sincalide and cholecystokinin-(27-33)

Key amino acids of the cholecystokinin (CCK) peptide for receptor binding are sulfated Y27, W30, D32, and F33-NH(2). Three-dimensional modeling showed that the CCK-A receptor (CCK-AR) antagonist devazepide penetrated into the transmembrane (TM) domains, whereas CCK was placed on the surface of the CCK-AR. Four types of rat CCK-AR cDNAs were transfected into CHO-K1 and COS-7 cells: normal CCK-AR cDNA transfected cells (wild type, WT); K120 substituted with V; K130V; and R352V. Binding of [3H]CCK-8 was observed in WT and K130V, but not in K120V and R352V. CCK caused Ca(2+) spiking in WT and K130V, whereas K120V and R352V had no effect. Three chimeras including the CCK-AR/3ibeta2 adrenergic receptor (beta2AR), 3Nibeta2AR, and 3Cibeta2AR were constructed. Two groups of point mutations in the CCK-AR3i were also made: Y252V, S274V, S281V, and S289V (non-phospho-acceptor Y or S); S260V, S264V, S271V, and S275V (phospho-acceptor S). WT and CCK-AR/3Cibeta2AR increased [Ca(2+)](i) in response to CCK; 3Nibeta2AR was vice versa. CCK failed to increase [IP(3)] in phospho-acceptor S to V without affecting binding. Non-phospho-acceptor S or Y to V showed normal response. Thus, Lys120 outside the TM2 and Arg352 outside the TM6 of the CCK-AR are amino acids interacting with Tyr[SO(3)H]27 and Asp32 of the CCK peptide for binding. Phospho-acceptor Ser groups in the CCK-AR 3Ni are amino acids for initiating cell signaling.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Calcium; CHO Cells; COS Cells; Cricetinae; Devazepide; Inositol 1,4,5-Trisphosphate; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Protein Binding; Radioligand Assay; Rats; Receptor, Cholecystokinin A; RNA, Messenger; Signal Transduction; Sincalide; Structure-Activity Relationship; Transfection

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.

Topics: Amino Acid Sequence; Amylases; Animals; Cattle; Molecular Sequence Data; Pancreas; Peptide Fragments; Phenylalanine; Rats; Receptors, Cholecystokinin; Sincalide; Structure-Activity Relationship

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac-CCK-7[Ac-Tyr(SO3H)-Met-Gly-Trp30-Met-Asp-Phe-NH2 (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK-A and CCK-B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp30 is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2-Nal30-Ac-CCK-7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK-A receptor antagonist MK-329. The extreme structural sensitivity of this anorectic activity is illustrated by the 1-naphthylalanine30 (19) and (benzo[b]thien-2-yl)alanine30 (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono- and bicyclic Trp30 replacements, including substituted phenylalanines, 3-quinolinylalanine, and 2-(5,6,7,8-tetrahydro)naphthylalanine, gave inactive compounds.

Topics: Amino Acid Sequence; Animals; Appetite Depressants; Blood Proteins; Cattle; Corpus Striatum; Molecular Sequence Data; Pancreas; Peptide Fragments; Rats; Sincalide; Structure-Activity Relationship; Tryptophan

The aim of this study was to determine the cholecystokinin (CCK) receptor subtype involved in the direct myogenic effect of CCK on pig ileum. Smooth muscle cells were dispersed from pig ileum circular muscle layer and incubated in the presence of various concentrations of CCK agonists and antagonists. Contraction was assessed by measuring the length of 50 cells and expressed as the percentage decrease in cell length from control. Maximal contraction varied between 19% +/- 3% (gastrin II, 10 nmol/L) and 26% +/- 3% [CCK octapeptide (CCK-8), 10 nmol/L]. EC50 for CCK tetrapeptide (CCK-4) was the same than for pentagastrin (30 pmol/L), which were more potent than CCK-8 (100 pmol/L) and unsulfated gastrin 17 (100 pmol/L), which in turn were more potent than unsulfated CCK heptapeptide (CCK-7; 300 pmol/L) and sulfated gastrin II (300 pmol/L). The maximal contraction induced by synthetic analogs of CCK was 22% +/- 1% for 1 nmol/L JMV 170 and 23% +/- 1% for 10 nmol/L JMV 180. EC50 was 10 pmol/L for JMV 170 and 800 pmol/L for JMV 180. Contraction induced by 10 nmol/L CCK was inhibited as follows: L 365,260 half maximal inhibition (IC50) = 1 nmol/L greater than L 364,718 (IC50 = 90 nmol/L) greater than proglumide (IC50 = 1 mumol/L). Contraction induced by 10 nmol/L unsulfated gastrin 17 was inhibited as follows: L 365,260 (IC50 = 1 pmol/L) greater than L 364,718 (IC50 = 60 nmol/L) greater than proglumide (IC50 = 4 mumol/L). Removal of Ca2+ from the extracellular medium did not alter the contraction induced by CCK-8 (10 nmol/L) but impaired the contraction induced by unsulfated gastrin 17 (10 nmol/L) -56% in Ca(2+)-free medium, -77% in Ca(2+)-free medium plus 2 mmol/L EGTA, and -70% in the presence of 1 mumol/L nifedipine. These results show that the CCK receptor of pig ileum smooth muscle cells is closely similar to the B receptor and is not dependent on an influx of extracellular Ca2+ to induce cell contraction. By contrast, gastrin could act through a specific receptor subtype, the "gastrin receptor," triggering a Ca2+ influx into the cell to induce cell contraction.

