sincalide and cholecystokinin-pentapeptide

Nitric oxide (NO) plays a major role in cardiopulmonary regulation as illustrated by the alterations of the NO system described in cardiopulmonary illnesses. Recent studies have found an association between panic disorder and cardiovascular death and illness, as well as pulmonary diseases. Our objective was to investigate whether pulmonary or systemic NO production was altered during induced panic attacks (PAs). We used a double-blind placebo-controlled crossover design with randomization of the order of an injection of placebo and pentagastrin, a cholecystokinin-B receptor agonist that induces PAs in healthy volunteers (HVs). A total of 17 HVs experienced a PA after pentagastrin challenge. Exhaled NO and NO metabolites were measured by chemiluminescence. During pentagastrin-induced PAs, HVs displayed significant decreases in plateau concentrations of NO exhaled, which were associated with proportional increases in minute ventilation. There were no significant changes in pulmonary or systemic NO production. These results suggest that the decrease in exhaled NO concentration observed during pentagastrin-induced PAs is related to the associated hyperventilation, rather than to any change in lung NO production. This study is the first to evaluate changes in NO measurements during acute anxiety.

Topics: Adult; Cross-Over Studies; Double-Blind Method; Fasting; Female; Food, Formulated; Humans; Lung; Male; Nitric Oxide; Panic; Panic Disorder; Peptide Fragments; Respiration; Sincalide; Time Factors

This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

Topics: Acetates; Bacillus; Catalysis; Chromatography, High Pressure Liquid; Enzymes, Immobilized; Esters; Glycine; Hydrolysis; Papain; Penicillin Amidase; Penicillin G; Pentagastrin; Peptide Fragments; Peptides; Sincalide; Solvents; Thermolysin

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small peptides. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis (CLN2). This disease is characterized by the accumulation of proteinaceous and autofluorescent material within the lysosomes of neurons, which undergo massive cell death during the course of the disease. The absence of TPP-I may result in the lysosomal accumulation of small peptides and proteins, which eventually compromises lysosomal functions critical to the survival of neurons. To investigate the metabolism of small peptides, we have studied the degradation of cholecystokinin-(29-33)-amide (GWMDF-NH2; cholecystokinin C-terminal pentapeptide) by lysosomal fractions isolated from mouse brain and several other tissues. GWMDF-NH2 is cleaved at only one peptide bond by brain lysosomes, to produce GWM and DF-NH2. Inhibitor studies demonstrate that this reaction is catalysed by TPP-I. In contrast, lysosomal fractions from other mouse tissues additionally cleave a second peptide bond to produce GW and MDF-NH2. Inhibitor studies indicate that this reaction is catalysed by dipeptidyl peptidase-I (DPP-I; cathepsin C). Inhibitors of TPP-I are sufficient to completely block the degradation of GWMDF-NH2 by brain, but inhibitors of both TPP-I and DPP-I are required to completely inhibit the degradation of GWMDF-NH2 by other mouse tissues. Enzyme assays confirm the low activity of DPP-I in brain. An unrelated neuropeptide, neuromedin B, is degraded by a pathway that is partially dependent on TPP-I. These results indicate that TPP-I is required for the partial or complete digestion of certain neuropeptides by brain lysosomes. In the absence of TPP-I, neuropeptides or their degradation products will accumulate in brain lysosomes and may contribute to the pathogenesis of CLN2. Other tissues are spared because they express another peptidase, DPP-I, which has extensive activity on peptides and can compensate for the loss of TPP-I.

Topics: Amino Acid Sequence; Aminopeptidases; Animals; Brain; Cells, Cultured; Child; Cholecystokinin; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Endopeptidases; Fibroblasts; Humans; Infant; Lysosomes; Mice; Neuronal Ceroid-Lipofuscinoses; Peptide Fragments; Peptides; Protease Inhibitors; Reference Values; Serine Proteases; Sincalide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tripeptidyl-Peptidase 1

