Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R₁ and MCH‐R₂, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R₁ was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R₁ and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R₁ (SLC‐1), the radioligand was [¹²⁵I]–[Tyr¹³]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [¹²⁵I]–[D‐Phe¹³, Tyr¹⁹]‐MCH served as radioligand. The bioassay used for MCH‐R₁ was intracellular Ca²⁺ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R₁ and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe¹³ in [Phe¹³, Tyr¹⁹]‐MCH was well tolerated by both MCH‐R₁ and B16 MCH‐R, change of configuration to D‐Phe¹³ in [D‐Phe¹³, Tyr¹⁹]‐MCH or [D‐Phe¹³]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R₁. By contrast, the D‐Phe¹³ residue increased the affinity of [D‐Phe¹³, Tyr¹⁹]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe¹³, Tyr¹⁹]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R₁ in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R₁.     

Synthesis and lodination of human (phenylalanine13, tyrosine19) melanin-concentrating hormone for radioreceptor assay

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr¹³ and Val¹⁹ of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe¹³, Tyr¹⁹] -MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmocstrategy and Polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [¹²⁵I]-[Phe¹³, Tyr¹⁹]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD ) was 1.18 × 10^?10 M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as α-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.     

Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the ¹²⁵I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that ¹²⁵I[Bpa⁰, Nle¹⁷]PACAP27, ¹²⁵I[Bpa⁶, Nle¹⁷]PACAP27 and ¹²⁵I[Nle¹⁷, Bpa²²]PACAP27 covalently labeled the Ser⁹⁸ - Met¹¹¹ segment, the Ser¹²⁴ - Glu¹²⁵ dipeptide and the Ser¹⁴¹ - Met¹⁷² fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.     

α-MSH Receptor Autoradiography on Mouse and Human Melanoma Tissue Sections and Biopsies

Abstract

MSH receptors and their binding characteristics of [¹²⁵I]-labelled derivatives of α-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was to determine the binding characteristics of α-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(¹²⁵I)Tyr²]-α-MSH or [(¹²⁵I)Tyr²,Nle⁴,D-Phe⁷]-α-MSH and specific α-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled α-MSH during incubation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: K_D = 1.87 and 1.31 nmol/l for B16 tumours and cells, respectively; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar binding properties of α-MSH radioligands to both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.     

Tetraamine‐modified octreotide and octreotate: labeling with 99mTc and preclinical comparison in AR4‐2J cells and AR4‐2J tumor‐bearing mice

Two somatostatin analogues, [^99mTc]Demotide and [^99mTc]Demotate 4, were compared with [^99mTc]Demotate 1, a previously reported somatostatin receptor subtype 2 (sst₂) targeting tracer. Conjugates were prepared by coupling an open‐chain tetraamine chelator to D ‐Phe¹ of [Tyr³]‐octreotide or [Tyr³]‐octreotate, respectively, via a p‐benzylaminodiglycolic acid spacer adopting solid‐phase peptide synthesis techniques. Peptide conjugates were collected in a highly pure form after chromatographic purification. Eventually, [^99mTc]Demotide and [^99mTc]Demotate 4 were obtained in ～1 Ci/µmol specific activity and >96% purity after labeling under alkaline conditions. Demotide and Demotate 4 exhibited similar high binding affinities for the sst₂ expressed in AR4‐2J cells with IC₅₀ values 0.16 and 0.10 nM, respectively. The (radio)metallated analogues [^99mTc]Demotide and [^99mTc]Demotate 4 showed equally high affinities to the sst₂ during saturation binding assays in AR4‐2J cell membranes (K_ds 0.08 and 0.07 nM, respectively). During incubation at 37 °C with AR4‐2J cells, the radiopeptides internalized effectively via a receptor‐mediated process, with [^99mTc]Demotate 4 exhibiting a faster internalization rate than [^99mTc]Demotide. After injection in athymic mice bearing sst₂‐expressing AR4‐2J tumors, the radiotracers showed high and specific uptake in the tumor (>25%ID/g at 1 h) and in the sst₂–positive organs. However, both [^99mTc]Demotide and [^99mTc]Demotate 4 showed unfavorably higher background activity, especially in the abdomen, in comparison to [^99mTc]Demotate 1 and are, therefore, less suited than [^99mTc]Demotate 1 for sst₂‐targeted tumor imaging in man. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

Pharmacological characterization of a Bombyx mori α‐adrenergic‐like octopamine receptor stably expressed in a mammalian cell line

