cgp-23996 and seglitide

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

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Brain; Cell Line; Cloning, Molecular; DNA; Dose-Response Relationship, Drug; Gene Expression; Humans; In Situ Hybridization; Luciferases; Male; Membranes; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Octreotide; Oligopeptides; Peptides, Cyclic; Radioligand Assay; Receptors, Somatostatin; Recombinant Fusion Proteins; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Somatostatin; Transfection

There is considerable controversy about the classification and nomenclature of somatostatin receptors. To date, five distinct receptor genes have been cloned and named chronologically according to their respective publication dates, but two were unfortunately given the same appellation (SSTR4). Consensually, a nomenclature for the recombinant receptors has been agreed according to IUPHAR guidelines (sst1, sst2, sst3, sst4, and sst5). However, a more informative classification is to be preferred for the future, employing all classification criteria in an integrated scheme. It is already apparent that the five recombinant receptors fall into two classes or groups, on the basis of not only structure but also pharmacological characteristics. One class (already referred to by some as SRIF1) appears to comprise sst2, sst3 and sst5 receptor subtypes. The other class (SRIF2) appears to comprise the other two recombinant receptor subtypes (sst1 and sst4). This promising approach is discussed but it is acknowledged that much more data from endogenous receptors in whole tissues are needed before further recommendations on somatostatin receptor nomenclature can be made.

Topics: Amino Acid Sequence; Molecular Sequence Data; Octreotide; Peptides, Cyclic; Receptors, Somatostatin; Recombinant Proteins; Somatostatin; Terminology as Topic

GH3 cells express receptors for the neuropeptide somatostatin (SRIF). In the present study, we have identified and characterized SRIF1 and SRIF2 receptors in GH3 cells using the radioligands [125I]MK 678 and [125I]CGP 23996. [125I]MK 678 binding to SRIF1 receptors was saturable and of high affinity and was potently inhibited by SRIF analogs with a rank order of potency of MK 678 > SRIF > SRIF 28 > CGP 23996. [125I]CGP 23996 binding to SRIF2 receptors was also saturable and of high affinity, and was potently inhibited by SRIF analogs with a rank order of potency of SRIF 28 > SRIF > CGP 23996, but was not inhibited by MK 678. Agonist pretreatment of GH3 cells differentially regulated SRIF1 and SRIF2 receptors. [125I]MK 678 binding to SRIF1 receptors was readily diminished after pre-exposure of GH3 cells to SRIF or MK 678. [125I]CGP 23996 binding to SRIF2 receptors was unaffected by pretreatment with MK 678 and was only partially affected by pretreatment with SRIF. [125I]MK 678 binding to SRIF1 receptors was abolished in the presence of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate, but [125I]CGP 23996 binding to SRIF2 receptors was unaffected. The SRIF1 receptor mediates inhibition of adenylyl cyclase activity, as SRIF and MK 678 inhibited forskolin-stimulated cyclic AMP accumulation in these cells to the same extent. GH3 cells are a unique model system for investigations of the pharmacological, biochemical and functional properties of these two receptor subclasses.

Topics: Animals; Cell Line; Colforsin; Cyclic AMP; GTP-Binding Proteins; Iodine Radioisotopes; Kinetics; Peptides, Cyclic; Pituitary Gland, Anterior; Rats; Receptors, Somatostatin; Sensitivity and Specificity; Somatostatin; Stimulation, Chemical; Temperature; Time Factors

Multiple somatostatin (SRIF) receptor subtypes, which mediate distinct biological actions of SRIF, are expressed in the rat central nervous system. In the present study, we examined the effects of local injections of SRIF and the SRIF analogs MK 678 and CGP 23996 into the anterior nucleus accumbens on locomotor activity. The binding of [125I]Tyr11-SRIF to membranes from rat nucleus accumbens was potently and monophasically inhibited by SRIF. MK 678 inhibited only 58% of specific [125I]Tyr11-SRIF binding, indicating that the nucleus accumbens expresses both SRIF1 (MK 678-sensitive) and SRIF2 (MK 678-insensitive) receptors. The inhibition of [125I]Tyr11-SRIF binding by CGP 23996 was best fit by a two-site model, and analysis indicated an approximately 100-fold selectivity of this peptide for SRIF receptor subtypes. Intra-accumbens injections of SRIF (3.2-100 ng/side) produced significant increases in locomotor activity with a maximal 212% increase relative to saline control. This effect was mediated by SRIF1 receptors, as MK 678 (1-320 ng/side) produced a dose-dependent significant increase in locomotor activity with a maximal 228% increase relative to saline control, comparable to that attained with 3 to 10 micrograms of d-amphetamine. In contrast, CGP 23996 did not affect locomotor activity at doses of 3.2 to 1000 ng/side. The retroenantiomer hexapeptide analog L363-572, which is 70-fold less potent than MK 678 to inhibit radioligand binding to SRIF1 receptors, did not affect locomotor activity at doses up to 100 ng/side. These results indicate that SRIF1 receptors mediate the locomotor-activating effects of SRIF in the nucleus accumbens of the rat.