A "free choice" two-bottle drinking test paradigm was implemented in naive adult male Wistar rats, resulting in a clear identification of rats drinking mainly water (water-preferring, WP rats) and rats spontaneously drinking also a consistent amount of a solution of cocaine (0.5 mg/ml water, cocaine-drinking, CD rats) or ethanol 10% v/v (ethanol-drinking, ED rats). Low, selective doses (5 micrograms/kg) of the specific cholecystokinin (CCK)-A receptor antagonist L-364,718 largely reduced the intake of ethanol 10% of ED rats only. In contrast, low, selective doses of GV-150013 (5 micrograms/kg) reduced significantly the consumption of cocaine of CD rats only. These results indicate that the CCK-A or B receptors are selectively involved in the modulation of alcohol or cocaine intake, respectively, and suggest an involvement of the CCKergic system in the drug-seeking behavior. WP rats and CD rats were then prepared for ex vivo electro-neurochemical analysis by means of differential pulse voltammetry (DPV) with micro-biosensors to monitor catechol, 5-hydroxyindole and peptidergic oxidation signals in the nucleus accumbens (nAcc). In this area, the peptidergic signal appeared to be related to the oxidation of endogenous CCK, which basal levels resulted higher in ED and CD rats than WP rats. Thus, the hypothesis that the endogenous tone of the CCK system is higher in the ED and CD rats than in the WP rats is proposed, and is supported by the observation that treatment with CCK-5 (CCK receptor agonist) selectively induced the WP rats to drink alcohol or cocaine. The selective effect of the CCK-antagonists on reducing the drug intake of ED or CD rats further supports this view, as it suggests that CCK antagonists may modify the individual sensitivity towards drugs of abuse set by the stimulating effect of high endogenous CCKergic tone over CCK-B or CCK-A receptors in spontaneous ED or CD rats, respectively. Therefore, the present data indicate that: i) Free-choice models may reveal the presence of individual sensitivity to alcohol or cocaine in naive rats; ii) the dopaminergic system is involved within the reward state, while peptidergic (CCKergic) activities modulate the drug-seeking state (craving state); iii) the CCK system could be a new target in the study of the drug dependency phenomenon. In particular, the data imply a CCK-A receptor mechanism in the regulation of individual sensitivity towards ethanol and a CCK-B receptor mechanism in the regulation of i

Topics: Adamantane; Alcohol Drinking; Alcoholism; Animals; Benzodiazepinones; Cocaine; Cocaine-Related Disorders; Drinking; Male; Peptide Fragments; Phenylurea Compounds; Rats; Rats, Wistar; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Receptors, Cholecystokinin; Sincalide

In an attempt to examine the structure-activity relationship of the cholecystokinin (CCK) peptide, we examined the structural motif of CCK truncated peptides responsible for rat pancreatic acinar amylase secretion and signal transduction. CCK-6 (Met28-Gly29-Trp30-Met31-Asp32-Phe33 -NH2), CCK-5 [CCK-(29-33)], and CCK-4 [CCK-(30-33)] caused monophasic amylase secretion with EC50 values of 3, 20, and 30 nM, respectively. CCK-7 [Tyr(SO3H)27 plus CCK-(28-33)] evoked biphasic secretion with an EC50 of 0.7 pM, whereas CCK-3 [CCK-(31-33)] had no effect, suggesting the importance of Tyr(SO3H)27 and Trp30. Whereas CCK-7 [Tyr(SO3H)27/ Met28] evoked biphasic amylase secretion, CCK-OPE [Tyr(SO3H)27/Nle28] and CCK-6 (Met28) caused monophasic secretion. Thus Tyr(SO3H)27/Met28 appears to be required for biphasic secretion. CCK-8-OH and CCK-5-OH did not cause amylase secretion and Ca2+ spiking, suggesting the importance of Phe33 amidation. Similar to CCK-8, CCK-6 and CCK-4 caused Ca2+ oscillations at low doses and large Ca2+ transients at high doses. In contrast, similar to JMV-180 and CCK-OPE, CCK-5 elicited Ca2+ oscillations at all concentrations. Phospholipase C (PLC) inhibitor inhibited amylase secretion induced by high doses of CCK-6 and CCK-4 but not by CCK-5. Protein tyrosine kinase (PTK) inhibitor only inhibited the action of high doses of CCK-4. Neither PLC inhibitor nor PTK inhibitor affected amylase secretion evoked by low doses of CCK-6, CCK-5, and CCK-4. In contrast to its actions on JMV-180 and CCK-OPE, phospholipase A2 (PLA2) inhibitor had no effect on the action evoked by all CCK short peptides. CCK-6, CCK-5, and CCK-4 caused a 2.1- to 3.2-fold increase in intracellular inositol 1,4,5-trisphosphate levels over basal. None of these CCK short peptides increased intracellular arachidonic acid levels. CCK-6 and CCK-5 did not stimulate PTK activity, whereas CCK-4 evoked a 3.2- to 5.3-fold increase over basal. We conclude that Tyr(SO3H)27, Trp30, and Phe33-CONH2 of the CCK peptide are key amino acids in evoking amylase secretion. At low doses, CCK-6, CCK-5, and CCK-4 utilize some yet to be identified pathways to evoke Ca2+ oscillations and amylase secretion. At high doses, CCK-6 and CCK-4 utilize PLC but not PLA2 pathways. CCK-4 possesses the minimal essential amino acids to fully activate the PTK pathway.

