Development of new potent agonists able to interact with two postulated subsites of the cholecystokinin CCK-B receptor

Summary Since the biochemical and pharmacological profile of BC 197 and BC 264, two CCK₈-derived agonists with high specificity for CCK-B receptors, suggests their potential interaction with two CCK-B receptor subsites, it appeared essential to design new series of compounds that would be able to discriminate between these two subsites. As CCK₄ is the shortest fragment of CCK which interacts selectively with CCK-B receptors, compounds derived from the C-terminal tetrapeptide domain of BC 264, Boc-Trp-(NMe)Nle-Asp-Phe-NH₂, and of the cyclic compound BC 197, were prepared. While RB 360 (N-(cycloamido)-α-Me(R)Trp-[(2S)-2-amino-9-((cycloamido)carbonyl)nonanoyl]-Asp-Phe-NH₂), like BC 197, has a CCK-B₁ profile with anxiogenic-like effects in the elevated plus-maze test, RB 400 (HOOC-CH₂-CO-Trp-(NMe)Nle-Asp-Phe-NH₂), like BC 264, seems to be a specific CCK-B₂ agonist, able to increase attention and/or memory processes in the Y-maze test.     

Conformational analysis of cholecystokinin fragments CCK4, CCK5, and CCK6 by 1H-NMR spectroscopy and fluorescence-transfer measurements

The confortmational behavior of the cholecystokinin-related fragments CCK₄, CCK₅, and CCK₆ as determined by ¹H-nmr spectroscopy in DMSO-d₆ and water and fluorescence-transfer measurements in aqueous medium are greatly dependent on the ionization states of these peptides. Under netral conditions, the backbones of CCK₅ and CCK₆ preferentially adopted folded forms with a β-turn including the four residues Gly-Trp-Met-Asp, probably stabilized by a hydrogen bond between the CO of Gly and the NH of Phe. In these structures, possible induced by an ionic interaction between the carboxylic group of Asp³² and the NH group of the N-terminal amino acid, the lateral chains of the various residues are quite distant from each other (15–16 Å). Under acidic conditions, extended structures without interactions between side chains predominate for CCK₅ and CCK₆, while for CCK₄, a conformational change drawing the Trp and Phe side chains in close proximity was shown by fluorescence. The conformations observed in aqueous medium at physiological pH are discussed in relation to the biological activity of these peptides.     

The Synergistic Roles of Cholecystokinin B and Dopamine D5 Receptors on the Regulation of Renal Sodium Excretion

Renal dopamine D₁-like receptors (D₁R and D₅R) and the gastrin receptor (CCK_BR) are involved in the maintenance of sodium homeostasis. The D₁R has been found to interact synergistically with CCK_BR in renal proximal tubule (RPT) cells to promote natriuresis and diuresis. D₅R, which has a higher affinity for dopamine than D₁R, has some constitutive activity. Hence, we sought to investigate the interaction between D₅R and CCK_BR in the regulation of renal sodium excretion. In present study, we found D₅R and CCK_BR increase each other’s expression in a concentration- and time-dependent manner in the HK-2 cell, the specificity of which was verified in HEK293 cells heterologously expressing both human D₅R and CCK_BR and in RPT cells from a male normotensive human. The specificity of D₅R in the D₅R and CCK_BR interaction was verified further using a selective D₅R antagonist, LE-PM436. Also, D₅R and CCK_BR colocalize and co-immunoprecipitate in BALB/c mouse RPTs and human RPT cells. CCK_BR protein expression in plasma membrane-enriched fractions of renal cortex (PMFs) is greater in D₅R^-/- mice than D₅R^+/+ littermates and D₅R protein expression in PMFs is also greater in CCK_BR^-/- mice than CCK_BR^+/+ littermates. High salt diet, relative to normal salt diet, increased the expression of CCK_BR and D₅R proteins in PMFs. Disruption of CCK_BR in mice caused hypertension and decreased sodium excretion. The natriuresis in salt-loaded BALB/c mice was decreased by YF476, a CCK_BR antagonist and {"type":"entrez-protein","attrs":{"text":"Sch23390","term_id":"1052733334"}}Sch23390, a D₁R/D₅R antagonist. Furthermore, the natriuresis caused by gastrin was blocked by {"type":"entrez-protein","attrs":{"text":"Sch23390","term_id":"1052733334"}}Sch23390 while the natriuresis caused by fenoldopam, a D₁R/D₅R agonist, was blocked by YF476. Taken together, our findings indicate that CCK_BR and D₅R synergistically interact in the kidney, which may contribute to the maintenance of normal sodium balance following an increase in sodium intake.     

