Measurement of cholecystokinin octapeptide using a new specific radioimmunoassay

A peptide analogue of CCK-8 (Tyroc) which has a tyrosine in place of the amide group in the C-terminal end, has been used both for raising antisera and for iodination. The antisera produced by immunisation with Tyroc are directed towards the N-terminal end of the CCK-8 molecule. The assay system appears totally specific for the CCK-8 sulphated molecule and shows no significant cross-reaction with other molecular forms of CCK, or with the gastrins. The assay can detect changes between adjacent tubes of 0.25 fmol/tube CCK-8 with 95% confidence. The assay is robust, reliable and reproducible and can be used to measure tissue and plasma levels of CCK-8.     

Preparation of an N-acetyl-octapeptide of cholecystokinin: The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5–4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentratiion-dependen isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.     

Multiple treatment potentiates the anticonvulsive activity of cholecystokinin octapeptides

The dose-response curves for the anticonvulsive activity of sulfated and nonsulfated cholecystokinin octapeptide (CCK-8-SE and CCK-8-NS) against picrotoxin-induced (6 mg/kg SC) seizures were assessed either following or without pretreatment with a single high dose of CCK-8-SE or CCK-8-NS, to examine acute tolerance to the effect after IP injections in mice. As CCK-8-SE or CCK-8-NS pretreatment, a 1.6 μmole/kg dose was injected 2 hr prior to the second injection. No acute tolerance to the anticonvulsive activity was demonstrated, and CCK-8-NS pretreatment significantly potentiated its own anticonvulsive activity. Chronic (8-day) daily treatment with a 0.16 μmole/kg dose of CCK-8-SE or CCK-8-NS antagonized seizures by picrotoxin, presumably in a cumulative manner. To investigate the interactions of CCK octapeptides with other anticonvulsive agents, picrotoxin-induced seizures were antagonized with several doses of diazepam following or without acute, high-dose pretreatment with CCK-8-SE or CCK-8-NS. The two octapeptides only slightly modified the activity of diazepam: CCK-8-SE pretreatment displayed a tendency to antagonize it, while CCK-8-NS pretreatment to potentiate it. The results suggest that multiple treatment with CCK-8 induces sensitization of CCK receptors mediating anticonvulsive activity.     

Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons

The actions of cholecystokinin octapeptide (CCK) on the membrane properties of mouse spinal neurons grown in monolayer culture were examined using intracellular recording techniques. In a subpopulation of cells, application of CCK (0.2-100 micron) by pressure ejection from micropipettes produced a small (approximately 2 mV) membrane depolarization that was accompanied by a decrease in membrane conductance (approximately 11 percent). These effects were associated with an enhanced tendency of the cells to generate action potentials when stimulated with intracellular depolarizing current. The unsulfated analog of CCK, which possesses weak biological activity in the gut, had little or no effect on cultured spinal neurons. A number of differences were noted between the responses to CCK and the excitatory amino acid glutamate. First, the effects of CCK were more delayed in onset (approximately 17 sec) and prolonged in duration (approximately 124 sec). Second, the depolarizations produced by glutamate were of larger magnitude and associated with variable effects on membrane conductance. Third, the response to CCK showed tachyphylaxis with repeated applications whereas glutamate remained effective as often as it was applied. It is concluded that CCK facilitates the excitability of spinal neurons in a manner distinct from that of the conventional excitant glutamate.     

Hypermotility induced by vasoactive intestinal peptide in the rat: its reciprocal action to cholecystokinin octapeptide

Intracerebroventricular administration of vasoactive intestinal peptide (VIP) shortened the duration of pentobarbital-induced sleep and produced significant hypermotility in the rat. Although hypermotility induced by methamphetamine was not potentiated by central administration of VIP, L-DOPA-induced hypermotility in pargyline-pretreated rats was markedly enhanced by VIP and this hypermotility was suppressed by simultaneous administration of cholecystokinin octapeptide (CCK-8) in a dose-related manner. Apomorphine-induced hypermotility was also potentiated by VIP. These results suggest that VIP may stimulate postsynaptic dopaminergic receptor, causing an increase in motility, and that a possible reciprocal interaction exists between VIP and CCK-8.     

