CCK-8-stimulated insulin secretion in vivo is mediated by CCKA receptors.

The effects of the cholecystokinin A (CCKA) receptor antagonist, L-364,718, and the CCKB receptor antagonist, L-365,260, on CCK-8-stimulated insulin secretion were studied in vivo in the mouse. It was found that CCK-8-stimulated insulin secretion was suppressed by L-364,718 at a low dose level (0.078 mumol/kg). In contrast, L-365,260 caused a partial inhibition of CCK-8-stimulated insulin release only at the high dose level (24 mumol/kg). It is concluded that the CCK-8-stimulated insulin release in vivo is mediated by CCK receptors of the CCKA subtype.     

L364,718 antagonizes the cholecystokinin-induced suppression of locomotor activity

To determine the role of CCK-A receptors in the cholecystokinin (CCK)-induced suppression of locomotor activity in the rat, the ability of the selective CCK-A receptor antagonist L364,718 to block these responses was investigated. Cholecystokinin octapeptide (CCK8) (10, 100 micrograms/kg IP) and caerulein (1, 5, 10 micrograms/kg IP) produced marked reductions in locomotor activity whereas cholecystokinin tetrapeptide (CCK4) (100 micrograms/kg IP) was without effect. The reductions in activity produced by CCK8 (10 micrograms/kg) and caerulein (10 micrograms/kg) were antagonized by L364,718 (100 micrograms/kg IP). In an open field test CCK8 (10 micrograms/kg IP) reduced locomotor activity and total number of rears and increased pause duration. These effects of CCK8 on open-field behaviour were also antagonized by L364,718 (100 micrograms/kg IP). It is concluded that L364,718 is a potent antagonist of the actions of CCK8 and caerulein on locomotor activity, suggesting that the effects of these peptides are mediated by a CCK-A receptor.     

Effect of intracerebroventricular and systemic injections of caerulein,a CCK analogue,on electrical self-stimulation and its interaction with the CCKA receptor antagonist,L-364,718 (MK-329)

Systemic administration of caerulein (10–100 µg/kg SC), a potent analogue of cholecystokinin, caused a profound dose-related depression of variable-interval self-stimulation, followed by progressive recovery within 60 min. Intracerebroventricular injection of caerulein (3–1000 ng) was not more effective than systemic injection, while injections into the nucleus accumbens (3–100 ng bilaterally) were without detectable effect. Systemic injections of L-364,718 (70–700 µg/kg IP), a specific competitive antagonist of CCK_A (peripheral-type) receptors, had no effect on self-stimulation when given alone. When given in combination with caerulein, L-364,718 (200 µg/kg IP) significantly reduced the inhibitory effect of caerulein (30 µg/kg SC); however, this dose, and higher doses of L-364,718, failed to confer complete protection. It is concluded that self-stimulation performance may be subject to modulation by CCK receptors distributed predominantly in the peripheral nervous system and that some but not all of these receptors are CCK_A receptors.     

L-365,260, a potent CCK-B/gastrin receptor antagonist, suppresses gastric acid secretion induced by histamine and bethanechol as well as pentagastrin in rats.

We evaluated the effects of a potent cholecystokinin (CCK)-B/gastrin receptor antagonist, L-365,260 (3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin - 3-yl)-N'-( 3-methylphenyl) urea); a selective CCK-A receptor antagonist, devazepide (L-364,718); and cimetidine on gastric acid secretion induced by pentagastrin, histamine and bethanechol in anesthetized rats. We also evaluated the effects of L-365,260 and cimetidine on acid secretion in pylorus-ligated rats. Intravenous administration of L-365,260, L-364,718 and cimetidine dose-dependently reduced acid secretion induced by pentagastrin (20 nmol/kg/hr), with ED50 values of 0.63, 19.1 and 2.5 mumol/kg, respectively. Of interest was the finding that L-365,260, like cimetidine, dose-dependently inhibited acid secretion induced by histamine (100 mumol/kg/hr) and bethanechol (5 mumol/kg/hr) with ED50 values of 5.9 and 4.3 mumol/kg, respectively. L-364,718, even at 30 mumol/kg, i.v., had only a slight effect on histamine- or bethanechol-induced acid secretion. Gastric acid secretion was suppressed by treatment with L-365,260 (3-100 mumol/kg, i.v.) and cimetidine (11.9-396.4 mumol/kg, i.v.) in pylorus-ligated rats, with ED50 values of 13.3 and 96.9 mumol/kg, respectively. These results indicate that L-365,260 suppresses acid secretion induced by histamine and bethanechol in rats and that the gastrin receptor plays an important role in acid secretion in pylorus-ligated rats.     

