gastrins and cholecystokinin-39

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Chromatography, High Pressure Liquid; Gastrins; Humans; Hydrolysis; In Vitro Techniques; Indicators and Reagents; Islets of Langerhans; Kinetics; Male; Molecular Sequence Data; Peptides; Protein Precursors; Rats; Serine Endopeptidases; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfates; Tyrosine; Water

A previous study has suggested the presence of two distinct binding sites for gasrin and cholecystokinin (CCK) in isolated non-parietal cells from rabbit gastric mucosa: a receptor which binds CCK-8 and CCK-39 with a high affinity and a receptor which binds gastrin and CCK-8 with the same high affinity and CCK-39 with a lower affinity. To characterize these receptors, their ability to induce phosphoinositide breakdown was investigated. Gastrin (HG-17), CCK-39 and CCK-8 induced [3H]-inositol phosphate ([3H]InsP) accumulation from [3H]inositol prelabelled cells with a high potency (EC50: 0.3-2.7 nM) but CCK-8 exhibited a higher efficacy than HG-17 or CCK-39. HG-17, CCK-8 and CCK-39 induced a rapid accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2) and [3H]inositol trisphosphate ([3H]InsP3) but CCK-8 caused a two times higher accumulation than HG-17 or CCK-39. Histamine- and somatostatin-containing cells appeared to be located in this non-parietal cells population. HG-17, CCK-8 and CCK-39 dose-dependently induced histamine release with the following order of potency: HG-17 = CCK-8 (EC50 approximately 0.2 nM) greater than CCK-39 (EC50 approximately 4 nM). In addition, HG-17 exhibited the highest efficacy. HG-17, CCK-8 and CCK-39 enhanced somatostatin-like immunoreactivity (SLI) release with the following order of potency: CCK-8 (EC50 approximately 0.1 nM) = CCK-39 greater than HG-17 (EC50 approximately 10 nM); CCK-8 and CCK-39 exhibited the highest efficacy. These results led us to the following conclusions: (i) existence of a "gastrin-type" and of a "CCK-type" receptor mediating phosphoinositide breakdown in these gastric non-parietal cells. CCK-8 interacts with both receptor-types with the same affinity; (ii) the release of histamine from histamine-containing cells could be induced following "gastrin-type" receptors activation; (iii) somatostatin release from D-cells present in this non-parietal cells population could be induced following "CCK-type" receptors activation.

Topics: Animals; Cholecystokinin; Gastric Mucosa; Gastrins; Histamine Release; Inositol Phosphates; Rabbits; Receptors, Cholecystokinin; Sincalide; Somatostatin; Tritium

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39-enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L-365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway.

Topics: Animals; Benzodiazepinones; Cholecystokinin; Devazepide; Gastric Mucosa; Gastrins; Histamine Release; Inositol Phosphates; Iodine Radioisotopes; Phenylurea Compounds; Rabbits; Receptors, Cholecystokinin; Somatostatin

The objective of the present study was to measure plasma levels of cholecystokinin (CCK-8 and CCK-33,39) as well as of gastrin during the menstrual cycle and pregnancy. Cholecystokinin and gastrin levels were measured by radioimmunoassay. Before being assayed for cholecystokinin, plasma samples were submitted to HPLC which allowed separation of gastrin and cholecystokinin as well as between CCK-8 and CCK-33,39. Fasting CCK levels were 5.2 +/- 0.6 and 7.1 +/- 0.9 pM during the follicular and luteal phases of the menstrual cycle, respectively. The difference was significant (P less than 0.05). CCK levels were 8.7 +/- 1.2, 10.1 +/- 1.6 and 10.4 +/- 1.2 pM during the first, second and third trimester, respectively. CCK levels during pregnancy were significantly higher than during the menstrual cycle. The ratio between CCK-33,39 and CCK-8 appeared to increase during pregnancy. Gastrin levels remained unchanged during the menstrual cycle and pregnancy. The role of the high levels of cholecystokinin may be to stimulate the exocrine and endocrine pancreatic function during pregnancy. Furthermore, since cholecystokinin inhibits gastric emptying, it may play a role in the sickness of early pregnancy.

Topics: Cholecystokinin; Chromatography, High Pressure Liquid; Female; Gastrins; Humans; Luteal Phase; Menstrual Cycle; Pregnancy; Pregnancy Trimester, First; Pregnancy Trimester, Second; Pregnancy Trimester, Third; Radioimmunoassay

A new specific method for determination of cholecystokinin, CCK-8, and CCK-33, 39 in rat plasma is described. Plasma CCK radioimmunoassay (RIA) is difficult, because of cross-reactivity with gastrin. In the rat, problems because of difficulties in separating gastrin from CCK by high performance liquid chromatography (HPLC) exist. These were solved by enzyme digestion of gastrin before HPLC separation of molecular variants of CCK from gastrin fragments. Cholecystokinin immunoreactive forms in the HPLC fractions were determined by an antibody, which recognises the carboxyl terminus of CCK and gastrin. Fasting concentrations of small (CCK-8) and large (CCK-33, 39) molecular forms of CCK averaged 1.9 (0.3) pM and were raised to 13.4 (3.8) pM in rats fed ad libitum. Cholecystokinin in lactating rats rose two-fold after suckling, compared with 2.8 fold in response to feeding. The basal ratio between CCK-8 and CCK-33, 39 was approximately 1:1, but increased in favour of CCK-8 after feeding and in response to suckling. Gastrin like immunoreactivity measured in unextracted plasma was found to rise after feeding, but was unchanged in response to suckling.