In vivo activities of peptide and pseudo-peptide analogs of the C-terminal octapeptide of cholecystokinin on pancreatic secretion in the rat

Most studies measuring the agonist and antagonist activities of CCK analogs and derivatives on the exocrine pancreas have been done with in vitro models. However, extrapolation to the in vivo situation may be sometimes hazardous, due to the catabolism of the peptides by circulating and tissue peptidases, and to their eventual interaction with various endogenous factors. The present experiments were organized to measure the efficacy and potency on pancreatic secretion of the rat in vivo of a series of CCK 8 analogs whose binding and activity had been previously measured on guinea-pig and rat isolated acini. The molecules tested were derivatives of Boc-(Nle 28-Nle 31)-CCK 26–33 (1), and comprised 2-phenylethylester derivatives, des-Phe derivatives, and a series of pseudo-peptides with a “reduced” bond CH2-NH replacing the peptide bond in position 28–29 to 32–33. They were perfused in anaesthetized rats, and the outputs of sodium, bicarbonate and total protein were measured. All of the derivatives studied had in vivo the same efficacy as (1) on the output of protein, and were 10 to 500 times less potent. For most compounds, the relative order of potencies measured in vivo was similar to that measured in vitro on amylase secretion by rat acini. However, the derivatives with reduced bonds in positions 28–29 and 29–30 were respectively 3 and 2 times less potent in vivo, relative to (1), while derivatives with reduced bonds in positions 30–31, 31–32 and 32–33 were 1.5 to 2.5 times more potent in vivo. The 2-phenylethylester derivatives were 7 and 9 times as potent in vitro as in vivo. The des-Phe derivative, which had in vitro antagonist properties on guinea-pig acini, and acted like a partial agonist on rat acini, was in vivo a complete agonist and was relatively 300 times as potent in vivo as in vitro. These results indicate that the metabolism of the peptides and/or their interaction with endogenous factors may change appreciably the effect of CCK derivatives on pancreatic secretion of the rat in vivo.     

Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTPγS as nucleotide activator, cholecystokinin as peptide hormone, and GDPβS and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (E_A/E_TOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant k₊₂, and a deactivation process (rate constant k_off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant k₂) and/or the dissociation of the intact nucleotide (rate constant k_?1), so that E_A/E_TOT = k₊₁/(k₊₁ + k₂ + k_?a). (2) The hormone CCK-8 increased the value of k₊₁ with GTP dose-dependently, from 0.2 to 10.9 min^?1. The value of k_?1 increased 0.01 to 0.3 min^?1 but the value of k₂ was unaltered at 7 min^?1, so that E_A/E_TOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 μg/ml allowed also a large increase in E_A/E_TOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the k_off value of the GTP-activated enzyme (from 7 min^? to 0.5 min^?1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of k₊₁ (from 0.2 to 0.8 min^?1) and the occurrence of a significant 0.3 min^?1 value for k_?1.     

Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): effects on motor behaviour, hexobarbital-induced sleep and harman-induced convulsions

Ten ceruletide analogues and cholecystokinin octapeptide (CCK-8) were compared with ceruletide regarding neuropharmacological effects in mice after subcutaneous administration. The effects under study were catalepsy, prolongation of hexobarbital-induced sleep and delay in onset of harman-induced convulsions. Ceruletide and several analogues were more potent than the reference drugs, diazepam and haloperidol. Desulfation, deamidation and shortening of the peptide chain by five amino acids strongly reduced or abolished the pharmacological activities of ceruletide. Other chemical modulations mostly weakened the efficacy, but to an unequal extent for the three effects, which altered the pharmacological selectivity.     

Inhibition of satiety by a cholecystokinin antagonist is independent of gastric emptying

The ability of a cholecystokinin antagonist Proglumide to inhibit satiety induced by intraperitoneal injections of cholecystokinin octapeptide (CCK-OP) and bombesin was examined in rats equipped with chronic gastric cannulae. Both CCK-OP and bombesin significantly suppressed sham feeding. Proglumide administered alone did not alter sham feeding but it abolished the suppression of feeding induced by CCK-OP. In contrast, Proglumide did not inhibit the effect of a low dose of bombesin, but partially inhibited satiety induced by a high dose of bombesin, thus confirming our previous findings. These results indicate that the effect of Proglumide is independent of its recently described effects on gastric emptying in rat.     

