Unsulfated CCK-8 Research Articles

Reciprocal effect of unsulfated and sulfated forms of cholecystokinin-octapeptide on gastric acid secretion in the rat

The sulfated and unsulfated forms of cholecystokinin-octapeptide (CCK-8) were compared, with respect to their effect on gastric acid secretion, in the rat. Unsulfated CCK-8 stimulated acid secretion in a dose-dependent manner, while the sulfated form was without stimulatory effect; thus, sulfation of the tyrosine residue in the seventh position from the C terminus completely abolished the gastrin-like action of CCK-8. Compared with pentagastrin and human gastrin II, unsulfated CCK-8 gave lower calculated maximal response. While sulfated CCK-8 given alone had no effect on acid secretion, it caused marked inhibition of the plateau response to submaximal pentagastrin. This inhibition was surmountable with higher doses of pentagastrin, suggesting a competitive type of inhibition. It is, therefore, concluded that lack of sulfation of the tyrosine residue in the seventh position does not exclude CCK-8 from occupying the gastrin receptor; but does prevent the hormone-receptor interaction that leads to the secretory response. These observations in the rat are different from those in the dog where desulfation of tyrosine renders the CCK analog, caerulein, ineffective in its ability to stimulate acid secretion.

Anxiogenic effect of central CCK administration is attenuated by chronic fluoxetine or ipsapirone treatment

The effect of chronic fluoxetine and ipsapirone treatment on the anxiogenic effect of centrally administered cholecystokinin (CCK) was studied in the social interaction test in male Sprague–Dawley rats. Intracerebroventricular injection of unsulfated CCK-8 significantly decreased total interaction time and locomotor activity and caused some increase in selfgrooming and a reduction in rearing behaviour in a familiar arena in low light conditions. The selective serotonin reuptake inhibitor antidepressant fluoxetine alone (5 mg/kg, i.p.) also had clear acute anxiogenic actions (decrease in total interaction time, locomotor activity, rearing, increase in selfgrooming) after single dosing, but all these effects were omitted after chronic (3 weeks) treatment. In contrast, a single injection of the 5-HT 1A receptor partial agonist ipsapirone (5 mg/kg, i.p.) alone had only motor effects (decrease in selfgrooming and rearing), and these effects were preserved after chronic treatment. Chronic fluoxetine treatment (5 mg/kg per day, 3 weeks) abolished the effects of CCK-8 (1 nmol/rat, i.c.v.). Chronic treatment with ipsapirone (5 mg/kg per day, 3 weeks) partially attenuated the effects of CCK-8 (1 nmol/rat, i.c.v.). Our studies provide further evidence for a 5-HT/CCK interaction in the regulation of anxiety.

On the mechanism(s) of cholecystokinin (CCK): receptor stimulation attenuates morphine dependence in mice.

In the present study, effect of cholecystokinin (CCK) agonists and on dependence to morphine in mice has been investigated. The influence of dopaminergic, adrenergic, cholinergic and serotonergic on attenuation of naloxone-induced jumping in morphine-dependent mice by CCK agonists were also considered. Mice were treated subcutaneously with morphine (50, 50 and 75 mg/kg) three times daily (10 a.m. 1 p.m. and 4 p.m.) for 3 days, and a last dose of morphine (50 mg/kg) was administered on the 4th day. Withdrawal syndrome (jumping) was precipitated by naloxone (5 mg/kg) which was administered intraperitoneally 2 hr after the last dose of morphine. To study effects of CCK receptor agonists, 10 injection of morphine (3 administrations each day) for dependence and a dose of 5 mg/kg of naloxone for withdrawal induction were employed. The CCK agonists CCK-8 (0.001-0.1 mg/kg), unsulfated CCK-8 (CCK-8U; 0.001-0.1 mg/kg) and caerulein (0.00001-0.01 mg/kg) were able to prevent withdrawal signs precipitated by naloxone (5 mg/kg). Sulpiride and pimozide increased response induced by CCK-8 agonists. The dopamine antagonists also attenuates jumping by themselves. SCH 23390 did not alter the CCK-8 effect, but decreased the jumping by itself. Phenoxybenzamine, propranolol, methysergide and atropine did not change the caerulein effect significantly. However, single administration of atropine increased and methysergide decreased jumping. It is concluded that CCK mechanism(s) may be involved in morphine dependence, and dopaminergic mechanism(s) may interact with CCK in attenuation of naloxone-induced jumping.

