CCK Peptides Research Articles

Cholecystokinin increases intracellular Ca 2+ concentration in cultured striatal neurons

Although it has been established that pancreatic cholecystokinin (CCK) receptors are coupled to phosphatidylinositol turnover, the events which follow activation of CCK receptors in the central nervous system have not received much attention. In this paper, changes in intracellular Ca 2+ concentration ([Ca 2+]i) in response to CCK peptides were measured in single cultured rat striatal neuron by fura-2 fluorometry. CCK peptides dose-dependently increased [Ca 2+]i in a monophasic manner. The order of the potencies of CCK peptides to increase [Ca 2+]i was as follows: caerulein > CCK-8 > desulfated CCK-8 > CCK-4. The effect of caerulein was completely blocked in a Ca 2+-depleted medium. In addition, ω-conotoxin GVIA completely inhibited the effect of caerulein, while neither nifedipine nor verapamil affected it. Our results indicate that CCK receptors couple to N-type voltage-sensitive Ca 2+ channels in cultured rat striatal neurons.

Boc-Trp-Orn(Z)-Asp-NH2 and derivatives: a new family of CCK antagonists

The respective roles of the benzyloxycarbonyl group (Z) and of the N-terminal tripeptide moiety in the antagonist properties of the cholecystokinin CCK8 analogue Boc-[Nle28,Orn(Z)31]CCK27-33 (Marseigne et al. J. Med. Chem. 1988, 31, 966.) were studied with the following derivatives: Boc-[Nle28,Orn(X)31]CCK27-33, Boc[Nle28,Orn(X)31]CCK27-32, Boc-[Orn(X)31]CCK30-33, and Boc-[Orn(X)31]CCK30-32 (X = Z, Boc, H). These derivatives, the synthesis of eight of which is reported here, were tested for their abilities to inhibit the binding of [3H]pCCK8 to guinea pig pancreatic and brain membranes and for their potencies in stimulating amylase release from guinea pig pancreatic acini. None of the Z derivatives produced amylase secretion, but they competitively antagonized the stimulation induced by CCK8. The deletion of the N-terminal tripeptide and/or Phe-NH2(33) residue did not play a key role in the recognition of peripheral receptors and in the activity of these peptides, whereas replacement of the Z group by a Boc group slightly decreased the affinities of the compounds for both pancreatic and brain binding sites and their potencies as peripheral antagonists. Moreover, the tetrapeptide Boc-Trp-Orn(Boc)-Asp-Phe-NH2 behaved as a partial agonist and analogues in which the Z or Boc groups on the ornithine residue were removed were full agonists. Interestingly, the short peptide derivative Boc-Trp-Orn(Z)-Asp-NH2 displayed the same affinity (KI = 2.0 +/- 0.2 x 10(-7] and the same antagonist activity (pA2 = 6.63) as its parent compound Boc-[Nle28,Orn(Z)31]CCK27-33. This tripeptide could be an interesting tool for studying the structural relationships between peptide and non-peptide CCK antagonists.

Expression and processing of procholecystokinin in a rat medullary thyroid carcinoma cell line

A rat medullary thyroid carcinoma cell line, CA-77, was shown to express the cholecystokinin (CCK) gene. Measurements using a library of sequence-specific radioimmunoassays before and after enzymic treatment of extracts and chromatographic fractions showed that the cells contained 1.0 pmol of alpha-carboxyamidated cholecystokinins/10(6) cells, 0.4 pmol of glycine-extended intermediates/10(6) cells and 1.0 pmol of further C-terminal-extended pro-CCK/10(6) cells. Gel chromatography and reverse-phase h.p.l.c. revealed both sulphated and nonsulphated CCK-8 in the cells. The growth medium contained in addition alpha-amidated CCK-33, glycine-extended CCK-8 and pro-CCK. Exposure to 0.1 microM-dexamethasone for 6 days increased the cellular content and secretion of all of the described CCK peptides by 2-3-fold. The increase was first noted after 3 days of treatment. Monensin inhibited the synthesis of alpha-carboxyamidated CCK and the secretion of all of the CCK forms measured. Colchicine at a low concentration (0.2 mumol/l) apparently increased the synthesis and secretion of alpha-carboxyamidated CCK, whereas higher concentrations inhibited CCK synthesis. Finally, chloroquine inhibited the alpha-carboxyamidation of CCK. We conclude that the CA-77 cell line is a useful tool for studies of the expression and post-translational processing of pro-CCK.