Topics: Animals; Calcium; Cholecystokinin; Dose-Response Relationship, Drug; Gastrins; Ileum; In Vitro Techniques; Male; Muscle Contraction; Pentagastrin; Peptide Fragments; Proglumide; Receptors, Cholecystokinin; Sincalide; Swine; Tetragastrin

We have tried to evaluate the significance of the tryptophan side chain residue and of the surrounding peptide bonds in the antagonist activity of cholecystokinin analogues lacking the C-terminal amide function and having a D-tryptophan. In order to perform this study, analogues of the C-terminal heptapeptide of cholecystokinin were synthesized by replacing the C-terminal phenylalanine residue with 2-phenylethyl alcohol and by either replacing the tryptophan residue with an alanine, a norleucine and a phenylalanine residue, or introducing a "reduced peptide bond" in the tryptophan 30 region. Most of these compounds were able to reproduce only part of the response of cholecystokinin in stimulating amylase release from rat pancreatic acini, as was already observed for 2-phenylethyl ester analogues of CCK. These results point out the key role of tryptophan 30 in the biological response of cholecystokinin.

Topics: Amino Acid Sequence; Amylases; Animals; Binding Sites; Brain; Dose-Response Relationship, Drug; Guinea Pigs; Magnetic Resonance Spectroscopy; Male; Membranes; Molecular Sequence Data; Oligopeptides; Pancreas; Peptide Fragments; Phenylethyl Alcohol; Rats; Receptors, Cholecystokinin; Sincalide

Our approach to design small molecule non-peptide analogues of the neuropeptide cholecystokinin (CCK) has led to the discovery of the CCK-B antagonist 'dipeptoids'. A representative member of this series of compounds, PD134308 [R-(R*,R*)]-4-[[2-[[3-(1H-Indol-3-yl)-2-methyl-1-oxo-2- [[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)carbonyl]amino]propyl] amino]-1-phenylethyl]amino]-4-oxobutanoic acid has high affinity (Ki = 1.7 nM) and selectivity for the CCK-B receptor (CCK-A/B ratio is 2500:1), is well absorbed and shows robust anxiolytic properties in several anxiogenic models in a dose related manner by both s.c. and oral routes of administration over the dose range 0.1-30 mg/Kg. The rational design of these dipeptoids from CCK 26-33 has involved the identification of the non-contiguous dipeptide fragment of CCK, Boc-Trp-Phe-NH2 with low micromolar affinity in binding assays. This dipeptide has been systematically chemically modified at the N- and C-terminal to increase CCK-B binding affinity 10,000-fold. These modifications include replacement of the L-tryptophan moiety by the non-genetically coded D-alpha-methyltryptophan residue. The modifications also enhance the stability of the molecule towards enzymatic and acid degradation and increase overall lipophilicity compared with the peptide in order to facilitate penetration of the blood-brain barrier.

Topics: Amino Acid Sequence; Animals; Binding Sites; Blood-Brain Barrier; Cerebral Cortex; Cholecystokinin; Drug Design; Indoles; Meglumine; Mice; Molecular Sequence Data; Peptide Fragments; Receptors, Cholecystokinin; Sincalide; Structure-Activity Relationship

In this work in vitro pharmacological profiles of two analogues of the C-terminal heptapeptide of cholecystokinin (CCK) were evaluated. The analogue Boc-[Nle28, Nle31]-CCK-7, a stable analogue of CCK-8, has the same activity profile as CCK-8, and was found to be very potent in stimulating amylase secretion, phospholipid breakdown and [Ca2+]i mobilization from rat pancreatic acini. It can be used as a probe for studying CCK-actions. The CCK-analogue Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester, (JMV180), which stimulates amylase secretion without inhibition at supramaximal concentrations, has different effects on phospholipid hydrolysis and [Ca2+]i mobilization, compared to CCK-8 and Boc-[Nle28, Nle31]-CCK-7. Compound JMV180 was unable to significantly promote phospholipid breakdown, and was only 50%-60% as efficacious as Boc-[Nle28, Nle31]-CCK-7 in promoting [Ca2+]i mobilization. These findings suggest that low affinity CCK-receptors might be responsible for the supra-maximal inhibition of amylase secretion, and are correlated with phospholipid breakdown and maximal [Ca2+]i mobilization.

Topics: Amylases; Animals; Calcium; In Vitro Techniques; Intracellular Fluid; Male; Pancreas; Peptide Fragments; Phospholipids; Rats; Rats, Inbred Strains; Receptors, Cholecystokinin; Sincalide