The effect of cholecystokinin octapeptide on pituitary-adrenal hormone secretion

The purpose of this investigation was to determine the influence of cholecystokinin octapeptide (CCK-OP) on pituitary-adrenal hormone secretion. CCK-OP at a dose of 5 μg/kg (i.p.) elevated plasma corticosterone from 27 to 43 μg/100 ml in one experiment and from 12 to 50 μg/100 ml in a second experiment: Lower doses of CCK-OP (0.5 μg/kg) elevated corticosterone from 12 μg/100 ml to 20 μg/100 ml. CCK-OP (1, 10, and 100 ng/ml) had no effect on ACTH-induced corticosterone released by isolated adrenal cells in vitro when tested in the presence of 50 pg of ACTH_1?24. 100 and 500 ng of CCK-OP resulted in an increased pituitary ACTH release equal to 123% (n.s.) and a 206% (P < 0.05) of control, respectively. In comparison, a $\text{3}\text{5}$ hypothalamic stalk median eminence equivalent increased ACTH release to 313% of control (P < 0.05). The exact mechanism of this CCK effect on pituitary ACTH release is unknown. Although it is likely that the direct effects on the pituitary in vitro represent a pharmacologic and not a physiologic effect of this peptide, in vivo doses are between doses used for pancreatic effects and satiety effects suggesting that there may be a physiologic stimulating action of this peptide on the hypothalamic-pituitary-adrenal axis but at a level above the adrenal and pituitary.     

Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): effects on isolated intestinal preparations and gallbladders of guinea pigs and mice

The smooth muscle stimulatory effects of cholecystokinin octapeptide (CCK-8), ceruletide (CER), ten analogues of CER, and carbachol were studied in isolated organs of the guinea pig and the mouse (stomach, ileum, duodenum, colon and gallbladder). On a molar basis, CCK-8 and CER had in all organs except stomach greater potency (lower EC50) than carbachol. The effectiveness (Emax) of CCK-8 and CER was in the gut less than that of carbachol, in the guinea pig gallbladder equal with and in the mouse gallbladder superior to that of carbachol. The alteration of peptide structure was virtually without influence on effectiveness; however, it greatly modified the potency and the organ selectivity of the effect. There was no clear-cut correlation between the potency to stimulate smooth muscle and to alter the behavior of the mouse.     

Characterization of cholecystokinin binding sites in rat cerebral cortex using a 125I-CCK-8 probe resistant to degradation

Specific binding sites for cholecystokinin (CCK) have been characterized in a particulate membrane fraction of rat cerebral cortex using a biologically active 125I-labeled derivative of the C-terminal octapeptide of CCK (CCK-8) prepared by reaction with the iodinated form of the imidoester (125IIE), methyl-p-hydroxybenzimidate. The time course of binding to cortical membranes was rapid, temperature dependent, and saturable. Half-maximal binding at 24 degrees C was reached in 30 min and full binding at 120 min. At 37 degrees C there was only a slight increase in 125IIE-CCK-8 bound after 15 min. The addition of a large excess of CCK-8 after 30 min of binding at 24 degrees C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of 125IIE-CCK-8 bound to membranes with increasing concentrations of CCK-8 and other structurally related peptides. CCK-8 displaced 50% of the radioligand at 4 nM, CCK-33 at 10 nM, and gastrin (desulfated CCK-8) at 60 nM. Secretin, a structurally unrelated peptide, was unable to displace the radioligand from cortical membranes at 1.0 microM. Finally, 125IIE-CCK-8 exposed to cortical membranes or to buffers that had previously contained such membranes for 60 min at 24 degrees C bound equally as well to fresh cortical membranes as control radioligand that had not been exposed to the same conditions. Thus the 125I-CCK-8 radioligand used in this study was highly resistant to degradative processes in rat brain tissue.     

Feeding and drinking responses in the golden hamster following treatment with cholecystokinin and angiotensin II

Drinking and feeding behaviours of female golden hamsters were examined following intracerebroventricular injections of angiotensin II or systemic and intracerebroventricular injections of cholecystokinin octapeptide. Injections of angiotensin II into the brain produced a dose-dependent drinking response in water repleted animals. Systemically, a low dose (0.5 microgram/kg body wt) of cholecystokinin was ineffective at reducing food intake of fasted animals during a 1 hr test. Larger peripheral doses (1.0 to 4.0 microgram/kg body wt), however, were effective at decreasing food intake. Injected in the lateral cerebral ventricle, nanogram doses of cholecystokinin decreased food consumption in a dose dependent manner. These results are discussed in relation to how these peptides regulate feeding and drinking behaviours in other species.     