Enhancement of morphine analgesia and prevention of morphine tolerance in the rat by the cholecystokinin antagonist L-364,718

The potent and selective non-peptide cholecystokinin (CCK) antagonist L-364,718 (0.5-2.0 mg/kg s.c.) enhanced the analgesia induced by acute morphine treatment in the rat tail flick test. Chronic treatment with L-364,718 (1.0 mg/kg) prevented the development of tolerance to morphine analgesia (after a 6 day period of morphine treatment) but did not influence the onset of opioid dependence. Since L-364,718 is considerably more potent in inhibiting CCK binding to peripheral tissues than to brain membranes its interaction with morphine is surprising. The exact locus of this interaction, or whether it involves 'peripheral-type' (CCK-A) or 'central-type' (CCK-B) receptors is not known.     

Inhibition of gastrin- and histamine-stimulated gastric acid secretion by gastrin and cholecystokinin antagonists in the rat

The gastric acid inhibitory activities of a peptide-like gastrin receptor antagonist, Boc-beta Ala-Trp-Leu-Asp-O(CH2)2-Ph-4-F (CH-486), a nonpeptide gastrin/CCK-B antagonist (L-365,260), and a CCK-A antagonist (L-364,718), were investigated in the gastric lumen-perfused anaesthetized rat. A single i.v. injection of CH-486, 100 mumol/kg, reduced acid secretion stimulated by pentagastrin, 15 micrograms kg/h, to unstimulated levels, with no recovery within 50 min. Histamine-, 0.1 mumol kg/min, and carbamylcholine-, 0.1 mg kg/h, stimulated acid secretion were also reduced to unstimulated levels by CH-486, 100 mumol/kg, although with these latter two stimulants the inhibition was transient. L-365,260 and L-364,718, 10 mumol/kg, significantly inhibited both pentagastrin- and histamine-stimulated acid secretion, the latter again transiently. We conclude that the non-selective nature of the gastric acid inhibitory activity of gastrin antagonists might allow novel approaches to control gastric acid secretion in peptic ulcer disease.     

Differential involvement of CCK-A and CCK-B receptors in the regulation of locomotor activity in the mouse

The influence of the CCK-A antagonist devazepide and the CCK-B/gastrin antagonist L-365,260 on the locomotor activity of mice was studied. Devazepide and L-365,260 had opposite effects on spontaneous locomotor activity, and on caerulein- and apomorphine-induced hypomotility in the mouse. Devazepide in high doses (0.1-1 mg/kg IP) reduced spontaneous motor activity, whereas L-365,260 at a high dose (1 mg/kg IP) increased the activity of mice. Devazepide (0.1-10 micrograms/kg) moderately antagonized the sedative effect of apomorphine (0.1 mg/kg SC) and caerulein (25 micrograms/kg SC), whereas L-365,260 (1-10 micrograms/kg) significantly potentiated the actions of dopamine and CCK agonists. Concomitant administration of caerulein (15 micrograms/kg SC) and apomorphine (0.1 mg/kg SC) caused an almost complete loss of locomotor activity in the mouse. Devazepide and L-365,260 (0.1-10 micrograms/kg) were completely ineffective against caerulein-induced potentiation of apomorphine hypomotility. Devazepide in high doses (0.1-1 mg/kg), reducing the spontaneous motor activity of mice, counteracted the motor excitation induced by d-amphetamine (5 mg/kg IP). The CCK agonist caerulein (100 micrograms/kg SC) had a similar antiamphetamine effect. Devazepide (1-100 micrograms/kg) and L-365,260 (1 micrograms/kg) reversed completely the antiamphetamine effect of caerulein. The results of present study reflect apparently distinct role of CCK-A and CCK-B receptors in the regulation of motor activity. The opposite effect of devazepide and L-365,260 on caerulein- and apomorphine-induced hypolocomotion is probably related to the antagonistic role of CCK-A and CCK-B receptor subtypes in the regulation of mesencephalic dopaminergic neurons. The antiamphetamine effect of caerulein is possibly linked to the stimulation of CCK-A receptors in the mouse brain, whereas the blockade of both subtypes of the CCK-8 receptor is involved in the antiamphetamine effect of devazepide.     