When pancreatic acini are incubated with increasing concentrations of cholecystokinin octapeptide (CCK-8) the dose-response curve for stimulation of enzyme secretion increases, reaches a maximum and then decreases. The upstroke of the dose-response curve reflects occupation of high-affinity, stimulatory CCK receptors (Kd 69 pM), whereas the downstroke of the dose-response curve reflects occupation of low-affinity inhibitory CCK receptors (Kd 10 nM). In the present study, we used dispersed acini from rat pancreas to examine the effects of CCK-JMV-180, an analogue of the C-terminal heptapeptide of CCK (CCK-7) having the structure BOC-Tyr(SO3) Ahx-Gly-Trp-Ahx-Asp2 phenylethyl ester. CCK-JMV-180 inhibited binding of 125I-labelled CCK-8 to pancreatic acini. Computer analysis of the dose-inhibition curve indicated that CCK-JMV-180 interacted with both classes of CCK receptor and had a Kd of 2.2 nM for high-affinity CCK receptors and a Kd of 19 nM for low-affinity CCK receptors. Occupation of high-affinity CCK receptors by CCK-JMV-180 caused a 14-fold increase in amylase secretion, the same increase caused by occupation of these high-affinity receptors by CCK-7 or CCK-8. Occupation of low-affinity CCK receptors by CCK-JMV-180 did not alter amylase secretion, in contrast to occupation of these low-affinity receptors by CCK-7 or CCK-8, each of which caused inhibition of amylase secretion. Furthermore, occupation of the low-affinity CCK receptors by CCK-JMV-180 reversed the inhibition of amylase secretion caused by a supramaximal concentration of CCK-8. The present results indicate that CCK-JMV-180 interacts with high-affinity CCK receptors and with low-affinity CCK receptors, and has a functionally distinct action at each class of receptor: CCK-JMV-180 is an agonist at the high-affinity receptors and a competitive antagonist at the low-affinity receptors.

Topics: Amylases; Animals; Binding, Competitive; Kinetics; Male; Pancreas; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Cholecystokinin; Sincalide

Syntheses of analogues of the C-terminal octa- and heptapeptide of cholecystokinin are described. These analogues were obtained by replacing the C-terminal phenylalanine residue by 2-phenylethyl alcohol or by 2-phenylethylamine derivatives and by replacing the tryptophan residue by a D-tryptophan. The CCK-derivatives were tested for their ability to inhibit binding of labeled CCK-8 to rat pancreatic acini and to guinea pig brain membranes, and for their action on stimulation of amylase release from rat pancreatic acini. Some of these derivatives appeared to exhibit only part of the CCK-activity on amylase release, the D-Trp analogues behaving as CCK-antagonists.

Topics: Amino Acid Sequence; Amylases; Animals; Biological Assay; Brain; Cell Membrane; Chemical Phenomena; Chemistry, Physical; Esters; Guinea Pigs; Molecular Sequence Data; Pancreas; Peptide Fragments; Phenethylamines; Rats; Sincalide; Structure-Activity Relationship

Cholecystokinin 7 (CCK 7), a synthetic analogue of cholecystokinin/pancreozymin (CCK 33), increased in a dose-dependent manner the tone of the guinea-pig ileal, gastric and gall bladder smooth muscle preparations. In all these preparations CCK 7 was more potent than CCK 8 and CCK 33 and all three cholecystokinins were more potent than acetylcholine (ACH). Atropine and tetrodotoxin (TTX) did not influence the CCK 7 action in fundus and gall bladder muscle strips but reduced non-competitively its effect in ileal muscle strips. Neither GE 410 nor dbcGMP affected the ACH and histamine (His) response of the muscle strips but both antagonists shifted the dose-response curve of CCK 7 to the right, GE 410 (cholecystokinin antagonist) being a much more potent antagonist of CCK 7 as compared to dbcGMP. In all muscle strips a competitive action on the CCK 7 responses was found for GE 410. In gastric muscle strips a competitive influence on the CCK 7 responses was found for dbcGMP at low concentration (1 x 10(-5)M) and a non-competitive influence at high concentration (5 x 10(-4)M). The results suggest that the contractile effects of CCK 7 in the isolated ileal smooth muscle are realized by cholinergic and direct myogenic mechanisms, whereas in the isolated gall bladder and gastric smooth muscles, by a direct myogenic mechanism only.

Topics: Animals; Atropine; Bucladesine; Female; Gallbladder; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle, Smooth; Peptide Fragments; Sincalide; Stomach; Tetrodotoxin

Possible biologically active (receptor-bound) conformations of peptides derived from cholecystokinin (CCK) have been deduced using conformational analysis combined with comparative studies of their biological specificities. Two peptides, the completely active carboxyl terminal heptapeptide from CCK (CCK-7), whose sequence is Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, and the carboxyl terminal heptapeptide from cerulein (CER-7) which has the same sequence as for CCK-7 except for replacement of Met 2 with a Thr 2, both stimulate peripheral receptors in gall bladder, pancreas, and pylorus in the gastrointestinal system. In contrast, two other very similar peptides, the last four residues of CCK (CCK-4) whose sequence is Trp-Met-Asp-Phe-NH2, and the carboxyl terminal hexapeptide of little gastrin (LGA-6, Tyr-Gly-Trp-Met-Asp-Phe-NH2, i.e., residue 2 deleted relative to CCK-7 and CER-7 sequences), interact specifically with gastrin receptors and not at all or very weakly with peripheral receptors. All of these peptides react with CCK receptors in the central nervous system, especially in forebrain. The results in the GI tract suggest that the peptides active on peripheral receptors adopt structures that are significantly different from those of the peptides that interact with gastrin receptors. We have generated all of the many low energy conformations for each of these peptides. By retaining only the conformations that are the same for peptides within the same group and then rejecting those resulting conformations that are the same for the peptides in the two different groups, we can greatly reduce the possible active conformations for the peptides within each class.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Ceruletide; Cholecystokinin; Enkephalin, Methionine; Gastrins; Models, Molecular; Molecular Sequence Data; Neuropeptides; Peptide Fragments; Peptides; Protein Conformation; Receptors, Cholecystokinin; Sequence Homology, Amino Acid; Sincalide; Tetragastrin