Changes in hypothalamic neuropeptide Y concentrations induced by cholecystokinin analogues

Neuropeptide Y (NPY) and cholecystokinin (CCK) are two peptides involved in opposite ways in the control of food intake. A possible interaction between NPY and CCK has not yet been well defined. Two CCK derivatives with agonistic and antagonistic properties were studied with regard to their effects on brain and plasma NPY levels. The CCK agonist decreased NPY levels in plasma and in the hypothalamus but not in the other brain areas assayed. The CCK antagonist reversed the agonist-induced decrease in both plasma and hypothalamus. These results suggest a negative relation between NPY and CCK peptides, which is not surprising given their opposite role in feeding regulation. The hypothalamus, a preferential site of this regulation, appears to be the brain area most involved in the NPY-CCK interaction. The plasma NPY level variations closely reflect the hypothalamic profile, suggesting a direct release of NPY by a mechanism that remains to be investigated.

Expression of the cholecystokinin gene in a human (small-cell) lung carcinoma cell-line

Expression of the cholecystokinin (CCK), gastrin and enkephalin A genes were studied by Northern blot analysis and a library of sequence-specific radioimmunoassays in human cell lines. The human small-cell lung carcinoma line (SCLC) U-1690 expressed moderate levels of CCK mRNA as compared to the human neuroepithelioma cell line SK-N-MC. Neither gastrin nor (pro)enkephalin A mRNAs were detectable in the U-1690 cell line. In contrast, the SCLC-line H-69 expressed Enk A but no CCK mRNA. The radioimmunoassays showed that the CCK mRNA transcript in the SCLC line U-1690 also is translated, and that preproCCK is processed into bioactive, carboxyamidated CCK peptides. Thus, the human small cell carcinoma cell line U-1690 is a useful model for studies of cell-specific CCK gene expression.

Procholecystokinin processing in rat cerebral cortex during development

Using a library of radioimmunoassays for essential sequences of procholecystokinin (proCCK), we have examined the post-translational processing in the rat cerebral cortex from fetal to adult state. The concentration of proCCK in the fetal cerebral cortex was 43 ± 7 pmol/g tissue (wet weight; mean ± S.E.M. ( n = 20)). It remained constant until day 21 post partum, after which it decreased to undetectable levels. In contrast, the concentration of fully processed, bioactive CCK peptides (i.e. α-carboxyamidated CCK) rose from 2 ± 1 pmol/g in the fetal cortex to 122 ± 21 pmol/g in the adult. A particularly steep increase occurred from day 7 post partum (13 ± 2 pmol/g) to day 21 (108 ± 11 pmol/g). The concentration of glycine-extended intermediates rose gradually from 8 ± 1 pmol/g in the fetal brain to 55 ± 6 pmol/g in the adult. Gel chromatography of cortical extracts from day 7, 21 and 100 confirmed the variable processing at the C-terminal amidation site. The results show that the CCK gene is expressed as proCCK already in the fetal brain. However, the covalent modifications of proCCK follow different time courses so that only a small fraction reaches maturation until the first week post partum. We conclude that expression of transmitter-active CCK peptides in the brain is largely regulated at the post-translational rather than at the transcriptional level.

CNS injection of CCK in rats: effects on real and sham feeding and gastric emptying