Topics: Animals; Cholecystokinin; Chromatography, High Pressure Liquid; Eating; Female; Gastric Mucosa; Gastrins; Intestinal Mucosa; Lactation; Methods; Pregnancy; Protein Precursors; Radioimmunoassay; Rats; Rats, Inbred Strains; Serine Endopeptidases; Sincalide

Molecular heterogeneity between cholecystokinin (CCK) present in humans and that present in the pig has been proposed. We recently demonstrated that CCK-8 exists in humans in form identical to the porcine peptide. The aims of this work were to evaluate the presence in human plasma of CCK forms larger than CCK-8 and to compare them with the well-characterized porcine forms. Antiserum (no. 4899) was raised in a New Zealand white rabbit immunized with porcine CCK-33 that had specificity for the 7 to 21 region of that peptide and that recognized molecules present in human plasma. To characterize these, postprandial human plasma was applied to an immunoaffinity column generated with this antiserum. Adsorbed peptides were eluted, concentrated on an octadecylsilane cartridge, separated by reversed-phase HPLC and gel filtration chromatography, and screened by cross-reacting and specific CCK and gastrin radioimmunoassays and CCK bioassay by quantification of amylase release by rat pancreatic acini. Two peptides were consistently identified that possessed CCK-like but not gastrin-specific immunoreactivity and CCK-like biological activity. These appeared to be similar in size to CCK-33 and intermediate in size between CCK-33 and CCK-8. Though analogous to porcine CCK based on antibody cross-reactivity and biological activity, the human peptides were heterogeneous from the porcine peptides based on differing chromatographic behavior.

Topics: Amylases; Animals; Antibody Specificity; Cholecystokinin; Chromatography, Gel; Chromatography, High Pressure Liquid; Eating; Female; Gastrins; Humans; Male; Molecular Weight; Peptide Fragments; Radioimmunoassay; Rats; Receptors, Cholecystokinin; Sincalide; Swine

Antisera raised against synthetic sulphated cholecystokinin (CCK 26-33) (n = 4) and against 30% pure porcine CCK (n = 11) were characterized by the use of different labelled CCK peptides. CCK 39 and CCK 26-33 were coupled to 125I by the chloramine-T method, while CCK 33 was conjugated to 125I-labelled hydroxyphenylpropionic acid-succinimide ester (Bolton-Hunter reagent). Antisera raised against CCK 26-33 bound to 125I-CCK 26-33 only. Of the antisera raised against 30% pure CCK, 2 bound to all 3 labels, 4 to 125I-BH-CCK 33 and 125I-CCK 26-33, 3 to 125I-CCK 39 and 125I-BH-CCK 33, 1 to 125I-CCK 26-33 only and 1 to 125I-BH-CCK 33 only. The antibodies reacting with 125I-CCK 26-33 bound also to 125I-gastrin 1-17. Different CCK labels bound to different binding sites in the same antiserum (antibody heterogeneity). The pattern of reactivity of the antiserum to CCK peptides was dependent on the type of label used. With these different labels, antibodies specific for CCK 39, for CCK 33 and CCK 39, for sulphated forms of CCK, and for all CCK peptides and gastrin could be detected. It is concluded that antisera raised against CCK should be characterized by use of different labelled CCK materials.

Topics: Animals; Binding Sites, Antibody; Cholecystokinin; Gastrins; Guinea Pigs; Humans; Immune Sera; Iodine Radioisotopes; Isotope Labeling; Peptide Fragments; Rabbits; Sincalide; Succinimides; Swine

Histamine accumulated in the ligated vagus nerve of the rat, both above and below the ligature; maximum accumulation was after 4 h. The finding is suggestive of axonal flow. Further evidence for histamine in peripheral nerves was obtained in experiments showing that the guinea-pig gut wall could be labelled with [3H]histamine. The experiments were carried out with isolated strips of stomach wall and taenia coli. Electrical stimulation released [3H]histamine from these specimens. The release could be blocked by Ca2+-free medium or by tetrodotoxin. The release was unaffected by vagal denervation or chemical sympathectomy (6-hydroxydopamine) but prevented by reserpinization. Gastrin-17 and cholecystokinin-39 released radioactivity by a tetrodotoxin-sensitive mechanism. The possible existence of a gastrin/cholecystokinin-sensitive neuronal pool of histamine in the gut wall offers a new perspective on the postulated role of histamine as a physiological stimulant of gastric acid secretion and might explain why H2-receptor antagonists block gastrin-stimulated acid secretion.

Topics: Animals; Cholecystokinin; Colon; Electric Stimulation; Gastrins; Guinea Pigs; Histamine; Neurons; Peptide Fragments; Stomach; Synaptic Transmission; Vagus Nerve