Protective effect of CR 1409 (cholecystokinin antagonist) on experimental pancreatitis in rats and mice

CR 1409, a glutaramic acid derivative with competitive cholecystokinin-antagonistic activity, was administered IP and evaluated in comparison with proglumide (the model CCK-receptor antagonist), gabexate (protease inhibitor) and PGE₂ (cytoprotective) on two different models of experimental pancreatitis. Acute pancreatitis was induced in mice by six IP injections of 50 μg/kg caerulein at hourly intervals. The drugs were administered 30 minutes before each caerulein administration. Blood samples and pancreata were collected 3 hours after the last caerulein injection. In the second experiment, pancreatitis was induced in rats by injecting 0.3 ml 6% sodium taurocholate interstitially into the pancreas. The drugs were administered twice, 30 minutes before and 3 hours after taurocholate. The animals were killed 6 hours after laparotomy and blood samples and pancreata were collected. CR 1409 exhibited on both pancreatitis models a protective effect in a dose range of 0.3–10 mg/kg. Proglumide exhibited a protective activity at higher doses (200–400 mg/kg). Gabexate and PGE₂ were effective only in pancreatitis induced by taurocholate in a dose range of 30–60 mg/kg and 60–130 μg/kg respectively. These results, showing a high protective effect of CR 1409 on different models of acute pancreatitis, suggest an important role of CCK in the pathogenesis of pancreatitis.     

In vitro degradation of the C-terminal octapeptide of cholecystokinin by 'enkephalinase A'

As the C-terminal octapeptide of cholecystokinin represents a putative neurotransmitter in the central nervous system, the membrane-bound enzymes involved in its inactivation were investigated. Two aminopeptidases, involved in the cleavage of enkephalins, and a metalloendopeptidase were identified in extracts of solubilized synaptic membranes. The metalloendopeptidase, which cleaves CCK-8 at the Trp30-Met31 bond, appeared to be indistinguishable from 'enkephalinase A1' on the basis of its chromatographic behaviour, sensitivity to inhibitors and relative affinities for Met- and Leu-enkephalins. This finding indicates that CCK-8 is inactivated in vitro by the same peptidases as enkephalins.     

八肽胆囊收缩素的结构与活性关系

本文采用液相多肽合成法制备了八肽胆囊收缩素(CCK_8)的六种类似物,并测定了它们诱导离体的豚鼠胆囊收缩的生物学活性。发现CCK_8的N-端乙酰化不改变其生物活性,脱去N-端氨基的CCK_8类似物即Suc-CCK_7与母体CCK_8相比,其活性明显增加。在Boc-CCK_7中,Met被NIe取代活性可完全保留,Gly~(29)被D-Ala取代后仍保留相当的活性,但Gly~(29)被β-Ala取代后则失去胆囊收缩活性;在Met~(28)-Gly~(29)区域引入刚性的r-内酰胺环作为构象限制,也导致活性完全丧失。     

Nucleus tractus solitarius lesions block the behavioral actions of cholecystokinin

Cholecystokinin (CCK) has been implicated as a signal for the syndrome of satiety in a variety of species. Several lines of evidence point to a peripheral site of action for the behavioral effects of CCK. Peripheral CCK receptors appear to activate a gut-brain pathway involving the sensory fibers of the vagus nerve. To investigate the central anatomical substrate of this visceral-behavioral control system, the terminal regions of the sensory tract of the vagus were lesioned. Radiofrequency lesions of the nucleus tractus solitarius abolished the effects of acute doses of CCK on exploratory behaviors. Sham lesions had no effect on baseline exploratory behaviors and did not influence the ability of CCK to decrease spontaneous exploratory behaviors. These findings delineate the first central site along the ascending sensory pathway which appears to mediate the satiety-related behavioral effects of CCK.     

Effects of unilateral intracerebroventricular microinjections of cholecystokinin (CCK) on circling behavior of rats

Unilateral intracerebroventricular (ICV) injections of a high dose of CCK₇ have been reported to elicit barrel rotations accompanied by contralateral postural asymmetry; there was no spontaneous locomotor activity other than barrel rolling. The aim of the present study was to investigate the effects of lower doses of CCK-peptides on circling behavior; it was reasoned that if ambulation was present following unilateral ICV administrations of lower doses of CCK, then the contralateral postural asymmetry previously reported might be expressed as contraversive circling. In the present study, spontaneous locomotor activity was observed following ICV injections of lower doses of CCK sulfated octapeptide (CCK₈), desulfated CCK octapeptide (dCCK₈) and CCK tetrapeptide (CCK₄). As postulated, contraversive circling was induced by CCK₈ (0.5–5000 ng, ICV); the two other CCK fragments, dCCK₈ and CCK₄, were inactive in this respect. In addition, the contraversive circling bias induced by CCK₈ (5.0 ng, ICV) was attenuated by co-injections of the CCK antagonist proglumide (10 and 100 ng) and by intraperitoneal injections of the dopamine (DA) antagonist haloperidol (0.05 and 0.1 mg/kg, 45 min prior to ICV CCK₈). These data suggest that the effect is mediated by CCK receptors and through a facilitatory influence on central DA function.