Topics: Aminobenzoates; Amylases; Animals; Calcium; Cells, Cultured; Chlorobenzoates; Cholecystokinin; Cinnamates; Enzyme Inhibitors; Estrenes; Kinetics; Male; ortho-Aminobenzoates; Pancreas; Peptide Fragments; Phosphodiesterase Inhibitors; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Sincalide; Tetragastrin; Type C Phospholipases

Cholecystokinin (CCK) is a peptide that can be found in the cerebral cortex in high concentrations and is involved in learning and memory as well as neurodegenerative processes. Cortical brain samples from 9 patients with Alzheimer's disease and 9 matched control cases were studied with respect to the concentrations of various molecular forms of CCK and the CCK receptor binding characteristics. No differences were found between patients and controls in any of these measures. Significant correlations were found between the concentrations of CCK-8 sulphated and the three nonsulphated CCK peptides measured. In addition, the concentrations of CCK-4 and CCK-5 showed a highly significant and positive correlation.

Topics: Age of Onset; Aged; Aged, 80 and over; Alzheimer Disease; Analysis of Variance; Case-Control Studies; Cholecystokinin; Female; Gyrus Cinguli; Humans; Linear Models; Male; Middle Aged; Peptide Fragments; Receptors, Cholecystokinin; Sincalide; Tetragastrin

A serine endopeptidase was characterized as a major inactivating enzyme for endogenous cholecystokinin (CCK) in brain. CCK-8 released by depolarization of slices of rat cerebral cortex, as measured by its immunoreactivity (CCK-ir), undergoes extensive degradation (approximately 85% of the amount released) before reaching the incubation medium. However, recovery of CCK-ir is enhanced up to 3-fold in the presence of serine-alkylating reagents (i.e., phenylmethylsulfonyl fluoride) as well as selected active site-directed inactivators (i.e., peptide chloromethyl ketones) or transition-state inhibitors (i.e., peptide boronic acids) of serine peptidases. Among these compounds, elastase inhibitors were the most potent protecting agents, whereas trypsin or chymotrypsin inhibitors were ineffective. HPLC analysis of endogenous CCK-ir recovered in media of depolarized slices indicated that endogenous CCK-5 [CCK-(29-33)-pentapeptide] was the most abundant fragment and that its formation was strongly decreased in the presence of an elastase inhibitor. HPLC analysis of fragments formed upon incubation of exogenous CCK-8 [CCK-(26-33)-octapeptide] with brain slices showed CCK-5, Gly-Trp-Met, and Trp-Met to be major metabolites of CCK-8 whose formation was prevented or at least diminished in the presence of the elastase inhibitor. It is concluded that there is an elastase-like serine endopeptidase in brain that cleaves the two peptide bonds of CCK-8 where the carboxyl group is donated by a methionine residue and constitutes a major inactivation ectoenzyme for the neuropeptide.

Topics: Animals; Cerebral Cortex; Cholecystokinin; Chromatography, High Pressure Liquid; Peptide Fragments; Rats; Serine Endopeptidases; Serine Proteinase Inhibitors; Sincalide

The purpose of this study is to purify and to characterize chemically cholecystokinin (CCK)-like peptides present in brain and gut extracts that elute from gel filtration after the octapeptide. Canine small intestinal mucosa and brain were boiled in water and then extracted in cold trifluoroacetic acid, and cholecystokinin-like immunoreactivity was determined by carboxyl-terminal specific radioimmunoassay. Gel permeation chromatography on Sephadex G-50 revealed a form of CCK apparently smaller than CCK-8. This peptide was purified by immunoaffinity chromatography and three successive reverse phase high-performance liquid chromatography steps. Microsequence analysis showed that the amino terminal primary sequence of this small CCK was Gly-Trp-Met-Asp. Immunochemical and chromatographic analysis indicated that the carboxyl-terminal residue was Phe-NH2 and thus the full sequence is Gly-Trp-Met-Asp-Phe-NH2. An antibody that recognizes synthetic CCK-8, CCK-5, and CCK-4 equally did not reveal the presence of significant amounts of CCK-4. These results indicate that CCK-5 is the major CCK form smaller than the octapeptide present in brain (19% of total CCK immunoreactivity) and small intestine (7% of total). This finding, coupled with the demonstration by others that CCK-5 interacts with high-affinity brain CCK receptors, indicates that CCK-5 may play a physiological role in brain function.

Topics: Amino Acid Sequence; Animals; Brain Chemistry; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Dogs; Immunologic Tests; Intestinal Mucosa; Peptide Fragments; Radioimmunoassay; Sincalide

Topics: Gastrins; Magnetic Resonance Spectroscopy; Peptide Fragments; Protein Conformation; Sincalide; Spectrometry, Fluorescence; Structure-Activity Relationship; Tetragastrin