Cholecystokinin (CCK) released from the intestine during feeding may have a physiological role in satiety. There is also evidence that activation of central CCK-containing pathways is involved in the control of feeding behavior. This study was carried out to determine whether CCK-8 administered into the lateral cerebral ventricles (lv) of rats suppresses both sham feeding (SF) and real feeding (RF). Rats with lv guides and gastric cannulas ate a liquid diet with cannulas open (SF) or closed (RF) after lv (0, 0.05, 0.5 micrograms) or intraperitoneal (ip) (0, 4 micrograms/kg) injection of CCK-8. Both RF and SF were significantly decreased by ip CCK-8. RF was also decreased in a dose-related manner after lv CCK-8, but SF was not affected by lv CCK-8. Decreased feeding after ip CCK-8 may be due in part to its suppression of gastric emptying rate (GER). To determine whether central nervous system (CNS) CCK might also be involved in the control of gastric function, GER was measured after lv (0, 0.05, 0.5 micrograms) or ip (0, 4 micrograms/kg) injection of CCK-8. GER was significantly decreased after ip CCK-8, but lv CCK-8 had no effect on GER. Although both CNS and peripheral CCK peptide systems may be involved in satiety, CNS CCK appears to depend on concurrent peripheral nutrient-related stimuli in eliciting satiety.

A71378: a CCK agonist with high potency and selectivity for CCK-A receptors

Receptors for the brain and gut peptide cholecystokinin (CCK) have been classified into two classes, CCK-A and CCK-B. To date, peptide analogues with selectivity for the CCK-B receptors have been identified, and selective antagonists for CCK-A and CCK-B receptors have been reported as well; until now, there have been no reports of highly selective CCK-A agonists. Herein we describe the properties of A71378 [desamino-Try(SO3H)-Nle-Gly-Trp-Nle-(N-methyl)Asp-Phe-NH2], a highly selective CCK-A receptor ligand. Characterization of A71378 was carried out in the guinea pig pancreas, cortex, gastric gland, and ileum, as well as in NCI-H345 cells. The IC50 values of A71378 for the pancreatic CCK-A, cortical CCK-B, and gastrin receptor were 0.4 nM, 300 nM, and 1,200 nM, respectively. A71378 proved to be a potent agonist in eliciting pancreatic amylase secretion (EC50 = 0.16 nM) and ileal muscle contraction (EC50 = 3.7 nM). In contrast, A71378 was relatively weak (EC50 = 600 nM) in mobilizing intracellular calcium from NCI-H345 cells, which express CCK-B/gastrin receptors. The high potency and selectivity of A71378 for the CCK-A over CCK-B and gastrin receptors is unprecedented among CCK peptides. Studies on CCK-7 analogues indicate that N-methylation of the Asp residue is responsible for the observed selectivity for CCK-A receptors. This discovery of a selective CCK-A agonist should prove valuable for studies aimed at understanding the physiological roles of CCK-A receptors in the brain and periphery.

Non-sulphated cholecystokinin in human medullary thyroid carcinomas

The expression of gastrin/cholecystokinin (CCK) peptides and their precursors was examined in 16 medullary carcinomas of the human thyroid. Measurements with libraries of sequence-specific radioimmunoassays before and after enzymatic cleavage of extracts and chromatographic fractions showed that the carcinomas contained 1.7 pmol carboxyamidated CCK/g tissue (median; range 0.6-21.8 pmol/g), 0.9 pmol glycine-extended precursor/g (median; range less than 0.2-2.3 pmol/g) and 2.3 pmol further COOH-terminal-extended proCCK/g (median; range 0.9-6.2 pmol/g). Neither carboxyamidated gastrins nor any progastrins could be measured. Gel and reverse-phase chromatography revealed only small molecular forms, i.e. greater than 90% of the amidated immunoreactivity eluted like non-sulphated CCK-8 or CCK-7. The results show that human medullary thyroid carcinomas synthesize CCK peptides. The predominance of non-sulphated CCK is unusual. Taken together with earlier observations from dogs and pigs, our results raise the possibility that small non-sulphated CCK peptides modulate thyroid C-cell secretion in an autocrine manner.