Characterization of [3H](+/-)L-364,718 binding to solubilized cholecystokinin (CCK) receptors of rat pancreas

The binding of [3H](+/-)L-364,718 (3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl )-1H-indole-2-carboxamide), an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. [3H](+/-)L-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound [3H](+/-)L-364,718. Specific [3H](+/-)L-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.53 nM and a Bmax of 3.1 pmol/mg protein. Specific [3H](+/-)L-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific [3H](+/-)L-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10-100 times. Collectively, the data presented indicate that [3H](+/-)L-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by [3H](+/-)L-364,718 binding determinations revealed an estimated molecular weight of 400,000 daltons.     

Cholecystokinin and morphine-induced hypothermia.

The effects of cholecystokinin-8 sulfate (CCK-8), cholecystokinin-8 unsulfate (CCK-8U), cholecystokinin-4 (CCK-4), caerulein and morphine on mice core body temperature have been studied in the present work. Subcutaneous injection of different doses of caerulein (0.05, 0.1 and 0.5 mg/kg), CCK-8 (0.05, 0.1 and 0.25 mg/kg) and morphine (10, 20 and 30 mg/kg) induced hypothermia. CCK-8U and CCK-4 did not elicit any response. The hypothermic response induced by caerulein, a CCK-related decapeptide but not morphine was decreased by selective CCK(A) receptor antagonist MK-329. However, the hypothermia induced by morphine but not caerulein was reduced by opioid antagonist naloxone. When morphine plus caerulein was administered a higher hypothermia was induced. Pretreatment of animals with L-365 260, a selective CCK(B) receptor antagonist did not alter the hypothermia induced by the drugs. The response induced by combination of the both drugs was decreased by MK-329. Administration of CCK antagonists MK-329 and L-365 260 to mice did not exert any effect on temperature. It is concluded that the CCK(A) receptor mechanism may be involved in the hypothermic effect of CCK agonists or morphine, while opioid receptor mechanism is not involved in CCK receptor agonists' response.     

Memory impairments induced by peripherally administered cholecystokinin a-type receptor antagonists in rats

Intraperitoneal injection of cholecystokinin (CCK) A-type receptor antagonists, L-364,718 and CR 1409, caused dose-dependent memory impairments in passive and active avoidance responses and in Morris water pool test in rats. These antagonist effects were significant at a dose of 1 mg/kg, while with proglumide, 50–100 mg/kg were required to produce a similar memory deficit. The results suggest that loss of biologically active CCK octapeptide (CCK-8), not only in the brain but also in the peripheral tissues, causes memory impairmets in rats. © 1992 Wiley-Liss, Inc.     

Effect of metoclopramide, bethanechol and the cholecystokinin receptor antagonist, L-364,718, on gastric emptying in the rat

The prokinetic effects of metoclopramide, bethanechol and L-364,718 on a semisolid meal and solid pellet gastric emptying were evaluated and compared. Each compound increased the rate of meal emptying as measured 90 min post-dose. L-364,718, a non-peptide CCK antagonist, was the most potent of these three agents with statistically significant activity observed at 0.03, 0.1 and 0.3 mg/kg p.o. Only metoclopramide significantly enhanced pellet emptying in a dose-dependent manner (3-30 mg/kg p.o.). The effects of each test agent and the potential physiologic role of cholecystokinin in regulating gastric emptying are discussed.     

Cholecystokinin antagonists. Synthesis and biological evaluation of 3-substituted benzolactams

A series of 1,3-substituted benzolactams are reported that are potent nonpeptidal antagonists of the peptide hormone cholecystokinin (CCK). Design considerations were based upon the natural product CCK antagonist asperlicin and the potent benzodiazepine antagonist series exemplified by L-364,718 (1). Compound 19, the most potent compound in the benzolactam series, had an IC50 = 3 nM for inhibition of binding of 125I-CCK-8 to CCK receptors in rat pancreatic tissue, and its racemic analogue 8 was found to be orally active in inhibiting CCK-induced gastric emptying in mice, with an ED50 = 2.6 mg/kg po. The effects of ring size, substitution at positions 1 and 3, and stereochemistry at position 3 are discussed. Conformational studies of compound 19 and L-364,718 have delineated similarities that these molecules share in their core conformations and substituent orientations.     