Cholecystokinin (CCK)-33 is known to stimulate insulin secretion. Presently, using the perfused rat pancreas, we have characterized the active site in the CCK-33 molecule that is responsible for this effect by the use of different CCK fragments. We found that CCK-33, CCK-8 and CCK-7 (1 nM) all significantly stimulated insulin secretion in the presence of 4.4 mM or 6.7 mM glucose. However, CCK-7 was much less potent than the longer forms. In contrast, CCK-4, CCK-6 and CCK-33 (1-21) had no effect on insulin secretion. We conclude that the shortest CCK-form that stimulates insulin secretion at 1 nM is the C-terminal heptapeptide CCK-7. However, CCK-8 is much more potent than CCK-7 in this respect.

Topics: Animals; Binding Sites; Cholecystokinin; Insulin; Insulin Secretion; Male; Pancreas; Peptide Fragments; Perfusion; Rats; Rats, Inbred Strains; Sincalide; Structure-Activity Relationship; Tetragastrin; Time Factors

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

Topics: Analgesics; Animals; Chemical Phenomena; Chemistry; Mice; Peptide Fragments; Sincalide

In dispersed acini from guinea pig, mouse, or rat pancreas cholecystokinin-(27-33) is a full agonist, and removing the sulfate ester from the tyrosine residue in position 27 caused a 100- to 300-fold decrease in potency with no change in efficacy. In dispersed acini from mouse or rat pancreas, cholecystokinin-(27-32)-NH2 is a partial agonist, and removing the sulfate ester from the tyrosine in position 27 abolished the efficacy. The desulfated peptide was able, however, to interact with CCK receptors with a potency that was threefold less than that of cholecystokinin-(27-32)-NH2 and therefore functioned as a cholecystokinin receptor antagonist. In dispersed acini from guinea pig pancreas cholecystokinin-(27-32)-NH2 is a cholecystokinin receptor antagonist. Removing the sulfate ester from the tyrosine residue in position 27 of cholecystokinin(27-32)-NH2 caused a fourfold decrease in potency but did not abolish the ability of the peptide to interact with cholecystokinin receptors; therefore, desulfated cholecystokinin-(27-32)-NH2 functioned as a cholecystokinin receptor antagonist.

Topics: Amylases; Animals; Carboxylic Acids; Cholecystokinin; Esters; Guinea Pigs; Male; Mice; Pancreas; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Cholecystokinin; Sincalide; Structure-Activity Relationship; Sulfates

The actions of CCK-8 and putative CCK-8 antagonists were examined on synaptic transmission in the hippocampal slice preparation. CCK-8 (10(-7) M) caused a reversible depression of postsynaptic responses of the Schaffer collateral-CA1 synapse. CCK-mediated depression was unaffected by proglumide (10(-3) M) but was antagonized by a CCK analog (CCK27-33). The CCK analog alone had an excitatory effect on synaptic transmission. These results suggest that CCK has an inhibitory action on synaptic transmission.

Topics: Animals; Depression, Chemical; Evoked Potentials; Hippocampus; Peptide Fragments; Proglumide; Rats; Rats, Inbred Strains; Sincalide; Synaptic Transmission

The biologically active conformations of a series of four peptides [four cholecystokinin (CCK)-related peptides and enkephalin] in their interactions with gastrointestinal receptors have been deduced using conformational computational analysis. The two peptides that interact exclusively with peripheral-type CCK receptors are the heptapeptide COOH-terminal fragment from CCK (CCK-7) and the analogous sequence from cerulein (CER-7) in which threonine replaces the methionine proximal to the NH2 terminus. The two peptides that interact exclusively with the gastrin receptor in the stomach are the active COOH-terminal fragment of little gastrin and the COOH-terminal tetrapeptide sequence common to all of these peptides, CCK-4. We find that preferred conformations for the peripherally active peptides CCK-7 and CER-7 are principally beta-bends, whereas little gastrin and CCK-4 are fundamentally helical. In the class of lowest energy structures for both CCK-7 and CER-7, the aromatic rings of the tyrosine and phenylalanine lie close to one another whereas the tryptophan indole ring points in the opposite direction. This structure is superimposable on the structures of a set of rigid indolyl benzodiazepine derivatives that interact with complete specificity and high affinity with peripheral CCK receptors further suggesting that the computed beta-bends are the biologically active conformation. The biologically active conformation for CCK-4 and the little gastrin hexapeptide has also been deduced. By excluding conformations common to CCK-7 and CCK-4, which do not bond to each other's receptors, and then by selecting conformations in common to CCK-4 and the gastrin-related hexapeptide, which do bind to each other's receptors, we deduce that the biologically active conformation at the gastrin receptor is partly helical and one in which the indole of tryptophan and the aromatic ring of phenylalanine are close to one another while the methionine and aspartic acid side chains point in the opposite direction. These major differences in preferred structures between the common CCK-7/CER-7 peptides and the common CCK-4/little gastrin peptides explain the mutually exclusive activities of these two sets of peptides. We have observed that [Met]enkephalin strongly antagonizes the action of the naturally occurring peripherally active CCK-8 (CCK-7 with an NH2-terminal aspartic acid residue added). The computed lowest energy structures for this opiate peptide closely resemble key feat