Series of agonists and antagonists were examined for their actions on a Bombyx moriα‐adrenergic‐like octopamine receptor (OAR) stably expressed in HEK‐293 cells. The rank order of potency of the agonists was clonidine>naphazoline>tolazoline in Ca²⁺ mobilization assays, and that of the antagonists was chlorpromazine>yohimbine. These findings suggest that the B. mori OAR is more closely related to the class‐1 OAR in the intact tissue than to the other classes. N′‐(4‐Chloro‐o‐tolyl)‐N‐methylformamidine (DMCDM) and 2‐(2,6‐diethylphenylimino)imidazolidine (NC‐5) elevated the intracellular calcium concentration ([Ca²⁺]_i) with EC₅₀s of 92.8 µM and 15.2 nM, respectively. DMCDM and NC‐5 led to increases in intracellular cAMP concentration ([cAMP]_i) with EC₅₀s of 234 nM and 125 nM, respectively. The difference in DMCDM potencies between the cAMP and Ca²⁺ assays might be due to “functional selectivity.” The Ca²⁺ and cAMP assay results for DMCDM suggest that the elevation of [cAMP]_i, but not that of [Ca²⁺]_i, might account for the insecticidal effect of formamidine insecticides. © 2009 Wiley Periodicals, Inc.     

Stereoselective pharmacokinetics of stable isotope (+/−)‐[13C]‐pantoprazole: Implications for a rapid screening phenotype test of CYP2C19 activity

Aims: We have previously shown that the (±)‐[¹³C]‐pantoprazole breath test is a promising noninvasive probe of CYP2C19 activity. As part of that trial, plasma, breath test indices and CYP2C19 (*2, *3, and *17) genotype were collected. Here, we examined whether [¹³C]‐pantoprazole exhibits enantioselective pharmacokinetics and whether this enantioselectivity is correlated with indices of breath test. Methods: Plasma (−)‐ and (+)‐[¹³C]‐pantoprazole that were measured using a chiral HPLC were compared between CYP2C19 genotypes and correlated with breath test indices. Results: The AUC_(0‐∞) of (+)‐[¹³C]‐pantoprazole in PM (*2/*2, n = 4) was 10.1‐ and 5.6‐fold higher that EM (*1/*1or *17, n = 10) and IM (*1/*2or *3, n = 10) of CYP2C19, respectively (P < 0.001). The AUC_(0‐∞) of (−)‐[¹³C]‐pantoprazole only significantly differed between PMs and EMs (1.98‐fold; P = 0.05). The AUC_(0‐∞) ratio of (+)‐/(−)‐[¹³C]‐pantoprazole was 3.45, 0.77, and 0.67 in PM, IM, and EM genotypes, respectively. Breath test index, delta over baseline show significant correlation with AUC_(0‐∞) of (+)‐[¹³C]‐pantoprazole (Pearson's r = 0.62; P < 0.001). Conclusions: [¹³C]‐pantoprazole exhibits enantioselective elimination. (+)‐[¹³C]‐pantoprazole is more dependent on CYP2C19 metabolic status and may serve as a more attractive probe of CYP2C19 activity than (−)‐[¹³C]‐pantoprazole or the racemic mixture. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Nociceptin and its natural and specifically‐modified fragments: Structural studies

The vibrational structures of Nociceptin (FQ), its short bioactive fragments, and specifically‐modified [Tyr¹]FQ (1‐6), [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) fragments were characterized. We showed that in the solid state, all of the aforementioned peptides except FQ adopt mainly turn and disordered secondary structures with a small contribution from an antiparallel β‐sheet conformation. FQ (1‐11), FQ (7‐17) [His¹]FQ (1‐6), and [His^1,4]FQ (1‐6) have an α‐helical backbone arrangement that could also slightly influence their secondary structure. The adsorption behavior of these peptides on a colloidal silver surface in an aqueous solution (pH = ～8.3) was investigated by means of surface‐enhanced Raman scattering (SERS). All of the peptides, excluding FQ (7‐17), chemisorbed on the colloidal silver surfaces through a Phe⁴ residue, which for FQ, FQ (1‐11), FQ (1‐6), [Tyr¹]FQ (1‐6), and [His¹]FQ (1‐6) lies almost flat on this surface, while for FQ (1‐13) and FQ (1‐13)NH₂ adopts a slightly tilted orientation with respect to the surface. The Tyr¹ residue in [Tyr¹]FQ (1‐6) does not interact with the colloidal silver surface, suggesting that the Tyr¹ and Phe⁴ side chains are located on the opposite sides of the peptide backbone, which can be also true for His¹ and Phe⁴ in [His¹]FQ (1‐6). The lone pair of electrons on the oxygen atom of the ionized carbonyl group of FQ (1‐13) and FQ (7‐17) appears to be coordinated to the colloidal silver nanoparticles, whereas in the case of the remaining peptides, it only assists in the adsorption process, similar to the ? NH₂ group. We also showed that upon adsorption, the secondary structure of these peptides is altered. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1039–1054, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ‐AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)1