Cholecystokinin receptor agonists block the jumping behaviour precipitated in morphine-dependent mice by naloxone.

The aim of present study was to reveal the role of cholecystokinin (CCK) in the jumping behaviour induced by the opioid antagonist naloxone (30 mg/kg) after the acute administration of morphine (200 mg/kg) in mice. Treatment with caerulein (0.01-1 microg/kg), a nonselective agonist of CCK receptors, induced a large reduction of jumping frequency without parallel suppression of locomotor activity. The CCK(B) receptor agonist CCK tetrapeptide (CCK-4. 0.125-32 mg/kg) caused the same effect, but it happened at much higher doses (above 0.5 mg/kg). Devazepide (1 microg/kg), a preferential CCK(A) receptor antagonist, completely reversed the action of caerulein (0.1 gmg/kg) and CCK-4 (2 mg/kg). A preferential CCK(B) receptor antagonists LY 288,513 at a high dose (4 mg/kg) blocked the action of CCK-4, but not that of caerulein. Acetorphan (16-128 mg/kg), an inhibitor of enkephalin metabolism, did not block naloxone-precipitated jumping behaviour. However, the combination of subthreshold doses of caerulein (0.001 microg/kg) and CCK-4 (0.25 mg/kg) with acetorphan (64 mg/kg) potently antagonized the behaviour induced by naloxone. In conclusion, the antagonism of CCK agonists against naloxone-precipitated jumping behaviour is apparently mediated via the CCK(A) receptor subtype. The stimulation of CCK(A) receptors seems to increase the release of endogenous enkephalins.     

The cholecystokinin antagonist L-364,718 inhibits the action of cholecystokinin but not bombesin on rat pancreatic secretion in vivo

The action of an antagonist of peripheral CCK receptors, L-364,718 on CCK8 and bombesin-induced pancreatic exocrine secretion was studied in anaesthetized rats. Both CCK8 and bombesin increased the rate of flow and rate of enzyme secretion from the pancreas. Intravenous L-364,718 inhibited the actions of CCK8, but not those of bombesin. It is concluded that bombesin acts on the exocrine pancreas directly rather than through the release of CCK.     

Changes in motor activity and forebrain [propionyl-3H]propionylated-CCK-8 binding in mice after repeated administration of drugs affecting cholecystokinin receptors.

The effects of acute or repeated treatment of male albino BKW mice with caerulein, a cholecystokinin octapeptide (CCK-8) agonist, and with devazepide (MK-329) and L-365,260, antagonists at CCKA ('peripheral') and CCKB ('central') receptors respectively, on motor activity and [propionyl-3H]propionylated-CCK-8 ([3H]pCCK-8) binding were studied. Acute treatment with a large dose of caerulein (100 micrograms/kg s.c.) suppressed motor activity (line crossings and rearings) whereas devazepide (2 mg/kg i.p.) had the opposite action. L-365,260 (2 mg/kg i.p.) increased only the number of rearings. Tolerance developed to the locomotor effects of caerulein and devazepide when these same doses were administered once daily (caerulein) or twice daily (devazepide) for 10 days. Twice daily administration of L-365,260 (2 mg/kg) for 10 days did not significantly alter the locomotor activity of mice. The sedative effect of caerulein (20 micrograms/kg s.c.) was markedly reduced in mice receiving repeated injections of either a larger amount of caerulein (100 micrograms/kg) or devazepide but not after L-365,260. The stimulant effect of (+)-amphetamine (2 mg/kg s.c.) on motor activity was increased by subchronic administration of either devazepide or caerulein, but not by L-365,260. All three compounds (caerulein, devazepide and L-365,260) increased the number of [( 3H]pCCK-8 binding sites in mouse forebrain but the increase was only significant after L-365,260. The effects of long-term treatment with caerulein are probably related to the stimulation of CCKA receptors, whereas the paradoxically similar action of devazepide may be linked to the blockade of both subtypes of the CCK-8 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the Mechanism(s) of Cholecystokinin (CCK): Receptor Stimulation Attenuates Morphine Dependence in Mice

Abstract: 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.     