Topics: Amino Acid Sequence; Benzodiazepines; Ceruletide; Enkephalin, Methionine; Gastrins; Indoles; Models, Molecular; Peptide Fragments; Protein Binding; Protein Conformation; Receptors, Cholecystokinin; Receptors, Opioid; Sincalide; Tetragastrin

Suc-Tyr(SE)-Met-Gly-Trp-Met-Asp-beta-phenethylamide(410) has been studied for its ability to antagonize contractile responses of guinea pig gall bladder, ileum and stomach muscle strips to desamino-cholecystokinin-octapeptide (CCK-7) and cholecystokinin octapeptide (CCK-8). Both CCK-7 and CCK-8 at concentrations of 10(-11)M to 10(-7)M produced dose-dependent tonic contractions in all muscle strips. Suc-Tyr(SE)-Met-Gly-Trp-Met-Asp-beta-phenethylamide (10(-8)M-10(-5)M) inhibited reversibly in a dose-dependent manner the contractile responses to CCK-7 and CCK-8. At the same concentrations the antagonist shifted to the right in parallel to the dose-response curves for CCK-7 and CCK-8 without decreasing their maximum response. Analysis of the data after Schild gave pA2 values (410 potency as antagonist) of CCK-7 in gall bladder, ileum and stomach of 8.36; 8.0 and 7.56, respectively, and pA2 values of CCK-8 of 7.64; 8.94 and 8.52, respectively. The slope of the Schild plots for both CCKs did not differ significantly from the unity, which suggests that 410 is a competitive antagonist. The antagonistic action of 410 is reversible and appeared to be specific since at concentrations of 5 X 10(-6), it had no effect on contractile responses of the gall bladder, ileum and gastric muscle strips to acetylcholine or histamine.

Topics: Animals; Dose-Response Relationship, Drug; Gallbladder; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Peptide Fragments; Sincalide; Stomach

In the present study we have examined the abilities of cholecystokinin-(26-33)-amide [CCK-(26-33)-NH2, CCK-8], nonsulfated CCK-(26-33)-NH2 (desulfated CCK-8), CCK-(30-33)-NH2 (CCK-4), CCK-(26-33)-OH (deamidated CCK-8), and succinyl CCK-(27-31)-NH2 (Suc-Des-Asp6,Phe7-CCK-7) to stimulate exocrine pancreatic secretion from both isolated pancreatic acini and isolated perfused pancreas. We have also compared this action with their ability to cause insulin release. The modification of either the N- or C-terminal amino acid residues of CCK-8 decreased in potency, but the magnitude of the stimulation of enzyme secretion caused by a maximally effective peptide concentration was the same. The minimal effective concentration of CCK-8, desulfated CCK-8, and CCK-4 for insulin release from the isolated rat pancreas in the presence of 8.3 mM glucose was the same as that for pancreatic exocrine secretion. In contrast, the concentrations of deamidated CCK-8 and Suc-Des-Asp6,Phe7-CCK-7 required to produce insulin release were 5-10 times higher than those required to cause stimulation of pancreatic enzyme and juice secretion. It is concluded therefore that the N-terminal 4-amino acid residues or the C-terminal 2-amino acid residues of CCK-8 are not essential for biological activity but do contribute to its potency. In addition, the C-terminal 2-amino acid residues and an amide group in the C-terminal phenylalanine residue of CCK-8 appear to be important determinants of the insulin-releasing activity of the CCK peptides.

Topics: Amylases; Animals; Cholecystokinin; Insulin; Insulin Secretion; Pancreas; Peptide Fragments; Rats; Sincalide; Tetragastrin

The biological activity of bovine pancreatic polypeptide (BPP) on rat exocrine pancreatic secretion was compared in vivo and in vitro. In anesthetized rats prepared with a bile-pancreatic duct cannula, BPP inhibited cholecystokinin (CCK)-stimulated (10 IDU . kg-1 X h-1) protein secretion in a dose-related manner (P less than 0.001). CCK, from 5-20 IDU . kg-1 X h-1, did not alter the degree of inhibition by BPP at 40 micrograms . kg-1 X h-1, suggesting a nonsurmountable inhibition. Analogues of BPP, including rat pancreatic polypeptide, neuropeptide Y, peptide YY, and the C-terminal hexapeptide of PP, also inhibited CCK-stimulated protein secretion. To determine whether BPP acts directly on acinar cells to suppress enzyme secretion, in vitro studies were performed. BPP and its analogues did not suppress octapeptide of CCK (CCK-8)-stimulated amylase release from either isolated rat pancreatic acini or preparations of pancreatic lobules. Specific binding of 125I-BPP to pancreatic acini was also not observed. From our data we conclude that BPP acts to inhibit pancreatic enzyme secretion in the rat in a noncompetitive manner. Absence of an effect by BPP or its analogues in vitro coupled with an absence of 125I-BPP binding to acini suggest that the inhibitory action of PP on exocrine pancreatic function is mediated by indirect mechanisms.