Topics: Animals; Locomotion; Male; Motor Activity; Nucleus Accumbens; Peptides, Cyclic; Rats; Rats, Sprague-Dawley; Receptors, Somatostatin; Somatostatin

Sodium ions have been shown to reduce the binding of agonists to a number of G protein-linked receptors. They are believed to do so by interacting with aspartate residues in the second membrane-spanning region of these receptors to cause G protein uncoupling, resulting in a diminished affinity of the receptors for agonists. To investigate Na+ regulation of agonist binding to somatostatin receptors, Na+ was tested for its effect on the binding of agonists to cloned somatostatin receptor type 1 (SSTR1) and somatostatin receptor type 2 (SSTR2) stably expressed in Chinese hamster ovary cells. Na+ reduced agonist binding to SSTR2 but not to SSTR1. Because high affinity agonist binding to SSTR1 does not depend on G protein coupling but agonist binding to SSTR2 is reduced by guanosine-5'-(beta, gamma-imido)triphosphate and pertussis toxin treatment, the selective Na+ effect on SSTR2 is consistent with previous findings with other receptors showing that Na+ uncouples receptors from G proteins, thereby reducing the affinity of the receptors for agonists. Conversion of Asp89 to Asn89 in SSTR2 resulted in a mutant receptor whose affinity for agonists was not altered by Na+, indicating that Asp89 is involved in mediating the effects of Na+ on agonist binding to SSTR2. However, the affinities of the mutant and wild-type receptors for somatostatin were the same, and both guanosine-5'-O-(gamma-thio)triphosphate and pertussis toxin treatment reduced agonist binding to the mutant and wild-type receptors. These findings differ from the results of similar mutagenesis studies on other G protein-linked receptors, in that the mutant and wild-type SSTR2 forms associate with G proteins in similar ways. These results indicate that Asp89 acts in a novel manner to regulate agonist binding and G protein interaction with SSTR2.

Topics: Animals; Aspartic Acid; Base Sequence; Binding Sites; CHO Cells; Cricetinae; Cricetulus; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Peptides, Cyclic; Pertussis Toxin; Receptors, Somatostatin; Sodium; Somatostatin; Virulence Factors, Bordetella

Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane-bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK-678, such that SRIF1 receptors are sensitive to MK-678 and SRIF2 receptors are insensitive to MK-678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I-Tyr11-SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK-678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF1 receptor. The binding of 125I-MK-678 to SRIF1 receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS-201-995), and the hexapeptides (such as MK-678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP-23996-like analogues did not inhibit 125I-MK-678 binding to SRIF1 receptors. The binding of 125I-CGP-23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of less than 1, indicating that 125I-CGP-23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP-23996-like compounds produced only partial inhibitions of 125I-CGP-23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I-CGP-23996. 125I-Tyr11-SRIF binding and 125I-CGP-23996 binding to SRIF receptors were likewise only partially affected by 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a concentration that completely abolishes specific 125I-MK-678 binding to SRIF1 receptors. The component of 125I-CGP-23996 labeling that was sensitive to GTP gamma S was also MK-678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF1 receptor is sensitive to cyclic hexapeptides such as MK-678 and to GTP gamma S but insensitive to smaller CGP-23996-like compounds. The SRIF2 receptor is sensitive to the CGP-23996-like compounds and can be selectively labeled by 125I-CGP-23996 in the presence of high concentrations of the hexapeptides or GTP gamma S because, unlike the SRIF1 receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype-selective peptide analogues will be useful in the future c

Topics: Animals; Binding, Competitive; Brain; Guanosine 5'-O-(3-Thiotriphosphate); Osmolar Concentration; Peptides, Cyclic; Rats; Receptors, Somatostatin; Somatostatin

