Comparative studies on cholecystokinin receptors

• Main Author: Gaudreau, Pierrette
• Other Authors: [non identifié]
• Language: English
• Published: Université de Sherbrooke 1986
• Online Access: http://hdl.handle.net/11143/11736

1. A scheme of synthesis was developped for cholecystokinin (CCK 26-33, using solid-phase methodology and successfully applied to the synthesis of its C- and N-terminal fragments. 2. Using CCK 30-33 as model, it was found that deprotection of the ?-phenacyl ester, with a 1 M solution of sodium thiophenoxide in DMF, leads to the formation of an aminosuccylnyl peptide, prior to ammonolysis. 3. Selenophenol reagent successfully removes the ?-phenacyl ester on protected CCK 32-33 and on protected CCK 30-33 polymer prior to ammonolytic cleavage of peptides from the resin. 4. Treatment of Boc-Asp(?-OPac)-Tyr(0-2,4-Dnp)-Met-Gly-Trp(Nin-For)-Met-Asp(?-OPac)-Phe-polymer with a 1 M solution of selenophenol in DMF, leads to irreversible rearrangement of the 0-2,4-dinitrophenyl ether. 5. Undesirable side-reactions can be avoided by sequential deprotections and cleavage. The 0-2,4-dinitrophenyl ether is removed by thiolysis following by selenolysis of the ?-phenacyl esters. Cleavage of the peptide from the resin was brought about by ammonolysis in a 30% solution of ammonia in methanol. 6. The best approach found to sulfate the tyrosyl residue of CCK 26-33 and its fragments is the use of a 1.5 molar excess sulfur trioxide-pyridine complex solution in pyridine, on N?-protected peptides, followed removal of the tert-butyloxycarbonyl group with anhydrous TFA containing 2-methylindole as scavenger. 7. Purification of CCK 26-33 and its fragments by countercurrent distribution and high-pressure liquid chromatography gives homogeneous peptides by tlc, analytical HPLC and amino acid analysis. 8. Overall yields are 54-72%, being respectively 85-100% after ammonolytic cleavage, 75-85% after preparative HPLC on N?-protected peptides and 85% after HPLC on deprotected peptides. 9. Biochemical characterizations of brain CCK receptors were done with [3H] pentagastrin, N? [125I-desaminotyrosyl] CCK 26-33 and N? [125I-desaminotyrosyl] CCK 1-33 in rat striatum sections. The results indicate that the three radioligands bind to a finite number of sites with high affinity. Saturation curves, done with [3H] pentagastrin and N? [125I-desaminotyrosyl] CCK 26-33 indicate that both ligands bind to the same population of binding sites. Inhibition studies with the three radioligands, reveal similar ligand selectivity patterns (CCK 26-33 ? CCK 26-33NS > CCK 27-33 > CCK 27-33NS>CCK 28-33 ? CCK 29-33 > CCK 30-33>>>CCK 26-32 ?CCK 26-31). Cations and guanyl nucleotides influence specific [3H] pentagastrin, N? [125I-desaminotyrosyl] CCK 26-33 and N[?125I-desaminotyrosyl] CCK 1-33 binding to rat striatum sections. Putative CCK receptor antagonists proglumide, benzotript and N?-benzyloxycarbonyl-L-tryptophan inhibit specific [3H] pentagastrin, N?[125I-desaminotyrosyl] CCK 26-33 and N?[125I-desaminotyrosyl] CCK 1-33 binding at high concentrations (mmolar range). The binding sites labeled by [3H] pentagastrin, N?[125l-desaminotyrosyl] CCK 26-33 and N? [125I-desaminotyrosyl] CCK 1-33 possess the biochemical characteristics of a sole CCK receptor. 10. Comparative autoradiographic distribution of [3H] pentagastrin N?[125I-desaminotyrosyl] CCK 26-33 and N? [125I-desaminotyrosyl] CCK 1-33 show similar binding patterns in rat brain. The highest densities of receptor binding sites are seen in cerebral cortice, olfactory bulb, olfactory tubercle, nucleus accumbens, caudate-putamen, area CA4 of the Ammon's horn, presubiculum, parasubiculum, amygdala (amygdalo-hippocampal area, postero-medial cortical nucleus), thalamus (reticular, ventral lateral geniculate, paraventricular and posterior paraventricular nuclei), hypothalamus (supraoptic and ventromedial nuclei), parabigemminal nucleus and nucleus of the solitary tract. These results do not suggest the presence of CCK receptor subtypes in rat brain. 11. Studies on cellular localization of CCK receptors in relation with the nigro-striatal and mesolimbic dopaminergic pathways were done in rat. 6-hydroxydopamine lesions do not induce significant variations of 3H pentagastrin binding in the caudate-putamen, nucleus accumbens and substantia nigra. Ibotenic acid lesions decrease [3H] pentagastrin binding densities in the caudate-putamen and substantia nigra, indicating that the bulk of CCK receptors in these two area of the brain are not localized on dopaminergic neurons. 12. No significant changes in specific N? [125I-desaminotyrosyl] CCK 26-33 binding are seen in the rat cingulate cortex, frontal medial cortex, olfactory tubercle, nucleus accumbens and striatum after chronic haloperidol treatment. 13. A structure-activity study done with C- and N-terminal fragments of CCK 26-33 in guinea pig gallbladder and ileum shows the importance of the sulfate ester of the tyrosyl residue for maximal activity. The general order of potency in both tissues is CCK 26-33 CCK 1-33 > CCK 27-33 >>> CCK 26-33NS > CCK 27-33NS ? CCK 26-32 > CCK 28-33 > CCK 29-33 > CCK 30-33 > CCK 26-31. None of the CCK fragments have antagonistic properties. These results suggest that brain and peripheral CCK receptors binding proteins are différent. 14. Behavioral studies with C- and N-terminal fragments of CCK 26-33, in mice, indicate that CCK 27-33 is the minimal peptide sequence inducing the syndrome of reduced exploratory behavior. None of the inactive fragments antagonize the behavioral effects of CCK 26-33. The results might suggest that the behavioral effect of CCK 26-33 is mediated by a yet uncharacterized peripheral CCK receptor binding site. 15. Comparative autoradiographic distributions of N? [125I-desaminotyrosyl] CCK 26-33 in rat, guinea pig, squirrel monkey and normal human brain reveal that highest concentrations of binding sites are found in cerebral cortex, caudate nucleus, nucleus accumbens and olfactory tubercle.