Topics: Amylases; Animals; Bile; Dibutyryl Cyclic GMP; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Nerve Tissue Proteins; Neuropeptide Y; Pancreas; Pancreatic Polypeptide; Peptide Fragments; Peptide YY; Peptides; Proteins; Rats; Rats, Inbred Strains; Sincalide; Stimulation, Chemical

Topics: Animals; Fluorescent Antibody Technique; Lampreys; Neurons; Peptide Fragments; Serotonin; Sincalide; Spinal Cord

Truncal vagotomy results in diminished pancreatic protein secretion in response to intraduodenal fat. This diminished secretion may be due, at least in part, to interruption of the vagal reflexes between the intestine and the pancreas that work independently of cholecystokinin (CCK). In five dogs with chronic pancreatic fistulas, plasma CCK concentrations and pancreatic protein secretion in response to an intestinal stimulant (intraduodenal oleate) and to two exogenous peptides (bombesin and CCK-33) were compared before and after bilateral truncal vagotomy. Vagotomy decreased integrated protein secretion by about 50% in response to intraduodenal oleate. In contrast, protein output in response to parenteral stimuli increased after vagotomy. Integrated output of CCK in response to intraduodenal oleate or to exogenous bombesin or CCK was not significantly affected by vagotomy, but release of pancreatic polypeptide was decreased significantly in response to all stimuli after truncal vagotomy. These data provide evidence that truncal vagotomy decreases pancreatic protein secretion in response to intestinal stimulants by interrupting enteropancreatic reflexes mediated by the vagus, while maintaining normal (or supranormal) sensitivity of the pancreas to endogenous and exogenous CCK.

Topics: Animals; Bombesin; Cholecystokinin; Dogs; Duodenum; Female; Male; Oleic Acid; Oleic Acids; Pancreas; Pancreatic Fistula; Pancreatic Polypeptide; Peptide Fragments; Proteins; Reflex; Sincalide; Stimulation, Chemical; Vagotomy; Vagus Nerve

The influence of desaminoocta-pancreozymin (DAO-PZ) was investigated in high and low activity periods by intraperitoneal or intranasal application. DAO-PZ is a CCK-8 derivative in which the N-terminal amino acid (Asp) is exchanged for succinyl acid. A decreasing long lasting effect for 3 to 4 h was always observed in locomotor activity. A food reduction was also proved after i. p. and intranasal application. The effects correspond to those of CCK-8 reported in the literature. They are discussed in this respect.

Topics: Animals; Eating; Male; Motor Activity; Peptide Fragments; Rats; Rats, Inbred Strains; Sincalide; Time Factors

Cholecystokinin (CCK) is a gut peptide which induces a syndrome of satiety, including reduced food intake and reduced exploratory behaviors. To investigate the minimal active sequence within the octapeptide molecule for reducing exploratory behavior, all C- and N-terminal fragments of CCK 26-33 (CCK8) were synthesized. Only CCK26-33 [H-Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and CCK 27-33 [H-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] induced the behavioral effects within a physiological dose range. Smaller fragments as well as unsulfated CCK26-33 and CCK27-33 were inactive in doses as high as 10(-3) mol/kg. The requirement of the entire C-terminal heptapeptide for the behavioral effects of CCK is contrasted against the activity of smaller fragments at pancreatic, gastrointestinal and brain CCK receptors.

Topics: Amino Acid Sequence; Animals; Cholecystokinin; Exploratory Behavior; Feeding Behavior; Male; Mice; Molecular Weight; Peptide Fragments; Receptors, Cell Surface; Receptors, Cholecystokinin; Sincalide

The present study was done to compare endogenous cholecystokinin-33 release in response to physiologic stimuli (meal, fat) in pigs, dogs and man. Plasma levels of cholecystokinin-33 were monitored using a radioimmunoassay for cholecystokinin which detects only cholecystokinin-33 and cholecystokinin-39. Ingestion of a meal caused release of cholecystokinin-33 within five, 20 and 120 minutes in man, pigs and dogs, respectively. Intraduodenal administration of corn oil resulted in a significant release of cholecystokinin-33 within 20 minutes in dogs, pigs and man.

Topics: Adult; Animals; Cholecystokinin; Corn Oil; Dietary Fats; Dogs; Female; Food; Humans; Intubation, Gastrointestinal; Male; Oils; Peptide Fragments; Radioimmunoassay; Sincalide; Swine; Time Factors

In Zucker obese rats the response to the effects of CCK on food intake and pancreatic exocrine function are decreased. However, it is unknown whether the decreased responsiveness is due to decreased receptor number and/or sensitivity or abnormal circulating concentrations of CCK. In these experiments percent total binding of 125I-CCK-33 to pancreatic acini from obese rats was one-half that in lean rats when data was expressed on a per microgram DNA basis (19.6 +/- 5.1 vs. 47.4 +/- 11.4, p less than 0.01). In a second experiment while the maximally effective dose of CCK for stimulating amylase secretion from dispersed pancreatic acini was similar in obese and lean rats (10(-10) M), less amylase was secreted in obese rats across the dose range tested (p less than 0.001). In contrast, carbachol had similar potency and efficacy in stimulating amylase release from obese and lean pancreatic acini. The increase of pancreas size by use of a trypsin inhibitor was greater in lean than obese rats (p less than 0.03). In addition, stimulation of amylase release by CCK from obese trypsin inhibitor-treated compared with control obese rats was greater than that from lean trypsin inhibitor-treated compared with control lean rats (p less than 0.002). However, overall, stimulation of amylase secretion by CCK was only 36% of control (p less than 0.001) and by carbachol was only 20% of control (p less than 0.001). Thus, increased size by increased cell number was associated with decreased response per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amylases; Animals; Body Weight; Carbachol; Culture Techniques; DNA; Dose-Response Relationship, Drug; Esters; Female; Gabexate; Guanidines; Pancreas; Peptide Fragments; Rats; Rats, Zucker; Receptors, Cell Surface; Receptors, Cholecystokinin; Sincalide