Somatostatin receptor subtypes were labeled with the somatostatin analogs [125I]CGP 23996 and [125I]MK 678 and the distribution of these receptors in rat brain was investigated using quantitative autoradiographic techniques. [125I]CGP 23996 and [125I]MK 678 specifically label different populations of somatostatin receptors in rat brain. In a number of brain regions striking differences in the distribution of the somatostatin receptor subtypes labeled by each peptide were observed. High levels of binding sites for both [125I]CGP 23996 and [125I]MK 678 were present in the cerebral cortex, CA1 region and subiculum of the hippocampus. In contrast, high levels of [125I]MK 678 binding were found in the dentate gyrus of the hippocampus while few [125I]CGP 23996 binding sites were observed in this brain region. [125I]CGP 23996 binding was detected in the central region of the interpeduncular nucleus whereas the dorsal and lateral subnuclei of this brain area expressed mainly somatostatin receptors with high affinity for MK 678. The locus coeruleus and regions of the superior colliculus and hypothalamus selectively express [125I]MK 678-sensitive somatostatin receptors. Furthermore, limbic structures such as the lateral septum, the nucleus accumbens and ventromedial striatum had much higher levels of [125I]MK 678 binding sites than [125I]CGP 23996 binding sites. Differences in the expression of the somatostatin receptor subtypes were also detected in the substantia nigra. [125I]CGP 23996 binding was present in the pars reticulata but not the pars compacta whereas the reverse distribution for [125I]MK 678 binding sites was observed. The differential distribution of [125I]CGP 23996 and [125I]MK 678 binding sites in rat brain supports the hypothesis that these peptides selectively label different somatostatin receptor subtypes in the central nervous system.

Topics: Animals; Autoradiography; Brain; Iodine Radioisotopes; Kinetics; Male; Organ Specificity; Peptides, Cyclic; Rats; Rats, Inbred Strains; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin

To investigate whether somatostatin (SRIF) receptor subpopulations mediate different physiological actions of SRIF, we tested the effects of SRIF and the SRIF agonists MK 678 and CGP 23996 on different biological responses in rat neocortical neurons in culture. Neocortical cells in culture express SRIF receptors that can be labeled with 125I-MK 678 and 125I-CGP 23996. Pharmacological analysis of the binding sites indicates that the radioligands label SRIF receptor subtypes with distinct pharmacological characteristics. These receptor subpopulations are similar to those expressed in adult rat brain. SRIF, MK 678, and CGP 23996 are able to inhibit forskolin-stimulated adenylate cyclase activity in rat neocortical membranes by 25-30%. Furthermore, they inhibit a high voltage-activated Ca2+ current in rat neocortical neurons in culture by 25-35%. Both SRIF and MK 678 potentiate a delayed rectifier K+ current in rat neocortical neurons in culture by 25-30%. In contrast, high concentrations of CGP 23996 do not alter the K+ current. In cells that do not respond to CGP 23996, MK 678 increases the delayed rectifier K+ current. The findings of these studies indicate that rat neocortical neurons in culture express functionally distinct SRIF receptor subtypes that can be differentially activated by SRIF agonists.

Topics: Adenylyl Cyclases; Animals; Brain Chemistry; Calcium Channels; Cells, Cultured; Peptides, Cyclic; Potassium Channels; Rats; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin

Somatostatin (SRIF) is a neurotransmitter in the brain. Subtypes of SRIF receptors may mediate the diverse physiological actions of SRIF in the central nervous system. In the present study, the characteristics of subtypes of brain SRIF receptors were examined using two SRIF analogs. [125I]CGP 23996 and [125I]MK 678. [125I]CGP 23996 binds selectively to rat brain SRIF receptors in a saturable manner and with high affinity. [125I]CGP 23996 binding to brain SRIF receptors is inhibited by SRIF agonists with a rank order of potency of SRIF greater than cyclo (aha-Cys-Phe-D-Trp-Lys-Thr-Cys) greater than SMS 201-995 much greater than MK 678 = L-363,301. [125I]MK 678 labels rat brain SRIF receptors which are not detected by low nanomolar concentrations of [125I]CGP 23996. [125I]MK 678 binding to brain membranes is saturable and of high affinity with a Kd of 0.3 nM and a Bmax of 217 fmol/mg of protein in brain and a Kd of 0.17 nM and a Bmax of 211 fmol/mg of protein in anterior pituitary. [125I]MK 678 binding to brain SRIF receptors is blocked selectively by SRIF analogs. SRIF, SRIF 28, D-Trp8 SRIF, SMS 201-995, cyclo (aha-Cys-Phe-D-Trp-Lys-Thr-Cys) and MK 678 have similar potencies to inhibit [125I]MK 678 binding to brain SRIF receptors. The different rank order of potencies of SRIF analogs to inhibit [125I]CGP 23996 and [125I]MK 678 binding to brain SRIF receptors suggests that these radioligands interact with different subtypes of brain SRIF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain Chemistry; GTP-Binding Proteins; Male; Peptides, Cyclic; Rats; Rats, Inbred Strains; Receptors, Neurotransmitter; Receptors, Somatostatin; Somatostatin