Structure-activity and dose-effect relationships of the antagonism of picrotoxin-induced seizures by cholecystokinin, fragments and analogues of cholecystokinin in mice

Intraperitoneal administration of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and nonsulphated cholecystokinin octapeptide (CCK-8-NS) enhanced the latency of seizures induced by picrotoxin in mice. Experiments with N- and C-terminal fragments revealed that the C-terminal tetrapeptide (CCK-5-8) was the active centre of the CCK octapeptide molecule. The analogues CCK-8-SE and CCK-8-NS (dose range 0.2-6.4 mumol/kg) and caerulein dose range 0.1-0.8 mumol/kg) showed bell-shaped dose-effect curves, with the greatest maximum inhibition for CCK-8-NS. The peptide CCK-5-8 had weak anticonvulsant activity in comparison to the octapeptides, 3.2 mumol/kg and larger doses of the reference drug, diazepam, totally prevented picrotoxin-induced seizures and mortality. The maximum effect of the peptides tested was less than that of diazepam. Experiments with analogues and derivatives of CCK-5-8 demonstrated that the effectiveness of the beta-alanyl derivatives of CCK-5-8 were enhanced and that they were equipotent with CCK-8-SE. Of the CCK-2-8 analogues, Ser(SO3H)7-Ac-CCK-2-8-SE and Thr(SO3H)7-Ac-CCK-2-8-SE and Hyp(SO3H)-Ac-CCK-2-8-SE were slightly more active than CCK-8-SE.     

The CCK-A receptor antagonist, devazepide, blocks the anorectic action of CCK but not peripheral serotonin in rats.

A role has been proposed for cholecystokinin (CCK)-A-type receptors in mediating the anorectic action produced by serotonergic stimulation in rats. We examined the effect of pretreatment with the CCK-A antagonist devazepide (DVZ) on anorexia produced by peripheral administration of serotonin [5-hydroxytryptamine (5-HT)] or CCK-8 in 3-h food-deprived rats consuming a 30-min test meal of sweetened mash. The anorectic effect of CCK-8 (4.0 nmol/kg, IP) was antagonized in a dose-dependent manner by DVZ (0.03, 0.10, and 0.30 mumol/kg, IP), with even the lowest dose producing a significant reversal. Under identical testing conditions, a supramaximal dose of DVZ (.75 mumol/kg) did not attenuate the reductions in food intake produced by either a moderate (4.0 mumol/kg) or a high dose (10.0 mumol/kg) of 5-HT. These data confirm established findings that the anorectic action of peripheral CCK depends upon CCK-A receptors. However, peripherally administered 5-HT reduces food intake independently of CCKergic function.     

Characterization of neuronal cholecystokinin receptor by L-364,718 in Auerbach's plexus

The release of [3H]acetylcholine [( 3H]ACh) from Auerbach's plexus and the contraction of longitudinal muscle strips in response to the administration of cholecystokinin (CCK) were measured and recorded simultaneously. The peripheral CCK receptor antagonist, 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-1H- indole-2-carboxamide (L-364,718), antagonized the ACh releasing effect of CCK in a dose-dependent manner. The IC50 value and the dissociation constant (KD) were 41.0 +/- 2.0 pM and 0.06 +/- 0.01 nM, respectively. These results suggest that L-364,718 is a very potent antagonist of the neuronal CCK receptors.     

Mechanism of action of the anti-shock effect of CCK-8: influence of CCK antagonists and of sympatholytic drugs

In an experimental model of haemorrhagic shock that causes 100% mortality in rats within 30 min, the intravenous bolus injection (20 micrograms/kg) of sulfated cholecystokinin octapeptide (CCK-8) induces a prompt and sustained rise in blood pressure and pulse amplitude, all treated animals still surviving at the end of the experiment (2 h). This effect of CCK-8 is completely blocked by reserpine (5 mg/kg i.p.), significantly antagonized by prazosin (0.1 mg/kg i.v.) and yohimbine (1 mg/kg i.v.), and unaffected by practolol (15 mg/kg i.v.) and proglumide (0.2 mg/kg i.v.); it is completely antagonized by the intravenous (0.01-0.05 mg/kg), but not by the intracerebroventricular (0.002 mg/kg) injection of the 'peripheral' CCK antagonist, L-364,718. The present data indicate that the cardiovascular effects of CCK-8 in haemorrhagic shock involve peripheral CCK receptors, and require the functional integrity of the sympathetic nervous system.