Effects of administration of cholecystokinin into the VTA on DA overflow in nucleus accumbens and amygdala of freely moving rats.

The carboxyterminal octapeptide of cholecystokinin (CCK-8) coexists with dopamine (DA) in mesolimbic neurons of the ventral tegmental area (VTA). In the present study, in vivo microdialysis in freely moving rats was used to assess the relative effects of sulfated CCK-8 (CCK-8S), unsulfated CCK-8 (CCK-8US) and CCK tetrapeptide (CCK-4), focally injected into the VTA, on DA overflow in two mesolimbic DA/CCK-8S terminal regions, the nucleus accumbens and the amygdala. Consistent with electrophysiological findings, microinjection of CCK-8S, but not CCK-8US or CCK-4, elicited increases in DA overflow in both terminal regions. In the absence of anatomical evidence of CCK-containing fibers in the VTA region, it seems reasonable to conclude that the modulation of terminal DA overflow by CCK-8S through actions at the somatodendritic region represents a form of autoregulation of these cells. Whereas CCK-8US and CCK-4 are preferential CCK-B receptor agonists, CCK-8S binds non-selectively to CCK-A and CCK-B receptors. Thus, these results implicate CCK-A receptors in the stimulatory effects of CCK-8S on VTA DA neurons.

Electrophysiological effects of cholecystokinin on neurons in rat substantia nigra pars reticulata

The electrophysiological effects of car☐yterminal fragments of cholecystokinin (CCK) on neurons in the dorsal substantia nigra pars reticulata (SNr) were evaluated in anesthetized rats. Microiontophoretic administration of sulfated CCK octapeptide (CCK-8S, agonist for CCK-A and CCK-B receptors) and the selective CCK-B receptor agonists, CCK-4 and unsulfated CCK-8, inhibited the firing rates of a subpopulation of SNr neurons. This effect appears to be mediated by CCK-B receptors.

CCK receptor activation may prevent tolerance to morphine in mice

Different groups of mice were treated with morphine (50 mg/kg s.c.) once daily for 1, 2, 3, 4, or 5 days, in order to develop tolerance to the drug. The antinociceptive effect of morphine (9 mg/kg s.c.) was tested 24 h after each dose of the drug administration. Tolerance to morphine reached its peak on the 4th day. Daily pretreatment of animals for a period of 4 days with different doses of cholecystokinin octapeptide (CCK-8; 0.001, 0.01, 0.05 and 0.1 mg/kg s.c.), caerulein (0.0001, 0.001, 0.005 and 0.01 mg/kg s.c.) but not unsulfated cholecystokinin octapeptide (0.001, 0.01 or 0.1 mg/kg s.c.) 30 min before daily administration of morphine (50 mg/kg s.c.) prevented the development of tolerance. A group of animals received a single dose of caerulein (0.005 mg/kg), CCK-8 (0.01 mg/kg) or unsulfated CCK-8 (0.01 mg/kg) 30 min before morphine injection (50 mg/kg s.c.) on the 3rd or 4th day. In these animals, which were tested for antinociception on the 5th day, tolerance to the drug (3, 6 and 9 mg/kg s.c.) was also decreased by caerulein, CCK-8 but not unsulfated CCK-8. In a group of mice in which peptides were administered 30 min prior to the doses of morphine (3, 6 or 9 mg/kg s.c.) on the 5th day, similar results were obtained. The results of the present study indicate that activation of both CCK B and CCK A receptors may prevent the development of tolerance to morphine, and the sulfate group in the CCK-8 molecule may be essential for the tolerance inhibition.