Localization of the murine cholecystokinin A and B receptor genes

We have determined the chromosomal locations of the two cholecystokinin (CCK) receptor genes in the mouse. Genetic localization utilized an interspecific backcross panel formed from the cross (C57BL/6J x Mus spretus) F₁ x Mus spretus. Genomic DNAs from 94 individuals in the backcross were analyzed by Southern hybridization with rat CCK_A and CCK_B receptor cDNA probes. Unique map positions were determined by haplotype analysis with 650 previously mapped loci in the mouse backcross. The CCK_A receptor gene (Cckar) mapped to mouse Chromosome (Chr) 5, in tight linkage with the DNA marker D5Bir8. The CCK_B receptor gene (Cckbr) mapped to mouse Chr 7, tightly linked to the -hemoglobin locus (Hbb). This localization places Cckbr in the same region as the mouse obesity mutation tubby (tub), which also maps near Hbb (2.4±1.4 cM). Since CCK can function as a satiety factor when administered to rodents, localization of Cckbr near the tub mutation identifies this receptor as a possible candidate gene for this obesity mutation.     

Effects of high concentrations of secretagogues on the morphology and secretory activity of the pancreas: A role for microfilaments

Summary Cholecystokinin (CCK) and acetylcholine, at concentrations greater than those required for maximal pancreatic enzyme secretion, elicit a submaximal secretory response. The mechanism for this secretagogue-induced unresponsiveness is unknown. Using isolated pancreatic acini of the mouse, we now find that high concentrations of secretagogues also induce a profound alteration in acinar morphology, characterized by the formation of spherical protrusions on the basal surface of the cells. Since both the determination of cell shape and exocytosis may involve calcium and contractile proteins, we used a calcium-free medium and cytochalasin B (CB) to evaluate the importance of a contractile mechanism in the secretory and morphological effects of high concentrations of CCK-octapeptide (CCK₈). Incubation in a calcium-free medium partially blocked CCK-induced unresponsiveness, but brought about dissociation of the acini. CB at a concentration of 3 g/ml caused the disappearance of apical microfilaments and, most strikingly, completely prevented the morphological alteration induced by CCK₈. Furthermore, CB converted the biphasic dose-response curve for CCK₈-induced amylase release to a monophasic shape, such that the amylase release stimulated by a high concentration of CCK₈ (10 nM) was augmented. It is concluded, therefore, that a contractile process involving microfilaments may mediate secretagogue-induced unresponsiveness in pancreatic acinar cells.     

Synthesis of tantalum and niobium complexes that contain the diamidoamine ligand, [(3,4,5-F3C6H2NCH2CH2)2NMe], and the triamidoamine ligand, [(3,5-Cl2C6H3NCH2CH2)3N]

Several niobium and tantalum compounds were prepared that contain either the diamidoamine ligand, [(3,4,5-F₃C₆H₂NCH₂CH₂)₂NMe]²⁻ ([F₃N₂NMe]²⁻), or the triamidoamine ligand, [(3,5-Cl₂C₆H₃NCH₂CH₂)₃N]³⁻ ([Cl₂N₂NMe]³⁻). The former include [F₃N₂NMe]TaCl₃, [F₃N₂NMe]NbCl₃, [F₃N₂NMe]TaMe₃, [F₃N₂NMe]NbMe₃, [(F₃N₂NMe)TaMe₂][MeB(C₆F₅)₃], [F₃N₂NMe]Ta(CHSiMe₃)(CH₂SiMe₃), [F₃N₂NMe]Ta(CH₂-t-Bu)Cl₂, [F₃N₂NMe]Ta(CH-t-Bu)(CH₃), and [F₃N₂NMe]Ta(η²-C₂H₄)(CH₂CH₃). The latter include [Cl₂N₂NMe]TaCl₂, [Cl₂N₂NMe]TaMe₂, [Cl₂N₂NMe]Ta(η²-C₂H₄), and [Cl₂N₂NMe]Ta(η²-C₂H₂).X-ray diffraction studies were carried out on [F₃N₂NMe]Ta(CHSiMe₃)(CH₂SiMe₃), [F₃N₂NMe]Ta(η²-C₂H₄)(CH₂CH₃), and [Cl₂N₂NMe]TaMe_2..     

Growth and Characterization of Epitaxial Insulating CaF2 Layers on Silicon by MBE

In the course of the present work CaF₂ epitaxialfilms were grown on Si(111) substrates by means ofMBE. The Si substrates were chemically cleaned priorto insertion into the system. The final volatile oxidewas desorbed in situ by heating to 850. CaF₂ was evaporated from aKnudsen-type cell by use of a graphite crucible, whilethe growth temperature was held at 650 .RHEED (Reflection High Energy Electron Diffraction)has been used to monitor the film growth in situand to study the epitaxial quality. Also we have usedthe MeV He⁺ RBS channeling technique to look atdefects and to measure strain in the CaF₂ layer.Usually good crystallographic properties are achievedunder optimum growth conditions, with values of_min < 5%. Electrical properties aremeasured by use of a special MIS structure.     