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the ¹³C‐enrichment ratios (¹³C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l ‐[3‐¹³C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d ‐[1‐¹³C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐13² of chl a was labeled with the ¹³C‐carbon of l ‐[methyl‐¹³C]methionine derived from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐ [3‐¹³C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P3¹, C‐P4, C‐P6, C‐P7¹, C‐P8, C‐P10, C‐P11¹, C‐P12, C‐P14, C‐P15¹, and C‐P16 from ¹³C‐isoprene (2‐[1,2‐methyl,3‐¹³C₃]methyl‐1,3‐butadiene) generated from l ‐[3‐¹³C]alanine via [2‐¹³C]acetyl CoA.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

Redox‐triggered chiroptical switching activity of ruthenium(III)‐bis‐(β‐diketonato) complexes bearing a bipyridine‐helicene ligand

The charged, electroactive bipyridine‐helicene‐ruthenium(III) complex [ 4 ] ^{. +},PF₆^? has been prepared from 3‐(2‐pyridyl)‐4‐aza[6]helicene and a Ru‐bis‐(β‐diketonato)‐bis‐acetonitrile precursor (β‐diketonato: 2,2,6,6‐tetramethyl‐3,5‐heptanedionato). Its chiroptical properties (electronic circular dichroism and optical rotation) were studied both experimentally and theoretically and suggest the presence of 2 diastereoisomers, namely (P,Δ)‐ and (P,Λ)‐[ 4 ] ^{. +},PF₆^? (denoted jointly as (P,Δ*)‐[ 4 ] ^{. +},PF₆^?) and their mirror‐images (M,Λ)‐ and (M,Δ)‐[ 4 ] ^{. +},PF₆^? ((M,Δ*)‐[ 4 ] ^{. +},PF₆^?). The electrochemical reduction of (P,Δ*)‐[ 4 ] ^{. +},PF₆^? to neutral complex (P,Δ*)‐ 4 was performed and revealed strong changes in the UV‐vis and electronic circular dichroism spectra. A reversible redox‐triggered chiroptical switching process was then achieved.     

Metabolism of somatostatin and its analogues by the liver

The rate of degradation of ¹²⁵I-labelled [Tyr¹¹]somatostatin by isolated rat hepatocytes was similar to that of unlabelled somatostatin. Reaction was dependent upon cell concentration and temperature, being rapid at 37°C and negligible at 0°C. The apparent K_m for the overall degradation process was approximately the same for degradation by hepatocytes and by partially-purified liver plasma membranes. Extracellular breakdown of somatostatin, by proteases released from cells into the incubation medium, represented less than 10% of the cell-associated degradation. Homogenization of hepatocytes resulted in a 10–20-fold increase in the degrading ability of the cells. After incubation of ¹²⁵I-labelled [Tyr¹¹]somatostatin and ¹²⁵I-labelled [Tyr¹]somatostatin with hepatocytes, ¹²⁵I-labelled tyrosine was the major radioactive product identified in the incubation medium. The rate of release of ¹²⁵I-labelled tyrosine from the labelled [Tyr¹] analogue was approximately 11 times greater than from the labelled [Tyr¹¹] analogue. ¹²⁵I-labelled [Tyr¹¹]somatostatin bound to the cells in a non-saturable manner and approx. 70% of the cell-associated radioactivity could be dissociated by dilute acid. The rate of degradation of somatostatin was unchanged by reagents that inhibit the internalisation and lysosomal degradation of polypeptides by cell suspensions but was reduced by reagents that inhibit sulphydryl-dependent proteases. It is proposed that plasma-membrane associated proteolysis, involving both endo- and exopeptidases may represent the predominant degradative pathway of somatostatin in vivo.     

Pharmacological Characterization of Somatostatin Receptors in the Rat Cerebellum During Development