Regulation of human T lymphoblast growth by sensory neuropeptides: augmentation of cholecystokinin-induced inhibition of Molt-4 proliferation by somatostatin and vasoactive intestinal peptide in vitro

The effects on proliferation of Molt-4 lymphoblasts of cholecystokinin (CCK-8), somatostatin-14 (SS), vasoactive intestinal peptide (VIP) and substance P (SP) were investigated using different combinations of the peptides, peptide analogs and their antagonists. In vitro proliferation of the cells was measured by a colorimetric assay for cell growth and survival. Results indicate that SP and SP (3–11) stimulated, whereas CCK-8, VIP and SS inhibited, proliferation in a dose-dependent manner ( P < 0.05). Unsulfated CCK-8 had no effect on growth of Molt-4 lymphoblasts, and a specific antagonist of CCK, at a concentration 10 −6 M, diminished the inhibitory effect of CCK-8 on Molt lymphoblasts ( P < 0.05). This suggests that the inhibitory action of CCK-8 was mediated by peripheral-type CCK receptors. SS and VIP, at equimolar concentrations of 10 −6 M, significantly augmented the CCK-8-induced inhibition of Molt-4 lymphoblast proliferation. However, none of the inhibiting neuropeptides suppressed stimulation of Molt-4 lymphoblast proliferation in response to SP. These data suggest a role of sensory neuropeptides including CCK in modulating human T lymphoblast proliferation during neuroendocrine interactions with the immune system.

Effects of the CCK-A receptor antagonist CR 1409 on the activity of rat midbrain dopamine neurons

Previous studies have shown that acute intravenous treatment with sulfated cholecystokinin octapeptide (CCK-8S) but not unsulfated CCK-8 increases the number of spontaneously active midbrain dopamine (DA) neurons. This suggested that a peripheral-type (CCK-A) CCK receptor mediates this effect. Proglumide does not discriminate between CCK-A and CCK-B (central-type) receptors. In the present study, rats were treated acutely or repeatedly (14 days) with the selective CCK-A antagonist CR 1409. Repeated treatment with 5 mg/kg (IP) increased the number of spontaneously active DA cells in the A10 (ventral tegmental area) but not the A9 (substantia nigra zona compacta) region, which suggests that these DA populations are differentially affected by prolonged CCK-A receptor blockade. The sensitivity of impulse-regulating DA autoreceptors to the DA agonist quinpirole was not altered by CR 1409.

Cholecystokinin receptor subtypes and neuromodulation

The sulfated octapeptide of Cholecystokinin (CCK-8S) and CCK fragments have been administered to mice to determine the subtype and location of the CCK receptor that modulates the release of dopamine (DA) in brain. 1. Centrally (i.c.v.) or peripherally (s.c.) administered CCK8S lowers DA release, and to a lesser extent, metabolism, in the neostriatum and olfactory tubercle. 2. DA release is decreased when the CCK-B selective compounds, unsulfated CCK-8 (CCK-8U) or the butoxycarbonyl tetrapeptide of CCK (t-boc-CCK-4), are given i.c.v. but not when injected s.c. 3. The increase in DA release following amphetamine administration is attenuated by i.c.v. but not s.c. injections of t-boc-CCK-4 or CCK-8U and by CCK-8S given via either route. 4. None of the s.c. actions of CCK-8S are prevented by the CCK-A receptor antagonist, L 364,718. CCK-B receptors in brain mediate the suppression by CCK of basal and augmented DA release. CCK-B receptor agonists may be useful for the treatment of psychiatric conditions that result from excessive DA release.

Unsulfated CCK-8 not blocked by proglumide or CR 1409 in the mouse abdominal irritant-induced stretching assay: Possible central site of action

Previous studies have shown that unsulfated cholecystokinin octapeptide (CCK-8-U) shares with the sulfated octapeptide (CCK-8-S) and the carboxyl-terminal tetrapeptide (CCK-4) the ability to block abdominal irritant-induced stretching when administered intracerebroventricularly. The effects of CCK-8-U, however, are not naloxone-reversible and do not appear upon systemic administration. To assess the hypothesis that the antistretching effects of CCK-8-U are mediated by central-type (CCK-B), rather than peripheral-type (CCK-A) receptors, the present experiments examined the reversal of these effects by CR 1409, a CCK receptor antagonist with in vitro selectivity for CCK-A receptors, and by proglumide. Both antagonists, when administered ICV, blocked the antistretching effects of CCK-8-S and CCK-4 (ICV), but not those of CCK-8-U. CR 1409 was approximately 40 times more potent against CCK-8-S by the ICV route than subcutaneously, indicating a likely action on CCK-A receptors in the brain. The effects of morphine, bombesin and neurotensin (ICV) were not blocked by CR 1409, indicating specificity for CCK receptor-mediated effects. The antistretching effects of CCK-8-U do not appear to be mediated by CCK-A receptors, and the possibility of a CCK-B receptor site of action must be considered.

