented antagonists furnished an alphavbeta3-selective antagonist exhibiting inhibitory activity not only in a receptor-binding assay, but also in a cell adhesion assay.     

Novel gonadotropin-releasing hormone antagonists with substitutions at position 5

Gonadotropin-releasing hormone (GnRH) antagonists with high potency and improved duration of action are needed for potential clinical applications. We synthesized four new antagonists (2-5) of GnRH homologues to Azaline B (1), with a common core sequence of [Aph(X)5, D-Aph(Cbm)6]Azaline B. In these analogs, (X) contains hydrophobic aromatic moieties (like homoveratoyl in 2, homovanillyl in 3, 2,5-dimethoxyphenylacetyl in 4, and 3,5-dimethoxyphenylacetyl in 5) designed to improve the duration of action over that of Azaline B. These analogs were tested in vitro for their ability to antagonize the GnRH receptor and in vivo for duration of action in a castrated male rat assay. Analogs 2, 4, and 5 were potent in vitro, but were found to be short acting in vivo. However, analog 3 [Aph(Hvn)5,D-Aph(Cbm)6]Azaline B is a potent human GnRH receptor antagonist in vitro (IC50 1.47 nM) and exhibits a longer duration of action than azaline B.     

Urethane-induced somatostatin mediated inhibition of gastric acid: reversal by the somatostatin 2 receptor antagonist, PRL-2903.

Urethane increases the release of somatostatin (SRIF) which inhibits gastric acid secretion. The SRIF monoclonal antibody, CURE.S6 and the novel sst2 antagonist, PRL-2903 injected intravenously at maximal effective doses increased gastric acid secretion by 2 and 10 fold respectively from basal values within 30 min in urethane-anesthetized rats. Plasma gastrin levels were elevated 2.5 fold within 15 min by PRL-2903 (1.3 micromol/kg, i.v.). These data indicate that the low gastrin and acid secretion levels induced by urethane result from endogenous SRIF acting on sst2 and that PRL-2903 is a valuable SRIF antagonist to assess sst2 mediated events.     

Somatostatin-dependent adenylyl cyclase activity in nonactivated and mitogen-activated human T cells: evidence for uncoupling of sst3 receptor from adenylyl cyclase

Neuropeptide somatostatin (SRIF) has been shown to modulate interleukin-2 (IL-2) secretion by mitogen-activated T cells. In this study, we further analyzed the transduction pathways underlying SRIF actions on human Jurkat T cells and compared SRIF signaling between nonactivated and mitogen-activated cells. SRIF effects on adenylyl cyclase activity in the absence and presence of mitogens were addressed by using three different analogs: SRIF14, SRIF28, and SMS 201-995. In semipurified membrane preparations obtained from nonactivated cells, all analogs inhibited adenylyl cyclase. However, in membrane preparations obtained from mitogen-activated cells, the maximal inhibition of adenylyl cyclase mediated by SRIF14 and SRIF28 equaled only one third of that measured in the absence of mitogens, whereas SMS 201-995 was completely inactive. To assess the relevant mechanisms associated with different effects of SRIF on adenylyl cyclase activity in nonactivated and mitogen-activated T cells, we performed binding assays by using iodinated SRIF as a radioligand. These experiments suggested that both the number of receptors and their affinities were almost identical in either nonactivated or activated cells. RT-PCR analysis of the pattern of SRIF receptor expression showed that nonactivated as well as activated Jurkat cells expressed only mRNA corresponding to the sst3 receptor subtype. Altogether, these data point to a functional activation-associated uncoupling of sst3 receptors from adenylyl cyclase in human T cells, indicating a T-cell activation-induced alteration in the sst3 receptor transduction pathway.     

Somatostatin sst(2) receptors inhibit peristalsis in the rat and mouse jejunum

Somatostatin [somatotropin release-inhibitory factor (SRIF)] has widespread actions throughout the gastrointestinal tract, but the receptor mechanisms involved are not fully characterized. We have examined the effect of selective SRIF-receptor ligands on intestinal peristalsis by studying migrating motor complexes (MMCs) in isolated segments of jejunum from rats, mice, and sst(2)-receptor knockout mice. MMCs were recorded in 4- to 5-cm segments of jejunum mounted horizontally in vitro. MMCs occurred in rat and mouse jejunum with intervals of 104.4 +/- 10 and 131.2 +/- 8 s, respectively. SRIF, octreotide, and BIM-23027 increased the interval between MMCs, an effect fully or partially antagonized by the sst(2)-receptor antagonist Cyanamid154806. A non-sst(2) receptor-mediated component was evident in mouse as confirmed by the observation of an inhibitory action of SRIF in sst(2) knockout tissue. Blocking nitric oxide generation abolished the response to SRIF in rat but not mouse jejunum. sst(2) Receptors mediate inhibition of peristalsis in both rat and mouse jejunum, but a non-sst(2) component also exists in the mouse. Nitrergic mechanisms are differentially involved in rat and mouse jejunum.     