Abstract: Somatostatin (SRIF) receptors (SRIF-Rs) are transiently expressed in a germinative lamina of the rat cerebellum, the external granule cell layer. The appearance of SRIF-Rs coincides with the expression of SRIF-like immunoreactivity in the cerebellum. However, the cellular location of SRIF-Rs does not overlap with the distribution of SRIF-like immunoreactivity, with the latter being restricted to ascending fibers arising from the brainstem, to perikarya within the white matter, and to some Purkinje cells. The characterization of SRIF-Rs in the immature (13–day-old) rat cerebellum was conducted by means of binding experiments in membraneenriched preparations and autoradiography, using two radioligands, [¹²⁵I-Tyr⁰,D-Trp⁸]SRIF-14 ([¹²⁵I-Tyr⁰,d -Trp⁸]S14) and ^I25I-SMS 204–090. The pharmacological profile of cerebellar SRIF-Rs was compared with that of adult cortical SRIF-Rs. Saturation studies performed in 13–day-old rat cerebellum showed that the A'_D values for [¹²⁵I-Tyr⁰,D-Trp⁸]S14 and ¹²⁵I-SMS 204–090 binding were 0.35 ± 0.04 and 0.39 ± 0.01 nM, respectively. The corresponding B_max values were 52.7 ± 4.8 and 49.9 ± 5.3 fmol/mg of protein, a result indicating that radioligands with high specific radioactivity (2,000 Ci/mmol) bind to a single class of high-affinity sites (SSI). Competition studies showed that different D-Trp-sub-stituted analogs displaced [¹²⁵I-Tyr⁰,d -Trp⁸]S14 binding with Hill coefficients >1, a finding indicating the existence of different subtypes of binding sites. When [Tyr⁰,d -Trp⁸]S14 was used as a competitor, two sites were resolved by Scatchard analysis in both 13–day-old cerebellum and adult cerebral cortex. The higher-affinity sites correspond to the SSI subtype identified in saturation experiments, whereas the lower-affinity sites most likely correspond to the SS2 subtype. Ionic supplementation studies showed that divalent cations were required to obtain maximal specific binding on the SSI sites. In particular, Mn²⁺ was the most efficient cation for promoting binding of [¹²⁵I-Tyr⁰,d -Trp⁸]S14. Addition of GTP to the incubation buffer induced a marked reduction of specific binding. The results obtained by membrane binding assays were similar to those obtained by quantitative autoradiography, a result indicating that the microenvironment of SRIF-Rs was preserved in both types of tissue preparations. Receptors expressed in the developing rat cerebellum exhibited the same K_D and similar pharmacological profile as those observed in the adult rat cortex. These results show that SRIF-binding sites transiently expressed in the external granule cell layer of the cerebellum of young rats are indistinguishable from adult rat brain SRIF-Rs. The extremely high density of SRIF-Rs found in the external granule cell layer in 13–day-old rats suggests that SRIF may play a pivotal role in the proliferation and/or differentiation of these germinative cells.     

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-R_pc) and the different melanocortin (MC) receptors. Radioreceptor assays using [¹²⁵I]MCH, [¹²⁵I]α-MSH and [¹²⁵I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC₅₀s of α-MSH and NEI or NGE for [¹²⁵I]MCH displacement from mouse MCH-R_pc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC₅₀s for MCH and NEI or NGE for [¹²⁵I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [¹²⁵I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Bromolactamization: Key Step in the Stereoselective Synthesis of Enantiomerically Pure,cis‐Configured Perhydropyrroloquinoxalines

Compounds based on the pyrroloquinoxaline system can interact with serotonin 5‐HT₃, cannabinoid CB₁, and μ‐opioid receptors. Herein, a chiral pool synthesis of diastereomerically and enantiomerically pure bromolactam (S,R,R,R)‐ 14A is presented. Introduction of the cyclohexenyl ring at the N‐atom of (S)‐proline derivatives 8 or methyl (S)‐pyroglutamate ( 12 ) led to the N‐cyclohexenyl substituted pyrrolidine derivatives 4 and 13 , respectively. All attempts to cyclize the (S)‐proline derivatives 4 with a basic pyrrolidine N‐atom via [3 + 2] cycloaddition, aziridination, or bromolactamization failed. Fast aromatization occurred during treatment of cyclohexenamines under halolactamization conditions. In contrast, reaction of a 1:1 mixture of diastereomeric pyroglutamates (S,R)‐ 13bA and (S,S)‐ 13bB with LiO^tBu and NBS provided the tricyclic bromolactam (S,R,R,R)‐ 14A with high diastereoselectivity from (S,R)‐ 13bA , but did not transform the diastereomer (S,S)‐ 13bB . The different behavior of the diastereomeric pyroglutamates (S,R)‐ 13bA and (S,S)‐ 13bB is explained by different energetically favored conformations. Chirality 26:793–800, 2014. © 2014 Wiley Periodicals, Inc.     

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

One‐Pot Synthesis of New (1,3‐Thiazolo[5,4‐b]pyridin‐2‐yl)benzenediols and Their Antiproliferative Activities against Human Cancer Cell Lines

A one‐pot synthesis of new 4‐(1,3‐thiazolo[5,4‐b]pyridin‐2‐yl)benzene‐1,3‐diols has been described. The compounds were prepared by the reaction of sulfinylbis[(2,4‐dihydroxyphenyl)methanethione] derivatives, with various substituents in the aryl rings, with 2‐chloropyridin‐3‐amines. Their structures were deduced from IR and, ¹H‐ and ¹³C‐NMR spectroscopic, mass spectrometric, and elemental analyses. The antiproliferative properties of some of the products against human cancer cell lines were comparable to those of cisplatin. Structure? activity analysis showed that the presence of hydrophobic substituents in both heterocyclic fused and phenyl rings of the compounds improves their biological effects. Further, an additional OH group in the resorcinol moiety reduced the antiproliferative activity.