Cholecystokinin-8 suppressed 3H-etorphine binding to rat brain opiate receptors

Radio receptor assay (RRA) was adopted to analyse the influence of CCK-8 on 3H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 (1pM to 1 μM) suppressed the binding of 3H-etorphine. This effect was completely reversed by proglumide at 1 μM. Rosenthal analysis for saturation revealed two populations of 3H-etorphine binding sites. CCK-8 (1pM to 1μM) inhibited 3H-etorphine binding to the high affinity sites by an increase in Kd (up to +235%) and decrease in Bmax (up to −80%) without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 (10nM) was also completely reversed by proglumide at 1 μM. Unsulfated CCK-8 (100pM to 1 μM) produced only a slight increase in Kd of the high affinity sites (+64%) without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

Neuroleptic-like properties of cholecystokinin analogs: Distinctive mechanisms underlying similar behavioral profiles depending on the route of administration

Rats were trained to discriminate vehicle injections from intraperitoneal injections of 3 μg/kg caerulein, a cholecystokinin (CCK) neuropeptide analog. The reward that reinforced correct choices was an electrical brain stimulation self-administered by bar pressing. Dose-response quantitative generalization was obtained by using 1 and 2 μg/kg caerulein. Qualitative generalization to the vehicle occurred after injecting 10, 20 and 200 μg/kg unsulfated CCK-8, 10, 20 and 200 μg/kg CCK-4, 5 μg/kg CCK-8 and 1 μg/kg caerulein, neurotensin or bombesin and 200 μg/kg apomorphine or 320 μg/kg amphetamine. Total generalization to the caerulein cue was obtained with 20 μg/kg sulfated CCK-8 or gastrin 2–17, 25 μg/kg somatostatin, 50 μg/kg haloperidol and 2 mg/kg chlorpromazine. The previous 5 mg/kg injection of an antiemetic drug such as chlorhydrate of trimethobenzamide did not eliminate the discriminative properties of a subsequent injection of caerulein. Our data thus tend to show that IP injection of caerulein produces effects similar to those of IP neuroleptics.

Expression of receptors for cholecystokinin and other Ca2+-mobilizing hormones in Xenopus oocytes.

The expression of receptors for cholecystokinin (CCK) and other similar acting Ca2+-mobilizing hormones was studied in Xenopus laevis oocytes. Poly(A)+ RNA was prepared from pancreatic AR42J cells, which normally express receptors for CCK and bombesin and the RNA injected into oocytes. The presence of these pancreatic receptors on the oocytes was then demonstrated by hormone-induced mobilization of 45Ca2+. CCK receptors were present 1 day (maximum, 2 days) after injection of RNA and were generally proportional to the amount of poly(A)+ RNA injected (1-50 ng). Oocyte CCK receptors retained selectivity for CCK analogs (CCK8 greater than unsulfated CCK8 greater than CCK4) and were blocked by the specific CCK receptor antagonist CR 1409. When poly(A)+ RNA was subjected to size fractionation on sucrose gradients, activity-inducing CCK receptors showed a single peak centered at 3 kilobases. The generality of this oocyte system for expressing Ca2+-mobilizing hormone receptors was further shown by expression of a response to bombesin after injection of AR42J cell RNA and a response to vasopressin and angiotensin II when poly(A)+ RNA from rat liver was injected. No response to CCK was demonstrable after injection of liver RNA, demonstrating the specificity of this assay.

Pharmacological examination of cholecystokinin (CCK-8)-induced contractile activity in the rat isolated pylorus.