Somatostatin regulates duodenal cholecystokinin and somatostatin messenger RNA

The gastrointestinal peptides, cholecystokinin (CCK) and somatostatin, are produced by discrete endocrine cells in the mucosa of the small intestine. Although somatostatin may inhibit CCK secretion, the mechanism by which this occurs is unknown. The present study was designed to determine the effect of somatostatin on intestinal CCK and somatostatin mRNA levels. Rats, prepared with indwelling intraduodenal and jugular cannulas, were first fed an elemental diet that did not stimulate CCK release. Next, as a means of stimulating CCK secretion, soybean trypsin inhibitor was added to the diet and perfused intraduodenally at 50 mg/h for 24 h. Trypsin inhibitor caused an 11-fold increase in plasma CCK levels and a 2.4-fold increase in duodenal CCK mRNA levels. Simultaneous intravenous infusion of somatostatin-14 (1-100 micrograms.kg-1.h-1) reduced trypsin inhibitor-stimulated CCK levels by 50% and lowered trypsin inhibitor-stimulated CCK mRNA levels by 58%. Somatostatin infusion also reduced intestinal somatostatin mRNA levels stimulated by trypsin inhibitor but did not affect basal somatostatin mRNA levels. Duodenal CCK and somatostatin peptide concentrations did not change under any of the experimental conditions. These studies demonstrate that somatostatin reduces duodenal CCK mRNA levels stimulated by diet and suggest that somatostatin itself inhibits duodenal somatostatin gene expression.

Differential Cholecystokinin Gene Expression in Brain and Gut of the Fasted Rat

Cholecystokinin (CCK) gene expression has been compared in the brain and duodenum of control and 5 days fasted rats. To study transcription, CCK mRNA was quantified using a solution hybridization assay. Large and small molecular weight CCK peptides were separated using a sequential extraction process and subsequently quantified by radioimmunoassay. In the duodenum, a fall in weight was paralleled by a decrease in CCK mRNA and in the large forms of CCK peptides. Small molecular species of CCK peptides did not change. There was no change in weight, CCK transcriptional or translational products in the brain as a whole. These data indicate location-specific differential regulation of the products of CCK gene expression in the fasted rat.

Cholecystokinin peptides and bombesin reverse hemorrhagic shock in rats

Many independent experimental findings suggest that endogenous opioid systems play an important role in the pathophysiology of shock [l]. And indeed exogenous opioid receptor-antagonists, as well as neuropeptides purported to be endogenous, physiological antagonists of opioids, such as TRH, ACTH and aMSH, have been shown to improve cardiovascular function and survival in various shock states [l 31. Growing evidence suggests that cholecystokinin (CCK), too, may play a role as physiologic antagonist of opioids [4]. We investigated a possible effect of CCK peptides, and of bombesin, which is a potent releasing factor of CCK [5], in an experimental model of hemorrhagic shock, in rats.

Novel localizations of central- and peripheral-type cholecystokinin binding sites in Syrian hamster brain as determined by autoradiography

Neuronal cholecystokinin (CCK) binding sites were studied in the Syriam hamster, a species with unique CNS localizations of CCK immunoreactivity. Preliminary studies of hamster forebrain sections using 125I-Bolton-Hunter-(BH)-CCK-8 indicated that radioligand binding kinetics and receptor selectivity for various unlabelled CCK peptides were similar to those reported for other species. Autoradiographic visualization of CCK binding sites revealed that the highest binding densities were distributed in the cerebral cortex, olfactory bulb, dorsal vagal complex, raphe obscurus and cochlear nuclei. Moderate binding densities were present in the amygdala, hippocampus, hypothalamus, thalamus, central grey and medial vestibular nucleus. Comparison of autoradiograms generated from adjacent sections incubated in 125I-BH-CCK-8 with unlabelled sulfated or desulfated CCK-8 confirmed the presence of peripheral-type CCK binding sites (i.e. at which desulfated CCK-8 has week displacing activity) in the hamster dorsal vagal complex. Peripheral-type CCK binding sites were also distributed in regions of the hypothalamus (e.g. the magnocellular cell groups) where similar receptor selectivity had not been previously demonstrated. These observations provide further evidence of species differences in CCK receptor distribution and specify, which may account for species differences in responsiveness to intracranial administration of CCK peptides.

Transient expression of the cholecystokinin gene in male germ cells and accumulation of the peptide in the acrosomal granule: possible role of cholecystokinin in fertilization.