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

Desamino-cholecystokinin-octapeptide (CCK-7) increased the gall-bladder pressure and decreased the gastric pressure following i.v. infusion in conscious dogs. In small intestine and Heidenhain pouch CCK-7 exerted an initial excitatory effect followed by an inhibitory effect on intraluminal pressure and peristalsis. In all organs the effect of CCK-7 on pressure was correlated with a change of spike discharge. CCK-7 is more potent on gastrointestinal motor activity than a Boots preparation of pancreozymin. The results suggest that CCK-7 at doses within the physiological range could be involved in the regulation of gastrointestinal motility.

Topics: Animals; Cholecystokinin; Dogs; Gallbladder; Gastrointestinal Motility; Intestine, Small; Membrane Potentials; Muscle Contraction; Muscle, Smooth; Peptide Fragments; Pressure; Sincalide; Stomach; Vagotomy

1H NMR study of cholecystokinin fragment (CCK27-33) in (C2H3)2SO and in 2H2O at different pH shows that sulfated (CCK7) and non sulfated (NS-CCK7) peptides are under preferentially folded conformations characterized by a beta-turn including the sequence Gly-Trp-Met-Asp with a H-bond between the CO of Gly and the NH of Asp. This structure is probably stabilized by an ionic interaction between Tyr and Asp. Moreover, the N-terminal part of CCK7 forms a C7 structure with a weak H-bond between the CO of Gly and the NH of Trp. In this model all CCK7 hydrophobic side chains are in close vicinity, far from the hydrophilic sulfate group. Full interaction with brain CCK8 receptors could require both the sulfate group and the maintaining of conformational constraints.

Topics: Amino Acid Sequence; Cholecystokinin; Hydrogen-Ion Concentration; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Peptide Fragments; Protein Conformation; Sincalide; Structure-Activity Relationship; Sulfuric Acids

The heptapeptide carbobenzoxy-L-tyrosyl(O-sulfate)-L-methionylglycyl-L-tryptophyl-L-methionyl-L- aspartyl-beta-L-phenylalanine amine (Z-32-beta-Asp-CCK-27-33) was synthesized and tested for its ability to stimulate secretion from dispersed pancreatic acini in vitro, to increase protein secretion from cat pancreas in vivo, and to cause contraction of guinea pig gallbladder in situ. In increasing amylase secretion in vitro, the Z-32-beta-Asp-CCK-27-33 was equal in efficacy with but approximatively one-third as potent as the Boc-CCK-27-33, and when tested in vivo its activity is approximately 10 Ivy dog units (Idu)/microgram. In stimulation of the contraction of the gallbladder, it showed an activity lower than 1 Idu/microgram. This analogue has more pancreozyminic activity than cholecystokin-like activity. This seems to indicate different affinities for the two receptors.

Topics: Amylases; Anesthesia; Animals; Cats; Cholecystokinin; Gallbladder; Guinea Pigs; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Pancreas; Peptide Fragments; Proteins; Sincalide

In the C-terminal heptapeptide of cholecystokinin (-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2), replacing the aspartic acid residue by beta-aspartic acid did not alter the ability of the peptide to cause stimulation, desensitization, or residual stimulation of enzyme secretion from dispersed pancreatic acini. Replacing the tyrosine sulfate residue by hydroxynorleucine sulfate did not alter the ability of the heptapeptide to cause stimulation or desensitization, but caused a 50-fold decrease in the potency with which the peptide caused residual stimulation of enzyme secretion. These findings suggest that a modification of the N-terminal region of cholecystokinin heptapeptide, which does not alter the ability of the peptide to bind to its receptor on pancreatic acini and by so doing cause stimulation and desensitization of enzyme secretion, can increase the rate at which the bound peptide dissociates when the acini are washed and reincubated. This increased dissociation is reflected by a reduction in the potency with which the peptide causes residual stimulation of enzyme secretion.

Topics: Amylases; Animals; Aspartic Acid; Cholecystokinin; Guinea Pigs; Isomerism; Pancreas; Peptide Fragments; Sincalide; Structure-Activity Relationship

In hog terminal bile duct cholecystokinin peptides caused an activation of cyclic AMP-dependent protein kinase (A-PK) with cyclic AMP, followed by increase in Ca uptake of sarcoplasmic reticulum fraction (SR-F). By contrast, papaverine showed no activation of A-PK-induced Ca uptake by SR-F with cyclic AMP. The Ca uptake by SR-F was dependent on ATP and Mg2+, but the component phosphorylated was not the phosphoenzyme intermediate in Ca2+-ATPase. The effect of Ca uptake was blocked by the inclusion of a protein inhibitor of A-PK. The correlation coefficient between cyclic AMP-dependent SR-F phosphorylation and stimulated Ca uptake by the phosphorylated SR-F was 0.731 (P less than 0.01). These results suggest that one of the mechanisms by which CCK-4, CCK-8, and CCK-33 peptides relax isolated Oddi's sphincters of terminal bile ducts is activation of A-PK-induced Ca uptake by sarcoplasmic reticulum fraction and possibly also by plasma membrane.