Bis(cyclopentadienyl) zirconium(IV) amides as possible precursors for low pressure CVD and plasma-enhanced ALD

Low pressure chemical vapour deposition (LPCVD) of [ZrCp₂(NMe₂)₂] (1), [ZrCp₂(η²-MeNCH₂CH₂NMe)] (2), [ZrCp′₂(NMe₂)₂] (3) and [ZrCp′₂(NEt₂)₂] (4) (Cp = η⁵-cyclopentadienyl, Cp′ = η⁵-monomethylcyclopentadienyl), onto glass substrates at 600 °C, afforded highly reflective and adhesive films of zirconium carbide and amorphous carbon. Powder XRD indicated that the films were largely amorphous, although small, broad peaks accounting for ZrC and ZrO₂ were present, suggesting that the remaining carbon was due to amorphous deposits from the cyclopentadienyl ligands. SEM images showed an island-growth mechanism with distinct crevices between the concentric nodules. Plasma-enhanced atomic layer deposition (PEALD) of compounds 1 and 2 showed that the precursors were not sufficiently stable or volatile to give a good rate of film growth.     

X-ray structural characterization of some sterically bulky N-donor and N-alkyl Grignard reagents

[MgBr₂(thf)₃] (1) and [FisoMg(thf)Cl]₂ (2), (Fiso⁻ = [ArNC(H)NAr]⁻, ), [2-PyC(SiMe₃)₂Mg(thf)Cl]₂ (3), [2-PyC(SiMe₃)₂Mg(thf)Br]₂ (4), and [(2-PyC(SiMe₃)₂Mg(thf))₂(OEt)Cl] · Et₂O (5). (2-Py(SiMe₃)₂CH = 2-{bis-(trimethylsilyl)methyl}2-pyridine) were isolated as by-products from reactions involving organometallic species and magnesium or diethylmagnesium. All compounds were characterized by single crystal X-ray crystallography. Compounds (1) and (5) have trigonal bipyramidal magnesium centres, while compounds (2)-(4) have square pyramidal structures. Compound (1) is monomeric, while compounds (2)-(5) are dinuclear with magnesium centres bridged by two halides for (2)-(4), and a chloride and an ethoxy ligand in (5).     

Transition metal complexes of 2-amino-3,5-dihalopyridines: Synthesis, structures and magnetic properties of Cu(3,5-diCAP)2X2 and Cu(3,5-diBAP)2X2

A family of bis(2-amino-3,5-dihalopyridine)dihalocopper(II) compounds has been synthesized, including (3,5-diCAP)₂CuCl₂ (1), (3,5-diCAP)₂CuBr₂ (2), (3,5-diBAP)₂CuCl₂ (3), and (3,5-diBAP)₂CuBr₂ (4) [3,5-diCAP = 2-amino-3,5-dichloropyridine; 3,5-diBAP = 2-amino-3,5-dibromopyridine]. These complexes have been analyzed through single crystal X-ray diffraction and temperature dependant magnetic susceptibility. The compounds are all isostructural, forming bi-bridged chains with long Cu-X?Cu bridges in the crystal lattice. The two copper chloride compounds (1 and 3) exhibit weak antiferromagnetic interactions along these chains.     

Syntheses, crystal structure, spectroscopic characterization and antifungal activity of new N-R-sulfonyldithiocarbimate metal complexes

Five new compounds with the general formula of (Bu₄N)₂[M(RSO₂NCS₂)₂], where Bu₄N = tetrabutylammonium cation, (M = Ni, R = 4-FC₆H₄) (1), (M = Zn, R = 4-FC₆H₄, 4-ClC₆H₄, 4-BrC₆H₄, 4-IC₆H₄), (2), (3), (4) and (5), respectively, were obtained by the reaction of the appropriate potassium N-R-sulfonyldithiocarbimate (RSO₂NCS₂K₂) with nickel(II) chloride hexahydrate or zinc(II) acetate dihydrate in metanol:water 1:1. The elemental analyses and the IR data are consistent with the formation of the expected bis(dithiocarbimato)metal(II) complexes. The ¹H and ¹³C NMR spectra showed the signals for the tetrabutylammonium cation and the dithiocarbimate moieties. The compounds 1, 2 and 5 were also characterized by X-ray diffraction techniques. The nickel(II) is coordinated by two N-4-fluorophenylsulphonyldithiocarbimato(2-) ligands forming a planar coordination. The zinc(II) exhibits distorted tetrahedral configuration in compounds 2 and 5 due to the chelation effect of two sulfur atoms of the N-R-sulfonyldithiocarbimate ligands. The antifungal activities of the compounds were tested in vitro against Colletotrichum gloeosporioides, an important fungus that causes the plant disease known as anthracnose in fruit trees. All the complexes were active.