Expression of the gene encoding the neurotransmitter/neuromodulator cholecystokinin (CCK) was demonstrated in testis of several different species. Two testicular CCK mRNA transcripts of different sizes were detected, and studies on the ontogeny of CCK gene expression indicated that the gene was expressed in male germ cells. In situ hybridization revealed CCK mRNA-expressing cells in the peripheral parts of the seminiferous tubules. Biochemical identification showed that the majority of prepro-CCK products in the testis represented pro-CCK. Immunofluorescence studies revealed CCK-like peptides primarily in spermatocytes and spermatids of mouse, rat, and monkey. Immuno electron microscopy of monkey testis demonstrated CCK immunoreactivity in the acrosomal granule of spermatids. Hence, an interesting possibility is that CCK peptides can be released during the acrosome reaction and thus may be of importance in the fertilization process.

Processing of cholecystokinin by isolated liver cells.

Although hepatic uptake of cholecystokinin (CCK) has been demonstrated, the liver cell involved and the mechanism of uptake remain unclear. We have used dispersed rat hepatocytes, Kupffer cells, and hepatic endothelial cells to characterize uptake and metabolism of radiolabeled CCK peptides. Only rat hepatocytes showed significant uptake of 125I-labeled cholecystokinin octapeptide (125I-CCK-8). Peptide specificity of uptake by hepatocytes was similar to that seen in the isolated perfused rat liver, with extraction of 125I-CCK-8 being sevenfold greater than that of 125I-CCK-33. Uptake was saturable, as 10(-4) M CCK-4 inhibited uptake of 125I-CCK-8 by 85%. Uptake was rapid, temperature dependent, and extensive and was decreased by metabolic inhibition, a proteolytic enzyme (trypsin), organic anions (sulfobromophthalein and taurocholic acid), and an inhibitor of anion transport 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. In addition, uptake was dependent on extracellular anions but not on extracellular sodium, calcium, or magnesium. After uptake, hepatocytes released radiolabel in a time- and temperature-dependent manner, predominantly in metabolized forms. Thus the hepatocyte is the liver cell that extracts CCK by an active, anion-dependent process. The characteristics of the uptake process resemble those described for organic anions and small, cyclic peptides and suggest that small, linear peptides may undergo hepatocyte extraction by a similar mechanism.

Cholecystokinin evokes secretion of oxytocin and vasopressin from rat neural lobe independent of external calcium.

Cholecystokinin (CCK) and its receptors are abundantly represented in the central nervous system. However, a specific role or mechanism of action for CCK in this context has not been established. CCK coexists with oxytocin in magnocellular neurons of the hypothalamic-neurohypophysial system, sharing common neurosecretory vesicles with oxytocin in the neural lobe of the pituitary. The neural lobe, which consists primarily of oxytocin- and vasopressin-containing axons and nerve terminals and their surrounding glia, provides a relatively simple model system allowing for the study of the regulation of neurosecretion at the nerve terminal level, free from the complex array of synaptic effects present throughout the rest of the central nervous system. In this paper, we demonstrate the presence of high-affinity CCK binding sites in the rat neural lobe and show that activation of these receptors by the sulfated octapeptide, CCK-8, and related peptides causes potent secretion of oxytocin and vasopressin from the isolated nerve terminals. The secretagogue action of CCK-8, which is blocked by a CCK receptor antagonist (L-364,718), is independent of electrical stimulation and extracellular calcium and is blocked by an inhibitor of protein kinase C. Thus, the action of CCK on the neural lobe provides an example of peptide ligand-induced neurosecretion apparently mediated by second messengers rather than depolarization-induced calcium influx.

Properties of receptors for gastrin and CCK on gastric smooth muscle cells

Previous studies have demonstrated that cholecystokinin (CCK), gastrin, and structurally related peptides can interact with various types of receptors that can be distinguished by their relative affinities for agonists and antagonists. In the present study we examined the effect of gastrin, the COOH-terminal octapeptide of CCK (CCK-8), and the tetrapeptide of CCK (CG-4) on contraction of dispersed gastric smooth muscle cells from guinea pig and tested the ability of various CCK receptor antagonists to affect agonist-induced muscle cell contraction. For purposes of comparison we tested the effect of each antagonist on CCK-stimulated amylase secretion by pancreatic acini from guinea pig. On gastric smooth muscle cells, CCK-8, gastrin, and CG-4 were all full agonists. CCK-8 and gastrin were equipotent and CG-4 was 6,000-fold less potent. Each antagonist caused inhibition of CCK-stimulated contraction with relative potencies (IC50): L364,718 (4 microM) = CBZ-CCK-(27-32)-NH2 (3 microM) greater than proglumide analogue 10 (90 microM). Inhibition by each of the antagonists was competitive in nature, specific for CCK peptides, and each had the same IC50 whether contraction was stimulated by CCK-8, gastrin, or CG-4. Relative potencies (IC50) of the three antagonists for inhibiting CCK-stimulated amylase release from pancreatic acini were L364,718 (3 nM) greater than proglumide analogue 10 (200 nM) greater than CBZ-CCK-(27-32)-NH2 (3 microM). These results demonstrate that gastric smooth muscle cells possess receptors that differ from CCK receptors on pancreatic acini in terms of affinities for both agonists and certain antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin, gastrin and their precursors in pheochromocytomas.