Topics: Animals; Biliary Tract; Calcium; Cholecystokinin; Cyclic AMP; Enzyme Activation; In Vitro Techniques; Muscle, Smooth; Peptide Fragments; Protein Kinases; Sarcoplasmic Reticulum; Sincalide; Swine

Central (intracerebroventricular) and peripheral (intraperitoneal) injections of the octapeptide of cholecystokinin (CCK-8) were compared to determine the most effective route of administration to elicit satiety for food intake in the rat. Subdiaphragmatic bilateral vagotomy and spinal cordotomy (T2-T3) were also performed to investigate the importance of visceral nerves for the satiety effect. CCK-8 suppressed feeding and elicited satiety resting behavior when injected peripherally but it was less effective when injected centrally. The satiety effect of CCK-8 or CCK-33 following peripheral injections was blocked by vagotomy whereas spinal cordotomy had no effect. The results indicate that some component of the vagus is required to mediate the peripherally induced cholecystokinin satiety effect, but the splanchnic nerves are not necessary. The weak effect of CCK-8 following ventricular administration is additional evidence suggesting that cholecystokinin of intestinal origin acts in the periphery rather than directly on the brain to elicit its typically rapid satiety effect in rats.

Topics: Adrenergic Fibers; Afferent Pathways; Animals; Cholecystokinin; Eating; Injections, Intraperitoneal; Injections, Intraventricular; Male; Muridae; Peptide Fragments; Satiation; Satiety Response; Sincalide; Spinal Cord; Splanchnic Nerves; Vagus Nerve

Topics: Animals; Cholecystokinin; Evoked Potentials; Fluorescent Antibody Technique; Male; Neurons; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Dopamine; Sincalide; Substantia Nigra; Tegmentum Mesencephali; Tyrosine 3-Monooxygenase

In the C-terminal heptapeptide of cholecystokinin, replacement of the penultimate residue, aspartic acid, by beta-alanine caused a 300-fold decrease in the potency with which the peptide stimulated enzyme secretions, whereas replacement by glutamic acid caused a 1000-fold decrease in potency. The beta-alanine-substituted peptide was approximately ten times more potent when the n terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl, and the glutamic acid-substituted peptide was approximately twice as potent when the N terminus was blocked with t-butyloxycarbonyl than when it was blocked with benzyloxycarbonyl. Changes in the ability of the peptide to stimulate amylase secretion were accompanied by corresponding changes in the ability of the peptide to inhibit binding of 125I-labeled cholecystokinin. The magnitude of stimulation of enzyme secretion caused by a maximally effective peptide concentration was the same with each analogue as it was with the unaltered peptide. Replacing the aspartyl residue by beta-alanine or glutamic acid or replacing the N-terminal t-butyloxycarbonyl moiety by benzyloxycarbonyl caused an equivalent decrease in the ability of the peptide to stimulate enzyme secretion and its ability to cause residual stimulation of enzyme secretion. In contrast, the N-terminal desamino analogue of cholecystokinin heptapeptide was ten times less potent than the unaltered peptide in stimulating amylase secretion, but 100 times less potent than the unaltered peptide in causing residual stimulation of enzyme secretion.

Topics: Alanine; Amino Acid Sequence; Amino Acids; Amylases; Animals; Aspartic Acid; Cholecystokinin; Guinea Pigs; Male; Pancreas; Peptide Fragments; Receptors, Cell Surface; Receptors, Cholecystokinin; Sincalide; Structure-Activity Relationship

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20 degrees C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO3H)-Trp-Met-Asp-Phe-NH2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8, and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent Km for CCK-8 and CCK-4 was 40 microM and maximal activity on CCK-8 was observed at pH 7.0. Zn2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type (Ki = 2 microM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.

Topics: Animals; Brain; Cations, Divalent; Cholecystokinin; Chromatography, High Pressure Liquid; Gastrins; Kinetics; Peptide Fragments; Protease Inhibitors; Puromycin; Rats; Sincalide; Synaptosomes; Tetragastrin

We tested the synthetic C-terminal heptapeptide of cholecystokinin, which has the same biologic activity as cholecystokinin, and various synthetic analogs of the C-terminal heptapeptide for their abilities to increase amylase secretion from dispersed acini prepared from guinea-pig pancreas. We found that altering the chemical character of the amino acid in position 27 altered the potency with which the peptide stimulated amylase secretion but did not alter the efficacy of the peptide. We also found that, in the amino acid in position 27, the major function of the side-chain seems to be to position to the sulfate ester group at a proper distance from the backbone of the peptide chain, whereas the chemical structure of the side-chain per se seems to be of relatively minor importance.

Topics: Amino Acid Sequence; Amylases; Animals; Cholecystokinin; Dibutyryl Cyclic GMP; Guinea Pigs; Male; Pancreas; Peptide Fragments; Sincalide