Characterization of the locomotor depression produced by an A2-selective adenosine agonist

Adenosine analogs, such as N6-cyclohexyladenosine (CHA) that are selective for A1-adenosine receptors, and analogs, such as 5'-N-ethylcarboxamidoadenosine (NECA) that are active at both A1 and A2 receptors, cause a profound depression of locomotor activity in mice via a central mechanism. The depression is effectively reversed by non-selective adenosine antagonists such as theophylline. We report that 2-([2-aminoethylamino) carbonylethylphenylethylamino]-5'-N-ethylcarboxamidoadenosine (APEC), an amine derivative of the A2-selective agonist, CGS21680, is a potent locomotor depressant in mice. The in vivo pharmacology is consistent with A2-selectivity at a central site of action. Two parameters indicative of locomotor activity, horizontal activity and total distance travelled, were measured using a computerized activity monitor. From dose-response curves it was found that APEC (ED50 16 micrograms/kg) is more potent than CHA (ED50 60 micrograms/kg) and less potent than NECA (ED50 2 micrograms/kg). The locomotor depression by APEC was reversible by theophylline, but not by the A1-selective antagonists 8-cyclopentyltheophylline (CPT) and 8-cyclopentyl-1, 3-dipropyl-2-thioxanthine, nor by the peripheral antagonists 8-p-sulfophenyltheophylline (8-PST) and 1,3-dipropyl-8-p-sulfophenylxanthine. The locomotor activity depression elicited by NECA and CHA was reversed by A1-selective antagonists. These results suggest that the effects of APEC are due to stimulation of A2 adenosine receptors in the brain.     

Somatostatin receptor subtype 1-selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor and so far medical therapy has provided inconclusive results. In the human MTC cell line TT, expressing all somatostatin (SST) receptor subtypes, cell proliferation decreases with SST and SST receptor subtype 2 (sst(2)), but not sst(5), selective agonist treatment, whereas calcitonin (CT) expression and secretion are reduced by SST, but not by sst(2) and sst(5) agonists. The effectiveness of two new SST analogs, BIM-23926 and BIM-23745, selectively interacting with sst(1), was investigated in the TT cell line. DNA synthesis is significantly reduced by BIM-23926 (27-40% at 10(-10)-10(-6)M) and BIM-23745 (32-90% at 10(-8)-10(-6)M). Viable cell number is also significantly reduced by both BIM-23926 (40% at 10(-12)-10(-6)M) and BIM-23745 ( approximately 40% at 10(-10)-10(-6)M). Treatment with sst(1)-selective agonists significantly reduces CT secretion and gene expression, with a reduction of CREB phosphorylation. These findings suggest that potent sst(1)-selective agonists could have a therapeutic role in MTC.     

Somatostatin receptors

In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst(1)-sst(5)), one of which is represented by two splice variants (sst(2A) and sst(2B)). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst(2) and sst(5) receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

Assessment of substitution in the second pharmacophore of Dmt-Tic analogues

The Dmt-Tic pharmacophore exhibits potent δ-opioid receptor antagonism. Analogues with substitutions in the second pharmacophore with (1, 1′) or without a COOH function (2–9) were synthesized: several had high δ affinity (1′, 2, 7, and 9), but exhibited low to non-selectivity toward μ receptors similar to H-Dmt-Tic-amide and H-Dmt-Tic-ol. Functional bioactivity indicated high δ antagonism (pA₂ 7.4–7.9) (1′, 2, and 9) and modest μ agonism, pEC₅₀ (6.1–6.3) (1′, 2, 8, and 9), but with E_max values analogous to dermorphin. These Dmt-Tic analogues with mixed δ antagonist/μ agonist properties would appear to be better candidates as analgesics than pure μ agonists.     