Using sequence-specific radioimmunoassays before and after cleavage with trypsin and carboxypeptidase B, we have examined the occurrence and molecular nature of cholecystokinin (CCK) and gastrin peptides in bioactive (i.e. alpha-carboxyamidated) as well as non-amidated precursor forms in extracts from 13 human pheochromocytomas. All but one tumour contained amidated CCK, but only in moderate amounts (less than or equal to 20 pmol/g tissue). In contrast to the complete sulphation in tissues which normally produce CCK (the brain and small intestine), the amidated adrenal CCK peptides were poorly sulphated (less than or equal to 17%). Four pheochromocytomas, including the one without amidated CCK, contained between 28 and 0.2 pmol amidated gastrin/g, mainly in the form of sulphated gastrin-17. In addition, all tumours contained biosynthetic precursors of both CCK and gastrin. In most extracts there was more precursor than bioactive peptide(s), the progastrin concentration ranging up to 338 pmol/g. The results show that pheochromocytomas synthesize CCK and gastrin. The posttranslational processing differs, however, markedly from that of the principal CCK and gastrin producing tissues, with respect to both proteolytic cleavages and amino acid derivatization. This emphasizes that accurate quantitation in tumours requires assays which measure the translation products irrespective of their degree of processing.

Cholecystokinin and gastrin inhibit histamine stimulated aminopyrine uptake in isolated rabbit gastric glands.

In the present study we have analyzed if cholecystokinin (CCK) or gastrin (G) can inhibit acid production in isolated rabbit gastric glands as revealed by the aminopyrine technique. The results show that G 17 I, CCK 8 NS, CCK 8 S, ceruletide and CCK 39 significantly inhibit histamine induced aminopyrine accumulation. No significant inhibition was noted for G 4, G 34 and NT G 1-13. As a group the CCK peptides were more effective than the gastrin peptides in inhibiting the aminopyrine uptake. CCK 8 S and ceruletide, the most potent inhibitors, reduced histamine induced aminopyrine accumulation with an ED50 of 10(-9) and 10(-10) M respectively. These potencies are similar to those by which CCK peptides stimulate isolated pancreatic acini to secrete amylase. Inhibition evoked by CCK 8 S was most effective following 20-40 min of incubation time, possibly indicating that the effect is mediated by the release of an intermediate substance. The results may therefore indicate a role for cholecystokinin as a physiological inhibitor of acid secretion in the rabbit. The results may also contribute to explain why the potent gastric secretagogue gastrin per se fails to stimulate acid formation in gastric glands isolated from the rabbit.

Effect of a new CCK-receptor antagonist, CR 1409, on pancreatic growth induced by caerulein, CCK-8, bombesin and gastrin-releasing peptide in the rat.

The effect of a peripheral cholecystokinin (CCK)-receptor antagonist, CR 1409, on pancreatic growth has been studied in the rat. 1.8 nmol/kg CCK-8 or caerulein and 3.6 nmol/kg bombesin or gastrin-releasing peptide (GRP) administered subcutaneously 3 times daily for 4 successive days increased pancreatic weight and its content in protein, enzymes and RNA but not in DNA, suggesting cellular hypertrophy. CR 1409 (10 mg/kg) administered intragastrically 30 min prior to peptides prevented pancreatic growth due to CCK-8 or caerulein but not that induced by bombesin and GRP. It is concluded that bombesin and GRP act on the exocrine pancreas directly rather than through the release of CCK.