The actions of cholecystokinin (CCK) in the production of a satiety-like state have been suggested to be mediated via receptors for CCK which are located in the pylorus. We investigated the actions of CCK and other pharmacological agents upon the isolated rat pylorus in vitro. We used the change in isometric tension of the tissue preparation (contraction amplitude) as the measure of the effects of the pharmacological agents. Cholecystokinin COOH-terminal octapeptide (CCK-8) was observed to elicit contraction in a dose-dependent manner, with the half-maximal dose (ED50) in the vicinity of 1 nM. Rapid desensitization to CCK was observed. The contraction amplitude was atropine-independent, and was not significantly antagonized by a wide variety of other pharmacological agents. The Na+-channel blocker tetrodotoxin was without effect upon contractile amplitude, as was the K+-channel blocker 4-aminopyridine, except at very high concentrations. Neurotensin, bombesin, and the substance P and bombesin antagonist spantide all elicited contraction in the isolated tissue; neurotensin had a similar potency to CCK-8 and bombesin was 10-15-fold less potent than CCK-8. Unsulfated CCK-8 was at least 170-fold less potent than sulfated CCK-8 and tetragastrin was at least 500-fold less potent than CCK-8. These results suggest that pyloric CCK receptors, which appear to have a pharmacological profile typical of peripheral CCK receptors, may have a physiological role in the peptidergic control of gastric emptying in the rat.

Cholecystokinin-dopamine coexistence: electrophysiological actions corresponding to cholecystokinin receptor subtype

Cholecystokinin (CCK)-like peptides when administered intravenously produce 2 distinct actions on the single-unit activity of mesencephalic dopamine (DA) neurons in the rat: an excitatory action and a potentiation of the inhibitory effects of DA agonists. The ability of several CCK fragments that have been shown to bind selectively to the peripheral and/or the central CCK-binding sites were examined for their ability to induce either excitation or a potentiation of DA. Only sulfated CCK-8 was able to induce excitation of mesencephalic DA neurons, but both sulfated and unsulfated CCK-8, as well as CCK-4, potentiated the inhibitory effects of the DA agonist apomorphine (APO). CCK-3 failed to potentiate APO-induced inhibition. Both of these effects appeared to be confined to cell bodies in regions of the ventral tegmental area and substantia nigra, zona compacta that have been reported to contain both DA and CCK. Thus, CCK-like peptides that have been shown to bind to the high-affinity CCK binding site in brain potentiated the effects of DA. In contrast, the ability of CCK-like peptides to induce neuronal excitation corresponds with their affinity for the peripheral-type CCK binding site.

Antinociceptive profile of sulfated CCK-8: Comparison with CCK-4, unsulfated CCK-8 and other neuropeptides

The antinociceptive activity of sulfated cholecystokinin octapeptide (CCK-8-S) was characterized by comparison with two other endogenous forms, unsulfated CCK-8 (CCK-8-U) and the carboxyl tetrapeptidc fragment (CCK-4) and two other peptides present m the gut and brain: bombcsin and neurotensin. By the intracerebroventricular (i.c.v.) route, CCK-8-S was antinociceptive in the hot plate and phenylquinone-induced writhing assays, but CCK-8-U and CCK-4 were active only in the latter test. By systemic administration, CCK-8-S retained anti-writhing activity but CCK-8-U and CCK-4 did not. Therefore, CCK receptors in brain may be involved in the apparent antinociception produced by CCK-8-U and CCK-4. Bombesin produced potent antinociceptive activity, along with a distinct, head-scratching syndrome, in both the writhing and hot plate tests. Naloxone reversed bombesin-induced elevation of latencies of mouse jump but not the head-scratching syndrome, indicating that the analgesic effect in the hot plate test was independent of the scratching behaviour. Neurotensin, unlike CCK-8-S, elevated tail-flick latencies, and was more potent in the writhing than in the hot plate test. Several differences between CCK-8-S and opioid substances included the need for relatively large doses of naloxone to block the effects of CCK-8-S in the phenylquinone-induced writhing test and the lack of effect of CCK-8-S in the tail-flick test. Global sedation can account for some, but not all, of the effects of CCK-8-S. Despite previous suggestions that CCK-8-S has opiate antagonist properties, the present results and clinical reports suggest that this neuropeptide may possess clinically-relevant analgesic properties.

Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: Characterization of the cleavage by solubilized endopeptidase - 24.11

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly 29-Trp 30-Met 31 and Asp 32-Phe 33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.