Small molecule somatostatin receptor subtype-2 antagonists

The first potent small molecule sst2 antagonists are reported. Altering known sst2 agonist molecules yielded compounds with high sst2 binding affinity and full antagonist activity. Compound 7a, for example, displaced somatostatin binding to the sst2 receptor with an IC(50)=2.9 nM and antagonized somatostatin action with an IC(50)=29nM.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

Development of potent glucagon antagonists: structure-activity relationship study of glycine at position 4.

We examined the functional role of glycine at position 4 in the potent glucagon antagonist [desHis(1), Glu(9)]glucagon amide, by substituting the L- and D-enantiomers of alanine and leucine for Gly(4) in this antagonist. The methyl and isobutyl side-chain substituents were introduced to evaluate the preference shown by the glucagon receptor, if any, for the orientation of the N-terminal residues. The L-amino acids demonstrated only slightly better receptor recognition than the D-enantiomers. These results suggest that the Gly(4) residue in glucagon antagonists may be exposed to the outside of the receptor. The enhanced binding affinities of analogs 1 and 3 compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide, may have resulted from the strengthened hydrophobic patch in the N-terminal region and/or the increased propensity for a helical conformation due to the replacement of alanine and leucine for glycine. Thus, as a result of the increased receptor binding affinities, antagonist activities of analogs 1-4 were increased 10-fold compared with the parent antagonist, [desHis(1), Glu(9)]glucagon amide. These potent glucagon antagonists have among the highest pA(2) values of any glucagon analogs reported to date.     

Adenylyl Cyclase/cAMP System Involvement in the Antiangiogenic Effect of Somatostatin in the Retina. Results from Transgenic Mice

Neoangiogenesis is a response to retinal hypoxia that is inhibited by somatostatin (SRIF) through its subtype 2 receptor (sst2). Using a mouse model of hypoxia-induced retinopathy, we investigated whether inhibition of adenylyl cyclase (AC) is involved in SRIF anti-angiogenic actions. Hypoxia increased AC responsiveness in wild type (WT) retinas and in retinas lacking sst2, but not in sst2-overexpressing retinas. Hypoxia also altered AC isoform expression with different patterns depending on sst2 expression level. The AC VII isoform mRNA and protein resulted the most affected. Indeed, in hypoxia AC VII expression was enhanced in WT retinas and it was further increased in sst2-lacking retinas, whereas in sst2 overexpressing retinas the increase of AC VII was lower than in WT retinas. These data suggest an involvement of AC/cAMP in mediating both hypoxia-evoked retinal neoangiogenesis and SRIF protective actions. The AC VII isoform is a candidate to a main role in these mechanisms.     

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery.

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.     

Homo- and heterodimerization of somatostatin receptor subtypes. Inactivation of sst(3) receptor function by heterodimerization with sst(2A)

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst(2A) and sst(3) exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and sst(3). The sst(2A)-sst(3) heterodimer exhibited high affinity binding to somatostatin-14 and the sst(2)-selective ligand L-779,976 but not to the sst(3)-selective ligand L-796,778. Like the sst(2A) homodimer, the sst(2A)-sst(3) heterodimer stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding, inhibition of adenylyl cyclase, and activation of extracellular signal-regulated kinases after exposure to the sst(2)-selective ligand L-779,976. However, unlike the sst(3) homodimer, the sst(2A)-sst(3) heterodimer did not promote GTPgammaS binding, adenylyl cyclase inhibition, or extracellular signal-regulated kinase activation in the presence of the sst(3)-selective ligand L-796,778. Interestingly, during prolonged somatostatin-14 exposure, the sst(2A)-sst(3) heterodimer desensitized at a slower rate than the sst(2A) and sst(3) homodimers. Both sst(2A) and sst(3) homodimers underwent agonist-induced endocytosis in the presence of somatostatin-14. In contrast, the sst(2A)-sst(3) heterodimer separated at the plasma membrane, and only sst(2A) but not sst(3) underwent agonist-induced endocytosis after exposure to somatostatin-14. Together, heterodimerization of sst(2A) and sst(3) results in a new receptor with a pharmacological and functional profile resembling that of the sst(2A) receptor, however with a greater resistance to agonist-induced desensitization. Thus, inactivation of sst(3) receptor function by heterodimerization with sst(2A) or possibly other G protein-coupled receptors may explain some of the difficulties in detecting sst(3)-specific binding and signaling in mammalian tissues.